BACKGROUND:Genomic assays (GA) guide chemotherapy (CT) use in stage I-II endocrine receptor-positive (ER+)/HER2-negative (HER2-) breast cancer (BC). In tumors N0/intermediate-risk or N1/low-to-intermediate-risk, randomized trials with OncotypeDX® showed a CT benefit only for women aged≤ 50 years/premenopausal. Comparable data for the Prosigna® GA are lacking. METHODS:We retrospectively included 567 women aged≤ 50 years with stage I-II ER+ /HER2 - BC tested with Prosigna® across 10 hospitals in Spain/Italy (2014-2023). Patients received endocrine therapy (ET) with/without (neo)adjuvant CT. Event-free survival (EFS) was analyzed using Kaplan-Meier curves, log-rank tests, and Cox regression. Propensity score matching (PSM) was applied. 5-year EFS rates were numerically compared with those of OncotypeDX® trials. RESULTS:Of 567 patients, 73.7% were N0 and 26.3% N1, 39.7% were Prosigna risk-of-relapse (ROR)-low (RL), 33.0% ROR-intermediate (RI), 27.3% ROR-high (RH) and 48.3% received CT. CT independently improved EFS in N0/RI and N1/RL-RI (5-year EFS 97.9% vs. 86.6%; adjusted hazard ratio=0.07, p = 0.018). In premenopausal women, CT benefit persisted only when adjuvant gonadotropin-releasing hormone analogue was not administered (p = 0.008), especially in N0/RI (p = 0.023). Results were confirmed after PSM. CT-treated N0/RH showed similar EFS to CT-treated N0/RI+N1/RL-RI, while N1/RH showed poor prognosis despite CT use. 5-year EFS rates were generally consistent with OncotypeDX® trials. CONCLUSION:Prosigna can help identify young women with stage I-II ER+ /HER2- BC who gain benefit from (neo)adjuvant CT and those in need of further escalated treatments. In premenopausal N0/RI and N1/RL-RI disease, the effect of CT seems to be driven by ovarian function suppression. Prospective validation is required.

Hormone receptor-positive (HR+) breast cancer (BC) is characterized by a persistent risk of recurrence that continues for decades. Although many factors have been linked to recurrence, the clinical features associated with late relapse and patterns of distant metastases are less understood. Our study aimed to identify factors associated with early (< 5 years) and late (5–10 years) recurrence in a real-world cohort of HR+/human epidermal growth factor receptor type2-negative (HER2-) BC patients. Our study performed a retrospective analysis of stage I-III HR+/HER2- BC cases diagnosed between 1981 and 2022. Assessed variables included metastatic sites, early and late invasive recurrences. A total of 4,367 women with HR+/HER2- were included; 81.2

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.

