Abstract Background: Malignant effusions (MEs) are a frequent complication of advanced breast cancer (BC), reflecting tumor dissemination and associated with poor prognosis. Liquid biopsy (LB), particularly via next-generation sequencing (NGS) of circulating cell-free DNA (cfDNA), has emerged as a valuable tool for real-time monitoring and molecular characterization. While blood is the most common fluid used, MEs offer a rich source of tumor-derived DNA and cells. In addition to cfDNA profiling, tumor cells (TCs) can potentially be isolated and cultured from MEs, harnessing these fluids as a valuable source to develop patient-derived models for research and drug testing. We hypothesized that MEs are a viable source for both cfDNA mutation analysis and TC culture, and that identified molecular alterations may correlate with drug sensitivity. Methods: MEs were prospectively collected from patients with advanced BC. cfDNA was extracted from fluid samples and subjected to targeted NGS for somatic mutation profiling. Cytology was used to confirm malignancy. Parallel attempts were made to isolate and culture TCs from effusion samples. In three successfully established 3D cultures, cell viability assays were performed with targeted therapies based on detected mutations. Clinical follow-up data were collected to evaluate prognostic associations. Results: A total of 28 MEs were collected, including 21 pleural and 7 ascitic effusions. cfDNA was successfully extracted from all MEs, enabling comprehensive molecular testing with an 11-gene NGS panel. Molecular alterations were detected in 54% of patients. The mutational landscape revealed PIK3CA as the most mutated gene (25% of patients), followed by TP53 (21%). ESR1 D538G was detected in two patients following palbociclib/letrozole and capecitabine treatments, respectively. Additionally, alterations in ERBB2, KRAS, FGFR1/2, PTEN, ARID1A and a germline BRCA1 mutation were also identified. Paired cytological analysis was available in 15 samples, showing a concordance rate of 80% with molecular testing results. A PIK3CA mutation was detected in a cytology-negative case, highlighting the usefulness of effusions’ analysis in identifying and characterizing tumoral cells. In two cases with positive cytology, no pathogenic alterations were detected by NGS. However, TCs could be isolated from the MEs, further confirming their malignancy. Drug sensitivity assays were performed in three patient-derived 3D TCs culture. In a sample harboring a PTEN deletion, sensitivity to everolimus was demonstrated, with a 60% reduction of cell viability at 0.001 µM dose. Additionally, in a sample with FGFR1 amplification, paclitaxel showed a 70% reduction of cell viability at 0.1µM. Finally, in a ERBB2 and KRAS G13D mutated case, TCs showed resistance to alpelisib, palbociclib, ribociclib, elacestrant and letrozole. Conclusion: Malignant effusions represent a valuable and overlooked source of tumor material for molecular diagnostics in advanced breast cancer. Although cfDNA NGS and cytology show high correlation, NGS can detect tumoral content in samples that tested negative for cytology, and TCs’ isolation is able to confirm malignancy even in cases with no molecular alterations detected. The feasibility of culturing TCs from MEs and testing drug sensitivity further underscores the potential of this approach for personalized treatment. Integrating cfDNA genotyping, cytology, and TC functional assays from MEs could enhance clinical decision-making in late-stage breast cancer. Citation Format: E. Marín, S. García-Roman, S. Morales, J. Porcel, J. Soberino, A. Martínez-Bueno, C. Mayo de las Casas C, B. García-Peláez, R. Roman, M. Molina-Vila, C. Aguado, A. Velasco. Molecular Profiling of Malignant Effusions in Advanced Breast Cancer via cfDNA and Tumor Cells Analysis: A Complementary Approach to Guide Targeted Therapy 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 PS4-05-09.
Marín et al. (Tue,) studied this question.