Invasive Ductal Carcinoma Samples Research Articles

72P - Survivin gene expression in the primary tumor and circulating tumor cells – a new biomarker of tumor progression of breast cancer

Breast cancer is a leading cause of morbidity and mortality of the female population from malignant tumors. Distant metastases are the main cause of death of patients, a substrate for the development of which are circulating tumor cells (CTCs). However, the search for these cells alone is not sufficient to provide full information about the nature and course of tumor in a single patient. Determination of the expression of tumor-genes responsible for the different processes of tumor progression allows a more complete picture. Such genes include the gene survivin (BIRC5) family of inhibitors of apoptosis (IAP). Using real-time PCR we investigated the expression of the survivin gene in 36 samples of primary invasive ductal carcinoma of the breast, 10 samples of benign tumors - fibroadenoma of the breast, as well as 36 samples of peripheral blood of patients with breast cancer at various stages of tumor and stage specific treatment, and 10 healthy people as controls. In primary breast carcinoma we determined a high expression of survivin gene in all 36 samples with the average value (M ± m) 1.58 ± 0.31 (min – 1.19; max – 4.41). The highest figures were found in tumors of medium and high grade (G II-III) with lymphovenous invasion (LVSI +). In 2 of 3 samples of benign tumor expression of survivin was not found, and one was 0.015. In CTCs, isolated from peripheral blood of breast cancer patients, all 36 samples as determined by the gene expression of survivin with an average value (M ± m) 1.10 ± 0.19 (min – 0.36; max – 3.79). The level of expression of the control samples did not exceed 0.003. It should be noted that the maximum volume of expression was obtained in samples of tumor patients with stage N +, and especially M1, on TNM classification. Any legitimate expression of survivin, depending on the size of the tumor had been received. In patients, receiving chemotherapy average expression of survivin gene was obswerved, but it never approached the indicators of control. Determination of expression of the survivin gene in primary tumor and in CTCs may be one of the most promising markers of tumor progression and for monitoring of breast cancer therapy.

FISHtrees 3.0: Tumor Phylogenetics Using a Ploidy Probe.

Advances in fluorescence in situ hybridization (FISH) make it feasible to detect multiple copy-number changes in hundreds of cells of solid tumors. Studies using FISH, sequencing, and other technologies have revealed substantial intra-tumor heterogeneity. The evolution of subclones in tumors may be modeled by phylogenies. Tumors often harbor aneuploid or polyploid cell populations. Using a FISH probe to estimate changes in ploidy can guide the creation of trees that model changes in ploidy and individual gene copy-number variations. We present FISHtrees 3.0, which implements a ploidy-based tree building method based on mixed integer linear programming (MILP). The ploidy-based modeling in FISHtrees includes a new formulation of the problem of merging trees for changes of a single gene into trees modeling changes in multiple genes and the ploidy. When multiple samples are collected from each patient, varying over time or tumor regions, it is useful to evaluate similarities in tumor progression among the samples. Therefore, we further implemented in FISHtrees 3.0 a new method to build consensus graphs for multiple samples. We validate FISHtrees 3.0 on a simulated data and on FISH data from paired cases of cervical primary and metastatic tumors and on paired breast ductal carcinoma in situ (DCIS) and invasive ductal carcinoma (IDC). Tests on simulated data show improved accuracy of the ploidy-based approach relative to prior ploidyless methods. Tests on real data further demonstrate novel insights these methods offer into tumor progression processes. Trees for DCIS samples are significantly less complex than trees for paired IDC samples. Consensus graphs show substantial divergence among most paired samples from both sets. Low consensus between DCIS and IDC trees may help explain the difficulty in finding biomarkers that predict which DCIS cases are at most risk to progress to IDC. The FISHtrees software is available at ftp://ftp.ncbi.nih.gov/pub/FISHtrees.

Relationship between the expression of Notch1 and EZH2 and the prognosis of breast invasive ductal carcinoma.

