Breast Cancer Primary Tumors Research Articles

MicroRNA 100 sensitizes luminal A breast cancer cells to paclitaxel treatment in part by targeting mTOR.

Luminal A breast cancer usually responds to hormonal therapies but does not benefit from chemotherapies, including microtubule-targeted paclitaxel. MicroRNAs could play a role in mediating this differential response. In this study, we examined the role of micro RNA 100 (miR-100) in the sensitivity of breast cancer to paclitaxel treatment. We found that while miR-100 was downregulated in both human breast cancer primary tumors and cell lines, the degree of downregulation was greater in the luminal A subtype than in other subtypes. The IC50 of paclitaxel was much higher in luminal A than in basal-like breast cancer cell lines. Ectopic miR-100 expression in the MCF-7 luminal A cell line enhanced the effect of paclitaxel on cell cycle arrest, multinucleation, and apoptosis, while knockdown of miR-100 in the MDA-MB-231 basal-like line compromised these effects. Similarly, overexpression of miR-100 enhanced the effects of paclitaxel on tumorigenesis in MCF-7 cells. Rapamycin-mediated inhibition of the mammalian target of rapamycin (mTOR), a target of miR-100, also sensitized MCF-7 cells to paclitaxel. Gene set enrichment analysis showed that genes that are part of the known paclitaxel-sensitive signature had a significant expression correlation with miR-100 in breast cancer samples. In addition, patients with lower levels of miR-100 expression had worse overall survival. These results suggest that miR-100 plays a causal role in determining the sensitivity of breast cancers to paclitaxel treatment.

The variation and clinical significance of hormone receptors and Her-2 status from primary to metastatic lesions in breast cancer patients.

The objective of this study is to investigate how the change of hormone receptor (HR) and human epidermal growth factor receptor-2 (Her-2) status is related to patients' clinical features. One hundred ninety-three cases of patients treated at general hospital of PLA from 2000 to 2015 with advanced breast cancer were included. All patients developed recurrence that were re-biopsied and had complete pathological profile both at initial diagnosis and at relapse. HR status before and after relapse were available for all patients, while only 143 cases had Her-2 status at the two stages. The changes of ER, PR, and Her-2 status and their association with clincopathological factors and DFS were analyzed. The discordant rates of ER, PR, and Her-2 status between primary breast cancer and recurrent tumor were 34.2, 38.3, and 16.8%, respectively. At relapse, the rates of gain of ER and PR positivity were 10.9 and 13.5%, respectively; the rates of loss of ER and PR positivity were 23.3 and 24.9%. Loss of positivity was more frequent than gain of positivity (p ER < 0.000, p PR = 0.001). Among patients with Her-2 negative primary tumors, 15.4% acquired Her-2 positivity at relapse; and among Her-2 positive patients at initial diagnosis, 1.4% turned to Her-2 negative at relapse; gain of positivity was more frequent than loss of positivity (p < 0.000). Patients with tumor larger than 2cm in diameter were more likely to experience change of Her-2 status (25.0 vs 5.8%, p = 0.005). Yet, the change of ER/PR was not significantly associated with the size of primary tumor. Patients with ER positive recurrent disease and PR positive primary tumor had a DFS of more than 40months. Compared to patients who maintained PR negative, patients who gained PR positivity at relapse had significantly longer DFS by 8.5% (35.2 vs 26.7months, p = 0.024). Patients losing ER positivity at relapse had shorter DFS by 7.8months compared to those with stable ER positive tumors; patients gaining ER positivity experienced longer DFS by 8.3months; but both differences were not statistically significant. Loss of Her-2 positivity was associated with longer DFS by 13.8months as opposed to stable Her-2 status, without statistical significance. For patients with Her-2 negative primary tumor, the changes of Her-2 status were not associated with DFS. 34.2, 38.3, and 16.8% of breast cancer patients had their ER, PR, and Her-2 status changed after recurrence, and these changes of receptor status were associated with DFS to some degree. Gain of PR positivity at relapse was significantly correlated with longer DFS.

Chemoprevention of Breast Cancer by Dietary Polyphenols.

The review will discuss in detail the effects of polyphenols on breast cancer, including both the advantages and disadvantages of the applications of these natural compounds. First, we focus on the characterization of the main classes of polyphenols and then on in vitro and in vivo experiments carried out in breast cancer models. Since the therapeutic effects of the administration of a single type of polyphenol might be limited because of the reduced bioavailability of these drugs, investigations on combination of several polyphenols or polyphenols with conventional therapy will also be discussed. In addition, we present recent data focusing on clinical trials with polyphenols and new approaches with nanoparticles in breast cancer. Besides the clinical and translational findings this review systematically summarizes our current knowledge about the molecular mechanisms of anti-cancer effects of polyphenols, which are related to apoptosis, cell cycle regulation, plasma membrane receptors, signaling pathways and epigenetic mechanisms. At the same time the effects of polyphenols on primary tumor, metastasis and angiogenesis in breast cancer are discussed. The increasing enthusiasm regarding the combination of polyphenols and conventional therapy in breast cancer might lead to additional efforts to motivate further research in this field.

