Radioguided surgery has become a key component of contemporary breast cancer care, supporting less invasive approaches while maintaining oncologic safety. This narrative review summarizes current practice and recent developments in radioguided breast and axillary surgery, from established molecular imaging workflows to emerging three-dimensional and intraoperative technologies. Modern breast cancer management is increasingly shaped by tumor biology and the widespread use of neoadjuvant systemic therapy, which is transforming surgical decision-making and driving a shift toward personalized, patient-tailored pathways. In this context, radioguided techniques help maintain procedural accuracy despite therapy-induced changes in breast and nodal anatomy, enabling reliable lesion localization and targeted management of the axilla. We discuss sentinel lymph node strategies and de-escalation concepts, including targeted axillary dissection (TAD) after neoadjuvant therapy using marked nodes and selective removal approaches. We also review localization methods, including radioactive seed-based techniques, and the expanding role of molecular imaging-guided surgery to support intraoperative decision-making. Particular attention is paid to technologies aimed at improving surgical precision and margin assessment, including portable/freehand SPECT concepts and intraoperative PET/CT-based specimen imaging for immediate evaluation of excised tissue. Finally, we highlight how artificial intelligence and digital tools may enable workflow optimization, navigation, image interpretation, and decision support, accelerating the transition toward individualized treatment. Overall, integrating molecular information with real-time 3D guidance can help tailor breast and axillary management to each patient while reducing morbidity.

530 Background: Breast cancer (BC) is the most common malignancy among young women of childbearing age, with a rising incidence in this population. Pregnancy-associated breast cancer (PABC) is an aggressive entity linked to poor prognosis and elevated metastatic risk. Despite advances in BC research, the impact of pregnancy, breastfeeding, and postpartum mammary gland remodeling on tumor biology remains unclear, highlighting the need to explore their interaction with the tumor microenvironment for novel therapies. Methods: A gene expression and immune cell profiling analysis was conducted using the nCounter Breast Cancer 360 panel (NanoString) and CIBERSORTx (Newman 2019, Nat Biotechnol) on FFPE tumor samples from GEICAM/2017-07 EMBARCAM study (NCT04603820) PABC patients during the gestation (PABC_GS, n=21), breastfeeding (PABC_BF, n=21), and first-year postpartum (not during lactation period, PABC_FY, n=15) vs non-PABC tumours (n=49). Differential expression analysis per gene and biological signature was performed using the limma package. P-values were adjusted with the Benjamini-Yekutieli false discovery rate (FDR) method. Additionally, the LM22 matrix from CIBERSORTx was employed to quantify 22 immune cell types from normalized NanoString data. Statistical significance was set at 5%. Results: PABC clinical subtypes were 46% HR-positive/HER2-negative, 21% HER2-positive, and 33% triple-negative. Differential gene expression analysis identified similar significant enrichment pathways across all PABC groups compared to non-PABC: DNA repair-related signatures (HRD, BRCAness, BC p53) and BC proliferation (adj p<0.05), along with higher CDK4 expression and genomic risk (p<0.05). Conversely, key regulatory pathways such as apoptosis, TGF-Beta, and PD-L1 were downregulated (adj p<0.05). Nonetheless, it is noteworthy that the PABC_BF group showed a unique profile marked by increased immune activity and cell abundance (cytotoxic cells, CD8 T cells, T-reg, cytotoxicity), elevated SOX2 expression (adj p<0.05) and inflammatory chemokines levels (p<0.01) compared to non-PABC. The CIBERSORTx analysis supported these findings, demonstrating a significantly higher abundance of several immune cells in PABC_BF, remarkably CD8+ T-cells and T-regs, compared to all other groups (p<0.05). Conclusions: This GEICAM EMBARCAM sub-study reveals that PABC tumors display aggressive molecular features across all subtypes, contributing to poor prognosis. Notably, the breastfeeding-associated subset (PABC_BF) exhibits a highly active tumor-immune microenvironment with robust immune cell infiltration and inflammatory signalling, highlighting potential for targeted immunotherapy. These findings underscore the need for further clinical research to optimize immune-based strategies in PABC patients’ management. Clinical trial information: NCT04603820 .

