Abstract Background Based on DESTINY-Breast04 (HER2-low) and -06 (HER2-low/-ultralow) trials, T-DXd is approved for HER2-low (immunohistochemistry [IHC] 1+ or IHC 2+/in situ hybridization negative) or -ultralow (IHC 0 with membrane staining in ≤10% of tumor cells) mBC. Whole slide images (WSIs) from mBC biopsy samples scored HER2 IHC 0/1+ were rescored by pathologists and using digital pathology (DP) to evaluate concordance. Methods This retrospective real-world evidence study included 384 WSIs collected 2020-2023, stained with PATHWAY HER2 (4B5) assay scored as HER2 IHC 0 (n = 246) or 1+ (n = 138). Three pathologists each performed 2 blinded readings per WSI using 2023 ASCO/CAP guidelines; if readings differed, a reconciled score was used. Consensus was agreement by ≥2 of 3 pathologists. The same WSIs were analysed with 4 AI-computational DP tools in development. Concordance vs manual consensus was measured by overall percentage agreement (OPA) and Cohen κ, with review time recorded. Results Of 384 WSIs, 375 had aligned HER2 IHC scores by pathologist review; 9 had discordance. Among consensus cases, 2/3 agreement occurred in 154 WSIs (41.1%) and 3/3 in 221 (58.9%); 81 (21.6%) WSIs were reclassified as IHC 0 absent membrane staining, 85 (22.7%) as IHC 0 with membrane staining, 203 (51.4%) as IHC 1+, and 6 (1.6%) as IHC 2+. HER2 IHC rescoring results with the 4 DP tools are shown in Table 1. OPA (95% CI) between consensus and DP-assisted scores across all HER2 IHC score categories was 74% (69-78%), 73% (68-77%), 69% (64-74%), and 55% (50-60%). Cohen κ (95% CI) ranged from 0.33-0.59. Median review times were shorter with DP vs manual review. Conclusion Preliminary analysis suggests integrating AI-computational DP tools into HER2 IHC clinical workflows may reduce pathologist review time. Further analysis is underway to assess concordance of DP tools with manual scoring. Citation Format: Savitri Krishnamurthy, Dhanrajan Tiruchinapalli, Clara lam, Simon M. Collin, Rosemary Taylor, Linlin Luo, Anupriya Dutta, Ehab A. Elgabry, Michele S. Woo, Grace E. Kwon, Robert Egger, Jennifer A. Hipp, Lauren Brunner, Jeppe S. Thagaard, Thomas W. Ramsing, Henrik Høeg, Wonkyung Jung, Heon Song, Chang Ho Ahn, Vladimir Kravtsov, Patrick Frey, Ralf Banisch, Stella Redpath. Comparison of digital and artificial intelligence (AI)-computational algorithms for quantifying low/ultralow human epidermal growth factor receptor 2 (HER2) protein expression in metastatic breast cancer (mBC) from clinical samples [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 1455.

Inflammatory breast cancer (IBC), an aggressive and lethal breast cancer subtype, lacks unequivocal genomic differences or robust biomarkers that differentiate it from non-IBC. Here, TGIRT-seq revealed myriad differences in tumors, PBMC, and plasma RNAs that distinguished IBC patients from non-IBC patients and healthy donors across different tested breast cancer subtypes. By mapping reads to genome and transcriptome reference sequences and quantitating intron-to-exon read depth ratios (IDRs), we developed methods for parallel analysis of transcriptional and posttranscriptional gene regulation. This analysis identified numerous protein-coding genes in IBC patient tumors and PBMCs with high IDRs, suggesting rate-limiting RNA splicing that decreases mRNA production. Mirroring gene expression differences in tumors and PBMCs, overrepresented protein-coding gene RNAs in IBC patient plasma were largely intron RNA fragments, while those in non-IBC patient and healthy donor plasma were largely mRNA fragments. Our findings provide insights into IBC and should enable monitoring disease progression by liquid biopsy.

