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Inflammatory breast cancer (IBC) is a rare and aggressive disease that accounts for ~2–4% of all breast cancers. However, despite its low incidence rate, IBC is responsible for 7–10% of breast cancer-related mortality in Western countries. Thus, the discovery of robust biological targets and the development of more effective therapeutics in IBC are crucial. Despite major international efforts to understand IBC biology, genomic studies have not led to the discovery of distinct biological mechanisms in IBC that can be translated into novel therapeutic strategies. In this Review, we discuss these molecular profiling efforts and highlight other important aspects of IBC biology. We present the intrinsic characteristics of IBC, including stemness, metastatic potential and hormone receptor positivity; the extrinsic features of the IBC tumour microenvironment (TME), including various constituent cell types; and lastly, the communication between these intrinsic and extrinsic components. We summarize the latest perspectives on the key biological features of IBC, with particular emphasis on the TME as an important contributor to the aggressive nature of IBC. On the basis of the current understanding of IBC, we hope to develop the next generation of translational studies, which will lead to much-needed survival improvements in patients with this deadly disease.

PurposeThe purpose of the study was to evaluate protein expression of PD-L1 and CD20 as prognostic biomarkers of patient outcome in inflammatory breast cancer (IBC) samples.MethodsPD-L1 and CD20 protein expression was measured by immunohistochemistry in 221 pretreatment IBC biopsies. PD-L1 was assessed in tumor cells (PD-L1+ tumor cells) and tumor stromal infiltrating lymphocytes (PD-L1+ TILs); CD20 was scored in tumor-infiltrating B cells. Kaplan–Meier curves and Cox proportional hazard models were used for survival analysis.ResultsPD-L1+ tumor cells, PD-L1+ TILs, and CD20+ TILs were found in 8%, 66%, and 62% of IBC, respectively. PD-L1+ tumor cells strongly correlated with high TILs, pathological complete response (pCR), CD20+ TILs, but marginally with breast cancer-specific survival (BCSS, P = 0.057). PD-L1+ TILs strongly correlated with high TILs, CD20+ TILs, and longer disease-free survival (DFS) in all IBC and in triple-negative (TN) IBC (P < 0.035). IBC and TN IBC patients with tumors containing both CD20+ TILs and PD-L1+ TILs (CD20+TILs/PD-L1+TILs) showed longer DFS and improved BCSS (P < 0.002) than patients lacking both, or those with either CD20+ TILs or PD-L1+ TILs alone. In multivariate analyses, CD20+TILs/PD-L1+TILs status was an independent prognostic factor for DFS in IBC (hazard ratio (HR): 0.53, 95% CI 0.37–0.77) and TN IBC (HR: 0.39 95% CI 0.17–0.88), and for BCSS in IBC (HR: 0.60 95% CI 0.43–0.85) and TN IBC (HR: 0.38 95% CI 0.17–0.83).ConclusionCD20+TILs/PD-L1+TILs status represents an independent favorable prognostic factor in IBC and TN IBC, suggesting a critical role for B cells in antitumor immune responses. Anti-PD-1/PD-L1 and B cell-activating immunotherapies should be explored in these settings.

Inflammatory Breast Cancer (IBC) is a highly aggressive malignancy with distinct clinical and histopathological features whose molecular basis is unresolved. Here we describe a human IBC cell line, A3250, that recapitulates key IBC features in a mouse xenograft model, including skin erythema, diffuse tumor growth, dermal lymphatic invasion, and extensive metastases. A3250 cells express very high levels of the CCL2 chemokine and induce tumors enriched in macrophages. CCL2 knockdown leads to a striking reduction in macrophage densities, tumor proliferation, skin erythema, and metastasis. These results establish IBC-derived CCL2 as a key factor driving macrophage expansion, and indirectly tumor growth, with transcriptomic analysis demonstrating the activation of multiple inflammatory pathways. Finally, primary human IBCs exhibit macrophage infiltration and an enriched macrophage RNA signature. Thus, this human IBC model provides insight into the distinctive biology of IBC, and highlights potential therapeutic approaches to this deadly disease. Inflammatory breast cancer (IBC) is an aggressive form of breast cancer with a poor prognosis. Here the authors report the characterization of a human IBC cell line recapitulating the clinical and histopathological features of the human disease, and implicating its high level of CCL2 in macrophage infiltration and tumor progression.

