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.

BackgroundPregnancy-associated breast cancer (PrBC) poses complex challenges in diagnosis and treatment, particularly when associated with biologically aggressive subtypes and extensive nodal involvement. Management must be individualized, integrating oncologic urgency, fetal safety, and limited validated evidence in this unique setting.Case SummaryWe present the case of a 36-year-old woman diagnosed during the second trimester of pregnancy with HER2-positive (HER2+), node-positive (cT2[m]N3a) breast cancer (BC). After a multidisciplinary team discussion, patient initiated anthracycline- and taxane-based neoadjuvant chemotherapy during gestation. Given its contraindication during pregnancy, anti-HER2 therapy was added postpartum, and surgery included nipple-sparing mastectomy with targeted axillary dissection (TAD) of clipped nodes. Pathology revealed minimal residual invasive disease in the breast and a complete axillary response, allowing omission of axillary lymph node dissection (ALND). Genomic profiling with HER2DX supported high-risk disease and informed systemic therapy with delayed anti HER2 therapy, and conservative axillary management (TAD without ALND) in cT2N3 PrBC, without compromising fetal outcome. The patient subsequently received adjuvant chest wall and nodal region radiotherapy plus trastuzumab-emtansine (T-DM1).ConclusionThis case underscores the value of personalized, multidisciplinary management in PrBC, particularly in patients with high-risk biologic features and advanced nodal disease. Integrating clinical judgment, genomic tools, and adaptive strategies, while accounting for gestational limitations, can optimize oncologic outcomes without compromising fetal safety.

Trastuzumab, pertuzumab, and a taxane (THP) has been the standard first-line therapy for HER2+ advanced breast cancer for over a decade. With new regimens emerging, genomic tools like HER2DX may help identify patients who benefit durably from THP versus those requiring intensification. Here, baseline tumor tissue from 122 patients with HER2+ treated with THP in Poland was tested with HER2DX. A previously published Spanish real-world cohort (n = 93) was added to generate a combined cohort (n = 215). Univariable analyses were performed in the Polish cohort, and multivariable Cox and logistic regression models were applied to the combined cohort. A HER2DX metastatic prognostic score was trained on overall survival (OS) in the Spanish cohort and validated in the Polish cohort. In the Polish cohort, high ERBB2 mRNA scores were associated with significantly longer real-world progression-free survival (rwPFS) (33.8 vs. 17.9 months; hazard ratio [HR] 0.57; p = 0.022) and real-world overall survival (rwOS) (75.1 vs. 40.2; HR 0.48; p = 0.009). In the combined cohort, ERBB2 high-score tumors showed prolonged rwPFS (33.8 vs. 12.5; HR 0.50; p < 0.001) and rwOS (not reached vs. 37.1; HR 0.36; p < 0.001), and higher rwORR (84.4% vs. 52.0%; p < 0.001). Prognostic value was independent of clinical variables, including number of metastatic sites. Subgroup analyses showed particularly favorable outcomes in patients with <3 sites (median rwPFS 51.7 vs. 20.3 months). The HER2DX metastatic prognostic score outperformed ERBB2 alone in the validation cohort. In conclusion, the HER2DX ERBB2 mRNA score provides independent prognostic information in HER2+ advanced breast cancer treated with THP. The HER2DX metastatic prognostic score further improves prognostic accuracy.

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 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.

Abstract Introduction Immune check-point inhibitors (ICI) were a major breakthrough in cancer care, but optimal patient selection remains elusive in most tumors. Methods Overall 173 adult patients with metastatic solid tumors candidates to ICI in clinical trials at our Institution were prospectively recruited. Blood samples were collected at cycle 1 (C1D1) and 2 (C2D1) and until the occurrence of progressive disease (PD). C1D1 LIPI, RMH, PMHI, NLR, dNLR, PIPO and GRIm prognostic scores were calculated. The primary endpoint was identifying the best score to predict rapid PD (≤ 4 months) with ICI using logistic regressions accounting for tumor type, and receiving operators characteristics (ROC) with area under curve (AUC), accompanied by an extensive comparison of the score performances in the prediction of overall survival (OS), progression-free survival (PFS), overall response rates (ORR) and durable clinical benefit (DCB). Secondary objectives included describing study cohort outcomes and studying the association between the selected score at C1D1, C2D1 and its dynamics with OS and PFS. Results C1D1 LIPI was the best predictor of rapid PD, OS and PFS, regardless of cancer type, compared to other scores. No score was associated to ORR and only RMH to DCB. Baseline LIPI detected three categories of patients with significantly different OS (p < 0.001) and PFS (p = 0.013). The same was observed at C2D1 for OS and PFS (both p = 0.020). Significant LIPI class shifts were observed in the overall population (p < 0.001), rapid progressors (p = 0.029) and non-rapid progressors (p = 0.009). Retaining a good LIPI or experiencing a shift towards a better prognostic class was associated to improved OS (p = 0.009) and PFS (p = 0.006). C2D1 LIPI, but not C1D1, remained significantly associated to rapid PD in multivariable analysis. Conclusions LIPI may improve patient selection for ICI and guide treatment adjustments according to on-treatment dynamics in a pancancer context.

