Inflammatory Breast Cancer Tumor Cells Research Articles

Abstract P3-08-06: Monitoring Lymphovascular Invasion and Tumor Growth of Inflammatory Breast Cancer in a Murine Lymphatic Reporter Window Chamber Model

Abstract Background: Lymphovascular invasion (LVI) is a major route of metastatic dissemination and recent studies indicate its value as an independent prognostic indicator for advanced breast, colorectal, squamous cell, prostate, brain cancers. LVI is a clinicopathological hallmark of inflammatory breast cancer (IBC), an understudied and most lethal breast cancer. IBC is often misdiagnosed due to an absence of a solid mass and its unique presentation of diffuse tumor cell clusters/emboli in the dermal lymphatics. Widely used mammary tumor implantation models coupled with bioluminescence or fluorescence imaging to monitor tumor growth kinetics are ineffective for evaluating spatial and temporal changes in growth and migration patterns of individual tumor cells and clusters within their microenvironmental context. The goal of this study was to develop a murine model to simulate the unique clinicopathological features of IBC patients and to assess both qualitatively and quantitatively local tumor growth, motility, and LVI. Methods: To specifically facilitate visualization of lymphatic and endothelial vessels along with tumor-vessel interactions, we generated a transgenic nude mice model (ProxTom RFP Nu/Nu) wherein, the mice exhibit red, fluorescent lymphatics [tdTomato fluorophore under control of a Prox1 promoter, which encodes a transcription factor (prospero-related homeobox 1) necessary for the formation and maintenance of lymphatic vessels]. Next, we employed a surgical technique, wherein a window chamber is placed on the dorsal skinfold of mice, which allows for microscopic examination of implanted tumor cells and ability to track dynamic changes of the tumor in its local microenvironment from the time of implantation up to 10 days. Patient-derived IBC or PDX stably transfected to express green, red fluorescent and/or dual tagged with luciferase reporters were transplanted in mice bearing window chambers. Intravital fluorescence microscopy and IVIS imaging were used to serially quantify local tumor growth, motility, length density of lymph and blood vessels, and degree of tumor cell lymphatic invasion over 0-140h. Results: Multichannel optical imaging of the window chamber in the ProxTom RFP Nu/Nu mice demonstrated co-localization of IBC tumor cells and lymphatics. Diffuse tumor cells were observed along regions of lymphatic vessels both proximal and distal to the primary tumor site. However, measurement of blood and lymph vessel density showed no significant change over time. Next, these datasets were used for quantitative analysis by setting the tumor cell channel (GFP) at a threshold to count any clusters greater than 50 pixels2 (~0.0013mm2) and with greater than the mean + 2 standard deviations of the background signal while avoiding noise/artifacts from very small regions (<~0.001mm2). This allowed for computation of the total tumor area including average area of each cluster. In addition, the area moment, which describes the basic directional growth pattern of the tumor cells, was quantified by multiplying a cluster distance from the center of mass by its area. Conclusions: A key novel finding from structured illumination imaging data was the observation of LVI occurring early, similar with the clinical presentation in IBC patients. This model was able to effectively track tumor cluster migration. This approach of short-term longitudinal imaging time frame in studying transient or dynamic events of diffuse, collectively migrating tumor cells in the local environment and quantitative analysis of the tumor area, motility and vessel characteristics is an innovation that can be used to investigate other cancer cell types exhibiting LVI. Funding in part by DoD W81XWH-17-1-0297; W81XWH201053 (GRD); ACS Mission Boost grant MBG-20-141-01 (GRD) and NIH grant P30-CA014236 (Imaging Core/GMP) Citation Format: Gayathri R Devi, Dorababu Sannareddy, Alexandra Bennion, Ashlyn Rickard, Douglas C Rouse, Mark W Dewhirst, Gregory M Palmer. Monitoring Lymphovascular Invasion and Tumor Growth of Inflammatory Breast Cancer in a Murine Lymphatic Reporter Window Chamber Model [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P3-08-06.