We determined whether the coexpression of Notch1 and EZH2 influences the progression and prognosis of breast invasive ductal carcinoma. Using the χ(2) test, a significant difference was found between high and low expression of Notch1 in terms of lymph node, hormone receptor, and p53 expression (P < 0.05). Moreover, a significant difference was found between high and low expression of EZH2 in terms of tumor size, histologic grade, hormone receptor, and expression of Ki67 (P < 0.05). Using Pearson correlation analysis, we found a significant positive correlation between Notch1 and EZH2 expression in the tissue samples of breast invasive ductal carcinoma (P = 0.038). High Notch1 and EZH2 expression was associated with poor progression-free survival compared with low expression (PNotch1 = 0.000, 40.3 vs 48.9 months; PEZH2 = 0.000, 40.2 vs 49.9 months). Moreover, we found that high Notch1 and EZH2 expression was associated with poor overall survival compared with low expression (PNotch1 = 0.000, 51.2 vs 56.2 months; PEZH2 = 0.002, 51.7 vs 56.4 months). In conclusion, Notch1 and EZH2 coexpression contributes to the progression and prognosis of breast invasive ductal carcinoma.

Comparative proteomic analysis of ductal and lobular invasive breast carcinoma.

Breast cancer is the second most common cancer worldwide and the first among women. Invasive ductal carcinoma (IDC) and invasive lobular carcinoma (ILC) are the two major histological subtypes, and the clinical and molecular differences between them justify the search for new markers to distinguish them. As proteomic analysis allows for a powerful and analytical approach to identify potential biomarkers, we performed a comparative analysis of IDC and ILC samples by using two-dimensional electrophoresis and mass spectrometry. Twenty-three spots were identified corresponding to 10 proteins differentially expressed between the two subtypes. ACTB, ACTG, TPM3, TBA1A, TBA1B, VIME, TPIS, PDIA3, PDIA6, and VTDB were upregulated in ductal carcinoma compared to in lobular carcinoma samples. Overall, these 10 proteins have a key role in oncogenesis. Their specific functions and relevance in cancer initiation and progression are further discussed in this study. The identified peptides represent promising biomarkers for the differentiation of ductal and lobular breast cancer subtypes, and for future interventions based on tailored therapy.

Identifying Differences Between Normal and Invasive Ductal Carcinoma grades for Breast tissues By FT-IR Spectroscopy

Breast cancer is one of the most important malignant forms of cancers and it represents a great threat to life for women. FT- IR spectra ware taken for 98 samples of breast tissues that were previously histopathologically identified by pathologist experienced as: 63 normal (N) samples and 35 invasive ductal carcinoma (IDC) samples that were classified in different grades 19 G1 samples,7 G2samples and 9 G3 samples in Mid-IR frequency range between 400 cm-1 and 4000 cm-1. Many spectral differences were observed in the frequency regions N-H stretching, Amide bands, C-H vibrations, and 950-1400 cm-1. The aim of this study is analyze different grades of IDC spectroscopically to evaluate the efficiency of FT-IR spectroscopy to differentiate between these grades. The results show considerable decrease in the lipid and carbohydrate content with the carcinoma grades (from G1 to G3), while, protein, collagen and nucleic acid (DNA) reveal slight increase with the carcinoma grade, that will be useful in classifying three different nuclear grades. This study demonstrates that FT-IR spectroscopy is a promising tool for accurate, rapid diagnosis of breast canser.

Expression of CPEB4 in invasive ductal breast carcinoma and its prognostic significance.

