Liu, Y H

Background Tumor-infiltrating lymphocytes (TILs) are established determinants of clinical outcomes in breast cancer. 1–6 While stromal TILs (sTILs) are commonly evaluated due to easier scoring and higher interobserver reproducibility, intraepithelial TILs may provide additional insights into anti-tumor immunity. 7 We aimed to comprehensively characterize immunotype-associated cellular landscapes in triple-negative breast cancer (TNBC) and identify features associated with clinical outcomes. Methods 212 TNBC tumors were classified into three immunotypes based on immune infiltrate location and density: immune desert (ID), immune excluded (IE), and immune inflamed (IN). 8 Single-cell spatial transcriptomics was performed on 75 tumors to define the cellular composition and functional states of the tumor microenvironment across immunotypes. Findings were validated and integrated with bulk RNA sequencing data and clinical outcome datasets from FinXX, CALGB-40603, I-SPY 2, and Real-World Clinico-Genomic Data (RWCGD) cohorts. Results Patients with IN tumors had significantly improved outcomes compared with ID tumors. Despite high sTILs, IE tumors showed poor outcomes similar to ID tumors. Single-cell spatial analysis revealed that ID and IE tumors exhibited reduced major histocompatibility complex I/II expression and fewer tumor-resident plasmacytoid dendritic cells (pDCs) compared with IN tumors, which were enriched for interferon-alpha (IFNα) and interferon-gamma (IFNγ) responses. High IFN response scores were associated with favorable outcomes across multiple therapy types in independent datasets. Deconvolution of RWCGD bulk RNA-seq data confirmed that pDC abundance correlated with improved outcomes specifically in hormone receptor–negative subtypes. Conclusions Our study highlights pDCs and IFN signaling as hallmarks of effective anti-tumor immunity in TNBC. Immunotype-based profiling underscores the limited prognostic value of sTILs alone in immune-excluded tumors and supports pDCs and IFN pathways as potential biomarkers for prognosis and therapeutic development in TNBC.

564 Background: HER2+ ILC is rare (~5% of ILC), with limited data on long-term adjuvant trastuzumab outcomes. We investigated late recurrence and immune landscapes of HER2+ ILC vs invasive ductal carcinoma (IDC) in the phase III NCCTG N9831 trial (NCT00005970) and a real-world cohort. Methods: We evaluated 3304 early-stage HER2+ breast cancer patients (pts) enrolled in N9831 (122 ILC, 3182 IDC). Recurrence-free survival (RFS) and overall survival (OS) were estimated (Kaplan-Meier) and compared using multivariable Cox regression adjusting for age, tumor size, grade, hormone receptor (HR), and nodal status. Findings were validated in a real-world cohort of HER2+ pts (Caris Life Sciences CODEai platform). Immune microenvironments were profiled by WTS deconvolution (Quantiseq). Real-world overall survival was calculated from trastuzumab start to last contact. Results: In N9831, ILC pts were older (median 54 vs 49 y), more ER+ (74.6% vs 51.4%), and had more N3 disease (25.4% vs 12.7%) compared to IDC. ILC pts derived a comparable magnitude of benefit from adjuvant trastuzumab compared to the overall population (HR 0.58 vs 0.67). However, long-term outcomes diverged: while the 5-year RFS was similar (77.1% vs 82.7%), ILC pts experienced a steep decline between years 5-15 (77.1% to 57.7%; ~20% decline) vs IDC (82.7% to 72.7%; ~10% decline) after multivariable Cox analysis (Table). In the real-world early/advanced cohort (n=1328; 26 ILC vs 1302 non-ILC [subset IDC n=595]) treated with trastuzumab-based therapy, ILC pts had worse mOS (33.7 vs 68.6 m, HR 2.01, 95%CI 1.00-4.07, p=0.04) compared to non-ILC. However, there was no statistically significant difference when compared specifically to IDC (HR 1.77, 95% CI 0.87-3.61, p=0.11). ILC had: i) numerically lower tumor mutation burden (TMB)-high (4.2% vs 9.5%, p=0.38); ii) higher infiltration of B cells, Tregs, and M2 macrophages (p<0.05); iii) upregulated MHC class II expression (HLA-DOA, p<0.05); and iv) downregulated proliferative and DNA repair pathways (E2F, G2M, MYC; FDR <0.25) compared to non-ILC. Conclusions: HER2+ ILC carries late recurrence risk despite initial trastuzumab benefit. The unique immune landscape (Treg/M2 macrophage enrichment and lower proliferative signatures) distinguishes HER2+ ILC from IDC, highlighting the need for tailored strategies. Long-term RFS and OS in HER2-positive ILC vs IDC (N9831 trial). Population N 5-year% (95% CI) 10-year% (95% CI) 15-year% (95% CI) ILC 122 RFS 77.13% (69.9, 85.1) OS 84.04% (77.7, 90.9) RFS 70.57% (62.7, 79.4) OS 76.0% (68.7, 84.2) RFS 57.67% (44.2, 75.1) OS 65.22% (56.6, 75.2) IDC 3182 RFS 82.71% (81.4, 84.1) OS 90.18% (89.1, 91.2) RFS 77.45% (75.9, 78.9) OS 81.81% (80.4, 83.2) RFS 72.68% (70.77, 74.6) OS 76.68% (75, 78.4)

