Ki67 is a well-established proliferation marker in breast cancer. Current clinical use focuses on the proportion of Ki67-positive cells, ignoring spatial heterogeneity in expression. However intra-tumoral heterogeneity has demonstrated to be associated with worse outcome. We hypothesized that spatial Ki67 heterogeneity carries clinical information beyond conventional scoring and aimed to evaluate its added value for predicting pathological complete response (pCR) after neoadjuvant chemotherapy (NACT) and for stratifying recurrence risk using genomic expression profiling (GEP). Using digital image analysis (DIA), precise and spatial quantification of biomarker distribution is possible. We analyzed two retrospective breast cancer cohorts using an AI-assisted DIA pipeline. Tumor sections stained for ER, PR, Ki67, and HER2 were digitized and analyzed in QuPath. Using AI, individual tumor cells were recognized and four tumor regions (0.5mm x 0.5mm) with the highest tumor/stroma ratio were selected for analysis. Spatial Ki67 heterogeneity was quantified using the Morisita-Horn Index (MHI) after the Ki67-positive and Ki67-negative tumor cells were mapped using XY-coordinates and square tessellation (100×100 µm tiles) was applied. The MHI was used to compare the similarity in cell composition between all pairs of tiles within a region. MHI values range from 0 to 1, with higher values indicating a more uneven distribution of Ki67+ cells. We used logistic regression and model comparison with Akaike Information Criterion (AIC), likelihood ratio test (LRT) or Vuong test, to evaluate the predictive value of Ki67 heterogeneity. In the first cohort (n=45), spatial heterogeneity was assessed on pretreatment biopsies from patients treated with NACT. In the second cohort (n=79), heterogeneity was evaluated in HR+/HER2- breast cancer patients stratified as high or low risk of recurrence based on GEP. In the GEP cohort, both a higher proportion of Ki67- positive cells and greater Ki67 heterogeneity were significantly associated with high genomic risk. The median MHI was 0.24 (0.03–0.35) in the high-risk group compared to 0.14 (0.01–0.43) in the low-risk group (P = 0.008). This higher MHI indicates more heterogeneous regionally clustered Ki67 expression, suggesting biologically distinct proliferative zones. In multivariate models, Ki67 heterogeneity remained a significant predictor of high-risk classification (OR 0.22, P = 0.036). Furthermore, in nested model comparison using LRT, addition of Ki67 heterogeneity significantly improved the model for predicting genomic risk (P = 0.034). These findings were consistent across biopsy and resection specimens, highlighting the robustness of heterogeneity measures. In the NACT cohort, Ki67 heterogeneity was higher in patients who achieved pCR (median MHI 0.26 [0.17–0.35]) compared to those who did not (median MHI 0.22 [0.12–0.40], P = 0.023). In multivariate modeling, Ki67 heterogeneity emerged as an independent predictor of pCR (OR 23.5, P = 0.038), outperforming Ki67 density and improving model fit (AIC 31.6 vs. 36.1; P = 0.038). Finally, in both cohorts, DIA-derived Ki67 models slightly outperformed traditional pathologist scoring, although Vuong tests did not show a statistically significant difference. Spatial Ki67 heterogeneity provides additional prognostic and predictive value beyond conventional Ki67 scoring. This heterogeneity indicates distinct areas of higher proliferation, clinically relevant biological variation, not captured by simple percentage positivity. Although validation in larger, prospective cohorts is necessary before clinical implementation, DIA provides a more objective and reproducible alternative to manual scoring, particularly when incorporating spatial heterogeneity. C. Van Berckelaer, K. Zwaenepoel, L. Cox, D. Charlotte, D. Julie, E. Louise, H. Fleur, L. Evy, G. R. Devi, A. Ramadhan, S. Koljenovic, P. Van Dam. Ki67 Spatial Heterogeneity as a Predictive and Prognostic Marker in Breast Cancer: A Spatial Image Analysis Approach [abstract]. In: Proceedings of the San Antonio Breast Cancer Symposium 2025; 2025 Dec 9-12; San Antonio, TX. Philadelphia (PA): AACR; Clin Cancer Res 2026;32(4 Suppl):Abstract nr PS2-08-19.

