Inflammatory Breast Cancer Tumors Research Articles

Abstract PO1-25-04: PTN-positive luminal progenitors induce angiogenesis and metastasis in inflammatory breast cancer

Abstract Background Inflammatory breast cancer (IBC) is a lethal subtype of breast cancer characterized by rapidly arising diffuse erythema and edema of the overlying skin that was thought to contribute to rapid metastasis. Dissecting the intricate cellular ecosystems within tumors and affected skin and their complex interplay may be pivotal in unraveling the aggressive nature of IBC. Methods Integrative analysis containing single-cell RNA sequencing, bulk RNA sequencing, and whole exome sequencing, was performed on paired tumor and skin samples from 17 treatment-naive IBC patients and 5 non-IBC patients from multiple centers. Meanwhile, paraffin sections and serum specimens from two centers were conducted to validate the omics results. Additionally, public single-cell transcriptomic data of non-IBC tumors and normal mammary glands, RNA microarray data from IBC cases, and the Cancer Genome Atlas (TCGA) database were utilized to validate the characteristics of IBC microenvironment. The regulatory mechanism was verified by in vitro experiments. Results We delineated the landscapes of IBC tumor and affected skin microenvironment at the single-cell level and demonstrated that the luminal progenitors (LP) cells highly expressing pleiotrophin (PTN) were most significantly enriched in IBC tumors compared to non-IBC tumors and normal mammary glands, in two independent cohorts. Moreover, the serum concentrations of PTN were significantly higher in IBC patients compared with non-IBC patients. Notably, PTN secreted by PTN+ LP cells interacted with Neuropilin 1(NRP1) receptor on endothelial tip cells in both tumor and affected skin of IBC. Moreover, in vitro assay demonstrated knocking down the expression of NRP1 on human umbilical vein endothelial cells significantly blocked PTN-induced migration, tube formation and sprouting, which might be independent of VEGFA. In addition, two immature phenotypes of PVL_MMP9 and PVL_CCL19 cells recruited by endothelial tip cells via PDGFB/PDGFRB axis were accumulated in affected skin of IBC, which might be associated with promotion of malignant cell metastasis. Finally, we revealed that malignant cells in affected skin of IBC had a higher ability of epithelial-mesenchymal transition than IBC intratumoral malignant cells and identified enhanced crosstalk between immature PVL cells and malignant cells in the skin affected with IBC , through TNFSF12-TNFRSF12A, TGFB-TGFBR1/TGFBR2, MDK-SDC1/4/NCL/(ITGA6+ITGB1). Conclusion We identified a unique, non-malignant epithelial cell subpopulation PTN+ LP cells, which induce massive angiogenesis via the PTN-NRP1 axis, recruit the immature phenotype PVL cells accumulated in affected skin of IBC thus promoting tumor progression. Our findings offer comprehensive insights into the pathogenesis of IBC and uncover a previously unknown, promising therapeutic target. Patient of discovery cohort details Clinical and pathology details for inflammatory breast cancer(IBC) and non-IBC patients analysed by single-cell RNA sequencing(scRNA-seq), bulk RNA sequencing(bulk RNA-seq) and whole exome sequencing(WES) in this study. Clinicalpathological feature of validation cohort Citation Format: Mengmeng Zhang, Kaiwen Zhou, Ting Liu, Filipa Lynce, Filipa Lynce, Wendy Chen, Qianjun Chen, Xiaoping Li, Zhongyu Yuan, Juan Xu, Zhenhai Cai, Jianping Guo, Nan Shao, Ying Lin. PTN-positive luminal progenitors induce angiogenesis and metastasis in inflammatory breast cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO1-25-04.

Abstract PO1-14-10: Comprehensive clinicogenomic characterization of inflammatory breast cancer

Abstract BACKGROUND: Inflammatory breast cancer (IBC) is a rare type of breast cancer associated with a unique clinical presentation and overall poor outcomes, recognized as a distinct category by the AJCC staging system. The biological mechanisms driving the IBC phenotype are relatively undefined—partially due to a lack of comprehensive, large-scale genomic studies and limited clinical cohorts. Here, we report one of the largest, subtype-informed clinicogenomic characterizations of IBC to date. METHODS: A retrospective analysis of 2457 patients with metastatic breast cancer who underwent targeted tumor-only DNA-sequencing (OncoPanel, up to 447 cancer-associated genes) was performed at Dana-Farber Cancer Institute. Clinicopathologic, single nucleotide variant (SNV), copy number variant (CNV) and tumor mutational burden (TMB) comparisons were made between IBC and non-IBC cases. Median follow-up was 28.1 months. RESULTS: Our profiled cohort included 140 patients with IBC specimens (n = 68 primary tumors, 72 metastatic tumors) and 2317 patients with non-IBC specimens (n = 702 primaries, 65 local recurrences, 1550 metastases). Of these, 87.4% of patients were White, 4.7% Black, 3.6% Asian or Pacific Islander, and 4.3% other/unknown. Clinicopathologic differences between IBC and non-IBC cases were consistent with previous reports—including younger age at diagnosis of metastatic disease (51 vs 54 years, p = 0.04), and a higher proportion of grade 3, estrogen receptor-negative and HER2-positive tumors (p < 0.001). Among the hormone-receptor (HR)-positive subtype, IBC tumors showed a significant enrichment in Luminal B (LumB)-inferred disease (62.5% vs 39.8%, p < 0.005), defined as tumors with grade 3 or progesterone receptor staining < 10%. The most recurrent somatic alterations spanning all subtypes in IBC were TP53 (72%), ERBB2 (32%), PIK3CA (24%), CCND1 (12%), MYC (9%), FGFR1 (8%) and GATA3 (8%). A multivariate logistic regression analysis accounting for HR and HER2 status revealed a significant enrichment in TP53 SNVs in IBC vs non-IBC (OR 2.08 [95% CI 1.34-3.24], adjusted p-value 0.04). Frequency of TP53 alterations in IBC vs non-IBC cases was 85.1% vs 64.3% in HER2-positive and 50.0% vs 27.7% in HR-positive disease—with an enrichment of TP53 mutations in IBC LumB-inferred tumors vs non-LumB (64% vs 26%, p < 0.05). When comparing HR+ IBC LumB vs HR+ non-IBC LumB cases, TP53 mutations were again enriched (p < 0.05)—suggesting LumB-like histopathology is not the only driving feature of TP53 enrichment in HR+ IBC. TMB did not differ substantially between IBC and non-IBC and no other statistically significant enrichments were observed, including when grouping mutations into six canonical cancer pathways (cell cycle, Notch signaling, PI3K pathway, RTK/RAS signaling, TP53 pathway and WNT signaling). CONCLUSIONS: Taken together, this study provides a comprehensive landscape of somatic alterations in a large cohort of patients with metastatic IBC and non-IBC. Our data support a lack of major genomic differences other than enrichments in TP53 mutations and an associated LumB-like histopathology. These results both reinforce the importance of TP53 mutations in IBC biology and suggest additional analyses beyond somatic DNA-level changes are warranted. Future efforts with the DFCI IBC cohort will assess germline-somatic interactions, non-genomic or transcriptomic characterizations, and potential environmental influences to better understand the mechanisms driving this unique disease. Citation Format: Nolan Priedigkeit, Beth Harrison, Melissa Hughes, Robert Shue, Yvonne Li, Gregory Kirkner, Claire Remolano, Sarah Strauss, Janet Files, Anne-Marie Feeney, Ayesha Mohammed-Abreu, Ana Garrido-Castro, Romualdo Barroso-Sousa, Brittany Bychkovsky, Faina Nakhlis, Jennifer Bellon, Tari King, Bruce Johnson, Lynette Sholl, Deborah Dillon, Beth Overmoyer, Sara Tolaney, Andrew Cherniack, Nancy Lin, Filipa Lynce. Comprehensive clinicogenomic characterization of inflammatory breast cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO1-14-10.