Table S2 demonstrates multivariable Cox proportional hazards model, excluding patients who were pregnant at time of diagnosis

Abstract Background: Inflammatory breast cancer (IBC) is an aggressive form of primary breast cancer marked by rapid progression and a high propensity for metastasis. Despite its severity, no specific FDA-approved targeted therapies exist for IBC, highlighting the need for novel therapeutic strategies. Through analysis of metastatic xenograft-derived IBC sublines, we identified soluble E-cadherin (sEcad), a proteolytic extracellular fragment of full-length E-cadherin, as a protein associated with tumor aggressiveness and metastasis. Mass spectrometry-based proteomics further identified Protein Disulfide Isomerase A4 (PDIA4) as a novel sEcad-binding partner. We hypothesize that sEcad promotes IBC tumor progression via PDIA4-dependent mechanisms. Methods: sEcad was stably overexpresed in MDA-IBC3 and SUM149 IBC cells via lentiviral transduction, and PDIA4 was silenced using lentiviral shRNAs. Modified cells were injected into cleared mammary fat pads of SCID/Beige mice to assess tumor growth and progression. Protein-protein interactions were validated by co-immunoprecipitation. RNA-seq and GSEA were used to identify enriched pathways. Serum sEcad levels from IBC patients (n=301) and healthy donors (n=20) were measured by ELISA. Results: Serum sEcad levels were significantly higher in IBC patients than in healthy donors (P<0.0001). Elevated sEcad levels were associated with metastatic disease (p=0.0036), reduced overall survival (p=0.04), and increased risk of metastasis (0.004). Functionally, sEcad overexpression enhanced cell survival and colony formation in vitro, and mice injected with sEcad-overexpressing SUM149 or MDA-IBC3 cells exhibited accelerated tumor growth compared with controls (SUM149: p=0.007; MDA-IBC3: p=0.006). Mechanistically, endogenous and exogenous co-immunoprecipitation confirmed the interaction between sEcad and PDIA4. RNA-seq and GSEA revealed significant enrichment of NF-κB signaling in sEcad high cells. sEcad overexpression increased PDIA4 expression and NF-kB activation, whereas PDIA4 knockdown suppressed NF-κB activation and increased cell death. PDIA4 overexpression also enhanced NF-κB activation in IBC cells. In vivo, PDIA4 knockdown markedly reduced tumor incidence and tumor burden in sEcad-high IBC mouse models. Conclusions: Our study identifies sEcad functions as a clinically relevant biomarker and a driver of IBC progression via activation of PDIA4-dependent NF-kB signaling. These findings support sEcad and PDIA4 as mechanistically linked contributors to IBC and highlight this pathway as a potential therapeutic target. Citation Format: Xiaoding Hu, Kiros Haddish Tesfamariam, Isabella L. Rizzo, Emilly S. Villodre, Lan Thi Hanh Phi, Juhee Song, Yun Gong, Savitri Krishnamurthy, Wendy A. Woodward, Bisrat G. Debeb. PDIA4-NF-κB signaling mediates soluble E-cadherin-driven inflammatory breast cancer progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(8_Suppl):Abstract nr LB280.

Abstract Ductal carcinoma in situ (DCIS) is the most common form of preinvasive breast cancer. However, our knowledge of the DCIS tumor microenvironment (TME) remains limited. Here, we investigated the TME of estrogen receptor positive DCIS using fresh tissues samples from DCIS, invasive breast cancers and normal breast tissues. In total, 795,585 cells were analyzed from 153 patients by single cell RNA sequencing and 39 samples were profiled with spatial transcriptomics methods (Visium, Xenium). Our data identified reprogrammed vascular cells, fibroblasts and basal cells that encircled the DCIS regions, forming cancer stromal loops (CSL) that support the preinvasive cancer cells. The DCIS regions were highly immunosuppressive, and only lipomacrophages (APOC1+) and interferon T-cells infiltrated. The preinvasive cancer cells showed activation of cell cycling and interferon signaling, while reducing cell growth regulation, RNA processing, secretion and cell adhesion. Collectively, these data show extensive remodeling of the TME in DCIS that persists during invasive progression. Citation Format: Siyuan He, Aatish Thennavan, Runmin Wei, Shanshan Bai, Jianzhuo Li, Chante Bethell, Emi Sei, Mitchell Rao, Tapsi Kumar, Chenling Tang, Ivan Marin, Jessica Montalvan, Chandandeep Nagi, Bora Lim, Savitri Krishnamurthy, Alastair Thompson, Nicholas E. Navin. Reprogramming of the stromal and immune microenvironment in preinvasive breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 2259.