The anti-HER2 monoclonal antibody trastuzumab and new derivative formulations are the standard treatment for HER2-positive breast cancer. However, after 1 to 5 years of treatment, some patients acquire resistance to therapy, leading to relapse. The microRNA-449 family members were downregulated in HER2-positive breast cancer cell lines and low levels were associated with patients’ worse prognosis. Moreover, trastuzumab-resistant HER2-positive breast cancer cell lines showed lower microRNAs-449 and higher Fatty Acid Synthase (FASN) expression, compared to sensitive cell lines. The direct regulation of FASN by microRNA-449a and microRNA-449b-5p was demonstrated. Moreover, microRNAs-449 overexpression and FASN inhibition decreased cell proliferation and sensitized cells to trastuzumab treatment by inhibiting the PI3K/AKT signaling pathway. Together, these results suggest the microRNAs-449/FASN axis as a potential therapeutic target in combination with anti-HER2 agents to overcome trastuzumab resistance and to improve treatment response in HER2-positive breast cancer patients.

Background: Despite the activity of both chemotherapy (CT) and endocrine treatment in high-risk HR+/HER2-neg EBC, the risk of recurrence persists over time, highlighting the need for additional strategies to improve outcome. HER3-DXd is a potential first-in-class HER3-directed antibody-drug conjugate with activity in multiple breast cancer subtypes. In SOLTI TOT-HER3, a single dose of HER3-DXd was associated with increased CelTIL and clinical response in patients (pts) with HR+/HER2-neg EBC (Oliveira et al, Ann Oncol 2023). Methods: SOLTI VALENTINE (NCT05569811) is a parallel, randomized, non-comparative, open-label neoadjuvant study of HER3-DXd +/- LET or CT in pts with operable stage II-III high risk (Ki67 ≥ 20% and/or high genomic risk) HR+/HER2-neg EBC. Pts were randomized 2:2:1 to: (A) HER3-DXd 5.6 mg/kg every (Q) 21 days (D) for 6 cycles; (B) HER3-DXd plus QD LET (+/- LHRH agonist); (C) CT with 4 cycles of EC/AC (epirubicin 90 mg/m2 or doxorubicin 60 mg/m2 and cyclophosphamide 600 mg/m2 Q14 or 21 D) followed by weekly paclitaxel 80mg/m2 for 12 weeks. The primary endpoint was pathological complete response (pCR, ypT0/is ypN0) rate at surgery. Secondary endpoints included residual cancer burden (RCB), overall response rate (ORR), CelTIL score change, safety, invasive disease-free survival, and overall survival. Baseline, C2D1, and surgical tissue samples were collected for evaluation of response and translational endpoints. The sample size was not selected to formally compare treatment arms in terms of pCR rate, but to achieve a certain level of precision estimating pCR. Assuming a pCR of 20%, a total of 48 per arm would provide a precision estimation of +-11.3% using asymptotic two-sided 95%CI. Results: From Nov-2022 to Sep-2023, 122 pts were randomized to HER3-DXd (n=50), HER3-DXd + LET (n=48) and CT (n=24). Median age was 51 (29 - 82), 99.2% were females, and 52.9% pre/perimenopausal. Most pts had T2 (54.1%), N1 (70.5%), Stage II (64.0%), G2 (69.0%) tumors; median Ki67 was 35.0% (18.0; 90.0). Proportion of pts with Stage III tumors was 36.0%, 39.5% and 29.2% in Arms A, B, and C, respectively. The pCR rate was 4.0% (95%CI 0.5-13.7) in Arm A, 2.1% (95%CI 0.1-11.1) in Arm B, and 4.2% (95%CI 0.1-21.1) in Arm C. The RCB0/1 rate was 18.4%, 12.5% and 30.4%, and ORR was 72.0%, 81.3%, and 70.8%, in Arms A, B, and C, respectively. A significant change in CelTIL score from baseline to C2D1 was observed in HER3-DXd arms, but not in Arm C: median increase in CelTIL of 8.2 (p<0.0001) in Arm A, 7.4 (p=0.001) in Arm Band 3.8 (p=0.13) in Arm C. CelTIL change at C2D1 associated with radiological response in Arm A (p<0.0001) and B (p=0.002), but not in Arm C (p=0.30). A decrease in Ki67, as well as a switch from PAM50 Risk of Recurrence (ROR)-high/medium to ROR- low score and from PAM50 Luminal B subtype to Luminal A/Normal-like subtypes were observed in all treatment arms at C2D1, and was further enhanced at surgery. Treatment-emergent adverse events (TEAEs) grade≥3 were less frequent in the HER3-DXd arms (18.0% and 16.7% in Arms A and B, respectively), compared to Arm C (54.2%). The most common related TEAEs reported in Arms A/B were nausea (56%/72.9%), alopecia (52%/68.8%), fatigue (50%/68.8%), and diarrhea (42%/54.2%). Conclusions: In SOLTI VALENTINE, treatment with HER3-DXd, with or without LET, resulted in similar pCR rates to CT, while exhibiting a lower incidence of grade ≥3 TEAEs. CelTIL score at C2D1 correlated with response to HER3-DXd, but not to CT. The ongoing translational analysis, as well as the survival outcomes of VALENTINE, will provide further insights into the activity of HER3-DXd in EBC and clarify its potential role as a treatment strategy for high-risk HR+/HER2-neg breast cancer. Citation Format: Mafalda Oliveira, Tomás Pascual, Kepa Amillano Parraga, Javier Salvador Bofill, Santiago González-Santiago, Clara Martínz Vila, Josefina Cruz, Xavier González-Farré, Elena Galve, Pilar Sánchez Henarejos, Juan Miguel Cejalvo, Maria-Eva Pérez-Lopez, Montse Muñoz, Serafin Morales, Alba González-Haba, Maria Borrell Puy, Encarnación González, Juan A. Guerra, Sergio Hoyos, Antonia Perelló, Pablo Tolosa Ortega, Juan De la Haba, Yenlik Zheteyava, David W. Sternberg, Fumitaka Suto, Esther Pang, Dalila Sellami, Rodrigo Sánchez-Bayona, Guillermo Villacampa, Samyukta Chillara, Mariana Paes Dias, Juan M. Ferrero-Cafiero, Aleix Prat. Primary results of SOLTI VALENTINE: neoadjuvant randomized phase II trial of HER3-DXd alone or in combination with letrozole for high-risk hormone receptor positive (HR+)/HER2-negative (neg) early breast cancer (EBC) [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 LB1-06.