Influence of BCL2-938 C>A promoter polymorphism and BCL2 gene expression on the progression of breast cancer.

BCL2 (B-cell leukemia/lymphoma 2) gene functions as antiapoptotic regulatory element and known to be associated with tumorigenesis. The SNP-938 (C>A) (rs2279115), located in the inhibitory P2 promoter of the BCL2 gene, influences differential binding affinities of transcriptional factors thereby affecting BCL2 expression. The present study is an attempt to evaluate the association between BCL2(-938C>A) polymorphism and clinical characteristics of breast cancer patients as well as to analyze BCL2 expression and Ki67 proliferation index with respect to the genotypes. One hundred ten primary breast cancer tumor tissues were genotyped for -938 C>A polymorphism through PCR-RFLP method as well as evaluated for BCL2 expression and ki67 proliferation index by immunohistochemistry. Evaluation of apoptosis level was performed by flowcytometry. The results revealed that AA genotype was associated with an increased risk (AA Vs AC + CC) by 2.86-fold (p = 0.07) for breast cancer development which reflected in elevated A allele frequency also. AA genotype was found to be predominant among BCL2 positive tumors as compared to BCL2 negative tumors. Further, AA genotype was found to be associated with advanced stage tumors, node positive status, and high Ki67 proliferation index compared to CA and CC genotypes indicating that elevated expression of BCL2 gene in the presence of A allele might be associated with decreased apoptosis and enhanced proliferation rate. AA genotype of BCL2-938C>A polymorphism might influence BCL2 gene expression there by associated with elevated risk for breast cancer progression. Probably, failure of apoptosis due to enhanced expression and antiapoptotic protein BCL2 might promote malignant growth.

Baseline blood immunological profiling differentiates between Her2-breast cancer molecular subtypes: implications for immunomediated mechanisms of treatment response.

PurposeBreast cancer patients’ response to treatment is highly dependent on the primary tumor molecular features, with triple-negative breast tumors having the worst prognosis of all subtypes. According to the molecular features, tumors stimulate the microenvironment to induce distinct immune responses, baseline immune activation being associated with higher likelihood of pathologic response. In this study, we investigated the deconvolution of the immunological status of triple-negative tumors in comparison with luminal tumors and the association with patients’ clinicopathological characteristics.Patients and methodsGene expression of 84 inflammatory molecules and their receptors were analyzed in 40 peripheral blood samples from patients with Her2− primary breast cancer tumors. We studied the association of triple-negative phenotype with age, clinical stage, tumor size, lymph nodes, and menopausal status.ResultsWe observed that more patients with estrogen (ER)/progesterone (PR)-negative tumors had grade III, while more patients with ER/PR-positive tumors had grade II tumors. Gene expression analysis revealed a panel of 14 genes to have differential expression between the two groups: several interleukins: IL13, IL16, IL17C and IL17F, IL1A, IL3; interleukin receptors: IL10RB, IL5RA; chemokines: CXCL13 and CCL26; and cytokines: CSF2, IFNA2, OSM, TNSF13.ConclusionThe expression levels of these genes have been previously shown to be associated with reduced immunological status; indeed, the triple-negative breast cancer patients presented with lower counts of lymphocytes and eosinophils than the ER/PR-positive ones. These results contribute to a better understanding of the possible role of antitumor immune responses in mediating the clinical outcome.

New methods to improve the safety assessment of cryopreserved ovarian tissue for fertility preservation in breast cancer patients

To develop a novel molecular panel of markers to detect breast cancer (BC) disseminated malignant cells in ovarian tissue, and to improve the safety of ovarian tissue transplantation. Experimental study. University hospital. Ten ovarian biopsies from healthy patients, 13 biopsies with diagnosed BC metastasis, and 4 biopsies from primary BC tumor for designing a diagnostic panel of BC cell contamination; 60 ovarian biopsies from BC patients undergoing fertility preservation for validating the panel. Female nude mice. A novel panel for BC malignant cell detection by reverse-transcription polymerase chain reaction (RT-PCR), inmmunohistochemical analysis, in vitro invasion assay and xenotransplantation assayed in ovarian tissue from BC patients. Expression of GCDFP15, MGB1, SBEM, MUC1, WT-1, and NY-BR-01, selected as markers, assessed by quantitative RT-PCR in samples with confirmed BC metastasis. The most sensitive markers were confirmed by immunohistochemistry, and tested in vitro and in vivo. GCDFP15, MGB1, and SBEM were the most sensitive and specific markers to detect BC metastatic cells when at least one was expressed by quantitative RT-PCR. The panel was validated in 60 patients and confirmed in an in vitro invasion assay, where no invasive cells were observed. Samples negative for BC cells cannot develop disease when xenografted. GCDFP15, MGB1, and SBEM were the most sensitive molecules to create a diagnostic panel for BC malignant cell contamination, which may make ovarian tissue cryopreservation and transplantation a safe technique for fertility preservation in BC patients.