AimsCytoplasmic polyadenylation element binding proteins (CPEBs) are RNA-binding proteins that regulate translation by inducing cytoplasmic polyadenylation. CPEB4 has been reported in association with tumor growth, vascularization, and invasion in several cancers. To date, the expression of CPEB4 with clinical prognosis of breast cancer was never reported before. We aim to investigate the expression of CPEB4 and its prognostic significance in invasive ductal breast carcinoma.MethodsImmunohistochemical staining of CPEB4 and estrogen receptor, progesterone receptor, and human epidermal growth factor receptor was performed in 107 invasive ductal carcinoma (IDC) samples, and prognostic significance was evaluated.ResultsHigh expression of CPEB4 was observed in 48.6% of IDC samples. Elevated CPEB4 expression was possibly related to increased histological grading (P=0.037) and N stage (P<0.001). Patients with high expression of CPEB4 showed shorter overall survival (P=0.001). High CPEB4 expression was an independent prognostic factor for overall survival (P=0.022, hazard ratio =4.344, 95% confidence interval =1.235–15.283).ConclusionHigh CPEB4 expression is associated with increased histological grading and N stage, and it can serve as an independent prognostic factor in IDC.

Upregulation of RHOXF2 and ODF4 Expression in Breast Cancer Tissues.

During the past decade, the importance of biomarker discovery has been highlighted in many aspects of cancer research. Biomarkers may have a role in early detection of cancer, prognosis and survival evaluation as well as drug response. Cancer-testis antigens (CTAs) have gained attention as cancer biomarkers because of their expression in a wide variety of tumors and restricted expression in testis. The aim of this study was to find putative biomarkers for breast cancer. In this applied-descriptive study, the expression of 4 CTAs, namely acrosin binding protein (ACRBP), outer dense fiber 4 (ODF4), Rhox homeobox family member 2 (RHOXF2) and spermatogenesis associated 19 (SPATA19) were ana- lyzed at the transcript level in two breast cancer lines (MCF-7 and MDA-MB-231), 40 invasive ductal carcinoma samples and their adjacent normal tissues as well as 10 fibroadenoma samples by means of quantitative real-time reverse transcription polymerase chain reaction (RT-PCR). All four genes were expressed in both cell lines. Expression of ODF4 and RH- OXF2 was detected in 62.5% and 60% of breast cancer tissues but in 22.5 and 17.5% of normal tissues examined respectively. The expression of both RHOXF2 and ODF4 was upregulated in cancerous tissues compared with their normal adjacent tissues by 3.31 and 2.96-fold respectively. The expression of both genes was correlated with HER2/neu overexpression. RHOXF2 expression but not ODF4 was correlated with higher stages of tumors. However, no significant association was seen between expression patterns and estrogen and progesterone receptors status. ODF4 and RHOXF2 are proposed as putative breast cancer biomarkers at the transcript level. However, their expression at protein level should be evaluated in future studies.

The phosphorylation status of VASP at serine 322 can be predictive for aggressiveness of invasive ductal carcinoma.

Vasodilator-stimulated phosphoprotein (VASP) signaling is critical for dynamic actin reorganization processes that define the motile phenotype of cells. Here we show that VASP is generally highly expressed in normal breast tissue and breast cancer. We also show that the phosphorylation status of VASP at S322 can be predictive for breast cancer progression to an aggressive phenotype. Our data indicate that phosphorylation at S322 is gradually decreased from normal breast to DCIS, luminal/ER+, HER2+ and basal-like/TN phenotypes. Similarly, the expression levels of PKD2, the kinase that phosphorylates VASP at this site, are decreased in invasive ductal carcinoma samples of all three groups. Overall, the phosphorylation status of this residue may serve as an indicator of aggressiveness of breast tumors.

Abstract 4075: Proportion of breast cancer stem cells in fine needle aspirates co-relates with the marker of metastatic outcome TMEM