1052 Background: The CLEOPATRA and PERUSE trials established the combination of a taxane with the antiHER2 monoclonal antibodies trastuzumab and pertuzumab (HP) as the gold standard first-line treatment for HER2+ mBC. In both studies, the progression events reached a plateau after 4 years and up to 30% of pts remained long-term progression-free, hypothesizing HP maintenance can be safely discontinued. We therefore evaluated whether epigenomic-based ctDNA MRD analysis can potentially identify pts with a higher chance of permanent remission. PRE-PHENIX is a multi-center observational study that explores the prevalence of MRD measured by Guardant Reveal in HER2+ mBC pts on long-term first-line HP maintenance. Methods: A total of 40 pts with HER2+ mBC on first-line treatment with HP maintenance for a minimum of 4 years were included. Confirmation of no progressive disease by CT or PET-scan in the last 3 months previous to study entry was mandatory. Plasma samples were analysed using Guardant Reveal powered by the Guardant Infinity platform, a tissue-free epigenomic assay interrogating differentially methylated regions of DNA optimized to detect breast cancer DNA from normal cell-free DNA. Two ctDNA tests were performed on each patient within a 6 – 12-week interval. Additionally, 11 pts with confirmed disease progression on antiHER2 therapy for mBC were included as case controls. The primary objective was to establish the prevalence of positive MRD in both populations and the agreement between the two tests for the Long-Term responders. Results: Median age was 63.2 years (range 30.8 – 84.4). The median duration of first-line HP treatment was 6.9 years (range 4.2 - 11.1). At diagnosis, 26 pts (65%) presented with “de novo” mBC and 20 (50%) had visceral disease. The last radiological evaluation categorized 6 pts (15%) as having stable disease (SD), 2 pts (5%) with partial response (PR), and 32 pts (80%) with complete response (CR). Among the 11 pts with confirmed progression, 2 presented exclusive Central Nervous System (CNS) disease. Guardant Reveal identified MRD in 4 long-term responders (10%), 3 out of 6 pts (50%) with SD, 1 of 2 pts (50%) with PR, and no MRD among the 32 pts with CR. A perfect agreement was observed between the two tests (Kappa-index of 1). Ten out of the 11 pts (91%) with disease progression had MRD, including the two with exclusive CNS involvement. Conclusions: Our study demonstrates clinically significant performance of a tissue-free MRD test, Guardant Reveal, as a potential non-invasive monitoring tool to guide de-escalation strategies in pts HER2+ mBC pts with long-term remissions on HP treatment. A prospective study (PHENIX) to guide HP interruption by ctDNA monitoring is planned. This study was funded by a Fundación Contigo full grant (Spain) and Guardant Health.

