Targets In Inflammatory Breast Cancer Research Articles

XIAP overexpressing inflammatory breast cancer patients have high infiltration of immunosuppressive subsets and increased TNFR1 signaling targetable with Birinapant

ObjectiveTo assess the expression pattern of X-linked inhibitor of apoptosis protein (XIAP), a cellular stress sensor, and delineate the associated changes in the tumor immune microenvironment (TiME) for prognostic value and new therapeutic targets in inflammatory breast cancer (IBC). MethodsImmunohistochemistry was conducted to assess the spatial localization of immune subsets, XIAP, and PDL1 expression in IBC and non-inflammatory breast cancer (nIBC) pretreatment tumors (n = 142). Validation and further exploration were performed by gene expression analysis of patient tumors along with signaling studies in a co-culture model. ResultsHigh XIAP in 37/81 IBC patients correlated significantly with high PD-L1, increased infiltration of FOXP3+ Tregs, CD163+ tumor-associated macrophages (TAMs), low CD8/CD163 ratio in both tumor stroma (TS) and invasive margins (IM), and higher CD8+ T cells and CD79α+ B cells in the IM. Gene set enrichment analysis identified cellular stress response- and inflammation-related genes along with tumor necrosis factor receptor 1 (TNFR1) expression in high-XIAP IBC tumors. Induction of TNFR1 and XIAP was observed when patient-derived SUM149 IBC cells were co-cultured with human macrophage-conditioned media simulating TAMs, further demonstrating that the TNF-α signaling pathway is a likely candidate governing TAM-induced XIAP overexpression in IBC cells. Finally, addition of Birinapant, a pan IAP antagonist, induced cell death in the pro-survival cytokine-enriched conditions. ConclusionUsing immunophenotyping and gene expression analysis in patient biospecimens along with in silico modeling and a preclinical model with a pan-IAP antagonist, this study revealed an interplay between increased TAMs, TNF-α signaling, and XIAP activation during (immune) stress in IBC. These data demonstrate the potential of IAP antagonists as immunomodulators for improving IBC therapeutic regimens.

Advances in targets in inflammatory breast cancer.

Advances in targets in inflammatory breast cancer.

Abstract P4-08-19: Biomarker analysis: Multi-omics elucidation of Cohort 1 from a phase II study of a triple combination of Atezolizumab + cobimetinib + eribulin in patients with metastatic inflammatory breast cancer

Abstract Background: Inflammatory breast cancer (IBC) is a rare but aggressive tumor type accounting for up to 10% of breast cancer deaths. One-third of IBCs express high PD-L1 that can be targeted by atezolizumab (Az). MEK inhibitor cobimetinib (Co) not only inhibits the RAS-MAPK pathway but can further enhance immune-mediated killing. Thus, we hypothesize that Az+Co may enhance the efficacy of chemotherapy in metastatic IBC (mIBC). We opened a trial to test this hypothesis with a comprehensive multi-omics biomarker assessment. Patients and Methods: In a single-center, open-label phase II study, cohort 1 received one cycle of Az+ Co, followed by four cycles of Az+Co+eribulin (E) to induce a maximum clinical response, followed by Az+Co maintenance. Pre and Post one cycle of Az+Co tumors were collected for immunohistochemistry (IHC), multiplex immunofluorescence (mIF), whole-exome sequencing (WES), and RNA sequencing (RNAseq). Blood was collected for circulating tumor DNA (ctDNA). Results: Seventeen patients were enrolled in cohort 1. Seven had PR, and three had SD as the best responses. Fourteen had pre, and six had pre/post tumors. The levels of PD-L1 expression at pre/post were not associated with responses. WES revealed the median tumor mutation burden at pre- was 9mt/Mb. More than 50% had TP53 and PI3K pathway mutations at pre. RTK-RAS and Notch pathways were altered in 4/9 cases. PRDM9 and DPY19L2 single-gene mutations were commonly noted in pre. No cancer-associated gene aberration, including potential biomarkers of anti-PDL1 agent response was associated with clinical outcomes. Transcriptomic gene set enrichment analysis demonstrated a greater degree of TNFa and TGFb signaling, Oxphos, angiogenesis, and epithelial-to-mesenchymal transition (EMT) processes in tumors from patients with poor response. Immune profiling by RNAseq revealed two responders to have elevated effector memory T cells, NK T cells, myeloid dendritic cells, and M1 macrophage signatures in pre-samples, but post-samples were not available. mIF confirmed a higher frequency of NK-T cells. The ctDNA analysis from serially collected blood samples is ongoing. Discussion: In this comprehensive multi-omics analysis of pre-and-post-Az+Co, we observed several novel findings, while conventional biomarkers for Az and Co did not correlate with clinical responses. EMT, Oxphos, Notch, and chronic inflammation pathways, which are not previously well reported, were observed in this IBC cohort. These markers warrant further validation to see if they carry significance as therapeutic targets in IBC. Citation Format: Bora Lim, Angela Alexander, Jie S. Willey, Huiming Sun, Suyu Liu, Anisha B. Patel, Edwin Roger Parra, Cara Haymaker, Luisa Solis Soto, Alejandra Serrano, Baohua Sun, Cibelle Freitas Pinto Lima, Auriole Tamegnon, Renganayaki K. Pandurengan, Dzifa Douse, Jessica Lan, Luthra Raja, Randy Chu, Mark Knafl, Scott E. Woodman, Haifeng Zhu, Katja Shulze, Katherine Fedenko, Walter Darbonne, Naoto T. Ueno, Vicente Valero. Biomarker analysis: Multi-omics elucidation of Cohort 1 from a phase II study of a triple combination of Atezolizumab + cobimetinib + eribulin in patients with metastatic inflammatory breast cancer. [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 P4-08-19.