AbstractBackground: Postpartum diagnosis of breast cancer confers increased risk of metastasis and death, possibly because of protumorigenic pathways that occur after weaning. We sought to evaluate the impact of postpartum diagnosis on survival in patients with inflammatory breast cancer (IBC). Methods: We performed a retrospective analysis of patients with nonmetastatic IBC enrolled in a prospective registry (2007–2023). Kruskal–Wallis, χ2, and Fisher exact tests were used to compare between postpartum groups. The Kaplan–Meier method and log-rank tests were used for survival estimates. Results: Among 320 patients with IBC, 25 (7.8%) were nulliparous, 31 (9.7%) were 0 to 5 years postpartum, 26 (8.1%) were 6 to 10 years postpartum, 56 (17.5%) were 11 to 20 years postpartum, and 182 (56.9%) were >20 years postpartum. Groups were similar with respect to race/ethnicity, body mass index, grade, receptor subtype, stage, trimodality therapy receipt, and pathologic complete response rate. At a median follow-up of 77 months (95% confidence interval, 69.3–86.5), there were no significant differences in overall survival, progression-free survival, and locoregional and distant recurrence-free survival by postpartum status. On multivariable analysis, parity status and years postpartum were not significantly associated with survival outcomes after controlling for age, stage, and receptor subtype. Conclusions: In this cohort of patients with IBC with a high proportion of childbearing, postpartum diagnosis did not significantly affect survival outcomes. The aggressive biology of IBC may override any additional risks imposed by a postpartum diagnosis. Impact: Unlike in noninflammatory invasive breast cancer, postpartum diagnosis did not negatively affect prognosis in IBC. These findings highlight the continuing need to identify unique drivers of aggressive biology in IBC.

e12594 Background: Patients with Stage III triple negative (TN) inflammatory breast cancer (IBC) are treated based on standard-of-care practices inclusive of the Keynote 522 regimen (KN522). Outcomes, however, are inferior to what has been reported in the non-IBC setting. Patients achieving pathologic complete response (pCR) were compared to those not achieving pCR in a cohort of patients with IBC receiving KN522 to evaluate for predictors of response. Methods: RNA sequencing (RNA-seq) was performed on 26 samples from 23 patients with IBC undergoing KN522 in the neoadjuvant setting (N = 19 baseline pre-treatment, N = 7 surgical post treatment, N = 5 paired samples). BostonGene’s Breast Cancer Classifier (BCC) was utilized to determine intrinsic subtypes. Tumor microenvironment was determined by BostonGene’s Tumor Portrait assay. Results: Among patients achieving pCR in the studied cohort (N = 4), 75% (N = 3) and 25% (N = 1) patients had basal and luminal B subtype, respectively, while in the non-pCR group (N = 5), 30% (N = 3) were HER2-low. The pCR group demonstrated higher PD-L1 RNA-seq expression and a trend towards higher abundance of T cell subsets in the tumor cell microenvironment comparatively. Among non-pCR cases, deconvolution analysis revealed reduced tumor fractions following neoadjuvant systemic therapy. A shift in tumor portrait was observed in 3/5 samples, including increased fibrosis features in two cases and loss of immune component in one. These non-pCR cases showed increased angiogenesis and endothelial enrichment signatures, trending toward reduced matrix remodeling. No association was identified between immune-related adverse events (irAEs) and either pCR or BCC subtype. Conclusions: While the small sample size and molecular heterogeneity preclude our ability to draw definitive conclusions, our findings show that in TN IBC, response to neoadjuvant therapy correlated with high PD-L1 expression and T cell abundance, whereas non-responders demonstrated angiogenic and endothelial shifts. No association was observed between irAEs and pCR or BCC. These findings highlight potential biomarkers of response and progression in TN IBC; however, interpretation is limited by the small cohort size and molecular heterogeneity.

