Summary Tumor-infiltrating lymphocytes (TILs) are widely used to assess antitumor immunity in breast cancer but may not reflect the functional competence of adaptive immune responses. We show that immune organization, reflected by tertiary lymphoid structures (TLS) and coordinated humoral and cellular immune programs, represents a distinct dimension of tumor immunity beyond lymphocyte abundance. By integrating histologic, transcriptomic, spatial, and immune receptor profiling analyses across multiple breast cancer cohorts, we show that immune organization is associated with greater immune repertoire diversity, evidence of therapy-induced clonal selection, and improved clinical outcomes, independent of immune infiltration. Transcriptomic measures of immune organization retained independent prognostic value across external cohorts, whereas measures of immune infiltration did not. Furthermore, treatment-induced increases in immune organization, but not immune infiltration, were associated with therapeutic response. These findings identify immune organization as a dynamic and clinically measurable state of adaptive antitumor immunity with implications for prognosis, treatment monitoring, and therapeutic development in breast cancer. One Sentence Summary Spatially organized immune responses, rather than lymphocyte abundance alone, define clinically relevant antitumor immunity in breast cancer.

Tables S5-8 showing results from univariate analysis for ORR, CBR, PFS and OS

BACKGROUND:HER2DX genomic assay is a genomic tool developed for personalizing care in early-stage HER2-positive breast cancer. This real-world analysis examines its associations with pathological complete response (pCR) and invasive disease-free survival (IDFS) following trastuzumab- and pertuzumab-based neoadjuvant therapy. MATERIALS AND METHODS:Retrospective, observational, single-center study of 156 patients with stage I-III HER2-positive breast cancer treated with dual HER2 blockade-based neoadjuvant therapy between February 2015 and May 2022 at University Hospital A Coruña. HER2DX and clinicopathological variables were assessed in pretreatment tumor biopsies. Statistical analyses included Fisher's exact test, logistic regression, Cox proportional hazards models, and Kaplan-Meier estimates. RESULTS:HER2DX was performed in 111 tumors (71.2%). Overall pCR rate was 51.3%. Hormone receptor-positive disease was significantly associated with lower pCR (odds ratio [OR] 0.25, 95% CI 0.11-0.50; p < 0.001), whereas HER2 immunohistochemistry 3+ showed a non-significant trend toward higher pCR (OR 2.95, 95% CI 0.96-11.08; p = 0.075). Median IDFS was not reached, but hormone receptor-positive tumors showed better outcomes (hazard ratio [HR] 3.67, 95% CI 1.02-13.32; p = 0.004 by log-rank). The association between HER2DX pCR score and pCR appeared heterogeneous. The continuous score was significantly associated with pCR in univariable analysis, not in multivariable model. In contrast, medium-high HER2DX pCR category showed significantly higher pCR rates than low category (OR 4.83, 95% CI 1.72-14.65; p = 0.004). The continuous HER2DX risk score was independently associated with IDFS (HR 2.84, 95% CI 1.24-6.48; p = 0.010). CONCLUSIONS:In this cohort, HER2DX scores were associated with pCR and IDFS, supporting previous evidence and highlighting potential role in treatment stratification.

ABSTRACT The development of clinically effective CAR-T cell therapies for solid tumors requires careful optimization of receptor design, functional fitness, and manufacturability. While advancing low-affinity HER2-targeting CAR-T cells toward clinical application, we found that the candidate with the strongest in vivo antitumor activity—comprising a CD8α hinge and transmembrane region and a 4-1BB co-stimulatory domain—exhibited measurable tonic signaling. This basal antigen-independent signaling, likely driven by high CAR surface expression, was associated with increased apoptosis and reduced ex vivo expansion under research-grade manufacturing conditions. Modification of the transmembrane domain reduced CAR surface expression but did not alleviate tonic signaling and instead impaired antitumor activity. By contrast, transient pharmacologic inhibition of CAR signaling with dasatinib rescued expansion and reduced apoptosis in small-scale research cultures. Notably, these tonic-signaling-associated defects were largely absent during large-scale, GMP-compliant manufacturing, which enabled robust CAR-T cell expansion without additional benefit from dasatinib supplementation. Together, these findings show that tonic signaling is not inherently detrimental to CAR-T cell performance and that its functional consequences are highly dependent on manufacturing context. Our study underscores the importance of evaluating CAR candidates within clinically relevant production platforms and supports the advancement of this 4-1BB–based HER2-specific CAR-T cell product toward clinical testing.

