Abstract P4-01-27: Detection, isolation, and genetic characterization of circulating tumor cells from the cerebrospinal fluid of breast cancer patients with leptomeningeal metastasis

Abstract The gold standard to diagnose leptomeningeal metastasis (LM) is to identify malignant cells in the cerebrospinal fluid (CSF) or in a leptomeningeal biopsy. Using conventional cytology its sensitivity remains low with approximately 50% and 85% for the first and the second lumbar puncture, respectively. While rare cell capture technologies like the CellSearch system reach higher sensitivity than conventional cytology, they have limitations, including capturing only cells with sufficient EpCAM antigen expression, limited antibody customization, and high costs. Once diagnosed with LMs, to date most patients are still treated with untargeted therapies with limited effectiveness and significant side effects. To address this, we developed a workflow using Sievewell 370 K microwell chips. Each chip contains 370,000 hexagonal microwells (20 μm in diameter, 25 μm deep) with two 2 μm pores at the bottom. On the chip, individual cells are separated, identified with cytokeratin antibodies, and isolated as single cells via micromanipulation for molecular analysis. 15 CSF samples from 9 LM-patients were analyzed on the microwell chips and by conventional cytology in parallel. The isolated cells’ DNA was amplified by whole genome amplification and sequenced by low pass whole genome sequencing and panel sequencing on single cell level and compared to extracranial tissue biopsies. On-chip immunostaining achieved less than 5% cell loss and over 95% single cell isolation for the MCF7 breast cancer cell line. For 10 of the 15 CSF samples, concordant results were obtained by analysis on the microwell chips and conventional cytology: 1 sample was tumor cell-negative, 9 samples were tumor cell-positive. In the other 5 samples, tumor cells were detected by analysis on the microwell chip with either inconclusive (3 samples) or negative (2 samples) results obtained by cytological analysis. With the microwell chips, 5 to 10,000 (mean 1,218; median 116) CSF-CTCs were found per mL CSF. The detection of chromosomal aberrations confirmed the malignant origin of detected tumor cells, including those from samples that were tumor cell-negative in conventional cytology. A high level of clonality was observed among the tumor cells in the CSF of each patient, yet these CSF tumor cells often exhibited an aberration pattern with distinct differences compared to the corresponding extracranial metastases or primary tumors. In addition to mutations shared by corresponding tissue biopsies, panel sequencing revealed private mutations of CSF-CTCs in resistance related genes and tumor driver genes. In conclusion, the microwell-based CSF-CTC detection appears superior to conventional cytology. The high level of clonality indicates a very close evolutionary relationship within the CSF-CTCs and a monoclonal origin of LMs. Private mutations in resistance related genes and tumor driver genes could impact drug susceptibility and therapy resistance. Therefore, the CSF-based liquid biopsy should be considered for genetic profiling of LMs, potentially improving diagnosis, treatment monitoring, and targeted therapy selection. Citation Format: André Franken, Martin Schramm, Barbara Alberter, Jens Warfsmann, Bernhard Polzer, Franziska Meier-Stiegen, Natalia Krawczyk, Michael Sabel, Marion Rapp, Eugen Ruckhäberle, Tanja Fehm, Hans Neubauer. Detection, isolation, and genetic characterization of circulating tumor cells from the cerebrospinal fluid of breast cancer patients with leptomeningeal metastasis [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 P4-01-27.