1591 Background: The 21-gene OncotypeDX (ODX) recurrence score (RS) is the standard for chemotherapy decision-making in HR+/HER2- early breast cancer (EBC), but cost limits accessibility in resource-constrained settings. The Magee equations (MEs) use routine pathology features (Nottingham grade and IHC; ER/PR/HER2/Ki-67) to estimate RS. We performed a meta-analysis to validate MEs performance as a triage tool for ODX testing and a qualitative synthesis to identify sources of discordance, highlighting the transition toward objective digital pathology AI models. Methods: A systematic review identified validation studies comparing MEs with RS in HR+ EBC. Studies with sufficient data to construct a 2X2 contingency table were included; neoadjuvant trials were excluded. Primary outcomes were Negative Predictive Value (NPV) for identifying low-risk cases (RS <26 for the current TAILORx cutoff and RS<31 for the historical cutoff) and ODX test sparing rate (TSR). Data were pooled using random-effects models. Qualitative themes regarding interpretation variability were extracted from study results and discussion sections. Results: Thirteen studies met the inclusion criteria, representing 5396 patients in global cohorts from the USA, Mexico, Colombia, Jordan, France, Belgium, and Canada. For RS >26, Magee <18 achieved pooled NPV of 0.96 (95% CI: 0.94-0.97) and TSR 61% (95% CI:51%-69%). For RS >31, NPV increased to 0.99 (95% CI: 0.97-1.00) and TSR 52% (95% CI: 44%-59%). Pooled diagnostic performance for Magee <18 predicting RS <26 demonstrated 83% sensitivity and 71% specificity (AUC 0.81, LR+:2.53, LR-:0.28). Qualitative analysis of discordance identified: (1) Inter-observer variability: Nottingham mitotic counts and Ki-67 scoring were the primary drivers of score fluctuations. (2) IHC quantification: Variations in H-score calculation and Allred-to-H-score conversion introduced heterogeneity (3) Pre-analytical and biological factors: Inflammation and stromal proliferation occasionally inflated genomic RS; selection of non-representative blocks and intratumor heterogeneity further contributed to discordance (4) Technological solutions: Digital pathology and AI models emerge as solutions to standardize these variables. Conclusions: Magee-based triage provides a safe and cost-effective strategy for HR+ EBC risk stratification, with NPV up to 99% for excluding high-risk disease and sparing up to 61% of ODX testing. These results confirm a robust histopathological signal. Transitioning to objective digital pathomics provides a pathway to reduce manual subjectivity and democratize precision oncology globally. Pooled diagnostic accuracy of Magee equations by cutoff. Magee score and RS cutoff Number of studies Total patients Pooled NPV (95% CI) ODX test sparing rate (95% CI) Magee <18/ RS>26 8 4153 0.96 (0.94-0.97) 61% (51%-69%) Magee <18/ RS>31 5 1243 0.99 (0.97-1.00) 52% (44%-59%)