Therapeutic Target In Inflammatory Breast Cancer Research Articles

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.

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.

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.

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.

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

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.

Suppression of mesenchymal phenotype in inflammatory breast cancer by EGFR tyrosine kinase inhibitor erlotinib: a novel approach to suppressing metastasis.

Abstract Abstract #2042 Background: Inflammatory breast cancer (IBC) is a rare subtype well known for its propensity for rapid metastasis. The cause of this rapid metastasis in IBC is unknown. An immunohistochemical analysis of 44 cases of IBC showed HER2 overexpression in 48% of patients and epidermal growth factor receptor (EGFR) overexpression in 30% of patients. EGFR overexpression was the only poor prognostic factor: the 5-year overall survival rate was significantly lower for women with EGFR-positive disease than for women with EGFR-negative disease (P=0.01). HER2 overexpression was not a prognostic factor. The association between EGFR overexpression and increased risk of death indicated that EGFR may represent a potential therapeutic target in IBC. We hypothesized that suppression of the EGFR pathway inhibits proliferation and metastasis of IBC.
 Methods: SUM149 IBC cells, which express low levels of HER2 and high levels of EGFR, were treated with siRNA against EGFR and with the EGFR tyrosine kinase inhibitor erlotinib.
 Results: EGFR siRNA knockdown (but not non-targeting siRNA control) inhibited the proliferation of SUM149 cells. SUM149 cells were sensitive to EGFR tyrosine kinase inhibitor erlotinib in a 2-dimensional (2-D) culture system (median inhibitory concentration [IC50] = 0.90 µM). When we activated ERK by transfecting constitutively active MEK1 in SUM149 cells, the cells showed more resistance to erlotinib. Moreover, ERK siRNA knockdown sensitized SUM149 cells to erlotinib. Further, when we cultured the SUM149 cells in matrigel by using the 3-D culture system (100% matrigel in the bottom layer and cultured medium with 5% FBS and 2% matrigel in the top layer), erlotinib treatment changed the molecular phenotype of SUM149 cells from mesenchymal (a phenotype characterized by low beta-catenin expression and high vimentin and fibronectin expression) to epithelial (recovery of beta-catenin to the sites of cell-cell contacts; downregulation of fibronectin). This reversal of the mesenchymal phenotype, a hallmark of inhibition of epithelial-to-mesenchymal transition (EMT), was ERK dependent. Interestingly, the erlotinib concentration that inhibited the mesenchymal phenotype (0.1 mcM) was one log lower than the concentration that inhibited proliferation (1 mcM).
 Conclusion: Inhibition of tumor growth and EMT in SUM149 IBC cell lines is dependent on the ERK pathway through the EGFR pathway. The erlotinib dose needed to produce an anti-mesenchymal effect is much lower than the cytotoxic dose. Thus, we speculate ERK pathway to be important in inhibiting metastasis in IBC. We are currently investigating the effects of erlotinib in a xenograft model of IBC. Our study provides a rationale for developing novel treatment strategies targeting the EGFR and ERK pathways to inhibit the growth and metastasis of IBC. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 2042.

NF-kappa B genes have a major role in Inflammatory Breast Cancer

BackgroundIBC (Inflammatory Breast cancer) is a rare form of breast cancer with a particular phenotype. New molecular targets are needed to improve the treatment of this rapidly fatal disease. Given the role of NF-κB-related genes in cell proliferation, invasiveness, angiogenesis and inflammation, we postulated that they might be deregulated in IBC.MethodsWe measured the mRNA expression levels of 60 NF-κB-related genes by using real-time quantitative RT-PCR in a well-defined series of 35 IBCs, by comparison with 22 stage IIB and III non inflammatory breast cancers. Twenty-four distant metastases of breast cancer served as "poor prognosis" breast tumor controls.ResultsThirty-five (58%) of the 60 NF-κB-related genes were significantly upregulated in IBC compared with non IBC. The upregulated genes were NF-κB genes (NFKB1, RELA, IKBKG, NFKBIB, NFKB2, REL, CHUK), apoptosis genes (MCL1L, TNFAIP3/A20, GADD45B, FASLG, MCL1S, IER3L, TNFRSF10B/TRAILR2), immune response genes (CD40, CD48, TNFSF11/RANKL, TNFRSF11A/RANK, CCL2/MCP-1, CD40LG, IL15, GBP1), proliferation genes (CCND2, CCND3, CSF1R, CSF1, SOD2), tumor-promoting genes (CXCL12, SELE, TNC, VCAM1, ICAM1, PLAU/UPA) or angiogenesis genes (PTGS2/COX2, CXCL1/GRO1). Only two of these 35 genes (PTGS2/COX2 and CXCL1/GRO1)were also upregulated in breast cancer metastases. We identified a five-gene molecular signature that matched patient outcomes, consisting of IL8 and VEGF plus three NF-κB-unrelated genes that we had previously identified as prognostic markers in the same series of IBC.ConclusionThe NF-κB pathway appears to play a major role in IBC, possibly contributing to the unusual phenotype and aggressiveness of this form of breast cancer. Some upregulated NF-κB-related genes might serve as novel therapeutic targets in IBC.

Expression of growth factor and chemokine receptors: new insights in the biology of inflammatory breast cancer

Recent studies have indicated that expression of chemokine receptors CXCR4 and CCR7 could be an indicator of the metastatic potential of breast cancer. Expression of CXCR4 and CCR7 along with the biomarkers HER2-neu and epidermal growth factor receptor (EGFR) was investigated in inflammatory breast cancer (IBC) to evaluate their prognostic implications. CXCR4, CCR7, and EGFR were evaluated by immunohistochemical staining (IHC) of paraffin-embedded tissue sections. HER2-neu amplification was assessed by FISH and/or IHC. All patients received chemotherapy, surgery, and radiation. Forty-four cases diagnosed with IBC from 1994 to 2002 were included in the study. In all, 18 (40.9%) patients had positive CXCR4, 10 (22.7%) had positive CCR7, 21 (47.7%) had positive HER2-neu, and EGFR was positive in 12 of 40 patients (30%). The 5-year overall survival (OS) was 24.8% for CXCR4-positive disease versus 42.3% for CXCR4-negative patients (P = 0.53) and 20.0% for CCR7-positive disease versus 41.9% for CCR7-negative patients (P = 0.24). EGFR-positive disease had significantly worse OS compared with EGFR-negative disease (P = 0.01). These data demonstrate the expression of growth factor and chemokine receptors in IBC. The expression of these receptors is associated with increased risk of recurrence and death, and thus, they may represent potential therapeutic targets in IBC.