CASP9 As a Prognostic Biomarker and Promising Drug Target Plays a Pivotal Role in Inflammatory Breast Cancer.

Background Inflammatory breast cancer (IBC) is one of the most rare and aggressive subtypes of primary breast cancer (BC). Our study aimed to explore hub genes related to the pathogenesis of IBC, which could be considered as novel molecular biomarkers for IBC diagnosis and prognosis. Material and Methods. Two datasets from gene expression omnibus database (GEO) were selected. Enrichment analysis and protein-protein interaction (PPI) network for the DEGs were performed. We analyzed the prognostic values of hub genes in the Kaplan-Meier Plotter. Connectivity Map (CMap) and Comparative Toxicogenomics Database (CTD) was used to find candidate small molecules capable to reverse the gene status of IBC. Results 157 DEGs were selected in total. We constructed the PPI network with 154 nodes interconnected by 128 interactions. The KEGG pathway analysis indicated that the DEGs were enriched in apoptosis, pathways in cancer and insulin signaling pathway. PTEN, PSMF1, PSMC6, AURKB, FZR1, CASP9, CASP6, CASP8, BAD, AKR7A2, ZNF24, SSX2IP, SIGLEC1, MS4A4A, and VSIG4 were selected as hub genes based on the high degree of connectivity. Six hub genes (PSMC6, AURKB, CASP9, BAD, ZNF24, and SSX2IP) that were significantly associated with the prognosis of breast cancer. The expression of CASP9 protein was associated with prognosis and immune cells infiltration of breast cancer. CASP9- naringenin (NGE) is expected to be the most promising candidate gene-compound interaction for the treatment of IBC. Conclusion Taken together, CASP9 can be used as a prognostic biomarker and a novel therapeutic target in IBC.

MARCKS as a Potential Therapeutic Target in Inflammatory Breast Cancer.

Inflammatory breast cancer (IBC) is the most pro-metastatic form of breast cancer (BC). We previously demonstrated that protein overexpression of Myristoylated Alanine-Rich C Kinase Substrate (MARCKS) protein was associated with shorter survival in IBC patients. MARCKS has been associated with the PI3K/AKT pathway. MARCKS inhibitors are in development. Our objective was to investigate MARCKS, expressed preferentially in IBC that non-IBC (nIBC), as a novel potential therapeutic target for IBC. The biologic activity of MPS, a MARCKS peptide inhibitor, on cell proliferation, migration, invasion, and mammosphere formation was evaluated in IBC (SUM149 and SUM190) and nIBC (MDA-MB-231 and MCF7) cell lines, as well as its effects on protein expression in the PTEN/AKT and MAPK pathways. The prognostic relevance of MARCKS and phosphatase and tensin homolog (PTEN) protein expression as a surrogate marker of metastasis-free survival (MFS) was evaluated by immunohistochemistry (IHC) in a retrospective series of archival tumor samples derived from 180 IBC patients and 355 nIBC patients. In vitro MPS impaired cell proliferation, migration and invasion, and mammosphere formation in IBC cells. MARCKS inhibition upregulated PTEN and downregulated pAKT and pMAPK expression in IBC cells, but not in nIBC cells. By IHC, MARCKS expression and PTEN expression were negatively correlated in IBC samples and were associated with shorter MFS and longer MFS, respectively, in multivariate analysis. The combination of MARCKS-/PTEN+ protein status was associated with longer MFS in IBC patient only (p = 8.7 × 10−3), and mirrored the molecular profile (MARCKS-downregulated/PTEN-upregulated) of MPS-treated IBC cell lines. In conclusion, our results uncover a functional role of MARCKS implicated in IBC aggressiveness. Associated with the good-prognosis value of the MARCKS-/PTEN+ protein status that mirrors the molecular profile of MPS-treated IBC cell lines, our results suggest that MARCKS could be a potential therapeutic target in patients with MARCKS-positive IBC. Future preclinical studies using a larger panel of IBC cell lines, animal models and analysis of a larger series of clinical samples are warranted in order to validate our results.

Inflammatory Breast Cancer: Clinical Implications of Genomic Alterations and Mutational Profiling.