Abstract 3171: IL-25/IL-17E does not induce breast tumor cell apoptosis and both IL-17A and IL-25/IL-17E contribute to breast cancer survival and proliferation associated with generation of pro-oncogenic low molecular forms of cyclin E

Abstract Pro-inflammatory IL-17 cytokines were initially described for their pathogenic role in chronic inflammatory diseases and subsequent accumulating evidence indicated their involvement in carcinogenesis. In the present study we focused our work on IL-17A and IL-17E which are found in the breast tumoral microenvironment. Cell proliferation and cytotoxic assays as well as Quantitative PCR and Western Blot analysis of oncogenic pathways were performed using several breast cancer cell lines and primary tumor cells. We report that IL-17A and IL-17E receptor subunits (IL17RA, RB and RC chains) are up-regulated in breast cancers versus normal samples. Moreover, IL-17E, which is undetectable in most normal breast tissues tested, is expressed at higher levels in tumors. We studied the molecular signaling following the stimulation of cell lines with IL-17A and IL-17E. We found that both cytokines induced cell proliferation and activation of pathways involved in cells survival. Accordingly, IL-17A and IL-17E promoted resistance to Docetaxel but failed to induce apoptosis as previously reported for IL-17E. Interestingly, we also revealed that both cytokines induced the generation of tumorigenic low molecular weight forms of cyclin E (LMW-E), high levels of which correlate strongly with a poor survival in breast cancer patients. These results show for the first time some of the molecular pathways activated by IL-17A and IL-17E that may participate to their pro-oncogenic activity in breast cancers. Citation Format: Jérôme Giustiniani, Hervé Curé, Christian Garbar, Yacine Merrouche, Nathalie Bonnefoy, Jérémy Bastid, Gilles Alberici, Jean-François Eliaou, Armand Bensussan. IL-25/IL-17E does not induce breast tumor cell apoptosis and both IL-17A and IL-25/IL-17E contribute to breast cancer survival and proliferation associated with generation of pro-oncogenic low molecular forms of cyclin E. [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 3171. doi:10.1158/1538-7445.AM2015-3171

Abstract 3363: RAGE/S100A7/Stat3-axis enhances breast cancer growth and metastasis via modulating tumor microenvironment

Abstract The Receptor for Advanced Glycation Endproducts (RAGE), a multifunctional, multi-ligand receptor, has been shown to play an important role in inflammation. However, not much is known about its role in breast cancer growth and metastasis. In this report, we observed that RAGE expression is upregulated in triple negative breast cancer (TNBC) cell lines, primary tumors and lymph-node metastasis samples. RAGE-/- mice show reduced breast cancer growth. Additionally, blocking RAGE with neutralizing-antibody inhibited lung metastasis in an intracardiac mouse model. Further elucidation of RAGE-mediated mechanisms revealed that RAGE binds to S100A7 and mediates S100A7-induced cell migration, Stat3 and NF-ĸB activation. Our results also indicate that S100A7/RAGE axis-modulates invasion/migration through Stat3 dependent MMP9 activation. In addition, RAGE neutralizing antibody and soluble RAGE inhibited breast cancer progression and metastasis in the inducible mS100a7a15 mouse model system. We demonstrated that RAGE/S100A7 enhanced mammary hyperplasia, tumor growth and metastasis through Stat3 activation. Notably, our studies revealed that RAGE/mS100a7a15 modulates the breast tumor microenvironment through recruitment of phospho-Stat3/MMP9-positive tumor-associated macrophages. Our studies suggest that RAGE expression could be used as a novel biomarker for aggressive/invasive breast cancer, especially TNBC. Collectively, these findings suggest that RAGE/S100A7/Stat3-axis has a novel role in linking inflammation to the development of invasive/aggressive breast cancer including TNBC. Citation Format: Mohd W. Nasser, Nissar A. Wani, Janani Ravi, Grace A. Amponsah, Dinesh K. Ahirwar, Catherine A. Powell, Mohamad Elbaz, Helong Zhao, Konstantin Shilo, Ramesh K. Ganju. RAGE/S100A7/Stat3-axis enhances breast cancer growth and metastasis via modulating tumor microenvironment. [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 3363. doi:10.1158/1538-7445.AM2015-3363

