PARP inhibitors (PARPi) are effective in tumors with homologous recombination repair (HRR) deficiency (HRD), typically identified by germline/tumor mutations. However, genetic testing may miss intrinsic PARPi sensitivity and resistance. We evaluated strategies to improve detection and longitudinal monitoring of HRD across >500 tumor samples and >20 paired liquid biopsies, integrating genetic, genomic, and functional readouts. HRD was more frequent in high-grade ovarian cancer (HGOC; 52%) than in metastatic breast (mBC; 12%) or prostate cancer (mPC; 20%). Assay concordance was low-to-moderate, underscoring complementarity. RAD51 testing and the genomic instability score identified HRD in tumors lacking pathogenic HRR mutations (6% and 30% in mBC, 38% and 46% in HGOC, 14% and 27% in mPC, respectively). Longitudinal ctDNA sequencing revealed BRCA1/BRCA2 reversion mutations in >30% of post-PARPi mBC samples, which were associated with poor response to subsequent platinum therapy. These findings support the use of complementary HRD biomarkers in tissue and liquid biopsy to guide PARPi use and monitor response.

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.

CAR T-cell therapy has transformed the treatment landscape for blood cancers, yet its success in solid tumors is limited by poor trafficking, immunosuppressive tumor microenvironment, and antigen heterogeneity. “Armored” CAR T cells aim to overcome these barriers by delivering immune-boosting payloads, although achieving precise and safe tumor-specific control of such potent functions remains a critical and unresolved challenge. Here, we present a post-transcriptional regulatory platform that leverages endogenous microRNA (miRNA) circuits to control therapeutic transgene expression in CAR T cells. This approach embeds complementary miRNA target sites (miRTS) into payload-encoding transcripts, making their expression dictated by endogenous miRNA abundance. This design exploits miRNAs naturally expressed in resting T cells to enforce repression of payloads under non-activated conditions. Upon CAR-mediated antigen stimulation, these miRNAs are downregulated, enabling targeted de-repression selectively at the tumor site. To identify microRNAs with this activation-coupled behavior, we combined small-RNA sequencing with a functional screen. Transcriptomic profiling of HER2-28Z CAR T cells activated in vitro and of CAR tumor-infiltrating lymphocytes from treated mice revealed 58 microRNAs that are abundant at rest but diminish upon activation. Using an in vivo, library-based functional screen informed by these data, we prioritized miR-29c-3p, miR-32-3p, miR-150-5p, and miR-181a-5p as the most effective regulators. Incorporating the corresponding target sites into the 3’UTR of the transgene within armored CAR-T generated a miRNA-inducible platform that enabled strong transgene repression in resting T cells while activation-specific payload expression in vitro and in various solid tumor models in vivo. In these models, all candidate miRNAs significantly reduced circulating payload levels, with reductions ranging from 53% to 94% compared to constitutive payload expression, while simultaneously permitting intratumoral expression and enhancing tumor control and T-cell persistence. The platform proved highly versatile. Each miRNA candidate provided distinct repression and induction dynamics, and regulatory strength could be further tuned by varying the number or combination of miRTS, with additional sites enhancing repression. This flexibility enables tailoring to transgenes with diverse safety requirements. The system supported varied payloads, including secreted cytokines (IL-12, IL-18) and membrane-bound proteins such as a secondary CAR. It performed consistently across CARs with different antigen specificities and with both 4-1BB and CD28 signaling domains. Importantly, we show that our platform is compatible with CAR T cells derived from cancer patients, supporting its translational feasibility. Collectively, our results define a post-transcriptional, plug-and-play regulatory layer for precise, context-driven, and modular control of armored CAR T-cell therapies—addressing a central barrier to their safe and effective implementation in solid tumors. Nina Barceló-Genestar, Teresa Lobo-Jarné, Paula Barbao, Marta Gimenez, Guillem Colell, Fleur Chapus, Salut Colell, Maria Hernandez, Hugo Calderón, Ariel Galindo-Albarrán, Marco Antonio Mendoza-Parra, Luca Gattinoni, Aleix Prat, Alena Gros, Xavier M. Anguela, Alba Rodriguez-Garcia, Sonia Guedan. A versatile microRNA-based platform for activation-dependent regulation of armored CAR T cell payloads [abstract]. In: Proceedings of the AACR Immuno-Oncology Conference (AACR IO): Discovery and Innovation in Cancer Immunology: Revolutionizing Treatment through Immunotherapy; 2026 Feb 18-21; Los Angeles, CA. Philadelphia (PA): AACR; Cancer Immunol Res 2026;14(2 Suppl):Abstract nr C007.