Background:Inflammatory breast cancer (IBC) is a rare and highly aggressive form of breast cancer with an increased propensity to metastasize to distant organs including the brain. Higher serum levels of the calcium-binding proteins S100A8/A9, particularly of S100A9, have emerged as a clinically and biologically significant factor in aggressive breast cancers that are associated with poorer prognosis, tumor progression, and resistance to therapy. However, its contribution in IBC specifically remains undefined. Here, we investigated whether serum levels of S100A8/A9 predict outcomes in patients with IBC. Methods:Serum S100A8/A9 levels were measured in a cohort of 304 IBC patients using ELISA assay. S100A8/A9 levels were categorized by their third quartile value (S100A8/A9-low ≤ 3rd quartile; S100A8/A9-high > 3rd quartile). Overall survival (OS) and breast cancer-specific survival (BCSS) were analyzed with Kaplan-Meier curves, log-rank tests, and Cox proportional hazard regression models. The cumulative incidence of any metastases and the cumulative incidence of brain metastases were analyzed using Aalen-Johansen method, Gray test, and Fine-Gray models. Results:The median follow-up time was 64 months. Forty-six percent of patients had estrogen receptor (ER)-negative tumors, 61.3% were stage III-IV, 77% high grade, 16.8% received adjuvant chemotherapy and 53.6% received adjuvant radiation. On univariate analysis, S100A8/A9 levels, disease stage, ER status, PR status, HER2 status, adjuvant chemotherapy, and adjuvant radiation therapy were significantly associated with OS and BCSS. Patients with high S100A8/A9 serum levels had poor OS (P=0.01) and BCSS (P=0.007) and had a higher risk of developing brain metastasis (P=0.01) but not other metastasis. On multivariate analysis, high S100A8/A9 serum levels were independently associated with reduced OS (hazard ratio [HR]=1.7, 95% CI 1.1 to 2.6, P=0.01), reduced BCSS (HR=1.8, 95% CI 1.2 to 2.8, P=0.006), and increased cumulative incidence of developing brain metastasis (subdistribution hazard ratio (sHR)=1.8, 95% CI 1.1 to 3.0, P=0.03). Conclusions:In patients with IBC, high serum levels of S100A8/A9 are an independent prognostic factor for brain metastasis and poor clinical outcomes. These findings support the potential of S100A8/A9 as predictive biomarker for identifying increased risk of brain metastasis and unfavorable prognosis in patients with IBC.