This single-arm, phase II, preoperative window-of-opportunity trial (ClinicalTrials.gov Identifier: NCT03802604) investigated the efficacy and safety of talimogene laherparepvec (T-VEC), an oncolytic virus, with atezolizumab, an anti-PD-L1 antibody, in patients with breast cancer and radiologically and pathologically confirmed residual disease prior to surgery. Eligible patients had triple-negative breast cancer (TNBC) or hormone receptor-positive (HR+)/HER2-negative disease with a high proliferation index (Ki67 ≥ 20%) prior to neoadjuvant chemotherapy. Treatment consisted of one intratumoral injection of T-VEC (106 plaque-forming units [PFU]/mL), followed by four biweekly T-VEC doses (108 PFU/mL) plus atezolizumab (840 mg, intravenously). Among the 28 patients enrolled, 20 patients (71.4%) had HR+/HER2-negative and 8 patients (28.6%) had TNBC. At surgery, 7 patients (26.9%) achieved Residual Cancer Burden (RCB)-0/I (primary endpoint), 12 (46.2%) RCB-II and 7 (26.9%) RCB-III. Safety profile was favorable, with mostly low-grade adverse events and no serious events (secondary endpoint). Therapy induced immune modulation, including increased tumor-infiltrating lymphocytes, elevated PD-L1 expression, and enhanced immune-related gene signatures (exploratory endpoints). The trial met its pre-specified efficacy and safety endpoints. These findings support the feasibility of T-VEC plus atezolizumab as a  preoperative immunotherapy approach for managing HER2-negative residual disease post-neoadjuvant chemotherapy and warrant further exploration in larger trials.

patient-level raw gene expression data of FFPE tumor samples and best overall response rate

PURPOSE:Hormone receptor-positive (HR+), HER2-negative (HER2-) metastatic breast cancer (mBC) is biologically distinct from early-stage disease, with a higher prevalence of genomically defined nonluminal subtypes, particularly the HER2-enriched (HER2-E) and basal-like subtypes. These tumors are highly proliferative, less dependent on hormone signaling, and associated with poor outcomes and early resistance to endocrine therapy and CDK4/6 inhibition (CDK4/6i). This biological shift highlights the need for biomarker-driven strategies in the CDK4/6i-resistant setting. PATIENTS AND METHODS:The SOLTI-1716 TATEN trial (NCT04251169) is a phase II, single-arm study evaluating the combination of pembrolizumab and paclitaxel in patients with HR+/HER2- mBC classified as HER2-E or basal-like by PAM50 following progression on CDK4/6i. A total of 126 patients were screened using the PAM50 genomic assay; 20 with HER2-E or basal-like subtypes were enrolled and received pembrolizumab (200 mg every 3 weeks) and paclitaxel (80 mg/m2 weekly). RESULTS:The primary endpoint, overall response rate (ORR), was 61.1% [95% confidence interval (CI), 35.7-82.7], and the clinical benefit rate was 94.4% (95% CI, 72.7-99.9). The median progression-free survival was 8.1 months (95% CI, 5.9-10.4), and the median overall survival was 26 months [95% CI, 18-not reached (NR)]. Three patients achieved durable responses lasting ≥24 months. Gene expression analyses revealed that high expression of proliferation- and immune-related genes predicted improved ORR and survival, whereas luminal-related gene expression was associated with lower clinical benefit. CONCLUSIONS:These results suggest that chemo-immunotherapy may be an effective strategy in genomically defined nonluminal subtypes of HR+/HER2- mBC following CDK4/6i resistance and highlight the value of molecular subtyping to guide post-endocrine treatment decisions.