Simple SummaryInflammatory breast cancer (IBC) is an aggressive disease with high mortality rates. Nowadays, there is no targeted treatment for this tumor type. Based on this context, we investigated the molecular profile of this disease by using well-established methodologies (high-resolution microarray platform, targeted next-generation sequencing, and immunohistochemistry) that have proven potential to unveil cancer biomarkers. We found alterations related to IBC aggressiveness and metastasis (gains of MDM4, losses of CHL1, and high homologous recombination deficiency scores), and worse overall survival (variants in HR and mismatch repair genes). We also compared the mutational profiling of our cases with literature data, which includes both non-IBC and IBC cases, validating our findings. Overall, we describe genetic alterations with the potential to be used as prognostic or predictive biomarkers and ultimately improve IBC patients’ care.Inflammatory breast cancer (IBC) is a rare and aggressive type of breast cancer whose molecular basis is poorly understood. We performed a comprehensive molecular analysis of 24 IBC biopsies naïve of treatment, using a high-resolution microarray platform and targeted next-generation sequencing (105 cancer-related genes). The genes more frequently affected by gains were MYC (75%) and MDM4 (71%), while frequent losses encompassed TP53 (71%) and RB1 (58%). Increased MYC and MDM4 protein expression levels were detected in 18 cases. These genes have been related to IBC aggressiveness, and MDM4 is a potential therapeutic target in IBC. Functional enrichment analysis revealed genes associated with inflammatory regulation and immune response. High homologous recombination (HR) deficiency scores were detected in triple-negative and metastatic IBC cases. A high telomeric allelic imbalance score was found in patients having worse overall survival (OS). The mutational profiling was compared with non-IBC (TCGA, n = 250) and IBC (n = 118) from four datasets, validating our findings. Higher frequency of TP53 and BRCA2 variants were detected compared to non-IBC, while PIKC3A showed similar frequency. Variants in mismatch repair and HR genes were associated with worse OS. Our study provided a framework for improved diagnosis and therapeutic alternatives for this aggressive tumor type.

Poor Response to Neoadjuvant Chemotherapy Correlates with Mast Cell Infiltration in Inflammatory Breast Cancer.

Our understanding is limited concerning the tumor immune microenvironment of inflammatory breast cancer (IBC), an aggressive form of primary cancer with low rates of pathologic complete response to current neoadjuvant chemotherapy (NAC) regimens. We retrospectively identified pretreatment (N = 86) and matched posttreatment tissue (N = 27) from patients with stage III or de novo stage IV IBC who received NAC followed by a mastectomy. Immune profiling was performed including quantification of lymphoid and myeloid infiltrates by IHC and T-cell repertoire analysis. Thirty-four of 86 cases in this cohort (39.5%) achieved a pathologic complete response. Characterization of the tumor microenvironment revealed that having a lower pretreatment mast cell density was significantly associated with achieving a pathologic complete response to NAC (P = 0.004), with responders also having more stromal tumor-infiltrating lymphocytes (P = 0.035), CD8+ T cells (P = 0.047), and CD20+ B cells (P = 0.054). Spatial analysis showed close proximity of mast cells to CD8+ T cells, CD163+ monocytes/macrophages, and tumor cells when pathologic complete response was not achieved. PD-L1 positivity on tumor cells was found in fewer than 2% of cases and on immune cells in 27% of cases, but with no correlation to response. Our results highlight the strong association of mast cell infiltration with poor response to NAC, suggesting a mechanism of treatment resistance and a potential therapeutic target in IBC. Proximity of mast cells to immune and tumor cells may suggest immunosuppressive or tumor-promoting interactions of these mast cells.

Estrogen Non‐Genomic Signaling as a Potential Therapeutic Target in Inflammatory Breast Cancer

Breast cancer is the second leading cause of death in females in the United States2, being Inflammatory Breast Cancer (IBC) one of the most aggressive subtypes. The poor prognosis for patients with IBC emphasizes the need to better understand the molecular signature of this disease with the goal of developing effective targeted therapeutics. To this date there is no effective targeted therapeutics, especially for those patients that account for approximately 20–40% of IBC cases with triple‐negative breast cancer (TNBC) classification3. Importantly, several studies have shown that estrogen can exert non‐genomic effects in IBC and non‐IBC TNBC, mediated by the expression of an alternate isoform of estrogen receptor alpha, estrogen receptor alpha‐36, and GPR305,7,9. In this context, estrogen can activate non‐genomic signaling pathways involved in the acquisition of oncogenic phenotypes such as increased motility and invasion in IBC cells. Phytoestrogens, like coumestrol (Cou), are natural compounds found in plants, specifically soybeans. Also, it is a polyphenolic compound that is structurally similar to estradiol (E2) and several studies have shown that coumestrol inhibits cell proliferation and migration in TNBC. We hypothesize (1) that estrogen non‐genomic signaling has an active role in the aggressive metastatic phenotype of IBC, and (2) that Cou has anticancer activity by inhibiting estrogen non‐genomic signaling in IBC. To test this hypothesis, these cells were treated with E2 and activation of downstream kinases (ERK and AKT) was analyzed by Western blot. Immunofluorescence in IBC SUM149 cell line and Western Blot of breast cancer cell lines showed expression of estrogen receptors. In addition, preliminary observations demonstrated that upon E2 treatment there was an activation of ERK and AKT kinase in both IBC and TNBC cell line. Finally, treatment using previously reported IC50 (50μM)10–12 of Cou caused a decrease in cell viability of breast cancer cell lines. In summary, IBC cells are responsive to E2 treatment making this signaling pathway attractive for the development of innovative therapeutic strategies. Importantly, these Cou preliminary studies shows a potential anticancer activity in IBC cell lines.Support or Funding InformationThis project is supported by UPR Fondos Institucionales para la Investigación (FIPI to E. Peterson ) and RISE program (5R25GM061151‐17 to I. Jiménez).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Abstract P3-05-08: Lymphoid and myeloid cell characterization of inflammatory breast cancer tumor microenvironment and correlation to pathological complete response