Estrogen receptor-positive (ER+), HER2-negative (HER2-) metastatic breast cancer (MBC) shows variable outcomes after first-line CDK4/6 inhibitors (CDK4/6i) plus endocrine therapy (ET). The prognostic role of PAM50 intrinsic subtypes (IS) in this setting remains unestablished. We evaluated IS and biomarker profiles in the SOLTI-1801 CDK-PREDICT cohort, focusing on real-world second- and third-line progression-free survival (rwPFS-2L and rwPFS-3L). This multicenter observational study reports a post hoc secondary analysis of ER+ /HER2- MBC patients previously treated with first-line CDK4/6i plus ET. Baseline metastatic biopsies were molecularly profiled (PAM50, CCNE1, PDCD1) using the nCounter platform. rwPFS-2L and rwPFS-3L were defined from initiation of second- or third-line therapy to progression or death. Kaplan–Meier and Cox models assessed associations with clinical, molecular, and treatment variables. Among evaluable patients (n = 125 for rwPFS-2L; n = 95 for rwPFS-3L), Luminal A/B subtypes represented most cases, while advanced lines showed more aggressive profiles. Median rwPFS-2L was 7.2 months in luminal IS vs. 6.1 in non-luminal (HR 1.40; 95

Patients with triple-negative breast cancer (TNBC) who achieve pathologic complete response (pCR) to neoadjuvant systemic therapy have favorable survival, while those with residual disease have high recurrence risk. Stromal tumor infiltrating lymphocytes (sTILs) and TNBC-DX both predict pCR in TNBC. Whether these 2 biomarkers provide complementary information has not been tested. We evaluated sTILs and TNBC-DX in TNBC patients treated with docetaxel-carboplatin (TCb) on the MMJ-CAR-2014-01 study (NCT01560663) or TCb plus pembrolizumab (TCb+Pem) on the NeoPACT trial (NCT03639948). sTILs and TNBC-DX independently predicted pCR in patients treated with TCb+Pem. Patients with sTILs ≥ 30% and a TNBC-DX pCR-high genomic score achieved a pCR rate of 91.3% with TCb+Pem. An integrated classification incorporating sTILs and TNBC-DX identified approximately 40% of the NeoPACT cohort with a pCR rate exceeding 85%. The integrated classification was prognostic for event-free survival in patients treated with TCb+Pem. Integrating sTILs and TNBC-DX may facilitate chemoimmunotherapy escalation and de-escalation trials.

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.

