Deciphering the molecular biology of inflammatory breast cancer through molecular characterization of patient samples and preclinical models.

Inflammatory breast cancer is an aggressive subtype of breast cancer with dismal patient prognosis and a unique clinical presentation. In the past two decades, molecular profiling technologies have been used in order to gain insight into the molecular biology of IBC and to search for possible targets for treatment. Although a gene signature that accurately discriminates between IBC and nIBC patient samples and preclinical models was identified, the overall genomic and transcriptomic differences are small and ambiguous, mainly due to the limited sample sizes of the evaluated patient series and the failure to correct for confounding effects of the molecular subtypes. Nevertheless, data collected over the past 20 years by independent research groups increasingly support the existence of several IBC-specific biological characteristics. In this review, these features are classified as established, emerging and conceptual hallmarks based on the level of evidence reported in the literature. In addition, a synoptic model is proposed that integrates all hallmarks and that can explain how cancer cell intrinsic mechanisms (i.e. NF-κB activation, genomic instability, MYC-addiction, TGF-ß resistance, adaptive stress response, chromatin remodeling, epithelial-to-mesenchymal transition) can contribute to the establishment of the dynamic immune microenvironment associated with IBC. It stands to reason that future research projects are needed to further refine (parts of) this model and to investigate its clinical translatability.

Measurement of LAG-3 Expression Across Multiple Staining Platforms With the 17B4 Antibody Clone.

CONTEXT.­: An immunohistochemistry (IHC) assay developed to detect lymphocyte-activation gene 3 (LAG-3), a novel immune checkpoint inhibitor target, has demonstrated high analytic precision and interlaboratory reproducibility using a Leica staining platform, but it has not been investigated on other IHC staining platforms. OBJECTIVE.­: To evaluate the performance of LAG-3 IHC assays using the 17B4 antibody clone across widely used IHC staining platforms: Agilent/Dako Autostainer Link 48 and VENTANA BenchMark ULTRA compared to Leica BOND-RX (BOND-RX). DESIGN.­: Eighty formalin-fixed, paraffin-embedded melanoma tissue blocks were cut into consecutive sections and evaluated using staining platform-specific IHC assays with the 17B4 antibody clone. Duplicate testing was performed on the BOND-RX platform to assess intraplatform agreement. LAG-3 expression using a numeric score was evaluated by a pathologist and with a digital scoring algorithm. LAG-3 positivity was determined from manual scores using a 1% or greater cutoff. RESULTS.­: LAG-3 IHC staining patterns and intensities were visually similar across all 3 staining platforms. Spearman and Pearson correlations were 0.75 or greater for interplatform and BOND-RX intraplatform concordance when LAG-3 expression was evaluated with a numeric score determined by a pathologist. Correlation increased with a numeric score determined with a digital scoring algorithm (Spearman and Pearson correlations ≥0.88 for all comparisons). Overall percentage agreement was 77.5% or greater for interplatform and BOND-RX intraplatform comparisons when LAG-3 positivity was determined using a 1% or greater cutoff. CONCLUSIONS.­: Data presented here demonstrate that LAG-3 expression can be robustly and reproducibly assessed across 3 major commercial IHC staining platforms using the 17B4 antibody clone.

Comparative transcriptional analyses of preclinical models and patient samples reveal MYC and RELA driven expression patterns that define the molecular landscape of IBC.

Inflammatory breast cancer (IBC) is an aggressive disease for which the spectrum of preclinical models was rather limited in the past. More recently, novel cell lines and xenografts have been developed. This study evaluates the transcriptome of an extended series of IBC preclinical models and performed a comparative analysis with patient samples to determine the extent to which the current models recapitulate the molecular characteristics of IBC observed clinically. We demonstrate that the IBC preclinical models are exclusively estrogen receptor (ER)-negative and of the basal-like subtype, which reflects to some extent the predominance of these subtypes in patient samples. The IBC-specific 79-signature we previously reported was retrained and discriminated between IBC and non-IBC preclinical models, but with a relatively high rate of false positive predictions. Further analyses of gene expression profiles revealed important roles for cell proliferation, MYC transcriptional activity, and TNFɑ/NFκB in the biology of IBC. Patterns of MYC expression and transcriptional activity were further explored in patient samples, which revealed interactions with ESR1 expression that are contrasting in IBC and nIBC and notable given the comparatively poor outcomes of ER+ IBC. Our analyses also suggest important roles for NMYC, MXD3, MAX, and MLX in shaping MYC signaling in IBC. Overall, we demonstrate that the IBC preclinical models can be used to unravel cancer cell intrinsic molecular features, and thus constitute valuable research tools. Nevertheless, the current lack of ER-positive IBC models remains a major hurdle, particularly since interactions with the ER pathway appear to be relevant for IBC.