Abstract 974: FAK inhibitors VS-6063 and VS-4718 target cancer stem cells: Implications for TNBC sequential and combination therapies

Abstract Cancer stem cells (CSCs), a subpopulation of tumor cells characterized by tumor-initiating capability and resistance to chemotherapy, are an underlying cause of tumor progression and metastasis. In breast cancer, CSCs can be identified by aldehyde dehydrogenase 1 (ALDH) or CD44+/CD24- expression separately, although combination of ALDH, CD44+, and CD24- together may better describe the CSC population in breast cancer. Neoadjuvant chemotherapy has been shown to lead to an increase in CSCs in locally advanced breast cancer (Alamgeer et al., 2014, Br. Can. Res. 16:R14) which is associated with a significantly worse prognosis. It is also evident that stem-like features of tumor cells are present in metastatic breast cancer (Yu et al., 2013, Science 339:580) indicating that targeting of CSCs may be valuable in metastatic disease. VS-6063 and VS-4718 are orally bioavailable, potent and selective inhibitors of Focal Adhesion Kinase (FAK). These agents preferentially target CSCs in preclinical models and are currently in clinical development. Here we report that VS-6063 and VS-4718 preferentially target CSCs in multiple breast cancer models. Ex vivo treatment of explants from human breast tumors with either VS-6063 or VS-4718 decreased the proportion of CSCs in contrast to paclitaxel treatment which increased the proportion of CSCs. In an MDA-MB-231 mouse xenograft model, in vivo treatment with VS-6063 decreased CSCs by more than 6-fold as demonstrated by limiting dilution transplantation assay. Consistent with the notion that CSCs are responsible for cancer relapse after chemotherapy, VS-6063 and VS-4718 substantially delayed tumor growth following cessation of paclitaxel or cisplatin treatment in models of TNBC, including a patient-derived xenograft. Furthermore, using an imaging-based 4T1-luciferase TNBC orthotopic model, both VS-6063 and VS-4718 inhibited metastatic outgrowth or induced regression of metastases after primary tumor resection, whereas metastases progressed in all animals in the vehicle control group. A multiplex assay for multiple CSC markers (ALDH1, CD44 and CD24) was developed and validated in biopsies of primary patient tumors and matched lymph node biopsies taken pre- and post-neoadjuvant chemotherapy. CSCs, represented by the ALDH+ and/or CD44+/CD24- populations were present at detectable levels in a high fraction of triple negative breast cancer primary tumors. In summary, CSCs are readily detectable in primary breast cancers at surgery, and VS-6063 and VS-4718 diminish the CSC subpopulation in vitro, ex vivo and in xenograft models. This critical subpopulation of CSCs is detectable in residual tumors after neoadjuvant therapy, and may also be present in metastatic disease. CSC-targeted agents such as VS-6063 or VS-4718 should be clinically tested in these breast cancer settings to potentially delay time to relapse and improve patient outcome. Citation Format: Vihren N. Kolev, Kam Sprott, Qunli Xu, Jonathan A. Pachter, David T. Weaver. FAK inhibitors VS-6063 and VS-4718 target cancer stem cells: Implications for TNBC sequential and combination therapies. [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 974. doi:10.1158/1538-7445.AM2015-974

Circulating tumor cells in breast cancer beyond the genotype of primary tumor for tailored therapy.

Although TNM staging based on tumor, node lymph status and metastasis status-is the most widely used method in the clinic to classify breast cancer (BC) and assess prognosis, it offers limited information for different BC subgroups. Circulating tumor cells (CTCs) are regarded as minimal residual disease and are proven to have a strong relationship with BC. Detection of ≥5 CTCs per 7.5 mL in peripheral blood predicts poor prognosis in metastatic BC irrespective of other clinical parameters, whereas, in early-stage BC, detection of CK19(+) CTCs are also associated with poor prognosis. Increasing data and clinical trials show that CTCs can improve prognostic accuracy and help tailor treatment for patients with BC. However, heterogeneous CTCs in the process of an epithelial-mesenchymal transition (EMT) in BC makes it a challenge to detect these rare cells. Moreover, the genotypic and phenotypic features of CTCs are different from primary BC tumors. Molecular analysis of CTCs in BC may benefit patients by identifying those amenable to tailored therapy. We propose that CTCs should be used alongside the TNM staging system and the genotype of primary tumor to guide tailored BC diagnosis and treatment.

Specific upregulation of RHOA and RAC1 in cancer-associated fibroblasts found at primary tumor and lymph node metastatic sites in breast cancer.