Abstract PO5-09-10: Inflammatory Breast Cancer Patient Profile in a Population of Puerto Rican Women

Abstract Background: Breast cancer (BC) is the most frequently diagnosed cancer type, as well as the leading cause of cancer death among women in Puerto Rico (PR). Moreover, inflammatory breast cancer (IBC) is a highly aggressive BC subtype. The current study aimed to describe the patient profile and the clinicopathological characteristics of IBC tumors in Hispanic women living in Puerto Rico. Methods: This is a retrospective, population-based study using the Puerto Rico Central Cancer Registry (PRCCR) database and the Health Insurance Linkage Database (PRCCR-HILD) to identify and describe the patient profile of Puerto Rican women diagnosed with IBC from 2008 to 2018. Variables such as age at diagnosis, staging variables, tumor receptor subtypes, treatment received, and overall survival (OS) were studied. Statistical analysis methods were employed to describe the population, estimate survival curves and examine the risk of dying. Results: We identified a total of 51 patients. The mean age at diagnosis of IBC in the current study was 59 years old, which is older than the mean age at diagnosis of IBC for women in the mainland US, but younger than the mean age of women that are diagnosed with BC in PR (63 years old). A total of 62.8% of patients had no metastases at diagnosis and 64.7% were diagnosed with stage III disease. Most tumors presented with ER+/PR+/Her2- (21.6%), or a triple-negative (ER-/PR-/Her2-, 15.7%) tumor concordance. The OS during the 12 months post-diagnosis was 66%, whereas at 36 months, OS decreased to 39%. The triple-negative subtype had the worst survival at 36 months (36%). Women with stage IV disease and those with ER-/PR- tumor subtype had a higher risk of dying. Conclusions: This is the first research to describe the patient profile and characteristics of women diagnosed with IBC in Puerto Rico. Our results suggest that the Puerto Rican IBC patient population presents unique characteristics. This research increases awareness about this lethal disease in PR. Citation Format: Luz Arroyo-Cruz, Michelle Martinez-Montemayor. Inflammatory Breast Cancer Patient Profile in a Population of Puerto Rican Women [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO5-09-10.

Abstract 6203: An altered profile of ribosomal proteins and ERK-eIF4E translational control in an inflammatory breast cancer model of acquired resistance and reversal is associated with pathological complete response in patients

Abstract Background: Drug resistance is a major challenge in inflammatory breast cancer (IBC), a hyperproliferative subtype characterized by the presence of diffuse tumor cell clusters that show high level of heterogeneity. As the degree of tumor heterogeneity during cancer progression can cause evolution of cell states that can either compete or co-operate resulting in therapeutic failure, the study objective was to identify drug resistance networks that can serve as biomarkers or therapeutic targets. Methods: Integrated transcriptomic and proteomic profiling approach was conducted in a progression model of acquired drug resistance and its reversal comprised of a treatment-naïve patient-derived SUM149 cell line, along with two isogenic derivatives and representing drug-resistant (rSUM149) and resistance-reversal states (rrSUM149). Global gene expression analysis in IBC patients (n=87) recorded responses to neoadjuvant chemotherapy. Based on the data, targeted multikinase inhibitors were evaluated in viability and signaling assays. Results: We identified specific ribosomal proteins associated with resistance acquisition, which correlated with high levels of pERK, CDK1, XIAP, and SOD2. Conversely, the resistance reversal in rrSUM149 showed almost complete normalization to the profile of SUM149. However, VIPER analysis revealed proteins related to ribosomal processes (AGO2, Exportin 1, RPL5) that did not revert in rrSUM149, suggesting this pathway may have pleiotropic effects in governing drug resistance. Notably, genes linked to ribosomal processes were significantly enriched (P<0.001) among the overexpressed genes in IBC patients (n=87) who exhibited pCR to neoadjuvant chemotherapy. This is crucial as IBC tumors, including rSUM149, exhibit MAPK hyperactivation including in the presence of EGFR tyrosine kinase inhibitors. Treatment with Merestinib, a multikinase inhibitor, suppressed pERK, peIF4E, downstream targets like CDK1, and ribosomal protein IMP3 underscoring the impact on protein synthesis signaling crucial for countering translational dependence in cancer cells. Additionally, Merestinib synergized with EGFR-TKI (lapatinib) in increasing cell death in all three states. Conclusion: Overall, our study describes adaptive changes in response to both therapeutic stress and therapy break that have the potential to serve as biomarkers or extended for pharmacological interrogation toward precision medicine for this rare, understudied cancer. Furthermore, the ability of Merestinib, in clinical trials, to target translational dependence is attractive as cancer cells often appropriate these signaling pathways to gain tolerance to various stress stimuli. Support: NIH-R01CA264529; DoD Breast Cancer Breakthrough level 2 Award W81XWH2010153 Citation Format: Gayathri R. Devi, Pritha PAI, Matthew W. Foster, Dorababu S. Sannareddy, Seayoung Lee, Steven V. Laere. An altered profile of ribosomal proteins and ERK-eIF4E translational control in an inflammatory breast cancer model of acquired resistance and reversal is associated with pathological complete response in patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6203.