AIM:To assess oncological outcomes in early-stage, luminal A-like, low-risk, breast cancer patients aged 65 years or more after conserving surgery followed by endocrine therapy (ET) and/or whole breast irradiation (WBI) or no adjuvant therapy. METHODS:Data from patients treated in EUSOMA-certified breast units between 2010 and 2022 were collected. Univariable and multivariable analyses for local, nodal, distant recurrences, breast cancer specific survival (BCSS), and overall mortality were conducted. Potential treatment-related determinants included adjuvant ET alone, WBI alone, ET+WBI or no adjuvant therapy. RESULTS:Breast cancer patients (9660) from 72 Breast Units across 14 European countries were enrolled. Tumours were pT1 in 85.8 % of cases. All tumours were luminal A-like. All patients had negative nodes. Adjuvant ET alone was prescribed for 806 (8.3 %) and WBI alone was delivered to 386 (4.0 %); ET and WBI were combined (ET+WBI) in 8154 (84.4 %) patients. No adjuvant therapy was given to 314 patients (3.3 %). The median follow-up was 1.87 years (first quartile 0.81, third quartile 4.16), and the mean was 2.62 years (range 0.003-13.82 years). Compared with no adjuvant therapy, multivariable analysis showed ET+WBI significantly improved in-breast tumour recurrence-free survival (IBTRFS) (HR 0.28, CI 95 % 0.10 -0.79; p = 0.016) and BCSS (HR 0.12; CI 95 %: 0.03 -0.51; p = 0.004). ET alone (HR 0.57; CI 95 %: 0.35 -0.90; p = 0.017), WBI alone (HR 0.51; CI 95 %: 0.27 -0.95; p = 0.033) and both treatments combined (HR 0.26; CI 95 %: 0.16 -0.42; p < 0.001) significantly lowered mortality. CONCLUSIONS:Despite a short follow-up, results from this large series of low-risk breast cancer patients who had undergone conserving surgery, showed adjuvant treatments impacted positively upon outcomes.

Inflammatory breast cancer (IBC) is a rare, aggressive subtype of breast cancer with unfavorable prognosis. Despite its distinct clinical presentation and molecular features, the immune landscape of IBC and its role in driving the aggressive phenotype remain poorly understood. This study aimed to characterize the spatial immune landscape of IBC using digital image analysis (DIA), compare it with subtype-matched non-inflammatory breast cancer (nIBC), and evaluate prognostic implications of immune cell composition and localization. We examined pretreatment tumor samples from 161 IBC and 115 subtype-matched nIBC patients using an AI-assisted DIA pipeline. Immunostainings (H-DAB) were conducted using validated protocols for CD8 (cytotoxic T cells), FOXP3 (Tregs), CD79α (activated B cells), PDL1 (SP124), and CD163 (tumor-associated macrophages, TAMs). Slides were digitized and assessed using VISIOPHARM® software, enabling virtual multiplexing. We quantified DAB+ immune cells and their locations using XY coordinates to calculate density. Spatial colocalization was analyzed using ecological statistics, based on point pattern or quadrant analysis. In point pattern analysis, we counted immune cells within a 30 µm radius around CD8+ cells or tumor cells. For quadrant analysis, we applied the Morisita-Horn Index (MHI) after square tessellation (250×250 µm tiles). The MHI compares cell composition between tile pairs, ranging from 0 to 1, with higher values indicating greater dissimilarity. Associations with clinicopathological features and outcome were assessed using multivariate regression models. Transcriptomic validation was performed using Affymetrix gene expression data and consensusTME deconvolution. IBC showed higher CD163+ tumor-associated macrophage (TAM) infiltration than nIBC. Gene expression data confirmed the IHC findings, and pathway analysis linked high TAM density with inflammatory and proliferative pathways. The spatial distribution of immune cells was prognostically relevant, with high CD8+ T-cell (OR: 0.41, 95% CI: 0.22-0.76, P = 0.004) and low CD79α+ B-cell infiltration (OR: 3.19, 95% CI: 1.68-6.03, P < 0.001) correlating with improved overall survival in IBC. The ratio of CD8+ T cells to FOXP3+ regulatory T cells within tumor area was a significant prognostic indicator (OR: 0.34, 95% CI: 0.14-0.83, P = 0.018), whereas absolute densities of CD8+ or FOXP3+ cells alone did not associate with outcome. Beyond density alone, spatial colocalization of B cells with tumor cells emerged as a key prognostic factor. A high tumor colocalization index for CD79α+ B cells was associated with significantly worse survival (OR: 3.59, 95% CI: 1.31-9.84, P = 0.013). Similarly, greater spatial similarity between CD79α+ B cells and tumor cells, assessed via the MHI, was linked to poor outcome (OR: 2.61, 95% CI: 1.19-5.74, P = 0.017). Incorporating spatial B-cell features improved the prognostic model (P = 0.0005). This comprehensive spatial immune profiling effort in a large IBC cohort revealed the immunosuppressive nature of the IBC microenvironment and underscored the central role of TAMs in promoting its aggressive behavior. Spatial context, not immune cell abundance, was most predictive of outcome. High colocalization of CD79α+ B cells with tumor cells associated with poor survival, suggesting a tumor-promoting role. These findings demonstrate the value of spatial immunophenotyping beyond conventional quantification and support exploring TAM- and B cell-targeted therapies in IBC. C. Van Berckelaer, S. Van Laere, C. Vermeulen, M. Kockx, Y. Waumans, K. Mariën, F. Berditchevski, F. Bertucci, P. Vermeulen, L. Dirix, C. Colpaert, G. R. Devi, P. Van Dam. Spatial Immune Profiling in Inflammatory Breast Cancer Reveals a Central Role for Tumor-Associated Macrophages and a Spatial Context-Dependent Negative Prognostic Impact of B Cells [abstract]. In: Proceedings of the San Antonio Breast Cancer Symposium 2025; 2025 Dec 9-12; San Antonio, TX. Philadelphia (PA): AACR; Clin Cancer Res 2026;32(4 Suppl):Abstract nr PS3-12-19.