CAR-T cell therapy has achieved remarkable clinical success for the treatment of hematologic malignancies; however, durable and sustained regressions in patients with solid tumors remain uncommon. A major cause of resistance is CAR-T cell dysfunction driven by chronic antigen exposure within the tumor site. To uncover molecular drivers of this dysfunction, we developed an in vivo CRISPR-based screen using a xenograft model that recapitulates tumor escape due to loss of CAR-T cell activity. We hypothesized that targeted ablation of the identified candidate genes would enhance CAR-T cell efficacy against solid tumors. NSG mice bearing ovarian xenograft tumors were treated with a single dose of anti-HER2 CD28-based CAR-T cells. Tumor-infiltrating T cells (TILs) were isolated short after infusion (effective phase) and after therapy failure (dysfunctional phase). Bulk RNA-seq and single-cell RNA-seq analysis were performed on dysfunctional CAR-TILs compared to effective CAR-TILs and preinfusion product. Effective CAR-TILs retained functional activity but were already transcriptionally committed to dysfunction. In contrast, dysfunctional CAR-TILs displayed phenotypic and functional hallmarks of T-cell exhaustion and a transcriptional program resembling dysfunctional TILs from cancer patients. To uncover mediators of CAR-T cell dysfunction we selected 300 significantly upregulated genes in dysfunctional CAR-TILs, generated a CRISPR/Cas9 library, and performed an in vivo loss-of-function screen in mice bearing ovarian tumors. Using the MAGeCK algorithm, we identified 14 significantly enriched candidate genes, 10 of which were upregulated in CAR-T cells at early stages after treatment. Three of the candidates, including ZC3H12C, TG, and ITGB8 were selected for individual validation. Genetic ablation of all validated candidates enhanced CAR-T cell persistence and antitumor efficacy. Single-cell RNA-seq and ATAC-seq confirmed preferential expression of these genes in exhausted T cells in our model. Analysis of previously published single-cell datasets from human TILs validated the relevance of these candidate genes in TILs undergoing exhaustion in cancer patients. Top hit in our screen ZC3H12C (Regnase-3) was further characterized under chronic antigen stimulation. Genetic ablation of ZC3H12C enhanced HER2-CD28z CAR-T cell proliferation, cytotoxicity, and cytokine production in vitro, and improved antitumor responses and persistence in vivo. CAR-TILs from ZC3H12C-KO-treated mice showed superior functionality and reduced expression of exhaustion makers. Comparable results were observed in lung and pancreatic tumor models with Mesothelin-CD28z and CD19-BBz CARs. In conclusion, by using an in vivo custom CRISPR screen, guided by transcriptional data on dysfunctional CAR-TILs, we identified novel and promising gene targets for engineering CAR-T cells with enhanced efficacy against solid tumors. Paula Barbao, Alba Rodriguez-Garcia, Ariel Galindo-Albarrán, Marta Giménez-Alejandre, Pablo Clavero, Marta Botas, Teresa Lobo-Jarne, Guillem Colell, Joan Castellsagué, Guim Cascalló, Irene Andreu-Saumell, Marta Soria-Castellano, Berta Marzal, Beatriz Martin-Mur, Anna Esteve-Codina, Alvaro Urbano-Ispizua, Aleix Prat, Luca Gattinoni, Marco Antonio. Mendoza-Parra, Sonia Guedan. In vivo CRISPR-screen identifies determinants of early CAR-T cell dysfunction in solid tumors [abstract]. In: Proceedings of the AACR Immuno-Oncology Conference (AACR IO): Discovery and Innovation in Cancer Immunology: Revolutionizing Treatment through Immunotherapy; 2026 Feb 18-21; Los Angeles, CA. Philadelphia (PA): AACR; Cancer Immunol Res 2026;14(2 Suppl):Abstract nr C006.

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.

Abstract In PREDIX LumB patients with estrogen receptor positive and human epidermal growth factor receptor negative (ER + /HER2-) breast cancer > 20 mm and/or with lymph node metastasis were randomized 1:1 to receive either paclitaxel weekly for 12 weeks followed by palbociclib and endocrine therapy for 12 weeks (arm A), or the reverse sequence (arm B). Primary endpoint is objective radiologic response at 12 weeks (ORR12), and key secondary endpoints are ORR24, pathologic complete response, event-free survival, safety and correlative studies of tissue and circulating biomarkers. Whole exome sequencing and RNA sequencing were performed on baseline fresh frozen tissue samples. In total, 179 patients comprise the intention-to-treat population. There is no statistically significant difference between the two arms in ORR12 (59% vs 45%, p = 0.058). An exploratory gene expression analysis identified differentially expressed genes and gene sets between responders and non-responders at 12 weeks. A predictive signature, CDKPredX, comprising 31 genes related to proliferation, ER signaling and immune activity was developed to identify patients resistant to chemotherapy but responding to palbociclib plus endocrine therapy (pinteraction=0.03). The predictive signature was independently validated in the CORALLEEN trial (pinteraction=0.048). Clinicaltrials.gov identifier: NCT02603679