Tables S9-S17 showing mutivariate and bivariate analysis for ORR, CBR, PFS and OS

Predictive biomarkers for immune checkpoint inhibitors (ICIs) are largely identified retrospectively, but their prospective clinical utility remains unproven. Here, we report DUTRENEO, a prospective randomized phase 2 trial testing whether a retrospectively validated 18-gene bulk tumor inflammation signature (TIS) can guide neoadjuvant ICI therapy in muscle-invasive bladder cancer. The trial does not meet its primary endpoint, and this demonstrates that bulk gene-expression stratification does not sufficiently enrich for responders. To define the biology underlying this failure, we generate single-cell spatial transcriptomic profiles of 377 genes in ∼5.4 million cells across large tissue areas. Response is governed by spatial architectures invisible to bulk assays, including CD8+ T cell proximity to cancer cells, localized checkpoint co-expression within epithelial cancer-rich neighborhoods, and fibroblast-rich immune-excluded communities in non-responders. We provide a quantitative framework showing that ≥77 genes and ≥3-mm tissue diameter regions preserve predictive spatial signal at scalable throughput. The registration details of the trial are EudraCT 2017-002246-68.

PURPOSE:Based on the results from SOLTI-PATRICIA trial (NCT02448420) cohorts A/B, a direct comparison with standard-of-care treatments is needed to evaluate the efficacy and safety of palbociclib, trastuzumab, and endocrine therapy (ET) in PAM50 luminal A/B pretreated patients. PATIENTS AND METHODS:SOLTI-PATRICIA cohort C is a randomized, multicenter, prospective, open-label, phase II study. Pretreated patients with HER2-positive, hormone receptor-positive, and PAM50 luminal A/B advanced breast cancer were randomized 1:1 to receive either i) the triplet regimen or ii) trastuzumab-based treatment of physician's choice (TPC). Patients allocated in the TPC arm were eligible for re-randomization upon disease progression, if the inclusion criteria were still met. The primary endpoint was investigator-assessed progression-free survival (PFS) per RECIST version 1.1. RESULTS:A total of 264 participants were prescreened from August 2019 to 2023, and 73 patients were randomly assigned (including seven re-randomizations). In the TPC arm, 48.5% were treated with trastuzumab plus chemotherapy, 39.4% with trastuzumab emtansine, and 12.1% with trastuzumab plus ET. The triplet was associated with a significantly better PFS compared with TPC [stratified hazard ratio = 0.52; 95% confidence interval (CI), 0.29-0.95; two-sided P = 0.03]. PFS rates after 24 months were 24.0% with the triplet and 4.3% in the TPC arm. The overall response rate was 18.9% (95% CI, 8.6-35.7) and 7.1% (95% CI, 1.2-25.0), respectively. In the triplet arm, grade ≥3 adverse events occurred in 61.5% of patients, with neutropenia being the most frequent (53.9%). No permanent discontinuations due to toxicity were observed. CONCLUSIONS:Combining palbociclib, trastuzumab, and ET was safe and significantly improved PFS, compared with TPC, in previously treated patients with HER2-positive, PAM50 luminal A/B advanced breast cancer.

Supplementary Figure S1. Study design and tissue availability for biomarker analysis.

Supplementary Figure S1. Clinical trial design of the ELIPSE trial.

We present the first documented oncologic case of a type I Kounis syndrome (KS) following paclitaxel administration, in a very young patient with HER2-positive(+) early-stage breast cancer (BC). KS is a relatively rare acute coronary syndrome triggered by anaphylactic or hypersensitivity reactions, of which there is limited awareness among healthcare providers. It is subdivided in four subtypes depending on cardiac artery medical history. While no established management guidelines exist, its treatment requires addressing severe infusion reactions while ensuring proper myocardial perfusion. We hereby illustrate its successful acute management and report on how tumor genomics through the novel HER2DX assay helped re-defining the entire neo/adjuvant oncologic strategy. HER2DX integrates tumor size and nodal involvement with 27 genes' expression data tracking four biological BC-related and immunologic signatures so to estimate a prognostic and a predictive score. This report demonstrates how clinical and genomic data can be effectively integrated to optimize therapeutic decisions in HER2+ BC, offering a model for personalized care also in atypical and complex cases.

Supplementary Figure S4. Decision tree algorithm to construct the multiparameter classifier of response in TBCRC023.