A subpopulation of luminal progenitors secretes pleiotrophin to promote angiogenesis and metastasis in inflammatory breast cancer.

Inflammatory breast cancer (IBC) is a highly aggressive subtype of breast cancer characterized by rapidly arising diffuse erythema and edema. Genomic studies have not identified consistent alterations and mechanisms that differentiate IBC from non-IBC tumors, suggesting that the microenvironment could be a potential driver of IBC phenotypes. Here, using single-cell RNA sequencing, multiplex staining, and serum analysis in IBC patients, we identified enrichment of a subgroup of luminal progenitor (LP) cells containing high expression of the neurotropic cytokine pleiotrophin (PTN) in IBC tumors. PTN secreted by the LP cells promoted angiogenesis by directly interacting with the NRP1 receptor on endothelial tip cells located in both IBC tumors and the affected skin. NRP1 activation in tip cells led to recruitment of immature perivascular cells in the affected skin of IBC, which are correlated with increased angiogenesis and IBC metastasis. Together, these findings reveal a role for crosstalk between LPs, endothelial tip cells, and immature perivascular cells via PTN-NRP1 axis in the pathogenesis of IBC, which could lead to improved strategies for treating IBC.

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.

Inflammatory breast cancer: An overview about the histo-pathological aspect and diagnosis.

Inflammatory Breast Cancer (IBC) is a rare and aggressive form of locally advanced breast cancer, classified as stage T4d according to the tumor-node-metastasisstaging criteria. This subtype of breast cancer is known for its rapid progression and significantly lower survival rates compared to other forms of breast cancer. Despite its distinctive clinical features outlined by the World Health Organization, the histopathological characteristics of IBC remain not fully elucidated, presenting challenges in its diagnosis and treatment. Histologically, IBC tumors often exhibit a ductal phenotype, characterized by emboli composed of pleomorphic cells with a high nuclear grade. These emboli are predominantly found in the papillary and reticular dermis of the skin overlaying the breast, suggesting a primary involvement of the lymphatic vessels. The tumor microenvironmentin IBC is a complex network involving various cells such as macrophages, monocytes, and predominantly T CD8+lymphocytes, and elements including blood vessels and extracellular matrix molecules, which play a pivotal role in the aggressive nature of IBC. A significant aspect of IBC is the frequent loss of expression of hormone receptors like estrogen and progesterone receptors, a phenomenon that is still under active investigation. Moreover, the overexpression of ERBB2/HER2 and TP53 in IBC cases is a topic of ongoing debate, with studies indicating a higher prevalence in IBC compared to non-inflammatory breast cancer. This overview seeks to provide a comprehensive understanding of the histopathological features and diagnostic approaches to IBC, emphasizing the critical areas that require further research.

High-fat diet, but not duration of lactation, increases mammary gland lymphatic vessel function and subsequent growth of inflammatory breast cancer cells

Inflammatory breast cancer (IBC) presents as rapid-onset swelling and breast skin changes caused by tumor emboli in the breast and breast skin lymphatics. IBC has been linked with obesity and duration of breastfeeding, but how these factors affect IBC tumor progression is not clear. We modeled the simultaneous effects of diet and weaning in mice on in vivo lymphatic function; on IBC tumor growth; and on aspects of the mammary gland microenvironment before and after IBC (SUM149) xenograft inoculation. We hypothesized that weaning status and diet would have synergistic effects on lymphatic function and the breast microenvironment to enhance IBC tumor growth. Changes in lymphatic structure and function were characterized with in vivo near-infrared fluorescence (NIRF) imaging. Mice were fed either a high-fat diet (HFD; 60 kcal%) or a normal/low-fat diet (LFD; 10 kcal%), bred twice, and subjected to either normal-duration nursing (NW) or forced weaning (FW). SUM149 IBC tumors were implanted at 14 months; images were obtained before and after implantation. Multiparous mice fed HFD showed increased pre-tumor lymphatic pulsing in both the FW and NW groups relative to mice fed LFD. HFD promoted tumor growth independent of weaning time (P = 0.04). Pre-tumor lymphatic pulsing was associated with tumor volume at 8 weeks (P = 0.02) and was significantly correlated with expression of the lymphatic tracking ligand CCL21 (P = 0.05, Table 1). HFD significantly increased the numbers of monocyte-derived IBA1+, CD163+, and CD11c+ cells (P < 0.0001, P < 0.0001, P = 0.0005) in the contralateral, non-tumor-bearing mammary gland. Numbers of lymphangiogenic podoplanin+/IBA1+ macrophages were increased in the ducts of HFD and FW mice (all P < 0.003). HFD in nulliparous mice had a similar increase in lymphatic pulsing at 14 weeks (P = 0.006), indicating that this functional change was independent of parity. We conclude that HFD induced increases in mammary gland lymphatic function, assessed as pulsing rate before tumor initiation, and correlated with inflammation in the mammary gland and increased SUM149 tumor growth. The relationship between diet, lymphatic pulsing, and tumor growth warrants further investigation.