PURPOSE:The implementation of the next-generation sequencing (NGS) in clinical practice has improved the genetic diagnosis of Hereditary Breast and Ovarian Cancer Syndrome (HBOC). We aimed to evaluate the diagnostic outcomes of using an NGS cancer gene panel in clinical practice for patients selected based on personal and/or family history of breast, ovarian, prostate, melanoma, and other HBOC-associated cancers. METHODS:The study series included 2561 consecutive Spanish individuals referred for genetic testing, comprising 2445 cancer patients and 116 healthy individuals with family history of HBOC. Eleven HBOC susceptibility genes (BRCA1, BRCA2, PALB2, ATM, CHEK2, BARD1, BRIP1, RAD51C, RAD51D, TP53, and PTEN) and three Lynch Syndrome genes (MLH1, MSH2, and MSH6) available for opportunistic testing were analyzed using a commercial Hereditary Cancer Panel and an in-house bioinformatics pipeline. RESULTS:Overall, the diagnostic yield was 11.0% in cancer patients and 8.6% in healthy individuals with a family history of breast/ovarian cancer. Pathogenic variants in high-risk genes were more frequent in patients with multiple HBOC tumors and a family history of different HBOC cancers. Additionally, we diagnosed five families with Lynch syndrome through opportunistic testing. CONCLUSION:Testing cancer susceptibility genes using an agnostic strategy confers a diagnostic benefit for hereditary cancer syndromes compared to phenotype-driven test, without adding complexity to the study. The analysis of healthy individuals with a family history of HBOC detects pathogenic variants in a cost-efficient percentage of cases, resulting in a good alternative strategy when the index case is unavailable.

Introduction: Women diagnosed with breast cancer (BC) before age 40 (YWBC) experience a substantially higher risk of recurrence and mortality than their older counterparts. The global trends in BC incidence and mortality among YWBC are scarcely described, although recent studies suggest a rise among certain high-income countries. Material and methods: We conducted a retrospective analysis including all consecutive newly diagnosed BC patients (NBC) between January 2014 and December 2023 at Hospital Clinic of Barcelona, Spain. Clinical and immunohistochemistry characteristics (estrogen receptor [ER], progesterone receptor [PR], HER2 status and Ki67 expression) determined BC subrogated subtypes: hormone receptor-positive (HR+)/HER-2 negative (HER2-), HER2-positive (HER2+) and triple negative (TN). YWBC incidence (YI) was defined as the yearly ratio between YWBC and NBC. Our primary objective was to report the YI over the years and determine if there was any variation. We also assessed incidence differences among immunohistochemical subtypes, as well as the YI in very young BC pts (≤ 35 years, VYWBC). Incidence difference across years was evaluated using the Kruskal–Wallis test, with a significance p-value <0.05. Results: A total of 262 YWBC pts were included, accounting for 9.6% of NBC over the 10-year period, ranging from a minimum of 7.38% in 2020 to a maximum of 14.02% in 2018. When analyzing YI over the years, we observed an initial increase until 2018, followed by a decline and stabilization at a steady percentage, with no statistically significant differences (p=0.44). With a median age of 37 years (range: 18 to 40 years), the majority (76.33%) were diagnosed at early stages (i.e., stage II or less). Stages II and III represented 38.50%, whereas 6.11% were metastatic at diagnosis, without significant variations throughout the period (p=0.44). Most YWBC were ER-positive (66.41%), PR-positive (53.05%), HER2-negative (62.6%), and had a Ki67 >20% (52.29%). Most tumors were HR+ (54.23%) throughout the entire period, with minimal variation across the years. The proportion of HR+ vs HR-negative (HR-) tumors remained stable over the 10 years studied (p=0.44). HER2+ tumors accounted for 23.66% of YWBC, ranging from 10.34% (2023) to 43.48% (2018). On the other hand, TN tumors represented 21.76% of the cases, with a range from 5.56% (2014) to 41.38% (2023) during the period, again with no significant change. VYWBC represented a steady 4% of NBC, with a non-significant tendency to decrease in recent years (reaching 1.84% in 2023). Stages II and III represented 38.51%, without significant changes throughout the period (p=0.437). Across subtypes, HR+ tumors were 47.19%, HER2+ 24.57% and TN, 27.03%. Interestingly, among YWBC, VYWBC represented a third of the new cases, peaking in 2020, followed by a numerical non-significant reduction in incidence (p=0.44). Conclusions: YWBC incidence has remained stable over the years. In our population, the YI is consistent with previous studies, ranging from 5-10% depending on the country, and slightly higher than the YI reported for Spain (6.6% in 2022). Both in YWBC and VYWBC, no variation was observed regarding stage and subtypes over time. Overall, these results indicate that over the past ten years there have not been significant changes in the YI, nor a trend towards more aggressive phenotypes among YWBC at our institution. Citation Format: Francisco Javier Muñoz i Carrillo, Benjamin Walbaum, María Rey, Carme Crous, Clara Rodrigo, Esther Sanfeliu, Fara Brasó-Maristany, Isabel Garcia-Fructuoso, Elia Seguí, Raquel Gómez-Bravo, Barbara Adamo, Tomás Pascual, Olga Martínez-Sáez, Francesco Schettini, Núria Chic, Montserrat Muñoz, Aleix Prat, Maria Vidal. Clinico-Pathological Characteristics and Incidence Trends of Breast Cancer in Young Women: A 10-Year Single Institution Retrospective Study [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 P2-12-13.