Abstract P5-04-04: The spatial interactions between FOXP3+ Tregs, CD8+ cytotoxic T cells and tumor cells predict response to therapy and prognosis in inflammatory breast cancer

Abstract Background: Inflammatory breast cancer (IBC) is a rare and aggressive type of locally advanced breast cancer. A 79-gene signature, reported by our lab, is shaped by specific immune response programs and discriminates between IBC and non-IBC (nIBC). However, it remains an enigma how infiltrating immune cells are able to determine the IBC phenotype. Furthermore, the presence of immune cells like FOXP3+ Tregs and CD8+ cytotoxic T cells is associated with outcome in proliferative subtypes of breast cancer and the interaction between these cells plays a role in the functional immune response. Therefore, we aimed to assess the spatial associations between immune cells in IBC. Additionally, we used deep-learning to examine interactions between cancer and immune cells. Methodology: Immunostainings (Hematoxylin-DAB, H-DAB) were done according to well-validated protocols for CD8 (cytotoxic T-cells), FOXP3 (Tregs) and CD163 (tumor-associated macrophages, TAMs) in a large population of 134 IBC patients. All slides were digitalized and evaluated using VISIOPHARM® software, allowing virtual multiplexing. We quantified the number of DAB+ immune cells and each positive immune cell was located using XY coordinates. Spatial co-localization was examined using statistics developed for ecological studies based on point pattern and quadrant analysis. TILs were scored according to the TIL working group guidelines on H&E slides. Tumor cell coordinates were collected using a deep-learning algorithm applied to the CD8 stained slide. To perform deep-learning, we aligned two consecutive slides: one PanCK stained slide and one slide stained for CD8 (H-DAB). Using virtual multiplexing and the PanCK staining, we determined the tumor regions on the H-DAB stained slide. Subsequently, 18 images were incorporated to train the algorithm with more than 150.000 iterations (Deeplabv3+), after which the algorithm was evaluated in a test set of 12 images, approved and applied to all images to locate the tumor cells. Results: Most of the patients presented with a hormone receptor (HR) positive carcinoma (60.6%, n= 82/132). The presence of distant disease, HR status and TIL score were associated with overall survival (OS), but the density of the different immune cells or the CD8/FOXP3 ratio was not. However, using an effector index we demonstrated that patients with more FOXP3+ cells in a radius of 30 μm surrounding a CD8+ cell had a significant worse outcome (Median OS: 2.7 vs. 6.3 years, P= .01) and this remained significant in a multivariate model (HR: 2.85, P< .001). Complete pathological response (pCR) after neo-adjuvant chemotherapy was achieved by 28.7% (n= 27/94) of the patients with initially localized disease. Infiltration with CD8+ T cells (P= .005), TAMs (P= .008) and TILs (P= .002) predicted pCR, but a likelihood ratio test showed no difference between a model using CD8+ cells, TAMs or TILs. Interestingly, pCR was less often achieved in patients that had colocalization of FOXP3+ cells near the tumor cells (P= .003). This colocalization was determined using a Morista-Horn index and independent of the number of Tregs, CD8+ cells or TILs. The deep-learning algorithm had a mean dice coefficient in the test set of 0.83, indicating a good overlap between the tumor area determined by the AI on the CD8 slides and the area on the PanCK stained slides. Conclusions: We have created a deep learning algorithm that adequately detects IBC tumor cells on H-DAB stained images and show, in a large cohort of 132 patients, that the negative impact of Tregs appears to depend on the spatial arrangement. While solely the number of Tregs is not associated with pCR or OS, patients with FOXP3+ Tregs that cluster together near CD8+ cytotoxic T cells had a worse outcome and pCR was achieved more often in patients with less Tregs near the tumor cells. Citation Format: Christophe Van Berckelaer, Charlotte Rypens, Steven Van Laere, Koen Marien, Pieter-Jan van Dam, Peter Vermeulen, Luc Dirix, Mark Kockx, Cecile Colpaert, Peter van Dam. The spatial interactions between FOXP3+ Tregs, CD8+ cytotoxic T cells and tumor cells predict response to therapy and prognosis in inflammatory breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P5-04-04.