501 Background: In the NATALEE clinical trial, RIB + NSAI significantly improved invasive disease–free survival (iDFS) vs NSAI alone in patients (pts) with stage II/III HR+/HER2− EBC. We report on the prognostic and predictive value of baseline gene exp in NATALEE. Methods: Pts were randomized 1:1 to RIB + NSAI or NSAI alone. Men and premenopausal women received goserelin. Eligible pts had anatomical stage IIA (if N0 with additional risk factors [G3, or G2 with Ki67 ≥20% or high genomic risk] or N1 [1-3 axillary lymph nodes]), IIB, or III disease per AJCC (8th ed). Gene exp in surgical samples was profiled by NanoString BC360 panel. PAM50-based intrinsic subtype × treatment (tx) interaction was estimated by likelihood ratio test. Prognostic and predictive effects of PAM50 subtypes and genomic risk/proliferation signature scores created from gene exp data were assessed with Cox proportional hazards models. Differences in genomic risk and proliferation scores were analyzed by Wilcoxon test. Associations between gene exp and RIB benefit were estimated with Cox models. Results: In all, 3022 baseline surgical samples were analyzed. iDFS benefit with RIB + NSAI was consistent in the biomarker (hazard ratio [HR], 0.72) and intent-to-treat populations (HR, 0.71). PAM50 subtype distribution was comparable across tx arms and differed between N0 and N1-N3 in pts with luminal A (LumA; 50% vs 68%), luminal B (lumB; 41% vs 26%), and basal-like (BSL; 6.4% vs 2.5%) disease, with similar percentages for HER2-enriched (HER2E; 2.5% vs 3.0%). PAM50 subtypes were strongly prognostic (HRs vs LumA: LumB, 1.39; HER2E, 2.62; BSL, 3.92). RIB had benefit across all PAM50 subtypes (HRs: LumA, 0.77; LumB, 0.71; HER2E, 0.50; BSL, 0.42), with no significant subtype × tx interaction ( P = .34). Higher genomic risk signature or proliferation signature scores showed a trend for increased RIB benefit, again with no significant interaction. Pts with N0 vs N1-N3 had significantly higher genomic risk scores and proliferation scores. Exploratory analysis of the predictive effect of gene exp on RIB benefit identified several genes for which higher (eg, CEACAM6 , NOD2 , and GPX3) or lower exp (eg, GATA3 , SLC39A6 , and MAPT ) was associated with increased RIB benefit. Conclusions: In this analysis of NATALEE, which examined the largest dataset of surgical tumor samples from any adj CDK4/6i trial in HR+/HER2− EBC, RIB had benefit across all PAM50 subtypes, with a trend for increased benefit in pts with higher genomic risk signature or proliferation signature scores. Baseline exp levels of several genes were associated with differential RIB benefit in EBC, showing potential predictive and prognostic value. The findings reinforce the therapeutic benefit of RIB in combination with ET across HR+/HER2− EBC populations. Clinical trial information: NCT03701334 .

516 Background: In DESTINY-Breast05 (NCT04622319), T-DXd showed superior efficacy vs T-DM1 in patients (pts) with human epidermal growth factor receptor 2-postive (HER2+) early breast cancer (eBC) with residual invasive disease after neoadjuvant therapy (NAT) and high risk of recurrence—a population in which radiotherapy (RT) is broadly used. Safety was consistent with the established T-DXd profile; ILD remained an important risk, although most cases were low grade. Previous T-DXd pooled analyses suggested potential associations between ILD and clinical and demographic factors. We report additional safety outcomes stratified by these risk factors. Methods: Pts with residual invasive HER2+ eBC and at high risk of recurrence after NAT were randomized 1:1 to receive T-DXd 5.4 mg/kg (n = 818) or T-DM1 3.6 mg/kg (n = 817) once every 3 weeks for 14 cycles. All pts underwent low-dose non-contrast chest computed tomography (CT) at screening. Pts who received adjuvant RT (aRT; either concurrently with or sequentially before study therapy) underwent additional low-dose CT during and after treatment completion. CT scans showing ILD or RP underwent blinded central adjudication. Results: At data cutoff (July 2, 2025), 806 pts in the T-DXd arm and 801 pts in the T-DM1 arm received ≥1 dose of treatment; no pts were ongoing study treatment. In the T-DXd arm, 93.9% of pts received aRT vs 93.6% in the T-DM1 arm. Adjudicated drug-related (adj DR) ILD rates were generally consistent across global regions (Asia, Europe, North America [NA] + Australia, and Rest of World), though numerically lower in NA + Australia for T-DXd. In both arms, adj DR ILD rates were higher in pts from Japan (n = 147) vs outside Japan (n = 1460; T-DXd: 14.9% vs 8.9%; T-DM1: 6.7% vs 1.2%), Japan vs other Asian countries (T-DXd: 14.9% vs 9.6%; T-DM1: 6.7% vs 2.1%), and in pts with baseline moderate vs normal renal impairment (T-DXd: 14.3% vs 9.7%; T-DM1: 5.0% vs 1.0%). Adj DR ILD incidence was comparable across racial subgroups. In both arms, investigator-reported RP rates were higher in pts from Japan vs outside Japan (T-DXd: 47.1% vs 27.4%; T-DM1: 45.0% vs 27.3%). Most ILD/RP events recovered or were recovering with protocol-specific management. Recovery/resolution was observed in 64.5% vs 75.0% of adj DR ILD events and 38.2% vs 43.2% of RP events in the T-DXd and T-DM1 arms, respectively. Conclusions: Adj DR ILD and RP events were mostly low grade and reversible with protocol-specific management guidelines. Adj DR ILD rates were higher with T-DXd than T-DM1 and were influenced by country and baseline renal function, consistent with prior pooled analysis. These findings are consistent with, and further characterize, the safety profile of T-DXd in post-NAT HER2+ eBC. Clinical trial information: NCT04622319 .