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

e23344 Background: NTRK gene fusions have been described as oncogenic drivers in diverse adult and paediatric cancers, as they promote cell survival and proliferation. Larotrectinib is a first-in-class TRK inhibitor with proven activity in a broad range of solid tumors. This study aimed to describe the effectiveness of larotrectinib in patients with solid tumors harbouring NTRK fusions in Spain. Methods: SPAINTRK is an observational, retrospective study that included adult and paediatric patients with a confirmed diagnosis of solid neoplasms bearing NTRK fusions. All patients were treated with larotrectinib in the context of the compassionate drug use program between EMA approval and commercialisation in Spain (October 2019 – September 2023). Patients treated with larotrectinib in clinical trials were excluded. NTRK fusions were detected using NGS, FISH, and/or IHC, along with confirmatory molecular test. The primary endpoint was to determine the effectiveness of larotrectinib using Duration of Response (DoR). The secondary endpoints were objective response rate (ORR) and safety. Results: Between February and June 2025, 20 patients from ten months to 81 years were included, 15 adults and 5 paediatrics. Eight different solid tumor types with NTRK fusions were represented. NTRK fusions were reported in NTRK1 gene (40%), NTRK2 (25%) and NTRK3 (35%), 65% of patients were diagnosed using NGS. Larotrectinib was administered for a median of 13 months (95% CI: 8-21). 45% of patients received larotrectinib as first-line treatment while 55% had received previous treatments. After a median follow-up of 24.4 months (95% CI: 13.2-35), the median DoR was 24.5 months (95% CI: 11.1- not reached) and ORR was 60% (95% CI: 36.1-80.9). ORR was higher among pediatric patients (80%; 95% CI: 28.4-99.5). The highest ORR were seen among patients with infant fibrosarcoma (100%; 95% CI: 29.2-100) and lung tumors (80%; 95% CI: 28.44-99.5). After 1 year of treatment, 60% of patients remained progression-free and 75% of the patients who showed response maintained it. At data cutoff, 41.7% of the patients with a response were disease-free and/or remained on treatment. Treatment-related AEs were uncommon, with neutropenia and transaminitis being the most frequent, reported in up to 15% of patients each. Conclusions: Larotrectinib evoked long antitumor responses in solid tumours with NTRK fusion in the real world, regardless of the tumor type. No safety concerns were reported, even after long-term administration. Screening techniques such as NGS should be implemented, and although its use is increasing, broader access is required. Clinical trial information: NCT06837090 .

BACKGROUND & AIMS:Cyclin-dependent kinase 4/6 inhibitors (CDK4/6i)-ribociclib, abemaciclib, palbociclib-are the standard of care for metastatic breast cancer. Although hepatotoxicity is a frequent adverse event, data on CDK4/6i drug-induced liver injury (DILI) remain scarce. We aimed to define the incidence, clinical presentation, severity, management, and relapse of CDK4/6i-related DILI in a large real-world cohort. METHODS:Retrospective multicentre study including all patients who developed ≥grade 2 DILI (alanine aminotransferase/aspartate aminotransferase >3 × upper limit of normal) while receiving CDK4/6i between January/2018 and December/2022. Severity was assessed according to Common Terminology Criteria for Adverse Events version 5 (CTCAEv5) and DILI-International Expert Working Group criteria. RESULTS:Among 2,222 CDK4/6i-treated patients, 100 (4.5%, 95% CI 3.7-5.4%) developed DILI. Incidence was significantly higher for ribociclib (8.3%, 95% CI 6.3-10.7%) and abemaciclib (6.7%, 95% CI 4.6-9.3%) than for palbociclib (1.4%, 95% CI 0.8-2.2%) (Difference, p <0.001). Most cases were grade 2 or 3 (CTCAE) and mild (DILI-International Expert Working Group), although moderate DILI (bilirubin >2 × ULN, n = 12) occurred with all three CDK4/6i. The most frequent pattern was hepatocellular, whereas cholestatic was mainly observed with abemaciclib. Liver biopsy findings (n = 11) were heterogeneous and, together with biochemical results, did not support an immune-mediated mechanism as the primary driver. Corticosteroids (n = 20) did not impact time to grade 1 or alanine aminotransferase normalisation. DILI recurred in 28.2% of CDK4/6i rechallenges (n = 85). Among patients treated with ribociclib, rechallenge with an alternative CDK4/6i was associated with lower risk of recurrence (p = 0.010). CONCLUSIONS:DILI caused by ribociclib and abemaciclib are more common that with palbociclib. The absence of immune-mediated features and the limited benefit of corticosteroids question their routine use. After CDK4/6i reintroduction, DILI recurrence rate was 28%. Standardised management is needed, particularly following the approval of ribociclib and abemaciclib as adjuvant therapy. IMPACT AND IMPLICATIONS:Although hepatotoxicity is a frequent adverse event, real-world data on CDK4/6i DILI remain scarce. DILI associated with ribociclib and abemaciclib is more common than for palbociclib, although moderate DILI (bilirubin >2 × normality) was observed with the three drugs. The absence of immune-mediated features and the limited benefit of corticosteroids question their routine use in CDK4/6i DILI. Relapse of DILI after rechallenge with a CDK4/6i was 28%, with all cases presenting as mild. The increasing use of CDK4/6i (metastatic breast cancer and ribociclib and abemaciclib as adjuvant therapy) highlights the need of standardising the management of CDK4/6i DILI.