Inflammatory breast cancer (IBC) is a rare but aggressive subtype of breast cancer characterized by rapid progression and poor prognosis. Despite its distinct clinical presentation and molecular features, the immune landscape of IBC and its potential role in driving the aggressive phenotype remain poorly understood. This study aimed to characterize the spatial immune landscape of IBC, compare it with that of subtype-matched non-inflammatory breast cancer (nIBC), and evaluate the prognostic implications of immune cell composition and localization. We analyzed pre-treatment tumor samples from 161 IBC and 115 subtype-matched nIBC patients using immunohistochemistry (IHC) for CD8, FOXP3, CD79α, CD163, and PD-L1. Digital image analysis quantified the immune cell density and relative marker area in the tumor area (TA) and invasive margin (IM). Associations with clinicopathological features, pathological response to neoadjuvant chemotherapy (NACT), and survival were assessed using multivariate logistic regression and Cox proportional hazards models. Transcriptomic validation was performed using Affymetrix gene expression data and consensus TME deconvolution. IBC showed higher infiltration of CD163 + tumor-associated macrophages (TAMs) compared to nIBC. Gene expression data confirmed IHC findings, and pathway analysis linked high TAM density with inflammatory and proliferative pathways. The spatial distribution of immune cells was prognostically relevant, with high CD8 + T-cell infiltration (OR: 0.41, 95

PURPOSE:Inflammatory breast cancers (IBC) are characterized by diffuse clusters of cells found in dermal lymphatic vessels, known as tumor emboli. IBC needs a novel treatment because it is the most aggressive breast cancer form. We hypothesized that the interaction between tumor emboli and the tumor immune microenvironment (TiME) fosters survival signaling, leading to the aggressiveness of disease. METHODS:Ex vivo tumor emboli were generated from patient-derived cell lines cultured in a lymphatic-like platform and subjected to transcriptomic and proteomic analysis. Spatial immunophenotyping was performed on IBC patient samples with tumor emboli. A transgenic CX3cr1GFP murine model was generated for visualization of macrophages and tumor emboli within the TiME via a surgically implanted window chamber, enabling intravital imaging and targeting. RESULTS:Gene and protein analyses of tumor emboli cultures compared to 2D monolayer cultures revealed upregulation of TNFR signaling networks, CXCL8, and immune cell chemotaxis genes. Spatial immunophenotyping of tumor emboli in patient samples demonstrated high levels of CD163+ tumor-associated macrophages. Furthermore, intravital imaging of CX3cr1GFP mice confirmed macrophage movement toward tumor cell clusters. Finally, targeting macrophage-associated TNF-α-signaling using SMAC mimetic, Birinapant, inhibited the tumor emboli phenotype in vivo. CONCLUSIONS:This study, the first to our knowledge, identifies TNF-α signaling and macrophage infiltration in IBC tumor emboli. Therapeutic strategies targeting TNF-α signaling to induce cell death and reduced macrophage influence have the potential to improve IBC outcomes.