Abstract B44: Immune pathway dysregulations in inflammatory breast cancer health disparity

Abstract Background: Inflammatory breast cancer (IBC) is a distinct, aggressive and the most lethal form of breast cancer. Furthermore, data from population-based registries, when stratified by race and compared to other locally advanced breast cancers, suggest that IBC has a disproportionately high incidence, prevalence & shorter median survival time in African Americans (AA) compared to Caucasian American (CA) patients. IBC health disparities that are independent of external factors like differences in income, screening rates, and access to health care suggest a biological component to poor outcomes. IBC pathobiology is also unique wherein instead of a solid tumor, tightly packed tumor cell clusters/emboli are formed with a propensity for lymphatic dissemination. The underlying mechanisms for this extremely aggressive breast cancer phenotype are largely unknown and, therefore, present a major impediment to identifying the molecular underpinnings for health disparities in IBC patients. Methods and Results: In order to understand how IBC cells evade cell death signals in the host microenvironment, we conducted comparative analysis of the largest collection of untreated primary tumor samples of IBC (N=137), along with stage- and subtype-matched non-IBC (N=252) and normal breast tissue. Results reveal that pretreatment tumor tissue samples from IBC patients have a heightened expression of adaptive stress response genes comprised of high levels of the nuclear transcription factor, NFkappaB, and its target genes in the anti-apoptotic and immune/inflammatory pathways that correlate with cell survival. In addition, guided by analysis based on presence of tumor infiltrating leukocytes in IBC (N=69) and non-IBC (N=62) patient samples, we have identified a set of pathways and genes that have the ability to distinguish the immune responses in IBC and non-IBC patients in a subtype independent manner. In particular, interferon alpha/beta and NFκB signaling pathways were differentially regulated in IBC vs. non-IBC. Gene expression driven analysis of enrichment of leukocyte subsets reveal specific differences in plasma cells and effector memory CD8 cells in IBC bulk tumor samples. Preliminary data analysis of gene expression data sets from laser microdissected epithelium and stromal compartments of AA and CA breast cancer patients also show enrichment of plasma cells, effector memory CD4+, and M1 macrophages. Studies are ongoing to characterize these immune profiles that are unique and distinguishable in IBC patients and further identify race-related differences. Most importantly, we discovered that the constitutive NFκB hyperactivation observed in IBC tumor cells is driven by stress-mediated translational upregulation of the most potent anti-apoptotic protein, XIAP. Interestingly, XIAP:NFκB overexpressing cells are highly resistant to immunotherapy mediated cell death. These data strongly suggest that in IBC, host immune phenotype promotes outgrowth of cell death resistant population and specific tumor cell pro-survival signaling mechanisms that enhances invasion and lymphatic dissemination. We have developed novel in vitro IBC tumor emboli culture model and a transgenic mouse bearing red fluorescent lymphatic vasculature that allow recapitulating host factors to define tumor cell signaling mechanisms on immune effector function and the resultant invasive phenotype. Conclusions: As the incidence is rising and targets for therapy are scant, IBC has the potential to become a major public health disparity concern. This study reveals that the immune factors in the host microenvironment may interact with underlying IBC genetics to promote the aggressive nature of the tumor, and more specifically, these factors may be one of the sources of biologic variation between patients of different ethnicities leading to IBC disparity outcomes. Supported in part by Duke IBC Consortium, DCI NIH CAO14236 development funds (GRD), DoD W81XWH-13-1-0047 (GRD). Citation Format: Gayathri R. Devi, Eun-Sil Shelley Hwang, John Stewart, Michael A. Morse, Steven Van Laere. Immune pathway dysregulations in inflammatory breast cancer health disparity. [abstract]. In: Proceedings of the Ninth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2016 Sep 25-28; Fort Lauderdale, FL. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2017;26(2 Suppl):Abstract nr B44.

Abstract P6-12-14: Histone deacetylase inhibitor suberoylanilide hydroxamic acid targets breast cancer stem cells and inhibits metastasis

Abstract Introduction: Inflammatory breast cancer (IBC) is a distinct and aggressive subtype of locally advance breast cancer associated with increased aldehyde dehydrogenase 1 (ALDH1) positive cancer stem cells (CSCs). IBC is associated with a poor survival rate (40% 5-year survival), with few therapeutic strategies identified that effectively blocked the growth and metastasis of IBC. Our previous in vitro studies revealed that the pan-histone deacetylase inhibitor Suberoylanilide Hydroxamic Acid (SAHA) effectively targets self-renewal of IBC tumor cells. Materials and Methods: The IBC cell line, SUM149 was tagged with the Luciferase gene (SUM149-LUC) and their in vivo growth in immune compromised mice tested in both orthotopic and metastatic settings. Results: SUM149-LUC rapidly develop primary tumors and metastatic lesions at a variety of locations including lung, brain, bone, liver, reproductive organs and adrenal glands. SAHA effectively blocked growth of SUM149 primary tumors and inhibited metastasis, which was synergistic with the microtubule stabilizer, Paclitaxel. The therapeutic efficacy of SAHA was associated with a significant decrease in ALDH1+ CSCs populations. Conclusions: Collectively, these results suggest that strategies of combining agents such as SAHA that target CSCs with Paclitaxel that targets the bulk of proliferating tumor cells warrant further investigation for their potential effectiveness in IBC patients, who have the lowest survival of breast cancer patients and have few therapeutic options. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P6-12-14.