Hormone receptor-positive, human epidermal growth factor receptor 2-negative (HR + /HER2 −) breast cancer (BC) is the most frequently diagnosed subtype of BC, accounting for approximately 70

PURPOSE:HER2DX is a validated genomic assay used to support treatment decisions in early-stage HER2-positive (HER2+) breast cancer. It provides three scores: relapse risk, likelihood of pathologic complete response (pCR), and ERBB2 mRNA expression. This study aimed to evaluate the association between HER2DX and histopathologic features and assess its relationship with pCR after neoadjuvant therapy. EXPERIMENTAL DESIGN:Patients with newly diagnosed stage I to III HER2+ breast cancer were analyzed based on available HER2DX results during routine care in Spain (January 2022-June 2025). Centralized HER2DX testing was performed on formalin-fixed, paraffin-embedded tumor samples. Histopathologic analysis included tumor grade, hormone receptor status, histologic subtype, Ki67 index, HER2 IHC score, stromal tumor-infiltrating lymphocytes (TIL), tertiary lymphoid structures, and spatial immune distribution. Univariate and multivariable logistic regression analyses were conducted to identify factors associated with pCR after neoadjuvant trastuzumab-based therapy. RESULTS:A total of 410 HER2+ tumors were analyzed, and 250 patients received neoadjuvant trastuzumab-based therapy with available surgical outcomes (36% achieved a pCR). HER2DX pCR scores were significantly associated with all eight histopathologic features, whereas relapse risk and ERBB2 scores were associated with five and two, respectively. TIL correlated with the immune/immunoglobulin signature (r = 0.59), and Ki67 with the proliferation signature (r = 0.50). The HER2DX pCR score remained the only independent predictor of pCR in multivariable analysis (OR, 1.77; 95% confidence interval, 1.08-2.97; P = 0.030). CONCLUSIONS:HER2DX reflects key biological and pathologic features of HER2+ breast cancer and independently predicts pCR, supporting its utility for individualized treatment decision-making.

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.

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.

In advanced HER2-positive breast cancer, the standard taxane-trastuzumab-pertuzumab (THP) regimen faces competition from new therapies, emphasizing the need for biomarkers to guide treatment. This study evaluates the HER2DX ERBB2 mRNA score as a prognostic predictor, aiming to tailor treatment strategies. We retrospectively analyzed 94 patients treated with the THP regimen between 2010 and 2024. The HER2DX ERBB2 mRNA score was categorized as low (n = 14), medium (n = 20), or high (n = 60), and its correlation with progression-free survival (PFS) and overall survival (OS) was assessed using Cox regression models. The median follow-up was 31.5 months. Patients with ERBB2-high scores had significantly better median PFS (33.9 vs. 10.6 months, hazard ratio [HR] = 0.40, 95% CI: 0.24–0.69, p < 0.001) and OS (not reached vs. 30.8 months, HR = 0.26, 95% CI: 0.13–0.49, p < 0.001) compared to ERBB2-low patients. Based on these findings, further validation of this biomarker in tumor samples from the CLEOPATRA phase III trial is ongoing, which could help optimize treatment strategies in this population.