Abstract Background: Inflammatory breast cancer (IBC) is an aggressive form of breast cancer with poor response rates to current chemotherapy regimens. With recent successes of immune targeted therapies in other solid tumors and a growing understanding of how the immune tumor microenvironment (TME) affects non-IBC outcomes, we sought to characterize the immune TME in IBC to identify biomarkers of treatment response and potential targets for drug development. Methods: Pre-treatment core biopsy samples were identified from the MD Anderson Cancer Center IBC tissue bank from patients with stage III and de novo stage IV (with T4d) disease who received neoadjuvant chemotherapy (NAC) with intent to take to mastectomy. Lymphocytes were characterized by stromal tumor infiltrating lymphocyte (sTIL) quantification, CD8 T cell quantification, and T cell receptor sequencing. PD-L1 expression was assessed using DAKO 22C3 clone on tumor and immune cells. Myeloid cells were characterized using a multiplex immunohistochemistry approach, using CD68 and CD163 for macrophage markers, tryptase for mast cell marker, HLA-DR for class II antigen presentation marker, and cytokeratin as tumor marker. Spatial analyses were performed by determining probabilities of finding cell 1 of interest within 20 uM of cell 2 of interest and computing area under the curve for statistical comparison. Results: 91 patients with stage III (N=62) or de novo stage IV (n=29) disease were identified. Breast cancer subtype included 25 triple negative, 34 HER2+ and 32 HER2-HR+. 86 patients received a mastectomy, of whom 33 (38.4%) patients experienced a pathologic complete response (pCR). sTIL was higher in stage III tumors (11.9 vs 4.8%, p<0.001) and in those having a pCR (13.8 vs 7.3%, p=0.019). CD8 T cell density (available in 48 cases) similarly was higher in stage III patients (360.3 vs 178.8 counts/mm2, p=0.040) and pCR cases (452.3 vs 219.2 counts/mm2, p=0.080) but also higher in HER2+ disease (560.9 for HER2+ vs 239.9 counts/mm2, p=0.087 for TNBC and 153.6 counts/mm2, p=0.005 for HER2-HR+). T cell clonality (available in 32 cases) ranged from 0.004 to 0.242 but showed no correlation to tumor characteristics or response. PD-L1 complete tumor membranous expression was seen in only 1 of 47 cases, whereas PD-L1 positivity on immune cells was seen on 36.2% of cases; neither correlated to response. Myeloid cell assessment (available in 25 cases) showed higher mast cell infiltration in non-pCR cases (63.8 vs 26.8 counts/mm2, p=0.008) and spatial analysis (performed on 10 cases) identified that closer proximity of mast cells to CD8 T cells correlates with response (AUC 6.0 vs 2.2, p=0.017), suggesting a possible immunosuppressive mechanism. HLA-DR analysis demonstrated no difference by response as a single stain marker, but co-localization of HLA-DR with cell type shows higher HLA-DR expression on tumor cells in non-responders (14.6 vs 1.6%, p=0.031). Conclusions: Higher TIL and CD8 T cell density are correlated with improved responses to NAC in IBC. Mast cell infiltration and HLA-DR expression on tumor cells are inversely correlated to response and suggest possible mechanisms of resistance. Mast cells could present potential therapeutic target in IBC. Citation Format: Reddy SM, Reuben A, Jiang H, Roszik J, Tetzlaff MT, Reuben J, Wang L, Tsujikawa T, Barua S, Rao A, Villareal L, Wood A, Woodward W, Ueno NT, Krishnamurthy S, Wargo JA, Mittendorf EA. Lymphoid and myeloid cell characterization of inflammatory breast cancer tumor microenvironment and correlation to pathological complete response [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P3-05-08.

EGFR signaling promotes inflammation and cancer stem-like activity in inflammatory breast cancer.

Inflammatory breast cancer (IBC) is the most lethal and aggressive type of breast cancer, with a strong proclivity to metastasize, and IBC-specific targeted therapies have not yet been developed. Epidermal growth factor receptor (EGFR) has emerged as an important therapeutic target in IBC. However, the mechanism behind the therapeutic effect of EGFR targeted therapy is not well defined. Here, we report that EGFR regulates the IBC cell population that expresses cancer stem-like cell (CSC) markers through COX-2, a key mediator of inflammation whose expression correlates with worse outcome in IBC. The COX-2 pathway promoted IBC cell migration and invasion and the CSC marker-bearing population in vitro, and the inhibition of this pathway reduced IBC tumor growth in vivo. Mechanistically, we identified Nodal, a member of the TGFβ superfamily, as a potential driver of COX-2-regulated invasive capacity and the CSC phenotype of IBC cells. Our data indicate that the EGFR pathway regulates the expression of COX-2, which in turn regulates the expression of Nodal and the activation of Nodal signaling. Together, our findings demonstrate a novel connection between the EGFR/COX-2/Nodal signaling axis and CSC regulation in IBC, which has potential implications for new combination approaches with EGFR targeted therapy for patients with IBC.