Presence of anaplastic lymphoma kinase in inflammatory breast cancer

Although Inflammatory Breast Cancer (IBC) is recognized as the most metastatic variant of locally advanced breast cancer, the molecular basis for the distinct clinical presentation and accelerated program of metastasis of IBC is unknown. Reverse phase protein arrays revealed activation of the receptor tyrosine kinase, anaplastic lymphoma kinase (ALK) and biochemically-linked downstream signaling molecules including JAK1/STAT3, AKT, mTor, PDK1, and AMPKβ in pre-clinical models of IBC. To evaluate the clinical relevance of ALK in IBC, analysis of 25 IBC patient tumors using the FDA approved diagnostic test for ALK genetic abnormalities was performed. These studies revealed that 20/25 (80%) had either increased ALK copy number, low level ALK gene amplification, or ALK gene expression, with a prevalence of ALK alterations in basal-like IBC. One of 25 patients was identified as having an EML4-ALK translocation. The generality of gains in ALK copy number in basal-like breast tumors with IBC characteristics was demonstrated by analysis of 479 breast tumors using the TGCA data-base and our newly developed 79 IBC-like gene signature. The small molecule dual tyrosine kinase cMET/ALK inhibitor, Crizotinib (PF-02341066/Xalkori®, Pfizer Inc), induced both cytotoxicity (IC50 = 0.89 μM) and apoptosis, with abrogation of pALK signaling in IBC tumor cells and in FC-IBC01 tumor xenograft model, a new IBC model derived from pleural effusion cells isolated from an ALK+ IBC patient. Based on these studies, IBC patients are currently being evaluated for the presence of ALK genetic abnormalities and when eligible, are being enrolled into clinical trials evaluating ALK targeted therapeutics.Electronic supplementary materialThe online version of this article (doi:10.1186/2193-1801-2-497) contains supplementary material, which is available to authorized users.

Uncovering the Molecular Secrets of Inflammatory Breast Cancer Biology: An Integrated Analysis of Three Distinct Affymetrix Gene Expression Datasets

Inflammatory breast cancer (IBC) is a poorly characterized form of breast cancer. So far, the results of expression profiling in IBC are inconclusive due to various reasons including limited sample size. Here, we present the integration of three Affymetrix expression datasets collected through the World IBC Consortium allowing us to interrogate the molecular profile of IBC using the largest series of IBC samples ever reported. Affymetrix profiles (HGU133-series) from 137 patients with IBC and 252 patients with non-IBC (nIBC) were analyzed using unsupervised and supervised techniques. Samples were classified according to the molecular subtypes using the PAM50-algorithm. Regression models were used to delineate IBC-specific and molecular subtype-independent changes in gene expression, pathway, and transcription factor activation. Four robust IBC-sample clusters were identified, associated with the different molecular subtypes (P<0.001), all of which were identified in IBC with a similar prevalence as in nIBC, except for the luminal A subtype (19% vs. 42%; P<0.001) and the HER2-enriched subtype (22% vs. 9%; P<0.001). Supervised analysis identified and validated an IBC-specific, molecular subtype-independent 79-gene signature, which held independent prognostic value in a series of 871 nIBCs. Functional analysis revealed attenuated TGF-β signaling in IBC. We show that IBC is transcriptionally heterogeneous and that all molecular subtypes described in nIBC are detectable in IBC, albeit with a different frequency. The molecular profile of IBC, bearing molecular traits of aggressive breast tumor biology, shows attenuation of TGF-β signaling, potentially explaining the metastatic potential of IBC tumor cells in an unexpected manner.