Supplementary Figure S1 shows the expression of the 50 genes in the PAM50 signature, obtained from the 164-gene codeset.

Bioinspired nano/micromotors with drug delivery capabilities are emerging tools with the promising potential to treat numerous diseases. However, some major challenges must be overcome before reaching real biomedical applications. Above all, it is necessary to design engines that employ biocompatible and bioavailable fuels to induce efficient propulsion in biological environments. In addition, ideal nanomotors should also be capable of delivering the cargo on-command using selected stimuli. To tackle these challenges, we herein present the design and evaluation (both in vitro and in vivo) of a glucose-driven gated Janus nanomotor that performs on-demand anticancer drug delivery to treat solid tumors. The motor's nanoarchitectonics is based on the anisotropic conjunction of catalytic platinum nanodendrites (PtNds) and a mesoporous silica nanoparticle (acting as a nanocontainer for anticancer drug doxorubicin) capped with enzyme glucose oxidase (GOx). Autonomous nanomotor movement is achieved thanks to two catalytic components, GOx and PtNds, in a hybrid cascade reaction: GOx transforms glucose to give H2O2 that is subsequently catalyzed by PtNds into H2O and O2. Besides, gatekeeper moieties (GOx) respond to the presence of intracellular proteases, which induces doxorubicin delivery. Biological experiments with the nanomotor are carried out in cancer cell cultures, three-dimensional (3D) tumor models (spheroids), in vivo and in patient-derived organoids (PDOs). A strong anticancer effect is found and attributed to the synergistic combination glucose-induced propulsion, controlled drug delivery, elimination of glucose (by GOx), ROS production (H2O2 generation by GOx) and hypoxia reduction (O2 generated by PtNds). Taken together, this study advances the engineering of endogenously fueled nanomotors for in vivo operation and provides insights into the application of active particles in cancer therapy toward clinical application.