Abstract 1580: LCN2 is a therapeutic target against inflammatory breast cancer

Abstract Breast cancer (BC) is the most common cancer type accounting for 12% of annual cancer cases worldwide. In the United States, 30% of the newly diagnosed cancer cases in woman are BC. Inflammatory breast cancer (IBC) is a rare and aggressive subtype of BC that accounts for 1 to 5% of all types of BC. Due to its inflammatory characteristics and the absence of a palpable mass, IBC is usually confounded with a mastitis. Once properly diagnosed, IBC has already metastasized. A high portion of IBC tumors overexpress human epidermal growth factor receptor 2 (HER2). These IBC patients (HER2+) are treated with Trastuzumab, a monoclonal antibody. However, many IBCs become resistant to this therapy. Thus, there is not an optimal treatment against this horrendous disease. One potential target against IBC is Lipocalin-2 (LCN2), a secreted protein involved in iron homeostasis, immune responses, transport of siderophores, and epithelial cell differentiation. In many aggressive tumors, high levels of LCN2 have been associated with increased cancer cell motility, proliferation, angiogenesis, invasion, and metastasis. Our research team published that LCN2 is aberrantly abundant in IBCs compared with non-IBC patients and cell lines and showed that small interference RNAs (siRNAs) targeting LCN2 effectively reduced the cell proliferation and invasion ability of IBC cells. Therefore, to understand the mechanisms involved after LCN2 knockdown, we did a RNA-sequencing using IBC3 cells transfected with LCN2-siRNA or with a negative control siRNA, where we identified 138 dysregulated genes. Genes as SOX18, which correlates to distant metastasis and poor clinical prognosis in cancer, and as LRP4, which correlates to proliferating clinical features by activating the PI3K/AKT pathway were identified. We used the Ingenuity Pathway Analysis software to identify the pathways regulated after LCN2 knockdown and found 25 canonical pathways altered. These results will be further validated by qPCR and Western Blot analysis. Also, after the identification of LCN2 as a plausible target for therapy against IBC, we worked on the preparation of a Herceptin-conjugated liposome loaded with siRNA against LCN2, to improve internalization in HER2+ IBC cells. We developed a three step reaction to obtain trastuzumab DSPE-PEG(2000) maleimide conjugates used to prepare the liposomal formulation. Trastuzumab conjugated liposomes were prepared and characterized using dynamic light scattering to measure their size distribution. Our formulation displayed sizes of 18.3 and 73.7 nm which could correspond to free DSPE-PEG(2000) trastuzumab and the trastuzumab-liposome, respectively. There is ongoing work to determine the optimal timepoint for internalization of liposomes into IBC cell lines and further treatment with liposomes charged with siRNA LCN2 to treat cell lines and determine the biological effect with the aim of developing a targeted treatment for inflammatory breast cancer. Citation Format: Marienid Flores-Colon, Pablo E. Vivas-Mejia. LCN2 is a therapeutic target against inflammatory breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1580.

Copy Number Variation in Inflammatory Breast Cancer.

Identification of a unique genomic biomarker in de novo inflammatory breast cancer (IBC) may provide an insight into the biology of this aggressive disease. The goal of our study was to elucidate biomarkers associated with IBC. We examined breast biopsies collected from Dana-Farber Cancer Institute patients with IBC prior to initiating preoperative systemic treatment (30 samples were examined, of which 14 were eligible). Patients without available biopsies (n = 1), with insufficient tumor epithelial cells (n = 10), or insufficient DNA yield (n = 5) were excluded from the analysis. Molecular subtype and tumor grade were abstracted from a medical records' review. Ten IBC tumors were estrogen-receptor-positive (ER+) and human epidermal growth factor receptor 2 (HER2)-negative (n = 10 out of 14). Sufficient RNA and DNA were simultaneously extracted from 14 biopsy specimens using the Qiagen AllPrep Kit. RNA was amplified using the Sensation kit and profiled using the Affymetrix Human Transcriptome Array 2.0. DNA was profiled for genome-wide copy number variation (CNV) using the Affymetrix OncoScan Array and analyzed using the Nexus Chromosome Analysis Suite. Among the 14 eligible samples, we first confirmed biological concordance and quality control metrics using replicates and gene expression data. Second, we examined CNVs and gene expression change by IBC subtype. We identified significant CNVs in IBC patients after adjusting for multiple comparisons. Next, to assess whether the CNVs were unique to IBC, we compared the IBC CNV data to fresh-frozen non-IBC CNV data from The Cancer Genome Atlas (n = 388). On chromosome 7p11.2, we identified significant CN gain located at position 58,019,983-58,025,423 in 8 ER+ IBC samples compared to 338 non-IBC ER+ samples (region length: 5440 bp gain and 69,039 bp, False Discovery Rate (FDR) p-value = 3.12 × 10-10) and at position 57,950,944-58,025,423 in 3 TN-IBC samples compared to 50 non-IBC TN samples (74,479 base pair, gain, FDR p-value = 4.27 × 10-5; near the EGFR gene). We also observed significant CN loss on chromosome 21, located at position 9,648,315-9,764,385 (p-value = 4.27 × 10-5). Secondarily, differential gene expression in IBC patients with 7p11.2 CN gain compared to SUM149 were explored after FDR correction for multiple testing (p-value = 0.0016), but the results should be interpreted with caution due to the small sample size. Finally, the data presented are hypothesis-generating. Validation of CNVs that contribute to the unique presentation and biological features associated with IBC in larger datasets may lead to the optimization of treatment strategies.

Abstract P1-04-07: Xiap expression is associated with infiltration of cd163+ tumor-associated macrophages in the tumor micro-environment of inflammatory breast cancer