INTRODUCTION:In breast cancer care multidisciplinary meetings (MDMs) are fundamental. Recently, the increasing number of patients, the complexity of cancer treatments, the shortages of specialists, and constrained hospital budgets have underscored the urgency of optimizing MDM processes to ensure that every patient benefits from thorough and timely multidisciplinary evaluation. MATERIAL AND METHODS:The European Society of Breast Cancer Specialists (EUSOMA) set up a multidisciplinary working group of international experts to develop consensus-based recommendations to optimize MDM management. To obtain insights into the current state of MDM organization and management within Europe and elsewhere, the experts designed and conducted an international survey. RESULTS:Briefly, the survey showed that most centres held a weekly MDM, either in person, online or combining the modalities, which lasted for 1-2 h. Discussion times for each case varied with its complexity. Some differences emerged between European and non- European breast cancer centres. The recommendations for improving MDM management focused on: i) timing, venue, logistics, administrative support; ii) technologies/equipment; iii) documentation; iv) planning and preparation; v) structure; vi) minutes/reporting; and viii) other issues. CONCLUSION:Optimizing MDM ensures that each patient receives the most appropriate, guideline-aligned treatment, tailored to individual needs. With the present recommendations, EUSOMA wishes to support breast centres in improving and standardizing MDM management.

In pancreatic ductal adenocarcinoma (PDAC), immune checkpoint inhibitors have shown limited efficacy, and the role of the TIGIT axis remains underexplored. This study aimed to characterize TIGIT axis components on protein level and their relationship to PD-1/PD-L1 expression in matched blood and tumor samples from PDAC patients to identify immunosuppressive mechanisms and fuel future strategies for immune checkpoint co-targeting in PDAC patients. Fresh tumor and peripheral blood samples were collected from PDAC patients undergoing surgical resection. Flow cytometry was performed on tumor-infiltrating lymphocytes and PBMCs to assess expression of TIGIT, DNAM-1, TACTILE, and PD-1. Ligands CD111, CD112, CD113, and CD155 were analyzed using immunohistochemistry. Additional RNA expression analysis (TCGA/GTEx) was used to evaluate ligand distribution and gene expression profiles. TIGIT was highly upregulated on intratumoral CD8⁺ T cells and regulatory T cells, frequently co-expressed with PD-1. DNAM-1 expression was significantly reduced in tumors. However, contrasting pattern emerges with Tregs, which uniquely upregulate DNAM-1 in the PDAC TME. In addition, CD112 and CD155 were broadly expressed, including novel stromal CD112 localization. NK cells were nearly absent intratumorally, correlating with DNAM-1 downregulation. Our findings identify TIGIT as a promising immunotherapeutic target in PDAC and suggest that dual checkpoint blockade (TIGIT/PD-1), alongside restoration of DNAM-1 signaling, may overcome immune suppression. These results provide mechanistic rationale to inform future clinical trials in PDAC.