Abstract Background: Circulating immune profiles offer a noninvasive approach to monitor treatment response in breast cancer. Inflammatory breast cancer (IBC), although clinically distinct, shares resistance and immune evasion features among each other. To identify immune escape mechanisms and early predictors of response in IBC, we profiled longitudinal peripheral immune markers and evaluated their association with pathologic complete response (pCR) and residual cancer burden (RCB) across treatment regimens and subtypes and developed composite immune indices to improve prediction under class imbalance. Methods: Peripheral blood samples (n=93) from patients treated with neoadjuvant or induction therapy for IBC were analyzed by multiparameter flow cytometry and annotated for subtype, regimen, pCR, and RCB. Group comparisons used Mann-Whitney U (pCR) and Kruskal-Wallis (RCB). Predictive models included logistic regression and random forest classifiers with class-weighting. Composite indices included CD8 Exhaustion, CD8 Memory, NK-ADCC scores derived from z-scaled components. Model performance was assessed using 5-fold cross-validation with AUC, PR-AUC, and calibration. Planned extensions include leave-one-regimen validation, temporal analyses, and external evaluation using a pre-registered pipeline. Results: Univariate analyses identified significant associations between pCR and the CD8 Exhaustion Index (p≈0.007) and CD8 Memory Index (p≈0.038), with NK-ADCC trending (p≈ 0.078). Logistic regression and random forest models incorporating combined indices improved AUC from ∼0.70 when individually associated, to ∼0.92 ± 0.07 (5-fold CV), albeit with wide uncertainty due to only up to 5 pCR events. Individual markers linked to pCR included Stem_Memory_CD8, NKG2A_ADCC, and CTLA4_CD8, while CD56hi NK and NKG2A +CD56hi NK distinguished RCB gradients (p<0.05 before correction). No markers remained significant after FDR adjustment given sample size. Conclusions: Peripheral immune signatures, particularly combined CD8 exhaustion, memory indices, NK ADCC were associate with higher pCR and lower RCB. Composite indices outperform single markers and capture the balance between immune activation and suppression. Ongoing cross-cohort validation will assess generalizability across regimens and subtypes. Severe class imbalance (∼5/93 pCR) and small subgroup sizes constrain precision of our analysis. Findings are hypothesis-generating and warrant external validation in larger prospective cohorts. Citation Format: Hui Gao, Ranjan Upadhyay, Angela Alexander, Angela N. Marx, Megumi Kai, Chelain R. Goodman, Azadeh Nasrazadani, Savitri Krishnamurthy, Anthony Lucci, Rachel M. Layman, Sadia Saleem, Vicente Valero, Michael C. Stauder, Susie X. Sun, Gary J. Whitman, Miral M. Patel, Huong C. Le-Petross, Chasity L. Yajima, Lily Villarreal, Heather Lopez, Bora Lim. Peripheral immune indices predict pathologic complete response and residual cancer burden in inflammatory breast cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 3744.

1022 Background: Trastuzumab deruxtecan is approved in HER2-low (IHC 1+ or 2+/ISH negative) or -ultralow (IHC 0 with membrane staining in ≤10% of tumor cells) metastatic BC per DESTINY-Breast04 and -06 trials. Manual HER2 IHC scoring can be time-consuming and subjective. This retrospective, real-world study assessed manual scoring (ground truth) vs standalone AI-computational pathology-assisted (CPa) tools for scoring. Methods: Whole slide images (WSIs; N = 600, scanned with Aperio AT2) of BC samples stained with PATHWAY HER2 (4B5) assay, originally scored as HER2 IHC 0 (n = 400) or 1+ (n = 200), were rescored by 3 pathologists and using 4 CPa/AI tools in development as either IHC 0 absent membrane staining, 0 with membrane staining in ≤10% of cells, 1+, or 2+. Using 2023 ASCO/CAP guidelines, each pathologist performed 2 blinded readings per WSI; a reconciled score was used if the 2 readings differed. Manual consensus required ≥2/3 agreement. Concordance between pathologist manual consensus vs standalone CPa scoring was measured by positive and negative percentage agreement (PPA; NPA), overall percentage agreement (OPA), and Cohen κ, with review time recorded. Results: Of 600 WSIs, 586 (97.7%) had manual consensus. CPa tools were faster (Table) than manual scoring (manual median review time: 7.0 min; range, 3.0-11.5). PPA was high (≥92.5%) between manual consensus scoring and CPa tools; NPA across the tools was 79.1%, 68.9%, 67.4%, and 23.1%. Overall concordance varied across CPa tools; OPA ranged from 48.5% to 75.4% and Cohen κ from 0.26 to 0.61. Conclusions: Integrating CPa/AI tools as decision support aids for pathologists may reduce pathologist review time and augment pathologist inter-observer reproducibility, especially in HER2-ultralow identification. Refinement of CPa/AI algorithms in development may improve scoring to an even greater extent. DP tool (N = 586 a ) Review time, median (range), min PPA, b,c % (95% CI) NPA, b,d % (95% CI) OPA, b,e % (95% CI) Cohen κ e (95% CI) RV73X 2.7 (0.3-56.8) 94.9 (92.5-96.7) 79.1 (70.6-85.7) 75.4 (71.7-78.9) 0.61 (0.56-0.66) MQ52G 0.7 (0.1-8.5) 93.4 (90.9-95.4) 68.9 (60.5-76.2) 73.0 (69.3-76.6) 0.57 (0.52-0.63) KL84Q 3.1 (0.5-84.4) 92.5 (89.8-94.6) 67.4 (59.1-74.8) 69.5 (65.6-73.2) 0.54 (0.48-0.59) ZX19P 2.5 f (Not available) 99.1 (97.6-99.6) 23.1 (16.4-31.5) 48.5 (44.4-52.6) 0.26 (0.22-0.31) a Primary and metastatic samples from biopsies, effusions, fine needle aspirations, and surgical resection; due to the inbuilt pre-QC module, a few WSI outputs were not processed by RV73X and ZX19P. b Rounded to 1 decimal. c Agreement for consensus-positive cases (IHC 0 with membrane staining, 1+, or 2+). d Agreement for consensus-negative cases (IHC 0 absent membrane staining). e Agreement across all IHC scores. f Mean review time; median not available.