Abstract BACKGROUND: Inflammatory breast cancer (IBC) is a rare, but aggressive type of locally advanced breast cancer for which a specific immune response seems to be an important driver (Van Laere et al., CCR 2013). The tumor immune micro-environment (TIME) of IBC is characterized by an influx of tumor-associated macrophages (TAMs) and patients with spatial colocalization of TAMs with tumor cells seem to have a worse prognosis (Van Berckelaer et al., SABCS 2019). In contrast with TAMs, the presence of CD8+ T cells was associated with a better prognosis in IBC. Cytotoxic T cells mediate killing of the tumor by inducing apoptosis after activation of effector caspases-3/-7, regulated by the X-Linked Inhibitor of Apoptosis protein (XIAP). Recently, we reported that XIAP in breast cancer is associated with shorter survival and resistance to chemotherapy (Devi et al, Cancers 2021). In IBC, XIAP is translationally upregulated during cellular stress and induces, via NFkB, an immunosuppressive signaling (Evan et al, Cancer Res 2018). Therefore, we investigated the interplay between tumor cell driven XIAP expression and the TIME. METHODOLOGY: IBC patient clinicopathological variables (n=75) along with Affymetrix data (n=30/75) were collected. Immunostainings for XIAP, CD8 and CD163 (Hematoxylin-DAB) were done according to validated protocols. All slides were digitized and evaluated in Visiopharm to quantify the number of DAB+ immune cells and the relative marker area (RMA: the DAB+ area/total area of interest). Spatial interactions were examined using an effector index (EI) that counts the number of immune cells in a circle with a radius of 30 mcm around a CD8+ T cell or a tumor cell. We evaluated correlations between XIAP protein and mRNA expression, immune cell signatures (using CIBERSORT) &amp; gene-expression pathways as a validation of our IHC findings. RESULTS: High XIAP expression was detected in 45.3% of the IBC samples (n= 34/75). Although, XIAP expression was not associated with any clinicopathological parameters like grade, molecular subtype, or disease stage. High XIAP expressing tumors had more sTIL infiltration (P= 0.008), PDL1 expression (P= 0.012) and infiltration with TAMs (RMA: 1.03% (0.03% – 5.97%) vs. 2.73% (0.01% – 11.3%), P= 0.003). Furthermore, in these XIAP high samples TAMs were also located closer to the tumor cells (EI: 0.95 cells (0.06 – 3.48) vs. 2.04 cells (0.28 – 5.20), P&amp;lt; 0.001) and the CD8+ T cells (EI: 2.85 cells (0.26 – 8.85) vs. 5.13 cells (0.87 – 10.16), P&amp;lt; 0.001), while there was no correlation between XIAP and the total number of CD8+ cells. Although gene expression data analysis did not reveal any correlation between protein and mRNA expression of XIAP, there was a significant correlation between XIAP activation signature and protein expression (P&amp;lt;0.001). Furthermore, we discovered a strong association between XIAP protein and cellular stress response- &amp; inflammation-related gene sets. Finally, in the CIBERSORT data, we observed the strongest correlation between XIAP expression and M2 macrophages &amp; activated dendritic cells. CONCLUSION: This is the first study showing high XIAP expressing IBC tumors have increased infiltration of immunosuppressive TAMs, which co-localized near tumor and cytotoxic T cells. We also demonstrated an association between XIAP protein expression and inflammation related &amp; cellular stress response gene signatures. This is relevant as XIAP function depends on translational upregulation during cellular stress which leads to immunosuppressive signaling and an aggressive breast cancer phenotype. XIAP antagonists are in clinical trials and these data suggest potential benefit in targeting XIAP to enhance immunotherapeutic efficacy in IBC therapy. FUNDING: DoDs-W81XWH-17-1-0297,W81XWH-20-1-0153, ACS Mission Boost Grants(GRD) Citation Format: Christophe Van Berckelaer, Steven Van Laere, Joseph Gerardts, Luc Dirix, Michael Morse, Mark Kockx, François Bertucci, Peter Van Dam, Gayathri R Devi. Xiap expression is associated with infiltration of cd163+ tumor-associated macrophages in the tumor micro-environment of inflammatory breast cancer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P1-04-07.

Accumulation of amyloid beta (Aβ) and amyloid precursor protein (APP) in tumors formed by a mouse xenograft model of inflammatory breast cancer.

Accumulation of amyloid in breast cancer is a well‐known phenomenon, but only immunoglobulin light‐chain amyloidosis (AL) or transthyretin (TTR) amyloid had been detected in human breast tumor samples previously. We recently reported that another amyloidogenic peptide, amyloid beta (Aβ), is present in an aggregated form in animal and human high‐grade gliomas and suggested that it originates systemically from the blood, possibly generated by platelets. To study whether breast cancers are also associated with these Aβ peptides and in what form, we used a nude mouse model inoculated with triple‐negative inflammatory breast cancer cell (SUM‐149) xenografts, which develop noticeable tumors. Immunostaining with two types of specific antibodies for Aβ identified the clear presence of Aβ peptides associated with (a) carcinoma cells and (b) extracellular aggregated amyloid (also revealed by Congo red and thioflavin S staining). Aβ peptides, in both cells and in aggregated amyloid, were distributed in clear gradients, with maximum levels near blood vessels. We detected significant presence of amyloid precursor protein (APP) in the walls of blood vessels of tumor samples, as well as in carcinoma cells. Finally, we used ELISA to confirm the presence of elevated levels of mouse‐generated Aβ40 in tumors. We conclude that Aβ in inflammatory breast cancer tumors, at least in a mouse model, is always present and is concentrated near blood vessels. We also discuss here the possible pathways of Aβ accumulation in tumors and whether this phenomenon could represent the specific signature of high‐grade cancers.

Lipocalin 2 promotes inflammatory breast cancer tumorigenesis and skin invasion.

Inflammatory breast cancer (IBC) is an aggressive form of primary breast cancer characterized by rapid onset and high risk of metastasis and poor clinical outcomes. The biological basis for the aggressiveness of IBC is still not well understood and no IBC‐specific targeted therapies exist. In this study, we report that lipocalin 2 (LCN2), a small secreted glycoprotein belonging to the lipocalin superfamily, is expressed at significantly higher levels in IBC vs non‐IBC tumors, independently of molecular subtype. LCN2 levels were also significantly higher in IBC cell lines and in their culture media than in non‐IBC cell lines. High expression was associated with poor‐prognosis features and shorter overall survival in IBC patients. Depletion of LCN2 in IBC cell lines reduced colony formation, migration, and cancer stem cell populations in vitro and inhibited tumor growth, skin invasion, and brain metastasis in mouse models of IBC. Analysis of our proteomics data showed reduced expression of proteins involved in cell cycle and DNA repair in LCN2‐silenced IBC cells. Our findings support that LCN2 promotes IBC tumor aggressiveness and offer a new potential therapeutic target for IBC.

Progress for Immunotherapy in Inflammatory Breast Cancer and Emerging Barriers to Therapeutic Efficacy

Simple SummaryDespite recent advances in the treatment of other breast cancer subtypes, inflammatory breast cancer (IBC) remains a significant clinical challenge, with an overall 5-year survival rate of 39%. Though immunotherapy has shown remarkable efficacy in other difficult-to-treat cancers, such approaches have yet to show substantial therapeutic efficacy in IBC. Here, we summarize the known immune composition of IBC tumors, as well as past and present efforts to advance immunotherapy in the treatment of IBC.Inflammatory breast cancer (IBC) is a rare and aggressive subtype of breast cancer that carries a particularly poor prognosis. Despite the efficacy of immunotherapy in other difficult to treat forms of breast cancer, progress for immunotherapy in IBC has been difficult. Though immunotherapy has been under clinical investigation in IBC since the 1970s, few approaches have shown significant therapeutic efficacy, and no immunotherapy regimens are currently used in the treatment of IBC. Here, we provide a comprehensive summary of what is known about the immune composition of IBC tumors, clinical and basic science evidence describing the role for immune checkpoints such as PD-L1 in IBC pathobiology, as well as past and present attempts to advance ICIs in the treatment of IBC.