553 Background: Approximately one third of all TNBC diagnoses are stage 1. No validated biomarker is routinely utilized to guide treatment at this early stage. Methods: Samples from patients with stage I TNBC or ER-low (1-10%) breast cancer undergoing surgery at Dana-Farber/Brigham Cancer Center between 2016 and 2021 were identified. The 10-gene Core Immune Gene (CIG) signature and the 4-gene proliferation signature (both part of the TNBC-DX tool) were derived from extracted RNA. Central evaluation of sTILs was conducted at Dana-Farber, with 5% and 20% used as thresholds. All markers were tested for prediction of recurrence free survival (RFS) using the Kaplan-Meier method. Results: We identified 253 patients with stage I TNBC (n=218) or ER-low tumors (n=35) treated at Dana-Farber. Median age was 61 (31 – 85), most tumors were ductal (89%), high-grade (73%), 48% were >1 cm and 65% received chemotherapy. 5-year RFS in the overall cohort was 86.8%, with numerical variation by tumor size (T1a 100%, T1b 93.8%, T1c 81.7%, p=0.26). Gene signatures and sTILs were obtained for 117 and 123 patients, respectively (both for 110 patients), with their association with outcomes described in Table 1. Median follow-up was 3 years. A total of 20/117 patients (17.1%) had medium-high CIG score, with none experiencing RFS events prior to year 5. Similarly, no recurrence was observed prior to year 5 in 29 patients (24.8%) at the upper CIG quartile (vs 83-88% 5-year RFS in other quartiles). Worse outcomes were seen among patients in the upper quartile of proliferation (5-year RFS 83%, vs 88-100% in other quartiles). Overall, 33/123 patients (26.8%) had high sTILs (>20%) and experienced the highest 5-year RFS (97%, vs 78% if low sTILs). OS data will be presented. Conclusions: High expression of the 10-gene CIG immune signature or high sTILs are associated with numerically improved outcomes in patients with stage I TNBC that did not reach statistical significance, warranting further study as prognostic tools. 3- and 5-year recurrence free survival (RFS) according to gene signatures and sTILs. P values were obtained via Cox proportional hazard models. N 3-year RFS 5-year RFS CIG score - Low- Med-High 9720 91% (85%, 98%)100% (100%, 100%) 89% (80%, 98%)100% (100%, 100%) CIG score (quartiles)- ≤25%- 25-50%- 50-76%- >75% 49102929 94% (87%, 100%)83% (58%, 100%)87% (74%, 100%)100% (100%, 100%) 88% (74%, 100%)83% (58%, 100%)87% (74%, 100%)100% (100%, 100%) Proliferation score - Low- Med-High 5859 96% (89%, 100%)90% (82%, 99%) 90% (78%, 100%)90% (82%, 99%) Proliferation score (quartiles)- ≤25%- 25-50%- 50-76%- >75% 30292929 100% (100%, 100%)90% (77%, 100%)100% (100%, 100%)83% (68%, 100%) 88% (67%, 100%)90% (77%, 100%)100% (100%, 100%)83% (68%, 100%) sTILs- 1-5%- >5-20%- >20% 622833 94% (87%, 100%)91% (81%, 100%)97% (90%, 100%) 78% (63%, 98%)91% (81%, 100%)97% (90%, 100%)