Background Inflammatory breast cancer (IBC) is a rare and rapidly progressive form of invasive breast cancer. The aim of this study was to explore the clinical evolution, stromal tumour-infiltrating lymphocytes (sTIL) infiltration and programmed death-ligand 1 (PD-L1) expression in a large IBC cohort. Patients and methods Data were collected prospectively from patients with IBC as part of an international collaborative effort since 1996. In total, 143 patients with IBC starting treatment between June 1996 and December 2016 were included. Clinicopathological variables were collected, and sTIL were scored by two pathologists on standard H&E stained sections. PD-L1 expression was assessed using a validated PD-L1 (SP142) assay. A validation cohort of 64 patients with IBC was used to test our findings. Results Survival outcomes of IBC remained poor with a 5-year overall survival (OS) of 45.6%. OS was significantly better in patients with primary non-metastatic disease who received taxane-containing (neo)adjuvant therapy ( P =  0.01), had a hormone receptor-positive tumour ( P =  0.001) and had lower cN stage at diagnosis ( P =  0.001). PD-L1 positivity on immune cells (42.9%) was higher in IBC than in non-IBC in both our patient samples and the validation cohort. Furthermore, PD-L1 expression predicted pCR ( P =  0.002) and correlated with sTIL infiltration ( P <  0.001). sTIL infiltration of more than 10% of the stroma was a significant predictor of improved OS (HR 0.47, 95% CI 0.27–0.81, P =  0.006) in a multivariate model. Conclusions IBC is characterised by poor survival and high PD-L1 immunoreactivity on sTIL. This suggests a role for PD1/PD-L1 inhibitors in the treatment of IBC. Furthermore, we showed that PD-L1 expression predicts response to neo-adjuvant therapy and that sTIL have prognostic significance in IBC.

In patients with triple-negative breast cancer (TNBC), tumor-infiltrating lymphocytes (TILs) are associated with improved survival. Lehmann et al. identified 4 molecular subtypes of TNBC [basal-like (BL) 1, BL2, mesenchymal (M), and luminal androgen receptor (LAR)], and an immunomodulatory (IM) gene expression signature indicates the presence of TILs and modifies these subtypes. The association between TNBC subtype and TILs is not known. Also, the association between inflammatory breast cancer (IBC) and the presence of TILs is not known. Therefore, we studied the IM subtype distribution among different TNBC subtypes. We retrospectively analyzed patients with TNBC from the World IBC Consortium dataset. The molecular subtype and the IM signature [positive (IM+) or negative (IM-)] were analyzed. Fisher's exact test was used to analyze the distribution of positivity for the IM signature according to the TNBC molecular subtype and IBC status. There were 88 patients with TNBC in the dataset, and among them 39 patients (44%) had IBC and 49 (56%) had non-IBC. The frequency of IM+ cases differed by TNBC subtype (p = 0.001). The frequency of IM+ cases by subtype was as follows: BL1, 48% (14/29); BL2, 30% (3/10); LAR, 18% (3/17); and M, 0% (0/21) (in 11 patients, the subtype could not be determined). The frequency of IM+ cases did not differ between patients with IBC and non-IBC (23% and 33%, respectively; p = 0.35). In conclusion, the IM signature representing the underlying molecular correlate of TILs in the tumor may differ by TNBC subtype but not by IBC status.

Background Tumor-associated macrophages (TAMs) are key promoters of inflammatory breast cancer (IBC), the most aggressive form of breast cancer. The receptor tyrosine kinase AXL is highly expressed in various cancer types, including IBC, but its role in TAMs remains unexplored. Methods We examined the effects of AXL inhibitor TP-0903 on tumor growth and tumor microenvironment (TME) component M2 macrophages (CD206 + ) in IBC and triple-negative breast cancer mouse models using flow cytometry and immunohistochemical staining. Additionally, we knocked out AXL expression in human THP-1 monocytes and evaluated the effect of AXL signaling on immunosuppressive M2 macrophage polarization and IBC cell growth and migration. We then investigated the underlying mechanisms through RNA sequencing analysis. Last, we performed CIBERSORT deconvolution to analyze the association between AXL expression and tumor-infiltrating immune cell types in tumor samples from the Inflammatory Breast Cancer International Consortium. Results We found that inhibiting the AXL pathway significantly reduced IBC tumor growth and decreased CD206 + macrophage populations within tumors. Mechanistically, our in vitro data showed that AXL promoted M2 macrophage polarization and enhanced the secretion of immunosuppressive chemokines, including CCL20, CCL26, and epiregulin, via the transcription factor STAT6 and thereby accelerated IBC cell growth and migration. RNA sequencing analysis further indicated that AXL signaling in immunosuppressive M2 macrophages regulated the expression of molecules and cytokines, contributing to an immunosuppressive TME in IBC. Moreover, high AXL expression was correlated with larger populations of immunosuppressive immune cells but smaller populations of immunoactive immune cells in tissues from patients with IBC. Conclusions AXL signaling promotes IBC growth by inducing M2 macrophage polarization and driving the secretion of immunosuppressive molecules and cytokines via STAT6 signaling, thereby contributing to an immunosuppressive TME. Collectively, these findings highlight the potential of targeting AXL signaling as a novel therapeutic approach for IBC that warrants further investigation in clinical trials.