Inflammatory Breast Carcinoma: Elevated microRNA miR-181b-5p and Reduced miR-200b-3p, miR-200c-3p, and miR-203a-3p Expression as Potential Biomarkers with Diagnostic Value.

Inflammatory breast cancer (IBC) is a rare yet aggressive breast cancer variant, associated with a poor prognosis. The major challenge for IBC is misdiagnosis due to the lack of molecular biomarkers. We profiled dysregulated expression of microRNAs (miRNAs) in primary samples of IBC and non-IBC tumors using human breast cancer miRNA PCR array. We discovered that 28 miRNAs were dysregulated (10 were upregulated, while 18 were underexpressed) in IBC vs. non-IBC tumors. We identified 128 hub genes, which are putative targets of the differentially expressed miRNAs and modulate important cancer biological processes. Furthermore, our qPCR analysis independently verified a significantly upregulated expression of miR-181b-5p, whereas a significant downregulation of miR-200b-3p, miR-200c-3p, and miR-203a-3p was detected in IBC tumors. Receiver operating characteristic (ROC) curves implied that the four miRNAs individually had a diagnostic accuracy in discriminating patients with IBC from non-IBC and that miR-203a-3p had the highest diagnostic value with an AUC of 0.821. Interestingly, a combination of miR-181b-5p, miR-200b-3p, and miR-200c-3p robustly improved the diagnostic accuracy, with an area under the curve (AUC) of 0.897. Intriguingly, qPCR revealed that the expression of zinc finger E box-binding homeobox 2 (ZEB2) mRNA, the putative target of miR-200b-3p, miR-200c-3p, and miR-203a-3p, was upregulated in IBC tumors. Overall, this study identified a set of miRNAs serving as potential biomarkers with diagnostic relevance for IBC.

Induction of heparanase via IL-10 correlates with a high infiltration of CD163+ M2-type tumor-associated macrophages in inflammatory breast carcinomas

Inflammatory breast cancer (IBC) is the most aggressive and lethal form of breast cancer, characterized by a high infiltration of tumor-associated macrophages and poor prognosis. To identify new biomarkers and to elucidate the molecular mechanisms underlying IBC pathogenesis, we investigated the expression pattern of heparanase (HPSE) and its activator cathepsin L (CTSL). First, we quantitated the HPSE and CTSL mRNA levels in a cohort of breast cancer patients after curative surgery (20 IBC and 20-non-IBC). We discovered that both HPSE and CTSL mRNA levels were significantly induced in IBC tissue vis-à-vis non-IBC patients (p <0 .05 and p <0 .001, respectively). According to the molecular subtypes, HPSE mRNA levels were significantly higher in carcinoma tissues of triple negative (TN)-IBC as compared to TN-non-IBC (p <0 .05). Mechanistically, we discovered that pharmacological inhibition of HPSE activity resulted in a significant reduction of invasiveness in the IBC SUM149 cell line. Moreover, siRNA-mediated HPSE knockdown significantly downregulated the expression of the metastasis-related gene MMP2 and the cancer stem cell marker CD44. We also found that IBC tumors revealed robust heparanase immune-reactivity and CD163+ M2-type tumor-associated macrophages, with a positive correlation of both markers. Moreover, the secretome of axillary tributaries blood IBC CD14+ monocytes and the cytokine IL-10 significantly upregulated HPSE mRNA and protein expression in SUM149 cells. Intriguingly, massively elevated IL-10 mRNA expression with a trend of positive correlation with HPSE mRNA expression was detected in carcinoma tissue of IBC. Our findings highlight a possible role played by CD14+ monocytes and CD163+ M2-type tumor-associated macrophages in regulating HPSE expression possibly via IL-10. Overall, we suggest that heparanase, cathepsin L and CD14+ monocytes-derived IL-10 may play an important role in the pathogenesis of IBC and their targeting could have therapeutic implications.

Abstract P3-01-10: Ndrg1-egfr axis in inflammatory breast cancer tumorigenesis and brain metastasis