Background: The implementation of germline genetic testing is complex due to emerging therapeutic indications such as adjuvant Olaparib for BRCA-mutated breast cancer (BC). Understanding the clinical-pathological characteristics of individuals meeting testing criteria for genetic testing can provide insights into the factors associated with identifying pathogenic germline variants (PVs) and likely pathogenic germline variants (LPVs). This knowledge can enhance the precision of genetic counseling and testing strategies. In this study, we reviewed our single-center experience of testing selected patients meeting clinical and pathological features, to develop a progressive and more sustainable model on the way towards universal germline screening. Methods: We evaluated 1,060 consecutive individuals with a personal history of breast cancer (BC) who met regional criteria for germline testing at the Hospital Clinic of Barcelona between 2016 and 2022. We excluded individuals diagnosed before 2000 (n=70), those women with DCIS (n=60), and those with missing clinical information (n=18). Clinical-pathological and molecular characteristics between carriers of PV/LPV and non-carriers. Chi-square tests or Student’s t-tests were used to compare the distribution of variables between two groups. Logistic regression analysis was then employed to evaluate the association of each variable with PV/LPV. The significance level for all statistical analyses was set at a two-sided alpha of 0.05. Results: A total of 912 individuals were evaluated. Most cases were women (n=898, 98.5%) with a median age of 49 (range 24-88) and had a family history (n=706, 78.3%). The main reasons for testing were family aggregation (n=357, 39.1%), BC onset ≤40 yrs-old (n=199, 21.8%), and triple-negative breast cancer (TNBC) onset ≤60 yrs-old (n=162, 17.7%). The rate of PV/LPV was 14.9% (n=136), and 151 individuals (16.6%) had variants of unknown significance (VUS). Overall, 776 (85.1%) had no PV/LPV identified. A higher number of PV/LPVs were identified in the BRCA2 gene (n = 41, 30.1%), followed by BRCA1 (n=31, 22.8%), CHEK2 (n=17, 12.5%), ATM (n=15, 11.0%), PALB2 (n=13, 9.6%), BRIP1 (n=5, 3.7%), TP53 (n=5, 3.7%), BARD1 (n=2, 1.5%), MSH2 (n=2, 1.5%), PTEN (n=2, 1.5%), BAP1 (n=1, 0.7%), CDKN2A (n=1, 0.7%), and RAD51C (n=1, 0.7%). Notably, 2 individuals with a PV in BRCA2 had another PV (i.e., MSH6 and CDK2NA), and 2 individuals with a PV in PALB2 had another PV (i.e., both in CHEK2). Most VUS (n=151) were identified in ATM (n=38, 25.2%), BRCA2 (n=23, 15.2%), PALB2 (n=15, 9.9%), MSH6 (n=12, 7.9%), and BRCA1 (n=10, 6.6%). The BC subtype distribution was 62.4% (n=563) HR+/HER2-, 15.0% (n=141) HER2+, and 22.0% (n=198) TNBC. The clinical-pathological variables significantly associated in univariate analyses with the identification of PV/LPV were sex, age, personal history of other cancer types, advanced TNM stage, TNBC, and bilateral BC. In a multivariable analysis, all the previously mentioned variables remained significantly associated with the identification of PV/LPV. The highest odds ratios (OR) were found for the following 2-group variables: male (OR=4.8), stage IV versus stage I (OR=3.1), bilateral BC (OR=2.7), other personal histories of cancer (OR=2.2), and TNBC (OR=1.7). Age was considered a continuous variable, and for every 10-year increase, the odds of detecting a PV/LPV decreased by approximately 34%. Conclusions: Based on established historical testing criteria, specific clinicopathological features were significantly and independently associated with the detection of clinically significant variants. As we move towards universal germline screening for breast cancer, well-established information continues to help prioritize individuals who will benefit most from early detection of breast cancer susceptibility. Citation Format: Adela Rodriguez Hernandez Barbara Adamo, Fara Brasó-Maristany, Benedetta Conte, Olga Martínez-Sáez, Miriam Potrony, Lorena Moreno, Elia Grau, Esther Sanfeliu, Raquel Gómez, Isabel García, Beatrice Fatrini, Elia Segui, Maria Vidal, Montserrat Muñoz, Teresa Ramón y Cajal, Francesc Balaguer, Aleix Prat, Barbara Adamo. Towards Universal Germline Screening for Breast Cancer, Planning for a Sustainable Future: A Single-Center Retrospective Analysis [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 P1-03-01.

