Abstract PS1-08-12: Rewiring Treatment Pathways: How Molecular Profiling After CDK4/6 Inhibitor (CDK4/6i) Failure Shapes Future Therapy in Luminal HER2-Negative Breast Cancer

Abstract Introduction: The incorporation of CDK4/6 inhibitors has significantly improved outcomes in patients with Luminal HER2-negative metastatic breast cancer. Nonetheless, resistance invariably emerges, limiting long-term efficacy and challenging subsequent treatment decisions. The therapy of choice after progression to cyclin inhibitor is not clearly defined, therefore molecular profiling may offer a valuable opportunity to uncover resistance mechanisms and identify actionable genomic alterations. This study aimed to characterize molecular alterations of luminal breast cancers treated with CDK4/6i in the real world setting, with the goal of guiding future therapy selection and optimizing treatment sequencing. Methods: Tumor samples were obtained from patients with HR+/HER2− breast cancer treated with CDK 4/6i, either from metastatic lesions collected at progression or from primary tumors prior to systemic therapy initiation. Following histopathologic evaluation and manual microdissection, DNA was extracted from FFPE tumor slides and sequenced using a targeted NGS panel (Somatic GS Focus Panel). The panel interrogates SNVs and Indels across 25 cancer-relevant genes (AKT1, ALK, ATM, BRAF, BRCA1, BRCA2, EGFR, ERBB2, ESR1, FGFR3, IDH1, IDH2, KIT, KRAS, MET, NRAS, PALB2, PDGFRA, PIK3CA, POLE, PTEN, RET, and TP53) and detects high-level copy number alterations in PTEN, EGFR and ERBB2. Variants were interpreted by molecular pathologists within a certified molecular diagnostics workflow. Results: Of the 72 patients selected, 16 (22%) were excluded due to insufficient DNA quality obtained from tumor samples or due to technical sequencing failure, resulting in 56 successfully sequenced cases. Among these, 30/56 (54%) presented a mutation in one of the genes analyzed. Pathway-level analysis revealed mutations in the PI3K/AKT/mTOR signaling in 21 (38%) cases (15 PIK3CA, 2 AKT1, 4 PTEN). Also related to resistance to endocrine therapy, ESR1 mutations were present in 3 tumors (5%). We also observed alterations related to homologous recombination deficiency in 5 cases (10%), including 1 BRCA1mut, 2 BRCA2mut, 2 ATMmut. Additionally, TP53 mutations were detected in 6 cases (8%) and ERBB2 mutations in 5 samples (7%). These genomic profiles reflect distinct therapeutic resistance mechanisms and highlight targetable vulnerabilities that may indicate the value of using agents like SERDs, PI3K inhibitors, or therapies targeting DNA repair deficiencies. Conclusion: Post-progression molecular profiling following CDK4/6 inhibitor failure reveals a diverse spectrum of clinically relevant alterations in luminal HER2-negative breast cancer. The predominance of PIK3CA, TP53 and ESR1 mutations, along with alterations in canonical signaling pathways, supports the integration of genomic data into post-CDK4/6 therapeutic planning. These findings reinforce the importance of a precision oncology approach to optimize treatment sequencing and personalize care for patients with HR+/HER2− metastatic breast cancer. The high failure rate (22%) of tissue testing underscores the need for more effective methods to obtain this valuable information, such as liquid biopsies. Citation Format: L. Coelho de Mattos, A. Cicilini, N. Soldi, V. Basilio, M. Godner, L. Leite, A. Ribeiro, R. Souza, N. Pandolfi, M. Tavares, F. Pinto, A. Diniz, M. de Brot, F. Makdissi, S. Sanches, V. Lima, E. Santana dos Santos. Rewiring Treatment Pathways: How Molecular Profiling After CDK4/6 Inhibitor (CDK4/6i) Failure Shapes Future Therapy in Luminal HER2-Negative Breast Cancer abstract. In: Proceedings of the San Antonio Breast Cancer Symposium 2025; 2025 Dec 9-12; San Antonio, TX. Philadelphia (PA): AACR; Clin Cancer Res 2026;32(4 Suppl):Abstract nr PS1-08-12.