The importance of tumor-stromal cell interactions in breast tumor progression and invasion is well established. Here, an evaluation of differential genomic profiles of carcinoma-associated fibroblasts (CAFs) compared to fibroblasts derived from tissues adjacent to fibroadenomas (NAFs) revealed altered focal adhesion pathways. These data were validated through confocal assays. To verify the possible role of fibroblasts in lymph node invasion, we constructed a tissue microarray consisting of primary breast cancer samples and corresponding lymph node metastasis and compared the expression of adhesion markers RhoA and Rac1 in fibroblasts located at these different locations. Two distinct tissue microarrays were constructed from the stromal component of 43 primary tumors and matched lymph node samples, respectively. Fibroblasts were characterized for their expression of α-smooth muscle actin (α-SMA) and vimentin. Moreover, we verified the level of these proteins in the stromal compartment from normal adjacent tissue and in non-compromised lymph nodes. Our immunohistochemistry revealed that 59 % of fibroblasts associated with primary tumors and 41 % of the respective metastatic lymph nodes (p = 0.271) displayed positive staining for RhoA. In line with this, 57.1 % of fibroblasts associated with primary tumors presented Rac1-positive staining, and the frequency of co-positivity within the lymph nodes was 42.9 % (p = 0.16). Expression of RhoA and Rac1 was absent in fibroblasts of adjacent normal tissue and in compromised lymph nodes. Based on our findings that no significant changes were observed between primary and metastatic lymph nodes, we suggest that fibroblasts are active participants in the invasion of cancer cells to lymph nodes and support the hypothesis that metastatic tumor cells continue to depend on their microenvironment.

Abstract A41: Automated immunohistochemistry of phosphobiomarkers: Case study of MTOR (MLN0128) and PI3Kα (MLN1117) investigational inhibitors, single agent and in combination, on xenografts and mouse skin

Abstract Background: Immunohistochemistry provides visual context to protein modulation and is relied upon to elucidate mechanism of action, marker translocation and pharmacodynamics (PD) in drug discovery. Markers of the PI3K-MTOR pathway have been monitored in this way to aid in the clinical development of various inhibitors targeting this pathway. Here, assays were developed using preclinical xenograft PD studies to explore the feasibility of automated staining of pAKT 473, pS6, p4EBP1 and pRb. In preparation for clinical assessment of these markers in human skin samples; these markers were also examined in normal mouse skin. p4EBP1 was selected to examine the reproducibility of these automated assays and to explore the correlation between pathway inhibition in PD xenograft tissues and murine skin tissues. The automated staining technique was coupled with whole slide scanning and digital image analysis for quantitative comparison. Methods: Assays were chosen for evaluation based on known biology. FFPE PTEN-null breast cancer primary human tumor xenografts with high, medium and low biomarker expression, with companion skin samples, were selected for feasibility and reproducibility testing. Whole slide scanning and reproducibility analysis was executed using Aperio Scanscope XT and Spectrum software. Statistical analysis methods of mixed effect linear models and two sample t-tests were applied. Xenograft and skin samples from NCI-H1048 tumor bearing Nude mice treated with MTOR (MLN0128)and PI3Kα (MLN1117) investigational inhibitors were immunohistochemically stained for p4EBP1. Definiens object-based imaging software was used to isolate and analyze viable tumor area and exclude necrotic and stromal components in xenografts. A customized Definiens® algorithm was used on skin to identify the epithelial layer for analysis. Results: After evaluation, baseline biomarker expression in the xenograft and skin samples determined marker selection for future steps. Baseline p4EBP1 levels were high in both xenograft and skin. The reproducibility testing yielded consistent results in both xenograft and skin, validating autostaining use for PD studies. Phosphomarker p4EBP1 expression was inhibited after MLN0128 administration, with increased reduction after combination with MLN1117. Development of a novel analysis solution using Definiens software for skin samples permitted the area of interest to be restricted to the epithelial layer and excluded other epidermal features with similar morphometric characteristics. The p4EBP1 modulation was concordant between skin and tumor though more pronounced in skin, indicating that skin is a suitable surrogate tissue for determining modulation of this PI3K-MTOR pathway marker. Conclusions: Autostaining platforms provide reproducible results and stain consistently for PI3K-MTOR phosphomarkers on both xenografts and mouse skin samples. Utilizing p4EBP1, target effect was observed after MTOR and PI3Kα administration. This development and evaluation of the phosphobiomarker using automated staining technology and advanced image analysis supports preclinical PD assessment and combination clinical trials as well as influenced timepoint selection for clinical assessment. Citation Format: Anna Kreshock, Natalia Iartchouk, Yueying Cao, Jeffrey Szwaya, Feng Gao, Christopher Simpson, Evan Luongo, Kristine Burke, Esha Gangolli, Keisuke Kuida, Rachael Brake, Stephen Tirrell, Vaishali Shinde. Automated immunohistochemistry of phosphobiomarkers: Case study of MTOR (MLN0128) and PI3Kα (MLN1117) investigational inhibitors, single agent and in combination, on xenografts and mouse skin. [abstract]. In: Proceedings of the AACR Special Conference: Targeting the PI3K-mTOR Network in Cancer; Sep 14-17, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(7 Suppl):Abstract nr A41.