Inflammatory breast cancer (IBC) is the most insidious form of locally advanced breast cancer; about a third of patients have distant metastasis at initial staging. Emerging evidence suggests that host factors in the tumor microenvironment may interact with underlying IBC cells to make them aggressive. It is unknown whether immune cells associated to the IBC microenvironment play a role in this scenario to transiently promote epithelial to mesenchymal transition (EMT) in these cells. We hypothesized that soluble factors secreted by activated immune cells can induce an EMT in IBC and thus promote metastasis. In a pilot study of 16 breast cancer patients, TNF-α production by peripheral blood T cells was correlated with the detection of circulating tumor cells expressing EMT markers. In a variety of IBC model cell lines, soluble factors from activated T cells induced expression of EMT-related genes, including FN1, VIM, TGM2, ZEB1. Interestingly, although IBC cells exhibited increased invasion and migration following exposure to immune factors, the expression of E-cadherin (CDH1), a cell adhesion molecule, increased uniquely in IBC cell lines but not in non-IBC cell lines. A combination of TNF-α, IL-6, and TGF-β was able to recapitulate EMT induction in IBC, and conditioned media preloaded with neutralizing antibodies against these factors exhibited decreased EMT. These data suggest that release of cytokines by activated immune cells may contribute to the aggressiveness of IBC and highlight these factors as potential target mediators of immune-IBC interaction.

IntroductionWe hypothesized that breast tissue not involved by tumor in inflammatory breast cancer (IBC) patients contains intrinsic differences, including increased mammary stem cells and macrophage infiltration, which may promote the IBC phenotype.Materials and methodsNormal breast parenchyma ≥ 5 cm away from primary tumors was obtained from mastectomy specimens. This included an initial cohort of 8 IBC patients and 60 non-IBC patients followed by a validation cohort of 19 IBC patients and 25 non-IBC patients. Samples were immunostained for either CD44+CD49f+CD133/2+ mammary stem cell markers or the CD68 macrophage marker and correlated with IBC status. Quantitation of positive cells was determined using inForm software from PerkinElmer. We also examined the association between IBC status and previously published tumorigenic stem cell and IBC tumor signatures in the validation cohort samples.Results8 of 8 IBC samples expressed isolated CD44+CD49f+CD133/2+ stem cell marked cells in the initial cohort as opposed to 0/60 non-IBC samples (p = 0.001). Similarly, the median number of CD44+CD49f+CD133/2+ cells was significantly higher in the IBC validation cohort as opposed to the non-IBC validation cohort (25.7 vs. 14.2, p = 0.007). 7 of 8 IBC samples expressed CD68 + histologically confirmed macrophages in initial cohort as opposed to 12/48 non-IBC samples (p = 0.001). In the validation cohort, the median number of CD68 + cells in IBC was 3.7 versus 1.0 in the non-IBC cohort (p = 0.06). IBC normal tissue was positively associated with a tumorigenic stem cell signature (p = 0.02) and with a 79-gene IBC signature (p < 0.001).ConclusionsNormal tissue from IBC patients is enriched for both mammary stem cells and macrophages and has higher association with both a tumorigenic stem cell signature and IBC-specific tumor signature. Collectively, these data suggest that IBC normal tissue differs from non-IBC tissue. Whether these changes occur before the tumor develops or is induced by tumor warrants further investigation.