Abstract Cell surface biomarkers CD44, CD24 and CD133 and intracellular marker ALDH1 have recently been used to identify breast cancer stem cells. A very small number of these cancer stem cells can initiate a tumor growth and cause it to metastasize. The micro-anatomical sites of hematogenous dissemination of breast cancer cells called Tumor MicroEnvironment of Metastasis (TMEM) have been described in formalin-fixed paraffin-embedded invasive ductal carcinomas from patients. These sites are defined as the direct apposition of tumor cells and perivascular macrophages and are visualized using triple immunohistochemistry for CD68, CD31 and Mena to identify the macrophages, endothelial cells and invasive tumor cells respectively. The identification of TMEM sites is clinically important because TMEM density is a predictor of metastatic outcome. Migratory subpopulation of tumor cells has been shown to be enriched for cancer stem cells. We have performed flow cytometry to identify CD44high/CD24low cells, mRNA fluorescent in situ hybridization (FISH) and qRT-PCR to identify CD133 and ALDH1 expression in breast cancer cells in 50 samples of invasive ductal carcinoma obtained from patients’ cancer excisions by fine needle aspiration (FNA) before formalin fixation. FNA is a minimally invasive method that uses 26 gauge needles to collect discohesive cancer cells primarily by capillary action. The stem cell marker expression was then compared to TMEM scores obtained from the very same cancer excisions after formalin fixation and paraffin embedding. Our results show very strong correlation between the percentage of CD44high/CD24low cells with TMEM scores (r = 0.91), as well as the percentage of CD133 and ALDH1 expressing cells with TMEM scores (r = 0.88 and 0.86 respectively). We validated the results obtained by FISH with the direct qRT-PCR for CD133 and ALDH1 in the FNA samples. The correlation of CD133 and ALDH1 expression obtained by qRT-PCR with TMEM score was again very strong (r = 0.76 and 0.73 respectively). Our findings here and those previously reported for migratory tumor cells demonstrate that TMEM rich microenvironments are enriched for cancer stem cells and partially explain positive correlation of TMEM scores with metastasis in patients. They also suggest that the assessment of stem cell proportion in breast cancers can be assessed in FNA samples. The clinical value of stem cell assessment using this approach for prognostic and predictive is under investigation. Citation Format: Maja H. Oktay, Eli Grunblatt, Sweta Roy, Nathan Agi, Esther Adler, Joan G. Jones, John S. Condeelis, Sumanta Goswami. Proportion of breast cancer stem cells in fine needle aspirates co-relates with the marker of metastatic outcome TMEM. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4075. doi:10.1158/1538-7445.AM2015-4075

Zinc distribution within breast cancer tissue: A possible marker for histological grading?

Focusing on the trace metal zinc as a potential biomarker for breast cancer, the literature describes bulk zinc concentrations in breast cancer tissue to be higher than in normal tissue. From a histopathological point of view, cancer cells are intermingled with normal cells of the stroma within breast cancer tissues; therefore, bulk analysis cannot reflect this situation adequately. To address this problem, analysis of zinc distribution in histological sections is the method of choice. In the present study, nine samples of invasive ductal and lobular breast carcinoma of histological grade 1-3 were investigated, clearly differentiating between cancer and stroma areas. Zinc concentrations were determined by laser ablation inductively coupled plasma mass spectrometry applying a calibration technique based on spiked polyacrylamide gels. Direct comparison between hematoxylin- and eosin-stained tissues and zinc contour plots revealed that zinc is enriched in cancer tissue containing tumor cells in contrast to normal stroma. Moreover, zinc concentration in carcinomatous tissues directly correlates with the histological malignancy grade. Differentiation between carcinomatous tissue and stroma by determination of zinc content and the correlation of zinc concentration with the histological malignancy grade not only provides a key feature for clinical decision making for cancer therapy but also suggests the trace metal zinc as a potential biomarker for breast cancer.

PDIA3 and PDIA6 gene expression as an aggressiveness marker in primary ductal breast cancer.

Changes in the expression of the protein disulfide isomerase genes PDIA3 and PDIA6 may increase endoplasmic reticulum stress, leading to cellular instability and neoplasia. We evaluated the expression of PDIA3 and PDIA6 in invasive ductal carcinomas. Using reverse transcription-quantitative polymerase chain reaction, we compared the mRNA expression level in 45 samples of invasive ductal carcinoma with that in normal breast samples. Increased expression of the PDIA3 gene in carcinomas (P = 0.0009) was observed. In addition, PDIA3 expression was increased in tumors with lymph node metastasis (P = 0.009) and with grade III (P < 0.02). The PDIA6 gene showed higher expression levels in the presence of lymph node metastasis (U = 99.00, P = 0.0476) and lower expression for negative hormone receptors status (P = 0.0351). Our results suggest that alterations in PDIA3/6 expression levels may be involved in the breast carcinogenic process and should be further investigated as a marker of aggressiveness.