Table S1 demonstrates OS, PFS, and RFS by receptor subtype

Inflammatory breast cancer (IBC) is an aggressive breast cancer subtype characterized by tumor emboli, lymphovascular invasion (LVI), and early dissemination. Herein, we establish adaptive stress response (ASR) as a biologic feature linking stress adaptation to tumor emboli survival, lymphatic dissemination, therapeutic response, and disparities. Using a previously defined 226 ASR-related genes, complementary preclinical models of tumor emboli and lymphatic circulating cell clusters, and independent patient cohorts, we identified ASR genes enriched for XIAP-NFκB, oxidative stress response, inflammatory, and immune pathways. CXCL8 emerged as one of the most highly upregulated transcripts in tumor emboli and was shared across both models; however, CXCL8 , IL6 , and PTGS2 were downregulated in lymphatic circulating cell clusters and LVI-positive triple-negative IBC patients, suggesting dynamic remodeling of inflammatory signaling during dissemination. CYP4B1 was associated with ER status, LVI, and therapeutic response across multiple cohorts, implicating metabolic stress adaptation in dissemination. IL6 and PTGS2 were elevated in self-reported Black patients with triple-negative IBC compared to White patients. Pharmacologic inhibition of XIAP-NFκB and oxidative stress pathways suppressed tumor emboli formation. Collectively, these findings identify ASR signaling as a framework linking tumor emboli survival, dissemination, and therapeutic vulnerability in IBC.

Ex vivo digital microscopy uses light in the visible and adjacent spectra to obtain digital images of tissues. They are optical imaging techniques that allow the acquisition of digital images of tissues with minimal or no tissue preparation and are currently available for evaluation of fresh and/or fixed tissues. This review will provide an overview of the different types of ex vivo digital microscopy techniques, including confocal microscopy (CM), optical coherence tomography (OCT), stimulated Raman Spectroscopy (SRS), light sheet microscopy (LSM), microscopy with ultraviolet excitation (MUSE), structured illumination microscopy (SIM), and nonlinear microscopy (NLM). Except for OCT and SRS, all the other tissue imaging techniques require labeling of tissues with fluorescent dyes to obtain digital images. An advantage of several of these techniques, including fluorescence CM, SRS, LSM, MUSE, SIM, and NLM, is that they can produce hematoxylin and eosin-like images. The promising potential of ex vivo digital microscopy techniques in surgical pathology practice is supported by several retrospective and limited prospective studies. Applications of ex vivo digital microscopy techniques include real-time evaluation of fresh tissue at the bedside in clinics and radiology suites, as well as intraoperatively in pathology laboratories. These techniques have great potential for incorporation into standard-of-care surgical pathology practice.