MicroRNA expression profiling identifies decreased expression of miR-205 in inflammatory breast cancer

Inflammatory breast cancer is the most aggressive form of breast cancer. Identifying new biomarkers to be used as therapeutic targets is in urgent need. Messenger RNA expression profiling studies have indicated that inflammatory breast cancer is a transcriptionally heterogeneous disease, and specific molecular targets for inflammatory breast cancer have not been well established. We performed microRNA expression profiling in inflammatory breast cancer in comparison with locally advanced noninflammatory breast cancer in this study. Although many microRNAs were differentially expressed between normal breast tissue and tumor tissue, most of them did not show differential expression between inflammatory and noninflammatory tumor samples. However, by microarray analysis, quantitative reverse transcription PCR, and in situ hybridization, we showed that microRNA-205 expression was decreased not only in tumor compared with normal breast tissue, but also in inflammatory breast cancer compared with noninflammatory breast cancer. Lower expression of microRNA-205 correlated with worse distant metastasis-free survival and overall survival in our cohort. A small-scale immunohistochemistry analysis showed coexistence of decreased microRNA-205 expression and decreased E-cadherin expression in some ductal tumors. MicroRNA-205 may serve as a therapeutic target in advanced breast cancer including inflammatory breast cancer.

Abstract P4-13-18: A phase I study of romidepsin in combination with nab-paclitaxel in patients with metastatic HER-2 negative inflammatory breast cancer (IBC)

Abstract Inflammatory breast carcinoma (IBC) is the most aggressive form of breast cancer. The hallmark of IBC is regional extension into dermal lymphatics as tumor emboli causing breast edema and erythema. Pathologic characteristics of IBC include high grade, negative hormone receptor status and overexpression of HER2 and E-cadherin. The latter is the most attractive therapeutic target in IBC. In preclinical studies the histone deacetylase inhibitor, romidepsin targeted E-cadherin, affecting tumor emboli and increasing taxane sensitivity. Rationale: In vitro studies show that histone deacetylase inhibitors (HDACi) with taxanes provide synergy to enhance cell death. HDACi alter expression of AIRH1, a regulator of autophagy, typically silenced in breast cancer. In vitro treatment with HDACi induces expression of AIRH1, resulting in enhanced cell death with taxanes. In vitro studies of IBC have demonstrated the utility of HDACi and romidepsin in IBC cell lines. SAHA and romidespin, HDACis, inhibited self-renewal of IBC tumor spheroids from IBC cell lines. This trial combines romidepsin with a taxane proven in metastatic breast cancer to explore whether the combination will be effective in IBC. Design: This is a phase I trial to assess the safety of romidepsin plus nab-paclitaxel in patients with recurrent or metastatic IBC. The maximum tolerated dose (MTD) of romidepsin + weekly nab-paclitaxel was determined to define the dose for the phase II trial. Secondary objectives included describing the adverse event profile and assessing the overall response rate (ORR) and Clinical Benefit Rate (CBR). This study employed a 3+3 design. DLTs included febrile neutropenia or non-hematologic grade 3 or 4 toxicities. Patients were treated with nab-paclitaxel 100 mg/m2 iv with romidepsin, 7 mg/m2 iv (1st cohort) and 10 mg/m2 iv (2nd cohort), on days 1, 8, 15 of a 28 day cycle. Results: Nine patients were treated. The median age was 52. Three patients were treated in the first cohort. Two patients showed progressive disease (PD). One patient has had stable disease (SD) over 10 cycles and continues treatment. DLT was not reached at 7 mg. Toxicities related to romidepsin included neutropenia, anemia and fatigue. Six patients were treated in the 2nd cohort. Grade 3 hypophosphatemia, a DLT, was reached. One patient had complete response (CR). One patient had SD; four patients had PD. Toxicities related to romidepsin were anemia, neutropenia, GI upset, edema, hyperglycemia, fatigue, hypophosphatemia, pruritis, dry mouth, and increased lab values. The overall response rate (ORR) was 33% (3/9). The table below shows results. CohortResponse# Prior TherapiesMetastatic Sites# Cycles on study1PD2pleura, nodes51PD0lung31SD2lung, nodes102PD2pleura, nodes, soft tissue42PD0liver, nodes, bone22CR2 chemotherapy, 2 endocrinebone, nodes72SD0nodes52PD0lung,liver, nodes22PD0liver, bone, nodes2 Conclusions: This phase I trial shows that romidepsin and nab-paclitaxel are well-tolerated in patients with advanced IBC. The MTD and recommended dose of romidepsin is 10 mg/m2 with nab-paclitaxel 100 mg/ m2 days 1, 8, 15 of a 28 day cycle. A phase II trial is planned in recurrent HER negative IBC patients. Citation Format: Avery TP, Jaslow R, Basu-Mallick A, Zibelli A, Fellin F, Cristofanilli M. A phase I study of romidepsin in combination with nab-paclitaxel in patients with metastatic HER-2 negative inflammatory breast cancer (IBC). [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P4-13-18.