Purpose The impact of preoperative radiation therapy (RT) on early-stage breast cancer (BC) is underexplored but may significantly improve outcomes and offer new therapeutic strategies. The YOUNGSTER study aimed to characterize the molecular changes induced by preoperative RT across BC subtypes: luminal A, luminal B, human epidermal growth factor receptor 2 (HER2)-enriched, and basal-like. Methods and Materials This exploratory study enrolled 20 patients with early-stage BC who were eligible for breast-conserving surgery and had not received prior treatment. Biological changes were assessed between baseline, 3 to 5 days post-RT, and surgical samples. A preoperative RT boost (5 × 2.67 Gy/fraction) was administered, followed by a core needle biopsy. Patients then proceeded to either surgery 4 weeks (2-8 weeks) after RT or neoadjuvant therapy. Gene expression was analyzed using a 192-gene panel, alongside immunohistochemistry for Ki67 and CD68, tumor-infiltrating lymphocytes quantification, and γH2AX staining for DNA damage assessment. Results At baseline, PAM50 subtype distribution was luminal A (35%), luminal B (25%), HER2-enriched (20%), and basal-like (20%). Early post-RT samples showed significant downregulation of proliferation genes, PAM50 proliferation signature, and Ki67 immunohistochemistry staining, increased DNA damage, and macrophage marker upregulation. In primary surgery samples (2-8 weeks post-RT) (n = 13), adaptive immune markers showed significant upregulation, along with a 14-gene immunoglobulin signature increase. Conclusions Preoperative RT induces early and late biological changes in BC, with initial effects on proliferation reduction and DNA damage within days, followed by adaptive immune activation within weeks. RT may serve as an effective primer for immunotherapy, especially in higher-risk subtypes, supporting the potential for combinatorial approaches in BC management.