The Evolution and Prognostic Role of Tumour-Infiltrating Lymphocytes and Peripheral Blood-Based Biomarkers in Inflammatory Breast Cancer Patients Treated with Neoadjuvant Chemotherapy.

INTRODUCTION: Inflammatory breast cancer (IBC) is a rare but aggressive form of breast cancer (BC) in which the (prognostic) role of stromal tumour-infiltrating lymphocytes (sTIL) and the peripheral circulating immune cells in patients with residual disease (RD) after neo-adjuvant chemotherapy (NACT) is not clearly established. METHODOLOGY: To describe the evolution of sTIL and some peripheral inflammation markers (Neutrophil-to-lymphocyte ratio, Platelet-to-lymphocyte ratio and Lymphocyte-to-monocyte ratio) after NACT in IBC, we retrospectively collected clinicopathological variables for 125 stage III IBC patients. sTILs were scored by three different researchers on an H&E slide of the mastectomy specimen. A cohort of subtype-matched non-IBC breast cancer patients (nIBC) treated with NACT was included for comparison. RESULTS: There was no significant difference in the pre- and posttreatment sTIL scores between IBC and nIBC and in both groups the number of sTIL was significantly lower after NACT. However, the IBC phenotype did correlate with a stronger decrease of sTIL after NACT (OR: 0.25, 95% CI: 0.073-0.76, p = 0.018). The change in the peripheral immune markers was not significantly different between IBC and nIBC. After NACT, 75 patients had residual disease. In this group, a high number of sTIL before NACT (HR: 0.23, 95% CI: 0.05-1.02, p = 0.05) was prognostic for a longer OS, while a low number of sTIL after NACT (HR: 0.33, 95% CI: 0.11-0.98, p = 0.046) and a low residual cancer cellularity (HR: 0.20, 95% CI: 0.08-0.52, p < 0.001) was associated with a longer DFS. CONCLUSIONS: IBC is associated with a significantly stronger decrease of sTIL after NACT compared to nIBC. Furthermore, a high number of sTIL after NACT was associated with a worse prognosis in IBC.

Inflammatory breast cancer cells are characterized by abrogated TGFß1-dependent cell motility and SMAD3 activity.

PURPOSE: Inflammatory breast cancer (IBC) is an aggressive form of breast cancer with elevated metastatic potential, characterized by tumor emboli in dermal and parenchymal lymph vessels. This study has investigated the hypothesis that TGFß signaling is implicated in the molecular biology of IBC. METHODS: TGFß1-induced cell motility and gene expression patterns were investigated in three IBC and three non-IBC (nIBC) cell lines. Tissue samples from IBC and nIBC patients were investigated for the expression of nuclear SMAD2, SMAD3, and SMAD4. SMAD protein levels were related to gene expression data. RESULTS: TGFß1-induced cell motility was strongly abrogated in IBC cells (P = 0.003). Genes differentially expressed between IBC and nIBC cells post TGFß1 exposure revealed attenuated expression of SMAD3 transcriptional regulators, but overexpression of MYC target genes in IBC. IBC patient samples demonstrated a near absence of SMAD3 and -4 expression in the primary tumor compared to nIBC patient samples (P < 0.001) and a further reduction of staining intensity in tumor emboli. Integration of gene and protein expression data revealed that a substantial fraction of the IBC signature genes correlated with SMAD3 and these genes are indicative of attenuated SMAD3 signaling in IBC. CONCLUSION: We demonstrate attenuated SMAD3 transcriptional activity and SMAD protein expression in IBC, together with obliterated TGFß1-induced IBC cell motility. The further reduction of nuclear SMAD expression levels in tumor emboli suggests that the activity of these transcription factors is involved in the metastatic dissemination of IBC cells, possibly by enabling collective invasion after partial EMT.