Background: The optimal treatment strategy for patients (pts) with HR+/HER2- advanced breast cancer (ABC) progressing on first-line endocrine therapy (ET) and CDK4/6 inhibitors (CDK4/6i) is influenced by the molecular alterations driving resistance to this combination. However, financial and logistical challenges hinder the routine implementation of next-generation sequencing technologies, leading to disparities in patient care. To address these issues, the SOLTI-1903 HOPE study (NCT04497285) aims to assess the feasibility of a molecular screening program that actively involves pts with ABC, providing insights into the genomic landscape and improving access to matched-targeted therapies in Spain. Methods: The SOLTI-1903 HOPE study is a Spain-wide patient-centered study in which pts with ABC actively manage their enrollment, participation, and follow-up through a digital tool. Pts provide clinical data and tumor tissue samples for FoundationOne CDx (F1CDx) analysis. At disease progression (PD), pts undergo a liquid biopsy (LBx) at a local laboratory to be analyzed using the Guardant360 (G360) panel. Clinical and molecular outcomes are evaluated by a multidisciplinary Advisory Board, which interprets the observed alterations and recommends potential routine or clinical trial-based targeted therapies. The primary endpoint of this sub-analysis is to evaluate the genomic profile of pts with ABC who progressed on previous CDK4/6i. Results: Of the 163 pts enrolled in HOPE who received first-line ET + CDK4/6i, 119 experienced documented PD. Of the 98 pts with a LBx result after ET + CDK4/6i, 22 (22.4%) had the blood extraction immediately after progressing to this treatment; median time from PD on ET + CDK4/6i to LBx was 9.2 months (mo) (range 0.07-64.1). ctDNA was detected in 97 pts (98.9%) with a median of 3 genomic alterations per patient (IQR 2-5, range 1-22) and a median variant allele frequency of 0.7% per alteration (IQR 0.2-3.2%, range 0.02-86.6%). ESCAT level I-II alterations were identified in 71pts (73.2%). Most frequent mutations were PIK3CA (44.3%) and ESR1 (34%). BRCA1/2 mutations occurred in 11.3% of pts, ERBB2 in 6.2%, and PTEN in 7.2%. Notably, co-occurring mutations involving >2 targetable alterations were found in 24 pts (24.7%), predominantly involving alterations in ESR1 and PIK3CA/PTEN/AKT1 pathway (58.3%). Only 24 pts had F1CDx performed on a tissue sample obtained after first-line ET+CDK4/6i. Median time to tissue obtention was 12.1mo (range 0.23-47.8). Most frequent biopsy sites were liver (37.5%), breast (25%), and skin (16.7%). Median TMB was 3 (IQR 1-4, range 0-18), and the median number of genomic alterations per patient was 8.5 (IQR 5-10, range 2-16). ESCAT level I-II alterations were found in 16 pts (66.7%) and co-occurring targetable alterations in 4 (16.7%), with no significant differences vs. LBx (p=0.5 and p=0.4, respectively). Most frequent mutations were TP53 (50%), ESR1 (37.5%), and PIK3CA (29.2%). In 20 pts with both LBx and tissue samples obtained after ET+CDK4/6i, ESCAT I-II alterations were found in 60% and 70% with G360 and F1CDx, respectively (p=0.5), with a concordance rate of 73.3%. Among 114 pts with follow-up clinical data, 17/55 pts (30.9%) with PIK3CA mut received targeted therapy after inclusion in HOPE; 7/55 (12.7%) had received it previously. None of the patients with BRCA1/2, ERBB2, ESR1, AKT1, or PTEN mutations has received targeted therapy after enrollment. Conclusions: Over two-thirds of pts progressing on CDK4/6i present with ESCAT I-II level alterations detected via tumor or liquid biopsy. Notably, 15-25% of these pts exhibit co-occurring targetable alterations. In our study, the most frequently targeted alteration was PIK3CA mut, due to lack of approval for targeted therapies for other ESCAT I-II alterations in Spain during the study period. Addressing current barriers to targeted therapies is crucial for expanding options for pts with ABC. Citation Format: Elia Segui, Rubén Olivera-Salguero, Juan Miguel Cejalvo, Mafalda OliveiraPablo Tolosa, Pablo Tolosa, Maria Vidal, Marcos Malumbres, Joaquín Gavilá, Cristina Saura, Sonia Pernas, Rafael López, Mireia Margelí, Judith Balmaña, Montserrat Muñoz, Isabel Blancas, Valentina Boni, Eva Ciruelos, Elena Galve, Antonia Perelló, Raquel Gómez-Bravo, Isabel García-Fructuoso, Susana de la Cruz, Miguel de la Hoya, Teresa Manchón, Juan Manuel Ferrero-Cafiero, Helena Masanas, Rosa Olmos, Gema Rodríguez, Amparo Medina, Maria José Prieto, Aleix Prat, Ana Casas, Tomás Pascual. Real-World Molecular Profiling After CDK4/6 Inhibition in Advanced Breast Cancer: Analysis of the SOLTI-1903 HOPE 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 P1-10-05.