Exploring the Functional Disorder and Corresponding Key Transcription Factors in Intraductal Papillary Mucinous Neoplasms Progression.

This study has analyzed the gene expression patterns of an IPMN microarray dataset including normal pancreatic ductal tissue (NT), intraductal papillary mucinous adenoma (IPMA), intraductal papillary mucinous carcinoma (IPMC), and invasive ductal carcinoma (IDC) samples. And eight clusters of differentially expressed genes (DEGs) with similar expression pattern were detected by k-means clustering. Then a survey map of functional disorder in IPMN progression was established by functional enrichment analysis of these clusters. In addition, transcription factors (TFs) enrichment analysis was used to detect the key TFs in each cluster of DEGs, and three TFs (FLI1, ERG, and ESR1) were found to significantly regulate DEGs in cluster 1, and expression of these three TFs was validated by qRT-PCR. All these results indicated that these three TFs might play key roles in the early stages of IPMN progression.

Implications of the Notch1-Snail/Slug-epithelial to mesenchymal transition axis for lymph node metastasis in infiltrating ductal carcinoma

Emerging evidence suggests that activation of the Notch1 signaling pathway inducing epithelial to mesenchymal transition (EMT) mediated by Snail/Slug promotes invasion and metastasis of breast cancer cells in vitro. However, the implication of the Notch1-Snail/Slug-EMT axis in breast cancer patients remains unclear. A total of 200 formalin-fixed paraffin-embedded samples of invasive ductal carcinoma (IDC), and 37 adjacent non-neoplastic tissue (ANNT) samples from patients who had not been treated with neoadjuvant therapy were examined. Expression of Notch1, Slug, Snail, E-cadherin, N-cadherin, and vimentin was determined by immunohistochemistry on tissue microarrays (TMAs). The correlation between protein expression and clinicopathological characteristics of breast cancer patients was also evaluated. Results showed that a significantly high percentage of cases with high expression of Notch1 (74%, 148/200), Slug (36%, 72/200), Snail (62%, 124/200), and N-cadherin (77%, 153/200) and a low percentage of cases with high expression of E-cadherin (27%, 54/200) were observed in IDC compared to those in ANNTs. High Notch1, Slug, Snail, and N-cadherin expression and low E-cadherin expression in patients with IDC were significantly correlated with lymph node metastasis. In addition, correlation analysis results revealed that high Notch1 expression was significantly associated with high Slug, Snail, and N-cadherin expression and low E-cadherin expression in IDC. Furthermore, a high Snail expression was significantly associated with low E-cadherin expression, and a high Slug expression was found to be significantly associated with increased N-cadherin expression in patients with IDC. Hence, our study suggested that the Notch1-Snail/Slug-EMT axis may be implicated in the lymph node metastasis affecting patients with IDC.

Abstract 4372: MiR-449a promotes breast cancer progression by activating the NF-κB pathway