The authors studied the radiosensitization of breast cancer stem‐like cells in vitro after treatment with the most commonly used statin, simvastatin, and examined the influence on local control after postmastectomy radiation among inflammatory breast cancer patients taking statins. This work provides new insight on combination regimens for breast cancer treatment and radiosensitization of this clinically radioresistant disease. Reported rates of local failure after adjuvant radiation for women with inflammatory breast cancer (IBC) and triple‐negative non‐IBC are higher than those of women with receptor‐expressing non‐IBC. These high rates of locoregional recurrence are potentially influenced by the contribution of radioresistant cancer stem cells to these cancers. Statins have been shown to target stem cells and improve disease‐free survival among IBC patients. We examined simvastatin radiosensitization of multiple subtypes of breast cancer cell lines in vitro in monolayer and mammosphere‐based clonogenic assays and examined the therapeutic benefit of statin use on local control after postmastectomy radiation (PMRT) among IBC patients. We found that simvastatin radiosensitizes mammosphere‐initiating cells (MICs) of IBC cell lines (MDA‐IBC3, SUM149, SUM190) and of the metaplastic, non‐IBC triple‐negative receptor cell line (SUM159). However, simvastatin radioprotects MICs of non‐IBC cell lines MCF‐7 and SKBR3. In a retrospective clinical study of 519 IBC patients treated with PMRT, 53 patients used a statin. On univariate analysis, actuarial 3‐year local recurrence‐free survival (LRFS) was higher among statin users, and on multivariate analysis, triple negative breast cancer, absence of lymphatic invasion, neoadjuvant pathological tumor response to preoperative chemotherapy, and statin use were independently associated with higher LRFS. In conclusion, patients with IBC and triple‐negative non‐IBC breast cancer have the highest rates of local failure, and there are no available known radiosensitizers. We report significant improvement in local control after PMRT among statin users with IBC and significant radiosensitization across triple‐negative and IBC cell lines of multiple subtypes using simvastatin. These data suggest that simvastatin should be justified as a radiosensitizing agent by a prospective clinical trial.

Background Inflammatory breast cancer (IBC), of which HER2 + is the predominant subtype, is extremely aggressive and difficult to treat. Previous studies have suggested that targeting the tumour microenvironment (TME) may provide new directions for IBC diagnosis and treatment. Methods In this study, we used single-cell transcriptome technology (scRNA-seq) to investigate the molecular features of the TME of HER2 + IBC patients and performed a comprehensive and detailed comparison of the cellular components and molecular phenotypes of the TME between IBC patients and noninflammatory breast cancer (nIBC) patients to elucidate the cell types that are specifically enriched in the TME of IBC patients, as well as the molecular features that are responsible for the preferential remodelling of the cellular functional state in the TME. Results A total of 15,832 cells, including epithelial cells, endothelial cells, stromal cells, T cells, B cells, antibody secreting cells (ASCs) and myeloid cells, were obtained from tumour tissues from 3 HER2 + IBC patients for scRNA analysis. By comparing the TME with that of HER2 + nIBC patients in a public database, we found that the TME of HER2 + IBC patients had a greater level of lymphocyte infiltration than that of nIBC patients did, and an especially significant enrichment of ASCs (mainly plasmablasts or plasma cells). In the TME of HER2 + IBC patients, tumour-infiltrating T cells exhibited a dual molecular phenotype of high activation and high exhaustion, with tumour-infiltrating B cells preferring the extrafollicular developmental pathway, and tumour-infiltrating myeloid and mesenchymal cells exhibiting a greater immunosuppressive status. By performing a cellular interaction analysis, we revealed that PTN molecules were significantly overexpressed in HER2 + IBC tumour cells and that the cellular interactions mediated by these molecules were strongly correlated with the functional polarisation of the cellular components in the TME. We observed that HER2 + IBC tumour cells have an active interferon response and epithelial mesenchymal transition (EMT) signalling, and that their malignant process is strongly correlated with the inflammatory response. Moreover, we found that HER2 + IBC tumour-infiltrating B cells promoted necroptosis of endothelial cells through high expression of TNF, thus promoting inflammatory responses. Conclusion We found a strong correlation between high expression of PTN molecules in HER2 + IBC tumour cells and their highly invasive characteristics and highly immunosuppressive TME. These results suggest that HER2 + IBC tumour cells can promote an inflammatory response by upregulating the expression of TNF molecules in B cells via PTN molecules and that the enhanced inflammatory response in turn promotes tumour progression, a malignant cycle that shapes a more immunosuppressive TME. Therefore, diagnostic and therapeutic strategies targeting the PTN-TNF molecular axis may have considerable potential for development in HER2 + IBC patients.