INTRODUCTION:Sentinel lymph node biopsy (SLNB) after neoadjuvant treatment (NAT) is an increasing option for axillary surgery in patients responding to treatment, whether diagnosed as clinically node-negative (cN0) or node-positive (cN + ). This study evaluates SLNB trends in patients with NAT in a large European population. MATERIALS AND METHODS:Data sourced from EUSOMADB, collating prospectively collected data from certified European Breast Units, included 17,321 patients who have undergone NAT between 2010 and 2021. Of those, 9,226 and 8.095 are clinically N0 and cN1, respectively. RESULTS:During the study period, for cN0 patients, there has been a significant increase in the proportion of cases with SLNB, rising from 86% in the 2010-2015 period to 94% in the 2016-2021 period. Consequently, a decline in direct axillary dissection (AD) has been shown in both periods, dropping from 14% to 6% (p < 0.001). Similarly, in cN+ patients, SLNB increased from 25% to 40%, while direct AD decreased from 75% to 60% (p < 0.001). Regarding immunohistochemistry subtypes, higher SLNB rates were reported in triple-negative and HER2-enriched tumors. Nevertheless, SLNB rates rose significantly across all immunohistochemical subtypes (p < 0.001) between both periods. Multivariate analysis identified as statistically significant predictors of SLNB: surgery period (second period), molecular subtype (HER2-positive, triple-negative), breast-conserving surgery and type of NAT. CONCLUSION:This study evidences a substantial shift towards SLNB as the primary axillary surgery following NAT during the study period. This trend emphasizes a preference for less invasive procedures, likely due to the efficacy of neoadjuvant therapy in reducing axillary lymph node involvement.

Supplementary Figure 1: Correlation betweenSARS-CoV-2 anti-S1 IgG antibody titers and NT50 values against Wuhan and BA.1 Omicron strains post homologous or heterologous booster vaccination in cancer patients and healthy individuals.

Supplementary Figure 5: Spike-specific CD8+ T cell responses post homologous or heterologous booster vaccination in subcohorts.

Supplementary Figure 2: Virus neutralization test with 50% neutralization titers (NT50) defined as the sample dilution (reciprocal titer) conveying 50% neutralization in SARS-CoV-2 (strains 2019-nCoV-Italy-INMI1 and VLD20211207) infected wells.

BACKGROUND:The aim of this study was to assess clinicopathologic characteristics, treatment modalities used and outcome of patients with locally advanced breast cancer (LABC). MATERIALS AND METHODS:we searched the European Society of Breast Cancer Specialists (EUSOMA) data warehouse for clinically LABC cT4a-d - in the 2013-2022 timeframe. RESULTS:Of a total of 132269 patients, we identified 2427 patients with cT4abc BC (1.83 %) and 977 with inflammatory (T4d) BCs (0.74 %), of whom 542/2427 (20.1 %) and 251/977 (25.6 %), respectively, had metastatic disease at presentation (p = 0.054. Ninety percent of patients with cT4abcM0 and 88.8 % of patients with cT4dM0 disease had surgery (p = 0.369) and 90.9 % and 88.7 % (p = 0.187) endocrine therapy. Neoadjuvant chemotherapy (CT), adjuvant CT, biological drugs and radiotherapy were given in 33.2 %, 47.0 %, 67.1 % and 61.1 % of cT4abcM0 cases compared to 77.3 %, 80.4 %, 87.7 % and 80.2 % of cT4dM0 cases (all p < 0.001). Multivariable analysis showed that age <70 years, luminal A and HER-2 pure subtype, surgical treatment, radiotherapy, and systemic treatment (all p ≤ 0.034) were determinants of better overall survival (OS). Local recurrence rate (LRR) was significantly lower in patients receiving radiotherapy or endocrine therapy (p ≤ 0.012). Cox analyses showed no difference in OS or LRR between patients with cT4abc and those with T4d BCs, neither at univariable or multivariable analysis. CONCLUSIONS:The current study confirms well-known features of cLABCs and underscores the importance of multimodal treatment, which is often underused in these patients.

Cell therapies, including tumor antigen-loaded dendritic cells used as therapeutic cancer vaccines, offer treatment options for patients with malignancies. We evaluated the feasibility, safety, immunogenicity, and clinical activity of adjuvant vaccination with Wilms’ tumor protein (WT1) mRNA-electroporated autologous dendritic cells (WT1-mRNA/DC) in a single-arm phase I/II clinical study of patients with advanced solid tumors receiving standard therapy. Disease status and immune reactivity were evaluated after 8 weeks and 6 months. WT1-mRNA/DC vaccination was feasible in all patients, except one. Vaccination was well tolerated without evidence of systemic toxicity. The disease control rate and overall response rate among a total of 39 evaluable patients were 74.4