Abstract INTRODUCTION MicroRNAs (miRs) have been implicated in various human malignancies and are being developed as diagnostic, predictive, and prognostic biomarkers. Our previous global profiling studies found miR-449a to be over-expressed in archival lymph node-negative invasive ductal breast carcinoma samples (n=74) when compared to normal tissues. Furthermore, miR-449a expression level was significantly associated with relapse. The mechanism(s) by which miR-449a functions remains unknown, and the aim of the current study was therefore to elucidate the roles and mRNA targets of miR-449a in breast cancer. MATERIAL AND METHODS MiR-449a expression was evaluated in six human breast cancer cell lines (T47D, MDA-MB-468, MDA-MB-231, MCF-7, MDA-MB-453, and ZR75-1). Three of these cell lines, T47D, MMDA-MB-468, and MDA-MB-231, were selected and used to assess the biological effects of miR-449a on cell viability, clonogenicity, cell migration, and invasion. Downstream target genes were identified by combining in silico miRWalk prediction and cell line/patient sample microarray data. Targets were validated using qRT-PCR and luciferase reporter assays. RESULTS Consistent with our global profiling work, several (4/6) of the tested breast cancer cell lines over-expressed miR-449a. CDC20B, the miR-449a host gene and an essential regulator of cell division, was also over-expressed. Knockdown of miR-449a resulted in significantly decreased cell viability, clonogenic survival, migration, and invasion. MiR-449a candidate targets were then identified by integrating in silico prediction algorithms, cell line mRNA expression profiling (Affymetrix Human Genome U133 Plus 2.0), and clinical specimen mRNA expression data. Two genes, CRIP2 (Cysteine rich protein 2, a transcription factor) and PRKAG1 (Protein Kinase, AMP-Activated, Gamma 1 Non-Catalytic), were verified as miR-449a targets using qRT-PCR. Moreover, direct interactions between miR-449a and the 3′-UTRs of both CRIP2 and PRKAG1 were confirmed using luciferase reporter assays. Inhibition of CRIP2 by miR-449a allowed for NF-κB-induced transcription of survival, proliferation, and growth-related genes, providing further evidence for the role of miR-449a in breast cancer progression. CONCLUSIONS MiR-449a is over-expressed in breast cancer cells, promoting cellular proliferation, migration, and invasion. One mechanism by which miR-449a functions is to down-regulate CRIP2, leading to NF-kB activation and possible cancer progression. Further investigations into gene regulation by miR-449a may reveal other promising targets for the management of breast cancer. Citation Format: Wei Shi, Matthew Lee, Ryunosuke Kogo, Jeff Bruce, Christine How, Kenneth W. Yip, Fei-Fei Liu. MiR-449a promotes breast cancer progression by activating the NF-κB pathway. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4372. doi:10.1158/1538-7445.AM2014-4372

The glycomic profile of invasive ductal carcinoma of the breast is altered in patients with hypoxic regions: implications for tumor behavior.

Hypoxic areas in solid tumors are often associated with poor prognosis and resistance to chemotherapy. The aim of the study was to analyze the expression of galectin-1 (Gal-1), galectin-3 (Gal-3), sialic acid and b1-6 branched glycan structures in hypoxic environment of invasive ductal carcinoma (IDC) of the breast. We performed lectin histochemistry with phytohemag glutinin-L (L-PHA) and Sambucus nigra lectin (SNA); and immunohistochemistry for Gal-1, Gal-3, carbonic anhydrase IX, hypoxia-inducible factor, estrogen receptor (ER), progesterone receptor and human epidermal growth factor receptor type-2 for 86 IDC samples. Patients with markers positive for hypoxia were mostly ER-negative (p = 0.003) and presented with more nodal invasion than the non-hypoxic group (p = 0.0439). Concerning the glycobiological aspects, the hypoxic group expressed more of Gal-3 (p = 0.0021) and SNA ligands (p = 0.0498), however, there was no association between lectin- and galectin-staining and clinical and histopathological data. Our results suggest a change in the glycomic profile of patients within hypoxic regions of IDC. However, further studies are needed to evaluate the role of lectin- and galectin-ligands in tumor's hypoxic environment, as well as their potential to be used as therapeutic targets.

Expression of integrin α3β1 and cyclooxygenase-2 (COX2) are positively correlated in human breast cancer.