Genomic and expression analysis of microdissected inflammatory breast cancer

Inflammatory breast cancer (IBC) is a unique clinical entity characterized by rapid onset of erythema and swelling of the breast often without an obvious breast mass. Many studies have examined and compared gene expression between IBC and non-IBC (nIBC), repeatedly finding clusters associated with receptor subtype, but no consistent gene signature associated with IBC has been validated. Here we compared microdissected IBC tumor cells to microdissected nIBC tumor cells matched based on estrogen and HER-2/neu receptor status. Gene expression analysis and comparative genomic hybridization were performed. An IBC gene set and genomic set were identified using a training set and validated on the remaining data. The IBC gene set was further tested using data from IBC consortium samples and publicly available data. Receptor driven clusters were identified in IBC; however, no IBC-specific gene signature was identified. Fifteen genes were correlated between increased genomic copy number and gene overexpression data. An expression-guided gene set upregulated in the IBC training set clustered the validation set into two clusters independent of receptor subtype but segregated only 75 % of samples in each group into IBC or nIBC. In a larger consortium cohort and in published data, the gene set failed to optimally enrich for IBC samples. However, this gene set had a high negative predictive value for excluding the diagnosis of IBC in publicly available data (100 %). An IBC enriched genomic data set accurately identified 10/16 cases in the validation data set. Even with microdissection, no IBC-specific gene signature distinguishes IBC from nIBC. Using microdissected data, a validated gene set was identified that is associated with IBC tumor cells. Inflammatory breast cancer comparative genomic hybridization data are presented, but a validated genomic data set that identifies IBC is not demonstrated.

Abstract P4-06-03: Zinc Finger Nuclease Genome Engineering Reveal Multiple Functions of Secretory Leukocyte Peptidase Inhibitor in Regulating Pleuripotency of Cancer Stem Cells in Inflammatory Breast Cancer

Abstract Background: Inflammatory breast cancer (IBC) is the most aggressive and lethal variant of this disease and is known to be enriched for cells with a cancer stem cell phenotype. IBC is characterized by the presence of cell aggregates, defined as tumor emboli, that metastasize into skin and chest wall. The only documented biomarker of tumor emboli is the surface glycoprotein, E-cadherin. We previously demonstrated that IBC tumor emboli express the alarm anti-protease, secretory leukocyte peptidase inhibitor (SLPI), a metastasis related gene highly expressed in IBC patient tumors. Since the function of SLPI in IBC is unknown, the present studies used zinc finger technology to knockout (KO) copies of SLPI in the SUM149 IBC cell line to define the role of SLPI in IBC. Materials and Methods: Using CompoZr zinc finger nuclease (ZFN) technology (Sigma-Aldrich), SLPI KO cell lines were generated by disrupting all alleles (3) in exon 1 of SUM149 cells derived from an IBC primary tumor with a high CD44+cell population. The target-specific ZFNs bound DNA at a sequence-specific location and created double strand breaks repaired by non-homologous end joining, resulting in deletions at the SLPI locus. Single cell SLPI KO clones were isolated and serially passaged to establish stable cell lines. Functional assays were used to assess proliferation, invasion, tube formation and clonogenicity. Global transcriptional profiling was performed to identify genes and signaling pathways directly regulated by SLPI. Results: SUM149 SLPI KO clones did not produce SLPI protein and had a significantly slower turn-over time of 75 hrs compared with 24 hrs in SUM149 wild type clones. Loss of SLPI blocked invasion by 50%, and completely inhibited formation of tube-like structures, an activity defined as vasculogenic mimicry, characteristic of IBC. The loss of only 1 SLPI allele resulted in the inability of SUM149 cells to grow as anchorage independent clones in soft agar, commonly used as a predictor of in vivo tumorigenicity. SLPI directly regulated expression of multiple genes within the embryonic stem cell pleuripotency canonical pathway, including WNT, Frizzled, PDK-1, platelet derived growth factor receptor and sphingosine-1-phosphate receptor. Studies are underway to determine the specific role of SLPI in IBC tumor growth and metastasis. Conclusions: Our previous studies demonstrated that SLPI is expressed by IBC tumor emboli and can be used as a biomarker of tumor emboli in IBC core and skin punch biopsies. SLPI was found to not only regulate critical functional activities of IBC tumor cells but also to directly regulate genes within pathways critically important to maintenance of pleuripotency of tumors with a cancer stem cell phenotype. Collectively, these studies demonstrate the power and utility of the zinc finger technology, which enables the interrogation of tumor cells to discern the direct function and role of specific genes of interest. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P4-06-03.