Supplementary Figure S2 shows a forest plot comparing basal vs. non-basal subtype as defined by PAM50 or by IHC, and their associations with disease free survival on capecitabine vs. observation arm.

Supplementary Table S1 shows the gene list for the NanoString custom nCounter codeset included in the study. Supplementary Table S2 shows patient characteristics in the GEICAM/CIBOMA translational study cohort versus the original trial. Supplementary Table S3 shows the distribution of PAM50 intrinsic subtypes in the GEICAM/CIBOMA translational study cohort. Supplementary Table S4 shows the differential gene expression analysis performed on the 164 genes included in the custom codeset comparing PAM50 non-basal vs. basal-like subtype and their biological processes. Supplementary Table S5 shows multivariate survival analysis and interaction tests for the four genes and metagenes included in the prespecified hypotheses testing their association with DFS. Supplementary Table S6 shows multivariate survival analysis and interaction tests for the continuous expression of the 38 individual genes included in the prespecified hypotheses testing their association with DRFS. Supplementary Table S7 shows multivariate survival analysis and interaction tests for the continuous expression of the 38 individual genes included in the prespecified hypotheses testing their association with OS. Supplementary Table S8 shows multivariate survival analysis and interaction tests for the continuous expression of the 38 individual genes included in the prespecified hypotheses testing their association with DFS. Supplementary Table S9 shows multivariate survival analysis and interaction tests restricted to the PAM50 non-basal subgroup. Supplementary Table S10 shows Exploratory analysis for the prognostic capacity of the continuous expression of genes and metagenes restricted to the PAM50 non-basal subgroup.

Supplementary Figure S3 shows a forest plot of the exploratory categorical analysis of selected (meta)gene expression and their association with distant recurrence free survival on the capecitabine arm vs. observation.

Supplementary Data S1 shows exploratory analysis for the predictive capacity of the continuous expression of additional genes and metagenes included in the codeset. Supplementary Data S2 shows exploratory analysis for the prognostic capacity of the continuous expression of the selected genes and metagenes