NOTCH and DNA repair pathways are more frequently targeted by genomic alterations in inflammatory than in non-inflammatory breast cancers.

Inflammatory breast cancer (IBC) is the most pro-metastatic form of breast cancer. Better understanding of its pathophysiology and identification of actionable genetic alterations (AGAs) are crucial to improve systemic treatment. We aimed to define the DNA profiles of IBC vs noninflammatory breast cancer (non-IBC) clinical samples in terms of copy number alterations (CNAs), mutations, and AGAs. We applied targeted next-generation sequencing (tNGS) and array-comparative genomic hybridization (aCGH) to 57 IBC and 50 non-IBC samples and pooled these data with four public datasets profiled using NGS and aCGH, leading to a total of 101 IBC and 2351 non-IBC untreated primary tumors. The respective percentages of each molecular subtype [hormone receptor-positive (HR+)/HER2-, HER2+, and triple-negative] were 68%, 15%, and 17% in non-IBC vs 25%, 35%, and 40% in IBC. The comparisons were adjusted for both the molecular subtypes and the American Joint Committee on Cancer (AJCC) stage. The 10 most frequently altered genes in IBCs were TP53 (63%), HER2/ERBB2 (30%), MYC (27%), PIK3CA (21%), BRCA2 (14%), CCND1 (13%), GATA3 (13%), NOTCH1 (12%), FGFR1 (11%), and ARID1A (10%). The tumor mutational burden was higher in IBC than in non-IBC. We identified 96 genes with an alteration frequency (p < 5% and q < 20%) different between IBC and non-IBC, independently from the molecular subtypes and AJCC stage; 95 were more frequently altered in IBC, including TP53, genes involved in the DNA repair (BRCA2) and NOTCH pathways, and one (PIK3CA) was more frequently altered in non-IBC. Ninety-seven percent of IBCs displayed at least one AGA. This percentage was higher than in non-IBC (87%), notably for drugs targeting DNA repair, NOTCH signaling, and CDK4/6, whose pathways were more frequently altered (DNA repair) or activated (NOTCH and CDK4/6) in IBC than in non-IBC. The genomic landscape of IBC is different from that of non-IBC. Enriched AGAs in IBC may explain its aggressiveness and provide clinically relevant targets.

Inflammatory breast cancer tumor emboli express high levels of anti-apoptotic proteins: use of a quantitative high content and high-throughput 3D IBC spheroid assay to identify targeting strategies.

Inflammatory breast cancer (IBC) is one of the most lethal breast cancer variants; with existing therapy, 5-yr survival rate is only 35%. Current barriers to successful treatment of IBC include frequent infiltration and the presence of tumor cell clusters, termed tumor emboli, within the breast parenchyma and lymphatics. Prior studies have identified the role of anti-apoptotic signaling, in particular hyperactivation of NFκB and its target genes, in IBC pathobiology and therapeutic resistance. The objectives of this study were to: (1) determine if IBC tumor emboli express anti-apoptotic proteins and (2) develop a high content, multiparametric assay to assess the morphology of the IBC 3D spheroids and to optimize a high throughput format to screen for compounds that can inhibit the formation of the IBC tumor clusters/embolic structures. Immunohistochemical analysis of IBC patient tumor samples with documented tumor emboli revealed high NFκB (p65) staining along with expression of XIAP, a potent anti-apoptotic protein known to interact with NFκB signaling in enhancing survival of malignant cells. Subsequently, the high content assay developed allowed for simultaneous imaging and morphometric analysis, including count and viability of spheroids derived from SUM149, rSUM149 and SUM190 cells and its application to evaluate XIAP and NFκB inhibitory agents. We demonstrate the efficacy of the off-patent drug disulfiram when chelated with copper, which we had previously reported to inhibit NFκB signaling, was highly effective in disrupting both IBC spheroids and emboli grown in vitro. Taken together, these results identify a high-throughput approach to target tumor spheroid formation for drug discovery. Finally, disulfiram is a safe and approved drug for management of alcohol abuse, warranting its evaluation for repurposing in IBC therapy.