Abstract Background: Inflammatory breast cancer (IBC) is a rare (1-2%) but lethal form of primary breast cancer responsible for 10% of breast cancer-related deaths in the United States. IBC tumors grow aggressively and have a high risk of metastasis, particularly to the brain. Unraveling the pathways involved in IBC aggressiveness and metastasis could reveal new therapeutic targets. We recently identified NDRG1 (N-Myc downstream regulated gene1) in a newly isolated subline of MDA-IBC3 cells as a top candidate protein associated with enhanced propensity for brain metastasis. Although the metastasis suppressor properties of NDRG1 are widely described, its function in the pathobiology of IBC is unknown. Here we investigated the function of NDRG1 in tumorigenesis and metastatic progression to brain in IBC. Methods: NDRG1 gene expression was analyzed in large publically available breast cancer clinical datasets and the IBC Consortium dataset, and median expression level was used to stratify patients into NDRG1-high and NDRG1-low groups. Tissue microarrays of IBC patient samples were immunostained with anti-NDRG1 antibody. Stable knockdown (KD) of NDRG1 in IBC cell lines (HER2+: MDA-IBC3; TNBC: SUM149, BCX010) was obtained by lentiviral gene transfer. For in vivo studies, control and NDRG1 KD IBC cell lines were transplanted into cleared mammary fat pads of SCID/Beige mice and tumor growth monitored via caliper measurements. Reverse phase protein array analysis was used for proteomic profiling. Results: In several independent cohorts of patients with breast cancer (including The Cancer Genome Atlas), NDRG1 mRNA expression was significantly higher in ER-negative and high-grade breast tumors (p&amp;lt;0.0001) and was associated with poor overall survival (p=0.0003), relapse-free survival (p&amp;lt;0.0001), and metastasis-free survival (p=0.0012). NDRG1 expression was higher in more aggressive, basal-like and HER2+ subtypes than in hormone receptor-positive subtypes (p&amp;lt;0.0001), and was also higher in IBC vs. non-IBC tumors (p=0.007, IBC consortium dataset). Immunostaining showed NDRG1 was frequently expressed in IBC patient tumors (83%) and was associated with worse overall survival (OS, p=0.0129) and disease-specific survival (DSS, p=0.013). On multivariate analysis, NDRG1 was an independent prognostic factor for poor OS [HR 2.5 (95%CI, 1.3-4.7), p=0.0047] and DSS [HR 2.6 (95%CI 1.3-5.1), p=0.006]. Notably, NDRG1 expression was higher in patients with brain metastasis vs. primary tumors (GSE43837, p=0.018) and correlated with shorter time to development of brain metastasis (GSE2034, p=0.026). NDRG1-high sublines had enhanced propensity for brain metastasis as shown by higher incidence (100% vs. 44% for NDRG1-low sublines, p=0.01) and increased brain metastasis burden (p=0.0008). Invitro, silencing NDRG1 in IBC cell lines significantly reduced colony formation, migration, invasion, and cancer stem-like properties. In vivo, silencing of NDRG1 inhibited primary tumor growth of SUM149 (p&amp;lt;0.0001) and MDA-IBC3 (p=0.0426) xenografts. Proteomics analysis showed that NDRG1 correlated positively with activation of the EGFR/STAT3 signaling pathway. Mechanistically, NDRG1 knockdown in IBC cell lines reduced EGFR expression and suppressed phosphorylation of EGFR and STAT3, whereas NDRG1 overexpression increased EGFR expression and enhanced EGFR-STAT3 activation. Conclusions: We showed that high NDRG1 was associated with worse clinical outcomes in patients with IBC and that NDRG1 drives tumor progression in IBC potentially via activation of the EGFR-STAT3 signaling pathway. Targeting the NDRG1-EGFR axis may be a novel therapeutic approach in IBC and other aggressive breast cancers. Additional investigations are underway to determine the detailed mechanisms of NDRG1-mediated IBC tumorigenesis and brain metastasis Citation Format: Emilly Schlee Villodre, Xiaoding Hu, Richard Larson, Bedrich L Eckhardt, Yun Gong, Lei Huo, Juhee Song, Savitri Krishnamurthy, Nuhad Ibrahim, Naoto T Ueno, Debu Tripathy, Wendy A Woodward, Bisrat G Debeb. Ndrg1-egfr axis in inflammatory breast cancer tumorigenesis and brain metastasis [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 P3-01-10.

Abstract P6-06-02: An in vitro microfluidic tumor platform for modeling and investigating tumor stromal interactions in inflammatory breast cancer

Abstract Introduction: Inflammatory breast cancer (IBC) is an aggressive and rare disease with poor prognosis, accounting for 10% of breast cancer mortality [1]. A primary factor contributing to the bleak prognosis is the lack of IBC specific treatments. There are currently no IBC specific therapies due to a lack of IBC specific diagnostic and targeting markers. Efforts focused on identifying driver mutations and tumor targets have implicated tumor stroma including stromal cells such as macrophages in mediating IBC-like symptoms. This highlights the significance of understanding the interactions of tumor cells with the tumor stroma in greater detail and the knowledge would enable determination of targetable biology from these interactions which would facilitate development of IBC specific treatments and therapeutics. What is needed is a model to capture the complexity of IBC, identify critical spatial hetero-cellular interactions and target them successfully in a physiologically relevant and high-throughput manner. Approach: To address this need, we developed a 3D IBC microfluidic platform, unique in its simultaneous integration of functional blood vessels, tumor cells, macrophages, and type I collagen whose density, stiffness, and porosity mimics cancerous breast stroma. The platform will be used to study the influence of macrophage-tumor-endothelial interactions on 2 key critical features of IBC: vascular sprouting and formation of IBC emboli surrounded by vascular sprouts. Results: The 3D IBC microfluidic platform composed of MDA-IBC3 cells and a functional endothelial blood vessel demonstrated both vascular sprouting and emboli formation, key features of IBC tumors seen in IBC patient derived xenograft (PDX) models. Additionally, we observed vascular nesting of MDA-IBC3 emboli, recreating a characteristic IBC phenomenon observed in Mary-X PDX models. Incorporation of macrophages significantly increased the number of new vascular sprouts, sprouting rate and resulted in sprouts forming at earlier time points. Additionally, the presence of macrophages resulted in the formation of a significantly more porous collagen matrix (p&amp;lt;0.05), increased endothelial vessel permeability (p&amp;lt;0.05) and expression of proangiogenic factors, IL-8 and MMP9 (p&amp;lt;0.05, p&amp;lt;0.05) compared to platforms without macrophages. 3D platforms with macrophages also exhibited vascular nesting of MDA-IBC3 emboli but no significant differences in the number of nested emboli were detected. Finally, in 3D platforms with macrophages, we observed intravasation of MDA-IBC3 emboli which was absent in platforms without macrophages. Conclusion: IBC is an aggressive and invasive breast cancer with a poor prognosis linked to tumor-stroma interactions. Current preclinical to study IBC consist primarily of PDX models where determining the influence of specific signaling pathways and microenvironmental stimuli on tumor progression is challenging. Here we present a novel 3D microfluidic IBC platform to study tumor stromal interactions in a controlled manner. The MDA-IBC3 breast tumor platform demonstrated both vascular sprouting and emboli formation, key features of IBC seen in PDX models and the presence of macrophages increased both angiogenic sprouting and remodeling of the collagen matrix. The stark differences in the tumor platform response associated with macrophage presence strengthens the hypothesis of tumor stroma as a key player driving the aggressive nature of IBC and reveals a potential target for IBC therapeutics. [1] Fernandez, S.V., et al., Breast cancer research and treatment, 140(1): p. 23-33, 2013 Citation Format: Manasa Gadde, Caleb Phillips, Omar Rahal, Wendy Woodward, Marissa Rylander, Thomas Yankeelov. An in vitro microfluidic tumor platform for modeling and investigating tumor stromal interactions 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 P6-06-02.