Context.—:Prevalence data of human epidermal growth factor receptor 2 (HER2)-ultralow (immunohistochemistry [IHC] 0 with membrane staining in ≤10% of cells; HER2 IHC 0+) breast cancer are limited. Objective.—:To evaluate the proportion of HER2-ultralow among metastatic breast cancer samples originally scored HER2 IHC 0 (with or absent membrane staining) and 1+ and to evaluate interobserver concordance between pathologists' rescores and original scores. Design.—:Whole slide images (WSIs) from metastatic breast cancer biopsy specimens originally scored HER2 IHC 0 (with or absent membrane staining) and 1+ were rescored as HER2 IHC 0 absent membrane staining, IHC 0+ (with membrane staining), IHC 1+, or IHC 2+ by 3 pathologists. Each pathologist performed 2 blinded readings (washout period of ≥2 weeks). Results.—:Of the 246 IHC 0 WSIs, 78 (31.7%), 75 (30.5%), and 86 (35.0%) were rescored as HER2 IHC 0 absent membrane staining, 0+, and 1+, respectively. Of the 138 IHC 1+ WSIs, 3 (2.2%), 10 (7.2%), 117 (84.8%), and 6 (4.3%) were rescored as IHC 0 absent membrane staining, 0+, 1+, and 2+, respectively. Interobserver concordance was substantial (overall percentage agreement [OPA; 95% CI], 86.7% [82.9%-90.0%]; Cohen κ [95% CI], 0.781 [0.726-0.835]) to moderate (OPA [95% CI], 62.0% [59.9%-66.9%]; Cohen κ [95% CI], 0.441 [0.379-0.502]). Agreement between pathologists' consensus scores and original scores was moderate (OPA [95% CI], 72.1% [67.3%-76.5%]; Cohen κ [95% CI], 0.461 [0.381-0.541]). Conclusions.—:Scoring discrepancies highlight the potential need/benefits of training for evaluating HER2-low/HER2-ultralow cases in metastatic breast cancer, which could improve consistency in HER2 scoring by pathologists.

Abstract Background: Inflammatory breast cancer (IBC) is a rare but highly aggressive variant of breast cancer, accounting for 10% of breast cancer-related deaths. We have identified NDRG1, located on chromosome 8q24.3 near MYC within a commonly amplified region, as a key promoter of tumor growth and progression in IBC models and is associated with poor survival outcomes. Unlike many oncogenes, NDRG1 is rarely deleted or silenced and is more frequently expressed in IBC compared to non-IBC tumors. A recent study reported NDRG1 amplification in 42% of triple-negative IBC cases. We hypothesize that NDRG1 amplification in triple-negative IBC is associated with distinct gene expression profiles and enrichment of oncogenic pathways that contribute to the aggressive phenotype of the disease. Methods: RNA sequencing data from 19 triple-negative IBC tumors (8 NDRG1 amplified, 11 non-amplified) were analyzed using DEseq2 package. Heatmaps were generated to visualize clustering and expression differences. Gene Set Enrichment Analysis (GSEA) was performed using Hallmark and KEGG pathway databases. Publicly available TCGA and METABRIC breast cancer datasets were used to assess NDRG1 amplification and its association with mRNA/protein expression and survival outcomes. Results. Among the 19 triple-negative tumors analyzed, 8 (42%) exhibited NDRG1 amplification, which was significantly correlated with increased RNA expression (p = 0.05). Differential expression analysis identified CALCA, RHO, DPYSL5, GPR101, ZIC3, and IRS4 as the most upregulated, and SERPINA6, SCGB3A1, PI3, LRG1, DAPL1, MYEOV as the most downregulated in NDRG1-amplified tumors. GSEA revealed enrichment of DNA repair, cell cycle, mTOR signaling, and MYC target pathways, and downregulated pathways included interferon gamma response, estrogen response and NFkB pathway. TCGA and METABRIC analyses confirmed that NDRG1 amplification correlates with elevated mRNA expression (p<0.0001) and elevate protein levels (p<0.0001) and poorer overall survival (p=0.013, TCGA; p=0.0002, METABRIC). Conclusions: NDRG1 amplification marks a transcriptionally distinct subset of triple-negative IBC, enriched in proliferative and DNA repair pathways. These molecular features highlight its potential as a biomarker of aggressive disease biology and potential target for further mechanistic investigation and therapeutic intervention. Citation Format: Emilly S. Villodre, Ganiraju Manyam, Xiaoding Hu, Isabella L. Rizzo, Lan H. Phi, Kiros H. Tesfamariam, Azadeh Nasrazadani, Rachel M. Layman, Bora Lim, Vicente Valero, Savitri Krishnamurthy, The MDACC IBC Team, MDACC Rare Tumor Initiative Team, Jing Wang, Xiaoping Wang, Naoto Ueno, Wendy A. Woodward, Bisrat G. Debeb. Transcriptomic analysis of NDRG1 amplification in aggressive breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 1181.

