Abstract 1147: Development of a novel assay to detect circulating tumor cells in the cerebrospinal fluid of patients with leptomeningeal disease
Authors: Laura A. Huppert, Erin G. Bayer, Brock Bartels, Michelle Melisko, Mark Magbanua, Arturo B. Ramirez
Journal: Cancer Research (2025)

Abstract Background: Approximately 5-15% of patients with metastatic breast cancer (MBC) will develop leptomeningeal disease (LMD), characterized by the spread of tumor cells to the lining of the brain and spinal cord. Analysis of cerebrospinal fluid (CSF) is commonly used, however, is insufficiently sensitive for the diagnosis of LMD. Given the morbidity and poor prognosis after diagnosis of LMD, improving methods to detect rare CTCs in the CSF could lead to earlier intervention and improved patient outcomes. Methods: To address this unmet clinical need, we modified an existing CTC platform (RareCyte, Inc) validated for enumeration and isolation of single CTCs from blood, and performed technical validation and initial testing in patient CSF samples. First, we performed technical validation using mock CSF that was prepared by suspending approximately 5000 white blood cells into 5 mL of a 2% plasma solution in phosphate-buffered saline. Model circulating tumor cells (mCTCs) were generated by spiking in cultured BT474 ER+/HER2+ breast cancer cells into these mock CSF samples. Triplicate spike-in samples containing 100, 30, 10, and 3 BT474 cells were prepared and processed using a centrifugation workflow. The cells were subsequently stained with the RarePlex™ HER2/ER CTC panel assay and the slides were scanned using CyteFinder® to detect mCTCs and assess estrogen receptor and HER2 expression. We are in the process of clinical validation of this platform using CSF from patients with MBC and LMD (planned enrollment for initial pilot testing n=5 patients). Results: The average detection rate of mCTCs across all spike-in concentrations was 70%. We detected all mCTCs in the lowest spike-in samples (3 cells). The positive expression of ER and HER2 observed in mCTCs was consistent with the known status of these biomarkers in BT474 cells. The CSF tumor cell enumeration and assessment of ER and HER2 expression from patients with MBC and LMD will also be presented. Conclusions: We developed a novel process for detecting and characterizing CTCs in the CSF of patients with LMD. Testing in model systems demonstrated capabilities for detecting and biomarker expression analysis in rare mCTCs. Pending the method’s clinical validation using patient samples, we aim to detect and isolate individual CTCs for downstream molecular characterization. This platform could increase the sensitivity of LMD diagnosis which has the potential to have a positive impact on patient outcomes. Citation Format: Laura A. Huppert, Erin G. Bayer, Brock Bartels, Michelle Melisko, Mark Magbanua, Arturo B. Ramirez. Development of a novel assay to detect circulating tumor cells in the cerebrospinal fluid of patients with leptomeningeal disease [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 1147.