Inflammatory Lipid Sphingosine‐1‐phosphate Upregulates c‐reactive Protein via C/EBPßß and Potentiates Breast Cancer Progression

The present study aimed to elucidate the molecular link between S1P and CRP during the invasive process of breast epithelial cells. This is the first report showing that transcription of CRP was markedly activated by S1P in breast cells. Our data suggest that not only S1P treatment but also the endogenously produced S1P may upregulate CRP in breast carcinoma cells. Transcription factors CCAAT/enhancer‐binding protein beta (C/EBPß) and c‐fos were required for S1P‐induced CRP expression. Coupling of S1P3 to heterotrimeric Gαq triggered the expression of CRP, utilizing signaling pathways involving reactive oxygen species (ROS), Ca2+, and ERKs. S1P‐induced CRP expression was crucial for the transcriptional activation of matrix metalloproteinase (MMP)‐9 through ERKs, ROS and c‐fos, leading to breast cell invasion. Using a xenograft mice tumor model, we demonstrated that S1P induced CRP expression both in vitro and in vivo. Taken together, our findings have revealed a molecular basis for S1P‐induced transcriptional activation of CRP and its functional significance in the acquisition of the invasive phenotype of human breast epithelial cells under inflammatory conditions. Our findings may provide useful information on the identification of useful therapeutic targets for inflammatory breast cancer.

Biomarkers and Therapeutic Targets in Inflammatory Breast Cancer (IBC)

Inflammatory breast cancer (IBC) is the most aggressive type of breast cancer with survival rates far lower than other types of breast cancer. Patterns of development, invasion, and presentation are unique to IBC compared with other breast cancers. However, therapies targeted specifically to the treatment of IBC are lacking. Specific therapies, which address the unique features of IBC, are needed to improve prognosis for this type of breast cancer. The first step in developing improved treatments is to identify biomarkers and genes, which are preferentially expressed in IBC and to develop therapies targeted to these markers. In this paper, we discuss advances made in the studies of biomarkers and gene expression in IBC over the last 5 years. Some of the markers have proven to be prognostic or predictive of response to therapy. In some cases, therapies targeted for biomarkers are already used in the treatment of cancer and could be evaluated in IBC patients.

Abstract P6-12-11: Celecoxib inhibits the growth of IBC tumors by suppressing the regulation of cancer stem-like cells by nodal

Abstract Background: Inflammatory breast cancer (IBC) is an aggressive type of breast cancer with no known molecular targets for treatment. Although erythema is commonly associated with IBC, the molecular mechanism of inflammation in the pathogenesis of IBC remains unknown. We have previously shown that EGFR is an emerging target in IBC (Zhang D. et al Clin Can Res 2009). As crosstalk between EGFR and COX-2 plays an important role in the inflammatory response in several cancers, including breast cancer, we hypothesized that COX-2 promotes the tumorigenesis and metastasis of IBC cells. Methods: Using clinically derived IBC and non-IBC tumor samples, a Spearman's Rank correlation coefficient analysis was performed to analyze the expression levels of COX-2 and EGFR in IBC and non-IBC. The levels of COX-2 metabolites, prostaglandins (PGs) PGE2 and PGF2α, were measured in IBC and non-IBC cell lines by HPLC/MS method. Cell migration and invasion assays were performed using SUM149 and KPL-4 IBC cell lines treated with PGs or the COX-2 inhibitor, celecoxib. We evaluated the epithelial to mesenchymal transition (EMT)-like phenotype in 3D culture of SUM149 cells treated with celecoxib, and the stem-like population by mammosphere formation, and CD44+/CD24− and aldefluor+ population by FACS. We treated preclinical IBC xenograft mice with celecoxib and measured tumor growth, PGs levels, and the expression of EMT protein markers. Nodal, a stem cell regulator and potential biomarker for breast cancer progression, was evaluated in IBC cells following treatment with celecoxib and recombinant Nodal or transfection with Nodal cDNA. Results: EGFR and COX-2 expression levels positively correlated within IBC, but not non-IBC tumors. Elevated levels of PGE2 and PGF2α were identified in multiple IBC cell lines suggesting that COX activity is elevated within IBC compared to non-IBC cells. PGs altered EMT protein markers and promoted cell migration and invasion, while Celecoxib inhibited EMT and migration and invasion in SUM149 and KPL-4 cells. Celecoxib treatment inhibited tumor growth in mice, and downregulated the expression of EMT protein markers, including Nodal. Celecoxib decreased the stem-like CD44+/CD24−, and aldefluor+ population and the formation of mammospheres. Exogenous Nodal mitigated the effects of celecoxib on cell migration and invasion and the stem-like population in SUM149 cells. Conclusion: We conclude that activation of the COX-2 inflammatory signaling pathway is critical in the development and progression of IBC. This study provides a novel insight into how inflammation may regulate cancer stem cells via Nodal, and will guide future research into the development of stem cell targeted therapies for IBC. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P6-12-11.