Background: Trastuzumab-deruxtecan (T-DXd) has shown an unprecedented clinical benefit in advanced breast cancer. Despite its meaningful anticancer outcomes, around 15-20% of patients have to discontinue T-DXd due to related toxicity (mainly pneumonitis/interstitial lung disease). To date, there is no evidence of the potential impact of pharmacogenomic variants and the risk of treatment-related adverse events (TRAE) in breast cancer patients treated with T-DXd. Methods: The PROCURE project is a translational research study comprising 26 Spanish institutions that aims to analyze pharmacogenomic variants and risk of toxicity in patients with breast cancer treated with T-DXd. Eligible patients had to receive at least one dose of T-DXd for its current approved indications in advanced HER2+/HER2-low breast cancer, with a minimum follow-up of 3 weeks to collect data on early-onset toxicity. Patients who had discontinued the treatment were also eligible. Patients’ demographics and clinical variables were gathered from medical records in an electronic database. Emerging adverse events were registered following CTCAE V.5 guidelines. One blood sample was collected for DNA extraction. Pharmacogenomic analyses included the determination of UGT1A1*28 allele and a massive SNP genotyping array with over 1.9 million genetic markers and enriched in pharmacogenomics variants (Infinium Global Diversity Array with Enhanced PGx, Illumina). Results: A total of 329 female patients were enrolled in the study from July 2022 to March 2024 with available genetic information. At data cut-off (1 July 2024), clinical data was available for 315 (95.7%). The median age was 56 years and the median duration of T-DXd treatment was 12.0 months (95%CI 10.4–14.2). Regarding UGT1A1 genotypes, 36 patients (11.4%) were *28/*28 homozygous (poor metabolizers), 143 (45.3%) were *1/*28 heterozygous (intermediate metabolizers), 1 (0.3%) was *1/*37 and 135 (42.7%) were *1/*1 (wild type). According to UGT1A1 genotypes (in this order: *1/*1, *1/*28, *28/*28) we observed the following TRAE of any grade: neutropenia (14.0%, 15.3%, 8.3%), anemia (13.2%, 7.6%, 11.1%), thrombocytopenia (4.4%, 4.2%, 11.1%), diarrhea (19.9%, 16.7%, 16.7%), vomiting (16.2%, 14.6%, 13.9%), pneumonitis/ILD (10.3%, 8.3%, 8.3%), dose reductions (28.9%, 27.8%, 34.8%), and treatment discontinuation (28.9%, 26.7%, 17.4%). We observed a higher incidence of grade 3 gastrointestinal toxicity in poor metabolizers (*28/*28) compared to the other two genotypes: diarrhea (0.7%, 0.7%, 2.8%), and vomiting (0.7%, 0.0%, 5.6%). The incidence of drug-induced pneumonitis/ILD in our study was 9.2% (29/315). We did not find an association between the UGT1A1 variants and the incidence of pneumonitis. In the SNP array, 314 of 315 samples and >95% of SNVs passed quality control. A logistic regression analysis of pneumonitis/ILD revealed more than 25 markers with unadjusted p-value <5x10-5 (additive model). Detailed pharmacogenomic TRAE analyses will be presented during the conference. Conclusions: Descriptive analyses from the PROCURE project show a similar incidence of all grade TRAE irrespective of UGT1A1 allelic variants. We observed a higher incidence of grade 3 gastrointestinal toxicity in UGT1A1 *28/*28 carriers. Potential pharmacogenomic markers of ILD were identified by a massive SNP genotyping array in unadjusted analysis that warrants further research. Citation Format: Rodrigo Sánchez-Bayona, Javier de Nicolás-Hernández, Cristina Saura, Maria Gion, Juan Miguel Cejalvo, Elisenda Llabrés, Javier Cortés, Alejandro Falcón, Sonia Pernas, Alfonso Lopez de Sa, Maria Vidal, Carmen Hinojo, Teresa Curiel, Josefina Cruz, Virginia Martínez, Blanca Cantos, Maria Jose Echarri, Isabel Gallegos, Coralia Bueno, Ana Milena Vargas, Santiago Escrivá-de-Romaní, Bartomeu Fullana, Maria Angeles Cobos, Alicia Arenas, Laia Joval-Ramentol, Tomás Pascual, Carlos Valdivia, Guillermo Villacampa, Eva Ciruelos, Cristina Rodriguez-Antona. Pharmacogenomic variants and risk of adverse events in breast cancer patients treated with Trastuzumab-deruxtecan: results from the PROCURE project [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 P2-01-16.