Background: The identification of biomarkers for evaluating sensitivity to endocrine therapy in early breast cancer (EBC) is critical. Assessing the dynamic biological changes in the tumor caused by brief pre-operative endocrine therapy (POET) can guide decisions on reducing or intensifying treatment. In this study, we examined the molecular changes induced by short-term POET and their correlation with the treatment's effectiveness. Methods:This is a retrospective study of paired samples from patients (pts) with hormone receptor-positive and HER2-negative (HR+/HER2-) EBC treated at Hospital Clinic of Barcelona between 2014 and 2023 and from the letrozole arm of SOLTI-1501 VENTANA trial (Adamo et al. BCR 2019; NCT02802748). All pts received POET for 2 to 12 weeks prior to surgery, with tamoxifen or aromatase inhibitors (AI), administered according to menopausal status. RNA expression was assessed in baseline and surgery samples, including PAM50 and HER2DX signatures. Treatment response was defined as a value of Ki67≤10% at surgery. Logistic regression models explored the association between baseline gene expression and response. Gene expression changes were analyzed using paired SAM analysis and t-tests. Results: A total 111 pts with both baseline and surgery samples available were included. Median age was 63 years-old (61.8-66.4) and most of the tumors were cT1 (68.5%) and cN0 (97.3%) at diagnosis. 19 pts (17.1%) were premenopausal and 92 (82.9%) postmenopausal. The median baseline Ki67 was 18% (14-25%). After POET, the median Ki67 value was 4% (1-10). 81 pts (73%) reached Ki67≤10% at surgery and 41 (37%) Ki67≤ 2.7% (complete cell cycle arrest). At baseline, PAM50 molecular subtype distribution was: 79 pts (71.2%) Luminal A, 23 (20.7%) Luminal B, 4 (3.6%) HER2-enriched, 3 (2.7%) Normal-like, 2 (1.8%) Basal-like. At surgery, there were 82 (73.9%) Luminal A, 23 (20.7%) Normal-like, and 6 (5.4%) Basal-like tumors. In the univariate analysis, baseline clinicopathological variables associated with response were age (odds ratio [OR]=1.04, p=0.028), percentage of estrogen receptor (OR=1.03, p=0.025), Ki67 value (OR=0.95, p=0.003) and type of endocrine therapy (tamoxifen vs AI, OR=0.17, p<0.001). In terms of baseline gene expression, high Luminal A signature (OR=7.72, p<0.001) and luminal-related genes (e.g.: FOXA1 [OR=1.46, p=0.021] or ESR1 [OR=1.38, p=0.001]) were associated with response, while high Basal-like (OR=0.19, p=0.009), PAM50 proliferation (OR=0.30, p=0.005) and HER2DX proliferation (OR=0.24, p=0.037) signatures and proliferation-related genes (e.g.: MYBL2 [OR=0.58, p<0.003] or MKI67 [OR=0.71, p=0.004]) were associated with no response. After POET, all genes and signatures were up- or downregulated significantly. In particular, we observed a significant decrease of the PAM50 Luminal and proliferation signatures, as well as the HER2DX proliferation and luminal signatures, with an increase of the HER2DX immune (IGG) and HER2 amplicon signatures, both in responders and non-responders (FDR<5%). Conclusion: POET is a simple and secure treatment that can be administrated before surgery in HR+/HER2- EBC. The molecular profiling and dynamic evaluation of biological changes induced by POET could offer opportunities for a better understanding of the tumor´s sensitivity to endocrine therapy and could help guide and optimize treatment strategies in HR+/HER2- EBC. Citation Format: Raquel Gómez-Bravo, Barbara Adamo, Benjamin Walbaum, Esther Sanfeliu, Blanca González-Farré, Francesco Schettini, Olga Martínez-Sáez, Elia Seguí, Isabel García-Fructuoso, Paula Blasco, Oleguer Castillo, Ángela Aguirre, Valeria Sirenko, Pol Giménez, María Rey, Jordi Canes, Patricia Galván, Tomás Pascual, Maria Vidal, Adela Rodriguez Hernandez, Eva Ciruelos, Meritxell Bellet, Aleix Prat, Montserrat Muñoz, Fara Brasó-Maristany. Molecular effects of short pre-operative endocrine therapy in hormone receptor-positive and HER2-negative early breast cancer [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 P2-05-18.