Trastuzumab deruxtecan (T-DXd) is commonly used for treating metastatic breast cancer (MBC); however, traditional HER2 immunohistochemistry has largely failed to predict T-DXd activity. We reviewed survival outcomes and tested the reliability of multiple HER2 quantitative assays in predicting T-DXd's performance among 191 patients with MBC. We demonstrate that T-DXd's activity varies depending on the temporal evolution of HER2 immunohistochemical expression, with the longest activity observed among patients with HER2-positive disease or maintaining HER2-low disease across primary and metastatic settings. Quantitative HER2 assessment on pre-T-DXd samples showed that time-to-next treatment progressively increased by High Sensitivity-HER2 quartiles, Reverse Phase Protein Array HER2 quartiles, HER2DX ERBB2 mRNA scores and plasma-based DNADX HER2 signature tertiles (all with log-rank p < 0.05). Conversely, HER2 immunohistochemical subtypes showed limited predictive value for clinical outcomes. Additionally, elevated TOPO1 expression was associated with worse outcomes with T-DXd in HER2-negative breast cancer, suggesting potential relevance for payload-related markers in predicting T-DXd performance.

Background: Despite advances in endocrine therapy, patients with hormone receptor-positive (HR+)/human epidermal growth factor receptor 2-negative (HER2−) early breast cancer (eBC) remain at risk of recurrence (ROR). Chemotherapy is often used in high-risk cases, but not all patients benefit. Objectives: To evaluate whether chemotherapy can be safely omitted in patients with stage II, grade 2/3 HR+/HER2− eBC who convert to low genomic risk (ROR-low) following neoadjuvant ribociclib plus letrozole. Methods: RIBOLARIS (NCT05296746) is an open-label, multicenter phase II trial in patients with stage II, grade 2 or 3, HR+/HER2− eBC defined as high risk based on a Ki-67 index ⩾20% and/or high genomic risk by a validated gene expression signature. All patients receive six 28-day cycles of neoadjuvant ribociclib (600 mg/day, 3 weeks on/1 week off) plus letrozole (2.5 mg/day). Following surgery, patients with ROR-low or pathological complete response continue adjuvant ribociclib (400 mg/day for 30 months) and endocrine therapy (⩾5 years), whereas patients with ROR-medium/high receive standard chemotherapy followed by ribociclib and endocrine therapy. The primary endpoint is 5-year distant metastasis-free survival (DMFS) in the ROR-low cohort. Interim futility analyses are planned at 300 and 800 patient-years (PYs), with the primary analysis after 1300 PYs. The study targets enrollment of 1100 patients, anticipating 400 ROR-low cases, providing ⩾90% power to detect a 5-year DMFS of 96% versus a benchmark of 90.6% (MINDACT trial high-risk subgroup). Discussion: RIBOLARIS is the first prospective trial to evaluate a chemotherapy-free, ribociclib-based strategy in high-risk HR+/HER2− eBC based on neoadjuvant molecular response. By integrating genomic risk reclassification into treatment decision-making, this study aims to demonstrate that patients achieving ROR-low status can maintain excellent long-term outcomes without chemotherapy, while avoiding unnecessary exposure to adjuvant chemotherapy, and to highlight the role of tumor profiling after neoadjuvant endocrine therapy plus cyclin-dependent kinase 4 and 6 inhibitors in optimizing post-neoadjuvant therapy. Trial registration: NCT05296746; EudraCT# 2021-002322-24.

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