Background: Biomarkers after progression to CDK4/6 inhibitors plus endocrine therapy (CDKi+ET) are needed to guide the use of ET-based therapies versus chemotherapy (CT). Here, we explored the prognostic and predictive value of the 4 major intrinsic subtypes (IS) of breast cancer (i.e., Luminal A [LumA], Luminal B [LumB], HER2-enriched [HER2E], Basal-like [BL]) in tumor samples of patients with HR+/HER2- MBC progressing to CDKi+ET. Methods: This retrospective/prospective observational study included 63 patients with HR+/HER2- MBC treated at the Hospital Clinic of Barcelona between 2018-2024 with at least one line after CDKi+ET and an available tumor biopsy obtained at progression from CDK4/6 inhibition. The primary objective was to determine the progression-free survival (PFS) and overall survival (OS) after CDKi+ET, according to IS determined at progression from CDKi+ET. PFS and OS within luminal (Lum) vs. non-Lum IS according to type of therapy was explored. A paired biopsy (before starting CDKi+ET and at progression to CDKi+ET) was available in 39 (61.9%) cases. IS was assessed using a research-based PAM50 assay on the nCounter platform. Survival analyses were conducted with the Kaplan-Meier method and Cox regression models. Significance was established at p≤0.05. Results: The median age was 57.6 years. Overall, CDKi+ET had been administered in 1st, 2nd and ≥3rd line in 65.1%, 14.8% and 20.1% cases, with 54.0% patients progressing to ribociclib as CDKi and 57.1% progressing to letrozole as ET. Median PFS to CDKi+ET was 13.6 months (95% CI 10.2-19.2). In tumor samples obtained at CDKi+ET progression, 27 (42.9%) were Lum (LumA+LumB) and 36 (57.1%) non-Lum (HER2E+BL+normal-like). With a median follow-up of 35.2 months (95% CI 22.5-53.3) after progressing to CDKi+ET, PFS was 5.5 months (95% CI 3.8-7.9), and OS was 21.3 months (95% CI 16.8-28.7). Subtypes at progression to CDKi+ET were prognostic for PFS (p<0.001) and OS (p=0.004) with LumA tumors displaying the best median PFS (7.9 months) and OS (43.3 months), followed by LumB (5.4 and 23.8 months), HER2E (4.8 and 21.4), and BL (4.6 and 10.3). The PFS and OS hazard ratios (HR) between LumA versus others, adjusted for post-CDKi treatment, were 0.39 (p=0.032) and 0.49 (p=0.202), respectively. Patients with non-Lum tumors received more CT +/- targeted therapy (63.9% vs 18.5%) and less ET-based therapies (25.0% vs 66.6%) than patients with Lum tumors (p<0.01). Type of therapy (CT-based versus ET-based) was not found significantly associated with PFS (p=0.585) and OS (p=0.516). However, CT-based therapies within non-Lum disease showed better PFS compared to ET-based therapies (HR=0.44, p=0.039). No difference in OS was observed (p=0.266). Within Lum disease no difference in PFS and OS was observed according to therapy. Finally, in 39 paired tumor samples, subtype switching occurred in 61.9% of the cases. Tumor samples obtained at progression to CDKi+ET were significantly enriched in HER2E disease (51.3% vs 35.9%) and showed less Lum IS (41.0% vs 56.4%), with a consistent increase in the HER2E PAM50 score (p=0.005) and mRNA levels of genes associated to proliferation or HER2E biology, e.g. MKI67 (p=0.009) and FGFR4 (p=0.035), despite no ERBB2 mRNA levels’ changes (p=0.841). Similar findings were observed in a subgroup of baseline Lum tumors shifting to HER2E. Conclusions: Subtype switching towards less ET-sensitive IS, especially the HER2E, occurs under CDKi+ET and has prognostic value. Post-CDKi LumA disease showed the best outcomes, regardless of treatment type. This group of patients might be the ideal group to be treated with ET-based therapies. Non-Lum IS performed better with CT-based approaches. Overall, these findings suggest the necessity to profile tumor samples at progression to CDKi+ET to better tailor treatments. Citation Format: Isabel Garcia-Fructuoso, Fara Brasó-Maristany, Olga Martínez-Sáez, Raquel Gómez-Bravo, Sabrina Nucera, Elia Seguí, Oleguer Castillo, Paula Blasco, Valeria Sirenko, Angela Aguirre, Natalia Lorman-Carbó, Patricia Galván, Benjamín Walbaum, Esther Sanfeliu, Blanca Gonzalez-Farre, Tomás Pascual, Barbara Adamo, Maria Vidal, Montserrat Muñoz, Aleix Prat, Francesco Schettini. Intrinsic Subtype at Progression to CDK4/6 Inhibitors Plus Endocrine Therapy in Hormone Receptor-Positive/HER2-Negative Metastatic Breast Cancer (MBC) [abstract]. In: Proceedings of the San Antonio Breast Cancer Symposium 2024; 2024 Dec 10-13; San Antonio, TX. Philadelphia (PA): AACR; Clin Cancer Res 2025;31(12 Suppl):Abstract nr PS2-07.

