Abstract PS2-08-24: Detection of predictive mutations and HER2/neu expression from circulating tumor cells of metastatic breast cancer patients: new opportunities by analyzing high blood volumes obtained by diagnostic leukapheresis

Abstract Background: Tumor-derived material obtained through liquid biopsy—most commonly circulating tumor DNA (ctDNA)—has become a key biomarker for guiding targeted therapy in metastatic breast cancer. However, ctDNA is not released by all tumors and does not provide information on protein expression. In contrast, circulating tumor cells (CTCs) are intact cells that allow for multiparametric analyses, including both genomic profiling and protein-level assessments such as HER2 expression.Despite these advantages, the clinical utility of CTCs is often limited by their extremely low abundance. To enhance CTC yield, the diagnostic leukapheresis (DLA) offers a powerful alternative. By processing several liters of blood, DLA enables the density-based pre-enrichment of mononuclear cells (MNCs), including rare CTCs.In this study, we compared the utility of CTCs with ctDNA and tissue biopsies to assess their respective and complementary roles as biomarkers in metastatic breast cancer. Material 98 samples were collected prospectively.Single CTCs were isolated via micromanipulation, followed by whole genome amplification of genomic DNA. Targeted sequencing was performed using a next generation sequencing (NGS) panel covering predictive hotspot mutations in PIK3CA, ESR1, AKT1, and ERBB2, and results were compared with matched tissue biopsies. In parallel, ctDNA isolated from matched plasma samples was analyzed by digital droplet PCR for hotspot mutations in PIK3CA and AKT1.Additionally, from DLA products of eight patients, CTCs were enriched from samples adjusted to 2 × 109 MNCs using the flow enrichment target capture Halbach (FETCH) technology. Single CTCs were then isolated by fluorescence-activated cell sorting and analyzed by NGS. Results: Sequencing of single CTCs was successful in 100% of samples containing ≥18 CTCs. In contrast, samples with lower CTC counts (1-8 CTCs) showed a reduced success rate of 38.5%. In case CTC sequencing was successful, mutations identified in matched tissue biopsies were also detected in CTCs. Notably, in a substantial fraction of patients, additional mutations were identified in CTCs that were not detectable in tissue. In CTC-positive cases (≥1 CTC), a high concordance (82.6%) between detected mutations in CTCs and ctDNA was observed. However, ctDNA failed to detect 50% of the predictive PIK3CA and AKT1 mutations identified in tissue, resulting in a superior sensitivity of CTCs over ctDNA when ≥19 CTCs were available for analysis. As expected, application of DLA significantly increased CTC yield. While standard 7.5 mL blood samples yielded ≥19 CTCs in only 12.5% of patients (≥1 CTCs in 62.5%), DLA yielded ≥19 CTCs and resulted in successful sequencing in all 8 cases. In 62.5% of the DLA samples, mutations were identified that were often not detectable from standard CTC analysis due to low counts or absent CTCs in blood. HER2-positive CTCs were found in 37.5% of the DLA samples. Overall, actionable alterations were identified in CTCs from 87.5% of the DLA patients. Conclusion: If present in sufficient numbers, CTCs enable more sensitive detection of predictive mutations than ctDNA and additionally provide HER2 protein expression data. DLA is critical to achieve the required CTC yield. CTC analysis from DLA products offers a valuable complement to tissue biopsies, particularly when metastases are inaccessible, biopsies require considerable effort, or are declined by patients. Citation Format: A. Franken, D. Karayel, M. Rivandi, J. Oles, M. Sudarsanam, C. Driemel, R. P. Neves, N. Krawczyk, B. Jäger, F. Meier-Stiegen, N. Stamm, B. Alberter, B. Polzer, T. W. Friedl, S. Riethdorf, B. Volz, A. Koch, K. Schäfer, T. Rau, J. C. Fischer, E. Ruckhäberle, K. Pantel, W. Janni, N. H. Stoecklein, T. Fehm, H. Neubauer. Detection of predictive mutations and HER2/neu expression from circulating tumor cells of metastatic breast cancer patients: new opportunities by analyzing high blood volumes obtained by diagnostic leukapheresis 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 PS2-08-24.