BackgroundExpression of integrin α3β1 is associated with tumor progression, metastasis, and poor prognosis in several cancers, including breast cancer. Moreover, preclinical studies have revealed important pro-tumorigenic and pro-metastatic functions for this integrin, including tumor growth, survival, invasion, and paracrine induction of angiogenesis. Our previously published work in a preclinical breast cancer model showed that integrin α3β1 promotes expression of cyclooxygenase-2 (COX2/PTGS2), a known driver of breast cancer progression. However, the clinical significance of this regulation was unknown. The objective of the current study was to assess the clinical relevance of the relationship between integrin α3β1 and COX2 by testing for their correlated expression among various forms of human breast cancer.MethodsImmunohistochemistry was performed to assess co-expression of α3 and COX2 in specimens of human invasive ductal carcinoma (IDC), either on a commercial tissue microarray (n = 59 samples) or obtained from Albany Medical Center archives (n = 68 samples). Immunostaining intensity for the integrin α3 subunit or COX2 was scored, and Spearman’s rank correlation coefficient analysis was performed to assess their co-expression across and within different tumor subtypes or clinicopathologic criteria.ResultsAlthough expression of integrin α3 or COX2 varied among clinical IDC samples, a statistically significant, positive correlation was detected between α3 and COX2 in both tissue microarrays (rs = 0.49, p < 0.001, n = 59) and archived samples (rs = 0.59, p < 0.0001, n = 68). In both sample sets, this correlation was independent of hormone receptor status, histological grade, or disease stage.ConclusionsCOX2 and α3 are correlated in IDC independently of hormone receptor status or other clinicopathologic features, supporting the hypothesis that integrin α3β1 is a determinant of COX2 expression in human breast cancer. These results support the clinical relevance of α3β1-dependent COX2 gene expression that we reported previously in breast cancer cells. The findings also suggest that COX2-positive breast carcinomas of various subtypes might be vulnerable to therapeutic strategies that target α3β1, and that α3 expression might serve as an independent prognostic biomarker.

Emerging Patterns in the Distribution of Trace Elements in Ovarian, Invasive and In-Situ Breast Cancer

Breast cancer is the most common cancer and ovarian cancer is the 8th most common cancer affecting women worldwide. This study highlights the changes of trace element levels accompanied by the progression from ductal carcinoma in situ (DCIS) to invasive ductal carcinoma (IDC) of the breast, using micro probe Synchrotron Radiation X-ray Fluorescence (μSRXRF). The average values for the increase in Ca, Fe and Zn in tumour regions with respect to surrounding regions for the DCIS samples were significantly higher compared to the increase in the IDC samples (P <0.01).This study was also carried out to find a connection between ovarian cancer and breast cancer with respect to the cellular distribution of Ca, Cu, Fe, and Zn. For IDC, DCIS and ovarian cases, the statistical analysis reveals a significant increase in the levels of Ca, Cu and Zn concentrations in cancer tissue when compared to the normal surrounding tissue. For Fe, the differences between tumour regions with respect to surrounding regions were found to be not significant in IDC and ovarian cases. In DCIS cases, the results reveal a significant increase in the levels of Fe in cancer tissue when compared to the surrounding normal breast tissue (P <0.01).

Overexpression of miR‐20b inhibits the expression of HIF‐1α and STAT‐3 in invasive ductal carcinoma of the breast (1138.2)

It is known that the hypoxia inducible factor‐1α (HIF‐1α) and the signal transducer and activator of transcription 3 (STAT‐3) are targets of miR‐20b. The role played by microRNAs (miRNAs) in breast cancer has been mainly studied using tumor cells. In this study, we investigated the expression of miR‐20b in formalin‐fixed paraffin embedded (FFPE) samples, as also as HIF‐1α and STAT‐3, in samples of invasive ductal carcinoma (IDC). The expression of miR‐20b and the proteins HIF‐1α and STAT‐3 was evaluated by real time PCR and immunohistochemistry, respectively. We found that the overexpression of miR‐20b (3‐fold; p&lt;0.05) is accompanied by the downregulation of STAT‐3 (70%; p&lt;0.001) in FFPE samples of IDC. We also observed that HIF‐1α expression in IDC and control samples was not statistically significant. Our results suggest that miR‐20b regulates the expression of HIF‐1α and its transcription factor STAT‐3 in IDC. These findings are relevant since HIF‐1α expression has been correlated with increased intratumoral angiogenesis, cancer progression and poor prognosis in breast cancer.Grant Funding Source: Supported by FAPESP 2012/05235‐3