To explore the role of the immune system in early-stage HER2+ breast cancer (HER2+ BC), focusing on how T-cell receptor (TCR) dynamics relate to the prognostic 14 B-cell gene/IgG immune signature (IGG) included in the clinically available HER2DX genomic test. This study aims to clarify how TCR diversity and targeting of tumor-associated antigens (TAA) contribute to patient outcomes and could inform potential therapeutic strategies. TCR/BCR clones were identified by PCR amplification and deep sequencing (ImmunoSEQ) in 41 early-stage HER2+ BC samples. CDR3 sequences were cross-referenced with the VDJ database, excluding inconclusive matches (VDJdb score 0-1). Protein expression was analyzed by digital spatial profiling (GeoMx) in 23 samples. In 6 samples, TCR identification was performed through single-cell RNA sequencing (scRNAseq; Chromium). IGG expression in each sample was evaluated using the HER2DX assay and correlated with bulk RNA data from TCGA and MTBC datasets. Spearman’s correlation and Wilcoxon tests were used for statistical analysis (R software). Of the 12,575 TCR clones with predicted targets, 759 (5.9%) showed reliable matches (score 2-3). 53 of them (7%) recognized TAA, primarily MART1 (18.9%), followed by gp100, ABCD3, MAGEA6, KRAS, NY-ESO1, p53, TERT, and others. Most non-human epitopes belonged to common viruses such as Influenza A (31.2%), EBV (30.7%), and CMV (20%). IGG expression was correlated with the number of TCR templates (Cor: 0.47, p<0.01), TCR entropy (Cor: 0.60, p<0.001), and shared TCR clonotypes between samples (Cor: 0.48, p<0.01). IGG was higher in samples with TCR clones against TAA (p50 75.6 vs. 61.1, p=0.044). A positive correlation was observed between the number of clones targeting human and viral epitopes (Cor: 0.44, p=0.013). The scRNAseq data confirmed that IGG-high samples exhibit greater TCR polyclonality and a higher fraction of cytotoxic CD8+ T cells (p<0.05), with upregulated perforin and granzyme A/B expression. IGG correlated with CD27 (Cor: 0.47, p=0.025) and CD3 (Cor: 0.52, p=0.011) protein levels, as well as the IFN-γ signature in TCGA (Cor: 0.56, p<0.01) and MTBC (Cor: 0.71, p<0.01) data. Fibronectin correlated with PD1 (Cor: 0.61, p<0.01) and CTLA4 (Cor: 0.81, p<0.001) levels, and inversely with IGG (Cor: -0.50, p=0.017), indicating that IGG-low tumors might have a denser stroma and a more exhausted, less active immune infiltrate. Early-stage HER2+ BC with high IGG expression is characterized by a robust, polyclonal immune response, with TCR clones targeting both tumor and viral antigens. These findings suggest enhanced immune fitness and may explain IGG favorable prognostic value. The discovery of shared TCR clones across tumors may help identify immunogenic targets, providing new opportunities for developing immune-based treatments or engineered T-cell therapies. Víctor Albarrán-Fernández, Carlota Rubio-Pérez, Patricia Galván, Oleguer Castillo, Paula Blasco, Esther Sanfeliu, Anabel Martínez-Romero, Mercedes Marín, Patricia Villagrasa, Francisco Pardo, Laia Paré, Isabel García-Fructuoso, Raquel Gómez, Elia Seguí, Bárbara Adamo, Benjamin Walbaum, Olga Martínez-Saez, Tomás Pascual, María Vidal, Montserrat Muñoz, Sònia Guedan, Fara Brasó, Laura Angelats, Aleix Prat. Uncovering T-cell receptor clones and immunogenic targets in HER2DX-defined HER2-positive breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 5867.