Tumor stromal vascular endothelial growth factor A is predictive of poor outcome in inflammatory breast cancer

BackgroundInflammatory breast cancer (IBC) is a highly angiogenic disease; thus, antiangiogenic therapy should result in a clinical response. However, clinical trials have demonstrated only modest responses, and the reasons for these outcomes remain unknown. Therefore, the purpose of this retrospective study was to determine the prognostic value of protein levels of vascular endothelial growth factor (VEGF-A), one of the main targets of antiangiogenic therapy, and its receptors (VEGF-R1 and -R2) in IBC tumor specimens.Patients and MethodsSpecimens from IBC and normal breast tissues were obtained from Algerian patients. Tumor epithelial and stromal staining of VEGF-A, VEGF-R1, and VEGF-R2 was evaluated by immunohistochemical analysis in tumors and normal breast tissues; this expression was correlated with clinicopathological variables and breast cancer-specific survival (BCSS) and disease-free survival (DFS) duration.ResultsFrom a set of 117 IBC samples, we evaluated 103 ductal IBC tissues and 25 normal specimens. Significantly lower epithelial VEGF-A immunostaining was found in IBC tumor cells than in normal breast tissues (P <0.01), cytoplasmic VEGF-R1 and nuclear VEGF-R2 levels were slightly higher, and cytoplasmic VEGF-R2 levels were significantly higher (P = 0.04). Sixty-two percent of IBC tumors had high stromal VEGF-A expression. In univariate analysis, stromal VEGF-A levels predicted BCSS and DFS in IBC patients with estrogen receptor-positive (P <0.01 for both), progesterone receptor-positive (P = 0.04 and P = 0.03), HER2+ (P = 0.04 and P = 0.03), and lymph node involvement (P <0.01 for both). Strikingly, in a multivariate analysis, tumor stromal VEGF-A was identified as an independent predictor of poor BCSS (hazard ratio [HR]: 5.0; 95% CI: 2.0-12.3; P <0.01) and DFS (HR: 4.2; 95% CI: 1.7-10.3; P <0.01).ConclusionsTo our knowledge, this is the first study to demonstrate that tumor stromal VEGF-A expression is a valuable prognostic indicator of BCSS and DFS at diagnosis and can therefore be used to stratify IBC patients into low-risk and high-risk groups for death and relapses. High levels of tumor stromal VEGF-A may be useful for identifying IBC patients who will benefit from anti-angiogenic treatment.

Genomic Profiling of Pre-Clinical Models of Inflammatory Breast Cancer Identifies a Signature of Epithelial Plasticity and Suppression of TGFβ Signaling