Surgical Treatment of the Primary Tumor in Stage iv Breast Cancer

IntroductionThe aim of the study was to analyse the impact of locoregional surgery on survival of patients with stage IV breast cancer. Patients and methodsRetrospective study that included patients with breast cancer and synchronous metastases. Patients with ECOG above 2 and high-risk patients were excluded. The following variables were evaluated: age, tumour size, nodal involvement, histological type, histological grade, hormone receptor status, HER2 overexpression, number of affected organs, location of metastases, and surgical treatment. The impact of surgery and several clinical and pathologic variables on survival was analysed by Cox regression model. ResultsA total of 69 patients, of whom 36 (52.2%) underwent surgery (study group) were included. After a mean follow-up of 34 months, the median survival of the series was 55 months and no significant differences between the study group and the group of patients without surgery (P=.187) were found. Two factors associated with worse survival were identified: the number of organs with metastases (HR=1.69, CI 95%: 1.05–2.71) and triple negative breast cancer (HR=3.49, CI 95%: 1.39–8.74). Locoregional surgery, however, was not associated with survival. ConclusionsLocoregional surgical treatment was not associated with improved survival in patients with stage IV breast cancer. The number of organs with metastases and tumours were triple negative prognostic factors for survival.

Redox-Mediated Suberoylanilide Hydroxamic Acid Sensitivity in Breast Cancer.

Vorinostat (suberoylanilide hydroxamic acid; SAHA) is a histone deacetylase inhibitor (HDACi) approved in the clinics for the treatment of T-cell lymphoma and with the potential to be effective also in breast cancer. We investigated the responsiveness to SAHA in human breast primary tumors and cancer cell lines. We observed a differential response to drug treatment in both human breast primary tumors and cancer cell lines. Gene expression analysis of the breast cancer cell lines revealed that genes involved in cell adhesion and redox pathways, especially glutathione metabolism, were differentially expressed in the cell lines resistant to SAHA compared with the sensitive ones, indicating their possible association with drug resistance mechanisms. Notably, such an association was also observed in breast primary tumors. Indeed, addition of buthionine sulfoximine (BSO), a compound capable of depleting cellular glutathione, significantly enhanced the cytotoxicity of SAHA in both breast cancer cell lines and primary breast tumors. We identify and validate transcriptional differences in genes involved in redox pathways, which include potential predictive markers of sensitivity to SAHA. In breast cancer, it could be relevant to evaluate the expression of antioxidant genes that may favor tumor resistance as a factor to consider for potential clinical application and treatment with epigenetic drugs (HDACis).

Hormone receptors and HER2 expression in primary tumor and synchronous axillary lymph node metastasis in estrogen receptor positive breast cancer.

e11580 Background: The receptor status, Estrogen Receptor (ER), Progesterone Receptor (PR) and HER-2 can change between the primary (P) and the synchronous axillary lymph node (ALN) metastases. The...

Changes in glycoprotein expression between primary breast tumour and synchronous lymph node metastases or asynchronous distant metastases.

BackgroundBreast cancer is a very heterogeneous disease and some patients are cured by the surgical removal of the primary tumour whilst other patients suffer from metastasis and spreading of the disease, despite adjuvant therapy. A number of prognostic and treatment predictive factors have been identified such as tumour size, oestrogen (ER) and progesterone (PgR) receptor status, human epidermal growth factor receptor type 2 (HER2) status, histological grade, Ki67 and age. Lymph node involvement is also assessed during surgery to determine if the tumour has spread which requires dissection of the axilla and adjuvant treatment. The prognostic and treatment predictive factors assessing the nature of the tumour are all routinely based on the status of the primary tumour.ResultsWe have analysed a unique tumour set of fourteen primary breast cancer tumours with matched synchronous axillary lymph node metastases and a set of nine primary tumours with, later developed, matched distant metastases from different sites in the body. We used a pairwise tumour analysis (from the same individual) since the difference between the same tumour-type in different patients was greater. Glycopeptide capture was used in this study to selectively isolate and quantify N-linked glycopeptides from tumours mixtures and the captured glycopeptides were subjected to label-free quantitative tandem mass spectrometry analysis. Differentially expressed proteins between primary tumours and matched lymph node metastasis and distant metastasis were identified. Two of the top hits, ATPIF1 and tubulin β-chain were validated by immunohistochemistry to be differentially regulated.ConclusionsWe show that the expression of a large number of glycosylated proteins change between primary tumours and matched lymph node metastases and distant metastases, confirming that cancer cells undergo a molecular transformation during the spread to a secondary site. The proteins are part of important pathways such as cell adhesion, migration pathways and immune response giving insight into molecular changes needed for the tumour to spread. The large difference between primary tumours and lymph node and distant metastases also suggest that treatment should be based on the phenotype of the lymph node and distant metastases.Electronic supplementary materialThe online version of this article (doi:10.1186/s12014-015-9084-7) contains supplementary material, which is available to authorized users.