Does ovarian hormonal stimulation with gonadotropins combined with Letrozole modify breast cancer gene expression? Ovarian stimulation with gonadotropins and Letrozole in breast cancer patients appears biologically safe, with gene expression changes suggesting no negative impact on tumor characteristics. Fertility preservation protocols are commonly used in women diagnosed with breast cancer, with oocyte cryopreservation being the standard approach. The relationship between elevated estradiol levels and breast cancer development is well-established, leading to the use of Letrozole during controlled ovarian stimulation to minimize the increase of serum estradiol levels. Clinical follow-up studies in these patients show no increase in recurrence or worse prognosis after oocyte cryopreservation. However, there are no studies examining histological and gene expression changes in breast tumor tissue after ovarian stimulation with gonadotropins and Letrozole. This longitudinal, prospective, observational study included 21 premenopausal women under 40 years old diagnosed with early-stage breast cancer and eligible for oocyte cryopreservation. Patients were recruited from a single assisted reproduction unit between October 2020 and September 2024. Ovarian stimulation consisted of daily subcutaneous administration of recombinant follicle-stimulating hormone combined with an aromatase inhibitor (Letrozole) to stimulate multiple follicle recruitment while minimizing circulating estradiol levels. Breast tumor samples were obtained by core needle biopsy pre-stimulation and within 24–48 hours post-follicle aspiration and oocyte retrieval. Gene expression in formalin-fixed paraffin-embedded tumor samples was analyzed using a 72-gene nCounter panel, encompassing PAM50 subtypes and the Risk of Recurrence (ROR) score. Paired Significance Analysis of Microarrays (SAM) with a False Discovery Rate <5% and t-tests were used to assess significant gene expression changes. The mean age was 31.9 years (range: 23–39). The mean stimulation length was 10 days (range: 6–15), with peak estradiol levels of 516.4 ± 527.5 pg/mL on the trigger day. The mean number of retrieved oocytes was 8 (range: 2–23), and the mean interval between oocyte retrieval and biopsy was 1 day (range: 0–2). Histological analysis showed no significant differences in the Ki67 proliferation index (41.29% pre vs. 40.19% post, p = 0.768) or tumor-infiltrating lymphocytes (18.86% pre vs. 23.85% post, p = 0.172). Estradiol levels, stimulation length and gonadotropin dose showed no correlations with the ki67 index (p = 0.610, p = 0.839, p = 0.911). PAM50 subtype distribution remained stable pre- and post-stimulation: 23.8% Luminal A, 28.6% Luminal B, 19% HER2-enriched, 23.8% Basal-like, and 4.8% Normal-like. Eleven of 72 genes (15.3%) were significantly downregulated post-stimulation, including proliferation-related genes (CDC6, CENPF, EXO1, RRM2). Two cases showed borderline subtype shifts without ROR score changes (Luminal A ⇆ Luminal B). Five ROR score switches occurred: two intermediates to low, two high to intermediate and one from intermediate to high (Her2-enriched tumor). No correlations were found between gene expression and clinical parameters previously described. The sample size of the study was limited. Although a general decrease in gene expression was observed for certain genes after ovarian stimulation, one case showed an increased ROR. Expanding the sample size would help confirm whether such changes in gene expression and ROR are consistently observed. This is the first study to assess the impact of ovarian stimulation on breast tumor tissue, offering insights into its safety in women with breast cancer. These findings, along with existing knowledge of clinical follow-up after fertility preservation, may guide clinical decisions and improve reproductive counseling for newly diagnosed patients. No

TPS1123 Background: Sacituzumab Govitecan (SG) is a TROP2-directed antibody-drug conjugate (ADC) linked to a topoisomerase I inhibitor via a hydrolysable CL2A linker. It is approved for the treatment of metastatic triple-negative breast cancer (mTNBC) patients who have undergone at least two prior systemic therapies, including one for advanced disease, and of hormone receptor-positive (HR+)/HER2-negative metastatic breast cancer (mBC) patients after endocrine therapy (ET) and two systemic treatments. Currently, no biomarkers, including TROP2 protein expression, have been identified to predict SG response, highlighting the need to explore biomarkers of efficacy and to identify key resistance mechanisms to the drug. The ACROSS-TROP2 study aims to address this unmet medical need. Methods: ACROSS-TROP2 (NCT06236269) is a phase II, open-label, single-arm trial investigating SG in HR+/HER2-negative mBC patients. The study initially planned to enroll 50 pre- or post-menopausal female or male participants who progressed during or after treatment with CDK4/6 inhibitors and received up to one prior chemotherapy or ADC regimen for metastatic disease. Due to high recruitment rates and promising findings demonstrating ADC benefits in earlier treatment lines (Bardia et al., NEJM 2024), a protocol amendment was introduced to expand the sample size to 100 patients. Participants will receive SG at 10 mg/kg via IV infusion on Days 1 and 8 of each 21-day cycle until disease progression (PD). Fresh tumor biopsies will be obtained at baseline, after 2–3 weeks of treatment (C2D1), and at PD. The primary endpoint is to measure changes in the CelTIL score—a composite of tumor cellularity and tumor-infiltrating lymphocytes—between baseline and C2D1 biopsies, as CelTIL is associated with long-term efficacy. Secondary endpoints include overall response rate, progression-free survival, duration of response, time to response, safety, and tolerability. Correlative analyses of molecular markers in tissue and blood will be conducted to correlate biological findings (e.g., CelTIL, Ki67, TROP2, PD-1/PD-L1, PAM50) with clinicopathological data, evaluate the predictive value of early dynamic changes in ctDNA, identify genomic alterations linked to treatment response and resistance, and explore changes from baseline to PD to identify mechanisms of resistance. A paired t-test will assess whether the mean change in CelTIL score is statistically different from zero. The study has been approved in Spain and is actively enrolling participants at 10 sites within the SOLTI network. Previously presented at ESMO Breast 2024, FPN: 265TiP, Eva Ciruelos et al. - Reused with permission. Clinical trial information: NCT06236269 .