Abstract P5-17-06: Immune modulation with humanized anti-EGFR antibody panitumumab in an immunocompetent mouse model for inflammatory breast cancer

Abstract Background: Inflammatory breast cancer (IBC) is the most lethal and aggressive form of breast cancer and there are no approved targeted therapies specifically for IBC. Targeting the epidermal growth factor receptor (EGFR) pathway is a promising therapeutic target for patients with triple-negative IBC (TN-IBC) with a reported pathological complete response rate of 42% (JAMA Oncology, 2018). The tumor microenvironment (TME) is a critical contributor to the aggressiveness of IBC. Delineating cross-talk between EGFR-targeted therapies and TME components, which define IBC, could inform more efficient combination regimens and novel clinical trial designs for IBC. However, such studies have not been conducted due to the lack of a syngeneic IBC mouse model. Here we report the establishment of an IBC immunocompetent mouse model and the effects of panitumumab (PmAb) on IBC tumor growth and the TME. Methods: TN-IBC cell lines, SUM149 or FC-IBC-02, were mixed with 50% Matrigel and inoculated into mammary fat pads of hu-NSG-SGM3 mice engrafted with hematopoietic stem cells (The Jackson Laboratory). SUM149 tumor growth in hu-NSG-SGM3 mice treated with either IgG2 (isotype control, 4 mg/kg) or PmAb (1 mg/kg and 4 mg/kg) was measured. The percentages of TME components, including human CD4+ T, CD8+ T, regulatory T (Tregs), and natural killer (NK) cells, and M1 or M2 macrophages, in the peripheral blood and tumor tissues treated with IgG2 and PmAb for 7 weeks were measured by flow cytometry. Results: Hu-NSG-SGM3 mice supported the growth of TN-IBC SUM149 and FC-IBC-02 xenografts. These humanized mouse models were named SUM149-huSGM3 and FC-IBC-02-huSGM3, respectively. Analysis of the blood cells showed that SUM149-huSGM3 mice display human CD4+ T, CD8+ T, Tregs, M1 and M2 macrophages. T cell infiltration and M1 and M2 macrophages were also detected in SUM149-huSGM3 tumors. NK cells were not detected in both peripheral blood and tumors. PmAb treated SUM149-huSGM3 mice had significantly reduced SUM149 tumor growth, compared with mice that received the IgG2 control. PmAb treatment increased the percentage of CD8+ T cells and reduced the percentage of Tregs in peripheral blood. A similar analysis of tumor infiltrating lymphocytes isolated from each group showed an increase in percent CD8+ T cells in mice treated with PmAb. There were no significant changes of M1 or M2 macrophages following PmAb treatment. These results suggest that the increase in percentage of CD8+ T cells in peripheral blood and IBC tumors, and the decrease in percentage of Tregs in peripheral blood may contribute to the therapeutic efficacy of PmAb. Conclusion: We established the first immunocompetent mouse model to study the TME and immune response in IBC, which provides the premise for conducting a diversity of novel preclinical therapeutic studies. The mechanism of how immune responses of TN-IBC xenografts mediates the therapeutic efficacy of PmAb in IBC tumors needs to be further investigated. Our study also suggests that combination therapy with immune checkpoint inhibitors may potentiate the efficacy of anti-EGFR therapy in IBC. The therapeutic efficacy of PmAb and anti-PD-L1 combination in SUM149 humanized mice is in progress. Citation Format: Wang X, Shao S, Pearson T, Cheng Y, Reuben JM, Tripathy D, Ueno NT. Immune modulation with humanized anti-EGFR antibody panitumumab in an immunocompetent mouse model for inflammatory breast cancer [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P5-17-06.

Abstract P5-17-01: Patient-derived organoid models of inflammatory breast cancer

Abstract Background: Inflammatory Breast Cancer (IBC) is an aggressive subtype of breast cancer that is associated with an increased likelihood of early metastasis and a poor prognosis, accounting for approximately 10% of all breast cancer mortality. Currently, there is limited availability of cell line and patient-derived xenograft (PDX) models of IBC. As a result, there is an urgent need for additional model systems to facilitate our understanding of this disease. Organoids have been used to grow breast cancer cells with high efficiency, resulting in long-term cultures with genetic and phenotypic stability; however, the extent to which IBC can be modeled using this system is unknown. We undertook an evaluation of organoid culture technology for growing IBC patient samples, with the goal of generating a model of IBC that more faithfully recapitulates the key features of the disease. Methods: Tumor samples (taken from lumpectomy, mastectomy, PDX, or core biopsy samples), were digested to the organoid level using collagenase, and were grown in three dimensional cultures using a basement membrane extract and a fully-defined organoid medium, as described (Sachs et al. Cell 2018). First, we surveyed non-IBC metastatic breast cancer samples to determine the efficiency of growth of multiple breast cancer subtypes under these conditions. Then, we assessed the efficiency of growth of IBC samples taken after treatment, at the time of mastectomy. Co-culture experiments in which IBC organoids were grown in the presence of human endothelial cells were performed in three dimensional culture using a mixture of type I collagen and basement membrane extract. Results: Our success rate for establishing organoid cultures from breast cancer samples in general was 75% (33 out of 44 samples), and for triple-negative breast cancers, the success rate for establishment of organoid cultures was 100% (14 out of 14 samples). Expansion of ER+ tumor organoids (16 of 26 samples) was primarily restricted by a slow growth rate in culture, as compared to triple-negative tumor organoids. We were able to establish 12 breast cancer organoids from core biopsies of multiple metastatic sites, including brain, liver, bone, breast, and skin. We subsequently were able to establish organoid cultures from five out of five IBC tumor samples. Our IBC organoids preserved histopathologic features of the original tumor, and expressed key markers, such as E-cadherin, that are known to be associated with IBC. In addition, our IBC organoids interacted with human endothelial cells under co-culture conditions designed to model lymphovascular invasion, an essential feature of IBC. Conclusion: We have shown that organoid cultures can be established to propagate IBC tumors with high efficiency, preserve key pathologic features of IBC in vitro, and model interactions between tumor cells and the microenvironment. Thus, organoids are useful complements to existing IBC models, and can be used to identify potential therapeutic vulnerabilities associated with this particularly aggressive breast cancer subtype. Citation Format: Rosenbluth JM, Zervantonakis I, Boedicker M, Wagle N, Dillon D, Nakhlis F, Brugge JS, Overmoyer B. Patient-derived organoid models of inflammatory breast cancer [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P5-17-01.