Inflammatory lipid sphingosine-1-phosphate upregulates C-reactive protein via C/EBPβ and potentiates breast cancer progression

A crucial role of the inflammatory lipid sphingosine-1-phosphate (S1P) in breast cancer aggressiveness has been reported. Recent clinical studies have suggested that C-reactive protein (CRP) has a role in breast cancer development. However, limited information is available on the molecular basis for the expression of CRP and its functional significance in breast cell invasion. The present study aimed to elucidate the molecular link between S1P and CRP during the invasive process of breast epithelial cells. This is the first report showing that transcription of CRP was markedly activated by S1P in breast cells. Our data suggest that not only S1P treatment but also the endogenously produced S1P may upregulate CRP in breast carcinoma cells. Transcription factors CCAAT/enhancer-binding protein beta and c-fos were required for S1P-induced CRP expression. Coupling of S1P3 to heterotrimeric Gαq triggered the expression of CRP, utilizing signaling pathways involving reactive oxygen species (ROS), Ca(2+) and extracellular signal-related kinases (ERKs). S1P-induced CRP expression was crucial for the transcriptional activation of matrix metalloproteinase-9 through ERKs, ROS and c-fos, leading to breast cell invasion. Using a xenograft mice tumor model, we demonstrated that S1P induced CRP expression both in vitro and in vivo. Taken together, our findings have revealed a molecular basis for S1P-induced transcriptional activation of CRP and its functional significance in the acquisition of the invasive phenotype of human breast epithelial cells under inflammatory conditions. Our findings may provide useful information on the identification of useful therapeutic targets for inflammatory breast cancer.

Abstract 3051: The miR-200 family as a potential therapeutic target in inflammatory breast cancer.

Abstract Purpose: Inflammatory breast cancer (IBC) is the most lethal, aggressive variant of breast cancer characterized by rapid progression, local and distant metastases, younger age of onset, and poor overall survival. Despite advances in multimodal breast cancer care, no IBC specific therapy is available clinically, demonstrating a critical need to identify novel therapeutics that target the distinct biology of IBC. MicroRNAs (miRNAs) are small noncoding RNAs that regulate gene expression and have been implicated as potential therapeutic targets. Although the oncogenic or tumor-suppressor functions of a large number of miRNAs have been described in various solid tumors including breast cancer, the role of miRNAs in the biology of IBC is poorly understood. Herein, we investigated miRNAs differentially expressed in IBC, particularly miR-200 family miRNAs as potential therapeutic targets in IBC. Methods: MicroRNA microarray profiling was conducted in IBC and non-IBC cell lines, and microarray results were validated using qRT-PCR. MirZip lentiviral based mirRNA inhibition was used to stably knockdown miRNAs and the effects of specific miRNA knockdown on proliferation, EMT, colony and mammosphere formation was evaluated. For tumor progression and metastasis in vivo experiments, we injected 500,000 SUM149 miR-200a, miR-141 knockdown and control (N=15 for each group) cells into the cleared mammary fatpad of SCID/Beige mice. We also validated the expression of miR-200 miRNAs in IBC patient samples. Results: Differential expression of mirRNAs was observed in IBC vs. non-IBC cell lines with a significantly higher expression of all miR-200 family (miR-200a, miR-200b, miR-200c, miR-141 and miR-479) in IBC cell lines. These results were validated using qRT-PCR, with the IBC cell lines showing a >1,000-fold higher expression of miR-200s vs non-IBC aggressive cell lines (P<0.0001). Moreover, two known targets of the miR-200 family, Zeb1 and Zeb2, were significantly downregulated while E-cadherin was upregulated in the IBC samples. Importantly, these observations were validated in IBC patient samples which showed a significantly higher expression of all miRNA 200s compared to the normal breast tissues (P<0.001). In vitro, miR-200a and miR-141 knockdown significantly reduced both colony and mammosphere formation in the triple negative SUM149 cells (P<0.001) but did not have a significant effect on the HER2+ IBC cell lines SUM190 and MDA-IBC-3 (P>0.05) while it upregulated the EMT markers N-cadherin, Fibronectin, TGFβ, and Smad4 and reduced E-cadherin expression compared to transduced controls. In vivo, miR-200 knockdown resulted in an increase in tumor progression vs. controls (P<0.05, Day 63) while metastasis studies are undergoing. Conclusions: These data suggest that miR-200 family microRNAs play an important role in IBC tumor progression and altering miR-200 levels could be an efficacious approach to target IBC. Citation Format: Bisrat G. Debeb, Lara Lacerda, Richard Larson, Li Li, Wei Xu, Lei Huo, Caimiao Wei, Savitri Krishnamurthy, James Reuben, Naoto Ueno, Thomas Buchholz, Wendy A. Woodward, Morgan Welch Inflammatory Breast Cancer Research Program and Clinic. The miR-200 family as a potential therapeutic target in inflammatory breast cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3051. doi:10.1158/1538-7445.AM2013-3051

Abstract PD05-01: Next generation genomic sequencing (NGS) identifies molecular targets in inflammatory breast cancer (IBC)