Triptolide-induced in vitro and in vivo cytotoxicity in human breast cancer stem cells and primary breast cancer cells

We investigated the potential efficacy of the Chinese herbal extract triptolide for the treatment of human breast cancer by measuring the triptolide-induced cytotoxicity in cultures of human primary breast cancer cells (BCCs) and breast cancer stem cells (BCSCs) in vitro and in vivo. Human BCCs and BCSCs from invasive ductal carcinoma samples were cultured and treated with 0.1, 0.5 or 1.0 µM triptolide. Cell death and apoptosis were measured after 24, 48 and 72 h of treatment. Mammospheres were found to be highly tumorigenic when implanted subcutaneously in nude BALB/c mice. Triptolide was cytotoxic against both human primary BCCs and BCSCs in vitro (P<0.05), but the cytotoxicity was stronger against the BCCs. In response to 1 µM triptolide for 72 h, the apoptotic rates were approximately 60% for BCCs and 30% for BCSCs. The BCSCs exhibited a high formation rate of tumors when implanted subcutaneously in nude BALB/c mice. Triptolide treatment in vivo significantly inhibited tumor growth compared with mock treatment. In conclusion, the cytotoxicity of triptolide against BCCs and BCSCs in vitro and in vivo suggests that this natural diterpenoid triepoxide compound may have clinical applications for the suppression of breast tumor growth.

Abstract P5-05-10: Estrogenic pathways exist in TNBC and correlate with androgen receptor status

Abstract Triple negative carcinoma are a difficult to treat subtype of carcinomas due to the fact they do not express any of the markers at which targeted therapy is directed at such as ER, PR or HER2. Traditionally these carcinomas are referred to as estrogen receptor negative, however in reality the assessment of ER expression is limited to the ERa form; evaluation of ERβ expression in TNBC has been, on the whole, overlooked. A limited number of studies have suggested that in a large proportion of TNBC ERβ continues to be expressed. In this study we chose to focus on the ERβ1 subtype, as this has been previously suggested to be associated with a positive prognosis in TNBC. We sought to examine if there was an overlap between androgen receptor, ERβ1 and AKR1C3 (17βHSD5) expression as these have been suggested, at least in the prostate, to form a steroid metabolism pathway via which the inactivation of androgen can generate estrogenic signals. In addition we also assessed the presence of the canonical pathway of estrogen metabolism via intratumoral aromatase to see if this had a potential biological role in breast cancer. Following appropriate IRB approval a cohort of 78 samples of triple negative invasive ductal carcinoma from Tohoku University Hospital were collected. These samples were stained for ERβ1 and aromatase. ERβ1 samples were classified as positive or negative using a 10% labelling index criteria while aromatase was classified as positive if greater that 50% of carcinoma cells showed immunoreactivity. The expression pattern of both stains was then compared to a historical set of data regarding AR, AKR1C3 (17βHSD5 staining) and Ki67. In this comparison ERβ1 positivity and aromatase positivity was significantly and positively associated with AR positivity (Chi squared test p &amp;lt;0.01). ERβ1 however showed no relationship to either aromatase expression or 17βHSD5 expression. While both stains showed a trend towards lower Ki67 labelling indexes in conjunction with higher immunoreactivity, neither was significant. In a standard least squares model incorporating AR, ERβ1, 17βHSD5 and AROM positivity was the only factor that had an independent significant effect on Ki67 labelling index was that of AR positivity (Model p&amp;lt;0.05, AR effect p&amp;lt;0.01) with, as we have previously shown, AR positivity being associated with lower Ki-67 labelling index. These results suggest that while estrogenic pathways remain in TNBC, the dominant steroid metabolism pathway is that of the androgen receptor. However, further research is warranted to fully understand the roles of estrogenic pathways in triple negative disease. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P5-05-10.