Abstract Study background: Inflammatory Breast Cancer (IBC) is the most metastatic variant of breast cancer. Although IBC is recognized as a distinct variant of breast cancer, the molecular basis for the rapid progression of IBC remains largely undefined, in part due to the lack of preclinical models that recapitulate the human disease as well as a lack of comprehensive analysis of the preclinical models of IBC that are available. Methods: All available 7 pre-clinical models of IBC, including 2 new models, FC-IBC01 and FC-IBC02 developed from pleural effusion, were used to identify genes and molecular pathways that are selectively altered compared to non IBC breast tumor models. Laser capture micro dissection of biopsy tissue from core biopsy and skin punch biopsies were also analyzed by whole transcriptome analysis. Results: Whole transcriptome analysis defined 7 pre-clinical models of IBC as being within either the triple negative or ErbB2/Her-2 expressing subtypes, similar to the prevalence of these subtypes of breast cancer observed in IBC patients. Comparative analysis of the FC-IBC01, FC-IBC02 and Mary-X models of IBC demonstrated that each of these recapitulate the formation of tumor emboli with encircling lymphovasculogenesis. The majority (6/7) of the pre-clinical models of IBC express CDH1, which encodes for E-cadherin, which was associated with a loss of ZEB1, a transcriptional repressor of E-cadherin. The lack of ZEB1 expression was validated in a limited set of 4 skin punch biopsy samples from IBC patients that were isolated by laser capture micro-dissection, demonstrating concordance with loss of ZEB1 in pre-clinical models of IBC. Expression of other transcription factors involved in acquisition of a cancer stem cell phenotype, including Snai1, which encodes for Snail, SNAI2, which encodes for Slug and TWIST1, was retained in pre-clinical models of IBC. Maintenance of E-cadherin in pre-clinical models of IBC was associated with a loss of genes within the transforming growth factor beta (TGFβ signaling pathway, with expression of SMAD6, a known repressor of TGFβ. This is similar to a recent study reporting the persistence of E-cadherin and loss of TGFβ signaling in IBC patient tumors based on gene profiling of 3 independent data sets. Conclusion: The present studies provide first time comparison of gene signatures of 7 pre-clinical models of IBC, including our 2 newly developed pre-clinical models, FC-IBC01 and FC-IBC02, that recapitulate formation of tumor emboli with encircling lymphatic vessels, similar to that observed in biopsy tissues of IBC patients. We demonstrate that E-cadherin expression was associated with both loss of ZEB1 and diminished expression of multiple genes within the TGFβ signaling pathway, with retention of expression of transcription factors and surface markers consistent with maintenance of a cancer stem cell phenotype, as has been reported to be a characteristic of IBC tumors. Collectively, these observations provide first time characterization of the molecular signatures of all available pre-clinical models of IBC, and suggest that IBC has a signature of epithelial plasticity, with characteristics of their ability to undergo the mesenchymal to epithelial reverting transition. The loss of genes within the TGFβ signaling is also consistent with the tight cell: cell aggregation of IBC tumor cells within tumor emboli that exhibit “cohesive invasion”. The new pre-clinical models of IBC that recapitulate the human disease will serve as useful tools to accelerate our understanding of the molecular underpinnings and therapeutic targets of IBC as the most lethal form of breast cancer.

P1-02-03: The Reciprocal Roles of E-Cadherin and ZEB1 Demonstrate the Mesenchymal-Epithelial Transition as a Primary Characteristic of Inflammatory Breast Cancer.

Abstract Background: Inflammatory breast cancer (IBC) is a rare but very aggressive form of breast cancer. IBC is characterized by nests of tightly aggregated cells, defined as tumor emboli, that exhibit characteristics of cancer stem cells (CSCs). IBC tumor emboli express E-cadherin which is required to maintain their integrity and our recent evidence demonstrates that expression of E-cadherin by tumor emboli is associated with lack of ZEB1 expression, a transcriptional repressor of E-cadherin. This is at odds with the current hypothesis that metastatic progression is associated with the process of epithelial mesenchymal transition (EMT), with loss of E-cadherin and gain of transcription factors including ZEB1, acquisition of CSC characteristics and enhanced invasive capabilities. Materials and Methods: shRNA knockdown and over-expression methods, real time PCR arrays, western blotting, and in vitro assays to evaluate proliferation, invasion, growth in soft agar and clonogenicity and in vivo animal studies were used. Results: Expression of E-cadherin was reduced by shRNA and ZEB1 was expressed in SUM149 IBC tumor cells. Numerous EMT-related genes were upregulated with loss of E-cadherin and gain of ZEB1, including N-cadherin and vimentin. However, there were marginal differences in the in vitro parameters of proliferation, Matrigel invasion and anchorage independent growth in soft agar between SUM149-shECad or SUM149-ZEB1 clones and their respective vector control cells. The loss of E-cadherin and gain of ZEB1 altered the morphology of SUM149 cells when cultured under low adherence conditions permissive for the enrichment of CSC, exhibiting a reversion in grape-like morphology to more well defined spheres, which was accompanied by increased clonogenicity in both SUM149-shECad and SUM149-ZEB1 cells. The loss of E-cadherin and the gain of ZEB1 significantly inhibited tumor growth of cells injected in the mammary fat pad of NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ mice. Tumor volume at 56 days for E-cadherin vector control cells was 771.9 mm3 +/− 185.6 compared to shECadherin tumors, which was 13.6 mm3 +/− 7.2. Tumor volume of ZEB1 vector control tumors was 346.1 mm3 +/− 96 compared to volume of ZEB1 expressing tumors, which was 21.5 mm3 +/− 7.2.Conclusions: E-cadherin with lack of ZEB1 expression in IBC is consistent with a mesenchymal-epithelial transition (MET), consistent with the retention of the epithelial phenotype while maintaining a program of rapid metastasis and colonization of lymph nodes and distant organ sites. Furthermore, we demonstrate that the E-cadherin-ZEB1 axis is critical for the in vivo growth of IBC tumor cells. Although SUM149 cells are fully capable of undergoing an EMT process, which is under negative regulation by E-cadherin, the process of EMT does not drive in vivo tumor growth in IBC. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P1-02-03.