BACKGROUND:The brain is a common site of relapse in inflammatory breast cancer (IBC), an E-cadherin-positive, aggressive form of breast cancer. Elevated serum levels of soluble E-cadherin (sEcad), an 80-kDa fragment, correlated with poorer outcomes and increased brain metastases in patients with metastatic IBC. We hypothesize that sEcad is a driver of brain metastasis in IBC. METHODS:Serum sEcad levels from 348 IBC patients were quantified by ELISA. To examine sEcad function, we used recombinant sEcad protein and generated stable IBC cell lines by cloning and overexpressing Flag-tagged sEcad. Control and sEcad-overexpressing MDA-IBC3 and SUM149 cells were injected into SCID/Beige mice to evaluate brain metastasis burden and survival, and a brain-permeable CXCR2 inhibitor was also tested for efficacy in these models. RESULTS:Higher serum sEcad levels correlated with poorer overall survival, earlier metastasis, and increased brain metastasis. In vitro, recombinant sEcad and stable sEcad overexpression in IBC cell lines promoted invasion, resistance to anoikis, and activation of pro-survival NF-κβ signaling. In vivo, mice injected with sEcad-overexpressing IBC cells had increased metastatic burden and reduced overall and brain metastasis-free survival. Further, sEcad induced reactive astrocytosis through the CXCL1/CXCL8-CXCR2 axis, and treatment with a brain-permeable CXCR2 antagonist reduced metastatic burden and prolonged survival in the brain metastasis models. CONCLUSION:sEcad drives brain metastasis by promoting invasion and anoikis resistance in cancer cells and inducing an inflammatory brain microenvironment via a targetable CXCL1/CXCL8-CXCR2 axis. These findings uncover a novel and critical role for sEcad and highlight CXCR2 as a therapeutic target in patients with metastatic IBC.

Inflammatory breast cancer (IBC) is a rare yet highly aggressive subtype of breast cancer, characterized by distinct clinical features, rapid progression, and complex biology. Despite decades of research, patient outcomes remain poor, underscoring the need for innovative approaches. The International IBC Symposium 2025 convened global experts to discuss advances in the field and to outline strategic priorities for research and clinical care. This report summarizes the key themes discussed. A major topic was IBC biology, with particular emphasis on tumor emboli formation and tumor microenvironment interactions, which shape the distinctive characteristics of the disease. Updates in molecular and immune profiling were presented, illustrating how these data may guide personalized therapeutic approaches. From a therapeutic standpoint, the importance of trimodal therapy in patients with nonmetastatic IBC was reinforced, while highlighting the need to investigate novel agents, rational drug combinations, and biologically informed strategies to improve outcomes. Additional sessions addressed optimization of locoregional management, even for selected patients with metastatic disease. Experts also discussed emerging technologies, including spatial transcriptomics, artificial intelligence tools, and liquid biopsy, as promising methods to advance disease characterization, facilitate earlier detection, and refine disease monitoring. Finally, a common theme across presentations was the critical importance of expanding awareness and education, as well as fostering global collaboration through the IBC International Consortium to standardize diagnosis, accelerate translational research, and ensure equitable access to care. Collectively, these efforts aim to redefine the clinical trajectory of IBC and move the field closer to achieving durable disease control and, ultimately, curative outcomes.