Background: The HER2DX assay, a 27-gene test, was developed to provide prognostic information and predict treatment responses in patients with early-stage HER2+ breast cancer. This assay evaluates four gene expression signatures: immune/immunoglobulin (IGG), proliferation, luminal, and HER2, offering a comprehensive risk score, a likelihood estimates for pathological complete response (pCR) and ERBB2 expression levels. In this study, we evaluated the HER2DX assay in HER2+ DCIS, aiming to understand its biology and relationship with HER2+ invasive breast cancer. Methods: Standardized HER2DX genomic test was evaluated centrally on 28 formalin-fixed paraffin-embedded tumor samples of HER2+ DCIS across three hospitals in Spain. DCIS features such as nuclear grade, comedonecrosis, architectural pattern, tumor size and hormone receptor (HR) status were evaluated. Percentage (%) of stromal tumor infiltrating lymphocytes (TILs), their spatial distribution (i.e.: inflamed, desert or excluded), and presence of tertiary lymphoid structures (TLS, defined as spatially organized, non-encapsulated areas of immune cell aggregates with or without germinal center [GC]) were assessed on hematoxylin eosin slides. Descriptive statistics were used. Results: The HER2DX assay was evaluated in 28 cases of HER2+ DCIS. Most cases had a 3 nuclear grade (67.9%) and presented with comedonecrosis (60.7%). HR positivity (HR+) was found in 42.9% of cases, as determined by immunohistochemistry. The median tumor size was 30 mm, with a range from 5 mm to 90 mm. The median % of TILs was 20% (range 0.5-70%). TILs, as a continuous variable, showed a moderate correlation with the HER2DX IGG signature (Pearson correlation coefficient=0.43, p-value=0.023), and a tendency with the inflamed spatial distribution (p-value=0.063). TLS were identified in 82.1% of DCIS samples, and their presence was associated with higher expression of the IGG signature (52.2% TLS in IGG-high vs. 21.7% in IGG-low). Regarding the HER2DX luminal signature, 41.7% of cases were classified as luminal-high, associated with HR+ disease (p=0.025). For the HER2DX proliferation signature, 82.1% of cases were identified as proliferation-low. In HER2DX risk stratification, 100% of cases were categorized as low-risk, 46.4% were pCR-high, and 78.6% were ERBB2-high. Notably, only one (3.6%) HER2+ DCIS case was found to be ERBB2-low. Conclusions: The HER2DX assay revealed that all HER2+ DCIS cases were categorized as low-risk, with most cases showing high ERBB2 expression and a high predicted response to anti-HER2-based therapy. These findings underscore the underlying biology of HER2+ DCIS and its tumor immune microenvironment, indicating that HER2+ DCIS shares similar tumor biology with low-risk HER2+ breast cancer. Citation Format: Esther Sanfeliu, Anabel Martinez-Romero, Mercedes Marín-Aguilera, Vicente Marco, Felip Garcia, Vicente Peg, Blanca González-Farré, Ivonne Vazquez, Patricia Galván, Oleguer Castillo, Paula Blasco, Valeria Sirenko, Angela Aguirre, Laia Paré, Guillermo Villacampa, Antonio Martínez, Jesus Soberino, Aleix Prat, Fara Brasó-Maristany. HER2DX assay in HER2-positive (HER2+) breast ductal carcinoma in situ (DCIS) [abstract]. In: Proceedings of the San Antonio Breast Cancer Symposium 2024; 2024 Dec 10-13; San Antonio, TX. Philadelphia (PA): AACR; Clin Cancer Res 2025;31(12 Suppl):Abstract nr P5-07-18.

Supplementary Figure S3. Distribution of intrinsic subtypes in (A) TBCRC023 and (B) PAMELA cohort.