Abstract BACKGROUND: Inflammatory breast cancer (IBC) is the most aggressive form of invasive breast cancer and is associated with poor outcome in spite of optimal multidisciplinary management. The disease is associated poor response to neoadjuvant chemotherapy and a peculiar pattern of recurrence (chest wall, nodes, CNS). There currently few distinct molecular alterations for effective therapeutic targeting in the advance setting. The availability of comprehensive cancer genomic testing using Next-Generation Sequencing (NGS) technology may identify a number of genomic alterations not assessed by traditional methods and provide opportunities for more effective treatment selection particularly in aggressive and uncommon diseases such as IBC. METHODS: Twenty-three patients with refractory metastatic IBC were included in this analysis. All patients underwent confirmatory biopsy and/or tissue collection. Tissue from metastastic lesions of patients with recurrent IBC was submitted as slides or FFPE blocks. NGS was performed by Foundation Medicine, Cambridge, MA. The entire coding sequence of 182 cancer-related genes (3,230 exons) plus 37 introns from 14 genes often rearranged in cancer were sequenced to high depth (>500X) All classes of genomic alterations were detected. RESULTS: The site distribution and frequency of disease recurrence included: soft tissue (19) lung (4), bone and liver (3), and brain (2). The disease subtype classification based on the expression of ER, PR and HER-2 were as follow: ER−, PR−, Her-2−, Basal-like or TN (65%); ER+ and/or PR+, HER-2− (grade3) or HER-2+, Luminal B (22%); ER−, PR−, HER2+ (13%). Genomic alterations were identified in all patients with various frequencies. Genetic mutations included: P53 (DNA-binding domain, splice site), PI3KCA (H1047R, R441N, E545K, K111E,); RB1; PTEN (D107Y); EGFR (L858R); ERBB2 (V777L, S310F) in a patient with TN disease; ESR1 D538G. Gene amplifications included: MYC (26%); CCND1 (22%); ERBB2 (in a patient with TN disease), MLC-1, CCNE1, CK4, K-RAS. One patient had evidence of discordant genomic abnormalities in two simultaneous sites of recurrence (skin and pleura) suggesting disease heterogeneity. Three patients were effectively treated with genomic alterations-guided therapies. CONCLUSIONS: Genomic sequencing of advanced IBC is feasible, identifies potential therapeutic targets and can advance our understanding of the molecular alterations of this aggressive disease. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr PD05-01.

New Therapeutic Targets in Inflammatory Breast Cancer

Inflammatory breast cancer (IBC) is the most lethal variant of locally advanced breast cancer. Although recognized as a distinct clinical entity, there have been few advances in the development of pre-clinical models of IBC, and a lack of IBC-specific therapeutic targets translated into clinical utility to increase overall survival, which is currently 40 % at three years. By use of newly developed pre-clinical models of IBC and patient tumor tissues, E-cadherin, anaplastic lymphoma kinase (ALK), and HSP90 have been identified as targets relevant to IBC that are matched by therapeutics that are either currently in clinical trials or will be tested in clinical trials within the next year. These exciting results illustrate the advances that have been made in recent years in defining the molecular basis of IBC as a distinct disease and the significant strides made in identifying more effective strategies for treatment of patients with IBC.

Limited efficacy and significant toxicities of lapatinib (Lap) plus chemotherapy as neoadjuvant therapy (NAC) for HER2-positive inflammatory breast cancer (IBC) patients (Pts).

594 Background: IBC is a rare disease but the most aggressive form of locally advanced breast cancer with a higher frequency of HER2 neu amplification (HER2+) compared to non-IBC. The EGFR pathways are also important therapeutic targets in IBC. (Zhang D, 2009). Lap is an oral reversible dual kinase inhibitor of epidermal growth factor receptor of EGFR and HER2. Our objective was to determine the efficacy of Lap in combination with paclitaxel as NAC in pts with previously untreated HER2+ IBC. The primary end-point was to determine the rate of pathological complete response (pCR) defined as no residual invasive disease in the breast and the axillary lymph nodes or Residual Cancer Burden (RCB) of 0. (Symmans FW, 2008). The second endpoint was to assess general safety and cardiac toxicity of this combination therapy. Methods: From October 2008 to May 2011, 15 chemo-naïve pts were treated with Lap 1,250 mg/day as a single agent for 2 weeks, followed by 12 weeks of paclitaxel (80 mg/m2 weekly) plus Lap (1,000 mg/day), and finally with 4 cycles of FEC-75 mg/m2 regimen plus Lap (1,000 mg/day) before surgery. Among 12 first pts enrolled, 6 pts had grade 3 diarrhea (CTCAC v3.0) and the protocol was amended to reduce the Lap dose to 750 mg/day. After modified radical mastectomy (MRM), patients received radiation therapy and one year of adjuvant trastuzumab. Results: Median age was 53.8 (range, 39 -70), performance status was 0 (13 pts), 1 (2 pts), 4 pts had metastatic disease at diagnosis. 10 of 15 pts had MRM. One pt achieved pCR. Five pts did not complete the NAC due to grade 2 cardiomyopathy (1), liver dysfunction (2), diarrhea (1), and insurance denial after pt started treatment (1). The trial was stopped early due to severe toxicity (>15%) and lack of efficacy. Conclusions: Lap in combination with chemotherapy as NAC has a limited anti-tumor activity in pts with HER2+ IBC with a pCR rate of 6.6%. Lap and paclitaxel was associated with severe diarrhea toxicity and required multiple dose reductions of Lap. With the recent promising results in HER2+ NAC studies, the role of Lap should continue to be explored in combination with other anti-HER2 agents in IBC patients.