Abstract P1-07-23: Pathway activation mapping of metastatic breast cancer identifies potential organ-specific signatures: Implications for patient stratification to targeted treatment

Abstract Background: The development of distant metastases is the strongest prognostic factor associated with cancer mortality. Customization of treatment based on molecular profiling of a patient’s primary tumor has yielded promising results and has opened a new paradigm for treating patients with advanced disease. Nevertheless profiling the primary tumor may not reflect the actionable molecular drivers of the metastatic lesions. In the last few years a large number of genomic and proteomic studies have demonstrated that at the molecular level metastatic lesions significantly differ from matched primary tumors. The impact of metastasis site organ microenvironments on breast cancer primary tumors has been only partially explored. Since most targeted therapies work by modulating aberrantly-activated protein kinase signaling, the aim of this study was to utilize reverse phase protein arrays (RPPA) to explore whether metastatic lesions derived from different patients, but invading the same host target organ showed similarities in their signaling architecture and presented with organ-specific targetable signatures. Methods: Snap frozen material collected from 14 metastatic breast cancer patients enrolled in a prospective phase II trial ("Side Out II") were used for this analysis. Sites of metastasis were: liver (n=7), skin/chest wall (n=4), and lung (n=3). All samples were subjected to Laser Capture Microdissection (LCM) and RPPA to measure the activation/phosphorylation levels of 12 FDA approved drug targets and linked substrates. Results: Among the 7 patients with liver metastases, p70S6K, c-Abl, ERK 1/2 were highly phosphorylated in 5 cases (71.4%), ErbB2/HER2 in 4 cases (57.1%), and AKT in 3 cases (42.8%). On the contrary, of the 4 patients with skin/chest wall lesions 3 showed high activation of ErbB2/HER2, ErbB3/HER3, and ERK 1/2 (75.0%) and 2 of EGFR (50.0%). Of note, none of the skin/chest wall lesions showed activation of AKT and only 1 case presented with activated p70S6K (25.0%). Lung metastasis showed an overall low activation of all 12 analytes measured. Discussion: Although this data is based on a relatively small number of samples and needs further validation, our initial analysis revealed potentially important trends that could have an impact in the prioritization and selection of targeted agents for metastatic breast cancer trials. We found that the distribution of the 12 FDA approved drug targets and downstream substrates varies between different metastatic sites. Thus, the emergent hypothesis from this work is that breast cancer patients with liver metastases may be more susceptible to AKT-mTOR directed targeted therapeutics and patients with skin/chest wall metastasis may benefit from therapies that target EGFR and/or downstream ERK signaling. Citation Format: Mariaelena Pierobon, Stephen P Anthony, K Alex Reeder, Nicholas J Robert, Donald W Northfelt, Mohammad Jahanzeb, Linda Vocila, Julia D Wulfkuhle, Bryant Dunetz, Lance A Liotta, Emanuel F Petricoin. Pathway activation mapping of metastatic breast cancer identifies potential organ-specific signatures: Implications for patient stratification to targeted treatment [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P1-07-23.

Abstract P1-06-02: Identifying hypermethylated tumor suppressor genes in breast cancer with an in vivo total genome knockdown screen