Introduction: Breast cancer (BC) in very young women (VYBC), specifically those under 35 years old, presents biological and clinical differences compared to older patients with breast cancer (OBC). Previous studies have shown discordance in tumor characteristics and treatment responses between younger and older patients, notably within HR+/HER2- subtype. This study aims to elucidate the transcriptomic landscape of HR+/HER2- in VYBC compared to OBC, focusing on identifying age-associated molecular differences that could decipher personalized treatment strategies for these patients. Methods: This study included 49 BC patients treated at Hospital Clínico Universitario de Valencia. RNA sequencing was performed on formalin-fixed samples from 22 VYBC (age ≤35 years) and 27 OBC (≥50 years) patients. Differential expression analysis and gene set enrichment analysis (GSEA) were performed to identify differentially expressed genes. Deconvolution analysis using MCP-counter and quanTIseq algorithms was employed to evaluate tumor cellular composition. Histological examination described tumor infiltrating lymphocytes (TILs) and tertiary lymphoid structures (TLS). Statistical analyses were assessed using R (version 4.0.2). Results: The BCVY cohort included 12 (55%) luminal, and 10 (45%) non-luminal BCs while the OBC cohort were 17 (63%) luminal and 10 (37%) non-luminal patients. Transcriptomic analysis revealed significant age-associated differences particularly in HR+/HER2- subtype (133 and 106 genes down- and up-regulated (log2 fold change < ± 2, adjusted p value < 0.05). HR+/HER2- VYBC tumors exhibited greater proliferation signatures (p = 5.17e-05) and ki67 (IHC, p = 0.021), increased chromosomal instability (CIN70, p = 1e-06), and higher expression of immune-related gene signatures compared to OBC tumors. Interestingly, despite no differences in estrogen and progesterone receptor by IHC, lower expression was found at RNA level in HR+/HER2- VYBC patients (ESR1: p= 0.0027, PGR: p= 0.0476). Deconvolution analysis confirmed an enhanced infiltration of immune cells in HR+/HER2- VYBC tumors, identifying them as immunoreactive tumors, characterized by high infiltration of cytotoxic T lymphocytes and overexpression of immune checkpoint molecules such as PD-1 and its ligand PD-L1. Histological validation confirmed increased TILs and TLS in VYBC tumors. Finally, PAM50-based chemoendocrine score (CES) was also determined for HR+/HER2- samples showing higher levels of CES, which are predictive of better response to chemotherapy in HR+/HER2- VYBC patients. Conclusions: Our study provides a comprehensive understanding of the molecular landscape of HR+/HER2- BC in very young women. We found age-related gene expression changes not only in cancer cells but also in the tumor microenvironment. These data suggest that HR+/HER2- tumors from VYBC patients could be more endocrine-resistant and immunoreactive than those from OBC patients. Accordingly, personalized therapeutic strategies in this subgroup are needed. Citation Format: Marta Tapia, Iris Garrido-Cano, Juan Carbonell, Carlos Peña, Sandra Torres-Ruiz, Anna Ágreda-Roca, Cristina Tébar, Octavio Burgués, Cristina Hernando, Ana Lluch, Begoña Bermejo, María Teresa Martínez, Juan Miguel Cejalvo. Deciphering the transcriptomic landscape of early HR+/HER2- breast cancer in very young women [abstract]. In: Proceedings of the San Antonio Breast Cancer Symposium 2024; 2024 Dec 10-13; San Antonio, TX. Philadelphia (PA): AACR; Clin Cancer Res 2025;31(12 Suppl):Abstract nr P5-02-25.

Purpose: Advanced, pretreated triple-negative breast cancer (TNBC) has a dismal prognosis and lacks effective options beyond standard cytotoxics. We previously showed, via phosphoproteomic screening, that cyclin-dependent kinase 6 (CDK6) and ERK hyperactivation are linked to adverse outcomes and represent actionable targets. This prompted us to evaluate palbociclib and binimetinib in advanced TNBC after one or two prior therapies.Patients and Methods: Patients with increased ERK and/or CDK6 activity were eligible. Treatment consisted in daily binimetinib (45 mg twice a day) plus palbociclib (100 mg daily, days 1-21) in 28-day cycles. Palbociclib escalation to 125 mg was allowed in cycle 2. The primary objective was to demonstrate a 2.5 month-long progression-free survival (PFS), a 50% increase over the reference PFS for cytotoxics (1.7 months). Whole-exome sequencing was performed in tumor samples of the efficacy population.Results: Fifty-one patients were screened, of whom 50 were biomarker-positive. Twenty-four initiated treatment between June 2021 and July 2022. Toxicity was frequent, consisting mainly in fatigue, diarrhea, neutropenia, and ocular effects, requiring frequent dose interruptions or reductions. The primary objective was not met (median PFS 50 days). However, a bimodal PFS pattern emerged, with 13% of the patients achieving disease control lasting 4 to 13 months. Whole-exome sequencing revealed a distinct mutational landscape among long-term responders compared with early progressors.Conclusions: In this biomarker-enriched TNBC population, the combination of palbociclib and binimetinib showed limited activity and notable toxicity. Whereas CDK6 and ERK hyperactivation confirmed their prognostic role, they did not predict treatment benefit. Exploratory genomic findings suggest the existence of a biologically distinct subset of patients with prolonged benefit, encouraging further investigation.Significance: Previous studies showed that patients with early TNBC with increased CDK4/6 and ERK activity are at high risk of relapse. Preclinical data also suggest the benefit of combined inhibition of CDK4/6 and MEK, and novel therapies are needed for TNBC in the advanced disease setting. Despite the rationale and a biomarker-driven design, this combination was toxic and showed limited efficacy. This combination should not be further developed in this disease.

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 .