P2-05-04: Mapping the Specific Gene Families Activated in the Lymphangiogenesis and Vasculogenic Mimicry Exhibited by Inflammatory Breast Cancer.

Abstract Background: Inflammatory breast cancer (IBC) is the most metastatic variant of locally advanced breast cancer. Although IBC is diagnosed less commonly than other types of breast cancer, it is extremely aggressive, and accounts for a disproportionate number of breast cancer related deaths annually. IBC exhibits very specific patterns of lymphangiogenesis and vasculogenic mimicry, however detailed studies of the genes and proteins involved in these angiogenic processes are lacking. This study performed whole unbiased gene transcription studies with validation by protein arrays using all available pre-clinical cell lines and in vivo xenograft models of IBC, including a new model of IBC, FC-IBC01, which exhibits lymphovascular invasion, to identify the specific pathways involved in the distinctive angiogenesis observed in IBC. Materials and Methods: Real-time quantitative RT-PCR, cDNA microarray gene profiling, immunofluorescence with confocal imaging and protein arrays were used to examine differential expression of specific angiogenic gene families including VEGFA,B,C,D, VEGF Receptor genes, and ANG/TIE genes linked to angiogenesis and lymphangiogenesis. Results: Activity of the matrix metalloproteinase, MMP-2, is required for IBC tumor cells to undergo vasculogenic mimicry (VM), which is associated with a loss of TIMP-2, a well known inhibitor of angiogenesis. Therapeutics that target MMP activity can successfully inhibit this VM. Furthermore, pre-clinical models of IBC that form IBC tumor emboli exhibit lymphovascular invasion that is associated with distinct patterns of expression of genes that encode for distinct receptor tyrosine kinases that may represent important therapeutic targets for IBC. Discussion: Identification of the distinct angiogenic pathways that are activated in IBC provides insight into the therapeutic targets that may abrogate the distinct lymphovascular invasion and vasculogenic mimicry that are linked to the aggressive metastasis of IBC. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P2-05-04.

Persistent E-Cadherin Expression in Inflammatory Breast Cancer

E-cadherin is a transmembrane glycoprotein that mediates epithelial cell-to-cell adhesion. Because loss of E-cadherin expression results in disruption of cellular clusters, it has been postulated that E-cadherin functions as a tumor suppressor protein. The role of E-cadherin in inflammatory breast cancer (IBC), a distinct and highly aggressive form of breast cancer, is largely unknown. The aim of our study was to elucidate whether E-cadherin expression contributes to the development and progression of the IBC phenotype and to investigate any differences in E-cadherin expression between IBC and stage-matched non-IBC. Forty-two breast cancer cases (20 IBC and 22 non-IBC) were identified. Strict and well-accepted criteria were used for the diagnosis of IBC. Clinical and pathologic features were studied, and formalin-fixed, paraffin-embedded tissue sections were immunostained for E-cadherin, estrogen and progesterone receptors (ER and PR, respectively), and HER2/neu. Statistical analysis was performed using Fisher's exact test. All IBC uniformly expressed E-cadherin, whereas 15 of the 22 (68%) of the non-IBC expressed the protein (P = .006). Intralymphatic tumor emboli in the IBC cases were also all E-cadherin positive. Two IBC tumors demonstrated invasive lobular histology, and both cases were positive for E-cadherin. Of the non-IBC cases, three were invasive lobular carcinomas, and all were positive for E-cadherin. No association was found between E-cadherin expression and ER, PR status, or HER2/neu overexpression. Our study demonstrates that there is a strong association between E-cadherin expression and IBC and suggests that E-cadherin may be involved in the pathogenesis of this form of advanced breast cancer. In our study, we demonstrate that circulating IBC tumor cells strongly express E-cadherin, thereby providing an important exception to the positive association between E-cadherin loss and poor prognosis in breast cancer.