Abstract Changes in gene expression are required for the progression of breast cancer; and DNA methylation, which silences tumor suppressor genes, is often responsible for these changes. Several de-methylating/DNA synthesis inhibiting drugs (such as decitabine) are potential breast cancer therapies; however, for their application to be successful, we need to identify which patients would most benefit from these treatments. To accomplish this, the critical genes, which must be silenced by hypermethylation in order for breast cancer to progress, need to be identified. For this purpose, we have performed an in vivo total genome knockdown screen. A genome-wide lentiviral-based shRNA screen was performed with breast cancer cell line MDA-MB-231 tumors implanted in the mammary fat pads of female NOD/SCID mice, with or without decitabine treatment (50mg/kg). The resulting tumors were harvested and shRNA sequences retrieved and hybridized to a Decode microarray, allowing for the identification of shRNA sequences that were enriched or depleted under decitabine treatment. The enriched shRNA sequences likely target genes of two categories: 1) they are methylated in the tumor and when expressed have tumor suppressive function, or 2) they are required for decitabine-mediated DNA synthesis inhibition and apoptosis. To determine if the shRNA-targeted genes identified in the screen fall within category 1 or 2, further analysis was done using GEO datasets for methylation in breast cancer cell lines, primary tumors, and normal breast tissues. In replicates of 6 mice, 111 shRNA sequences were enriched more than 2-fold in 5/6 mice. Of the 111 genes, 29 genes showed significant enrichment (p&amp;lt;0.05) in the decitabine-treated tumors. Using the GEO datasets, 19 of those significantly enriched genes fit a tumor suppressor methylation profile and merit further investigation of their methylation status in the MDA-MB-231 cell line. The remaining enriched genes may play a role in DNA synthesis inhibition and apoptosis- this will be confirmed with in vitro apoptosis assays. DAVID analyses did not reveal any functional clustering of the genes of interest, but did implicate several pathways that involved a single potential hypermethylated tumor suppressor gene. We have identified several genes that are hypermethylated in breast cancer and are potentially involved in tumor progression. Confirmation experiments will reveal a list of genes that when found hypermethylated in patient tumors would identify candidate breast cancer patients who would benefit from decitabine treatment. Citation Format: Margaret Thomas, Krysta Coyle, Mohammad Sultan, Luzhe Pan, Dae-Gyun Ahn, Patrick Lee, Carman Giacomantonio, Paola Marcato. Identifying hypermethylated tumor suppressor genes in breast cancer with an in vivo total genome knockdown screen [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P1-06-02.

Electrophilic Nitroalkenes Cause Degradation of NFκB RelA Protein in Triple Negative Breast Cancer Cells

Triple negative breast cancer (TNBC), which lacks estrogen receptor (ER), progesterone receptor (PR) and Her2/Neu, is an aggressive and metastatic cancer. Current therapy for TNBC is limited, and it is an unmet need for novel therapeutic agents with improved efficacy. Electrophilic fatty acids are endogenously‐generated signaling mediators that modulate cell differentiation and proliferation via post‐translational modification of nucleophilic amino acids (Cys, His) of transcriptional regulatory proteins such as NFkB, PPARgamma, and Keap1/Nrf2. Preliminary studies have revealed that an exemplary electrophilic fatty acid nitroalkene, nitro‐oleic acid (OA‐NO2), displays potential therapeutic value in TNBC cells, by preferentially reducing the growth and viability of two human TNBC cell lines, but not normal breast cells. Essential to proliferation and survival, NFkB signaling is highly activated in ER‐negative breast cancer cells and primary tumors. Notably, NFkB RelA protein level was diminished in TNBC cells upon OA‐NO2 treatment. Therefore, it is hypothesized that electrophilic fatty acids reduce TNBC cell proliferation and survival through down‐regulation of NFkB expression and signaling. We have demonstrated that OA‐NO2 down‐regulates transcript levels of NFkB –regulated genes, cyclin D1 and the pro‐survival genes, bcl‐xL and survivin, in TNBC cells. Moreover, OA‐NO2 promotes RelA protein polyubiquitination via nitroalkylation of RelA in TNBC cells. Overall, this study will serve as a prelude to the clinical study of electrophilic nitrated lipids as therapeutic agents that may display a high selectivity for killing TNBC tumors.

Change in estrogen receptor, HER2, and Ki-67 status between primary breast cancer and ipsilateral breast cancer tumor recurrence

IntroductionChanges in the biological marker status between primary and recurrent tumors are observed in breast cancer. However, their clinical significance is still uncertain, especially for patients with ipsilateral breast tumor recurrence (IBTR) after breast-conserving surgery. Patients and methodsA total of 117 patients with IBTR without distant metastases were enrolled in this study. All patients were examined for estrogen receptor (ER), HER2, and Ki-67 in both the primary tumors and paired IBTR. We evaluated the impact of changes in these biomarkers between primary tumors and IBTR on the prognosis after IBTR. ResultsThere were no associations of changes in the ER, HER2 status with distant disease-free survival (DDFS) after surgical resection of IBTR, whereas the change in the Ki-67 status between the primary tumors and IBTR was significantly correlated with DDFS (unadjusted: p = 0.0094; adjusted: p = 0.013). Patients in the “increased or remained high” Ki-67 group had a significantly shorter DDFS than those in the “decreased or remained low” Ki-67 group (5-year DDFS: 55.5 vs. 79.3%, respectively, p = 0.0084 by log-rank test). ConclusionAn increased or persistently high Ki-67 status in the IBTR was significantly correlated with a poorer prognosis after IBTR.