Identification and enumeration of circulating tumor cells in the cerebrospinal fluid of breast cancer patients with central nervous system metastases

Abstract

The number of circulating tumor cells (CTCs) in the peripheral blood of metastatic breast cancer patients is now an established prognostic marker. While the central nervous system is a common site of metastasis in breast cancer, the standard marker for disease progression in this setting is cerebrospinal fluid (CSF) cytology. However, the significance of CSF cytology is unclear, requires large sample size, is insensitive and subjective, and sometimes yields equivocal results. Here, we report the detection of breast cancer cells in CSF using molecular markers by adapting the CellSearch system (Veridex). We used this platform to isolate and enumerate breast cancer cells in CSF of breast cancer patients with central nervous system (CNS) metastases. The number of CSF tumor cells correlated with tumor response to chemotherapy and were dynamically associated with disease burden. This CSF tumor cell detection method provides a semi-automated molecular analysis that vastly improves the sensitivity, reliability, objectivity, and accuracy of detecting CSF tumor cells compared to CSF cytology. CSF tumor cells may serve as a marker of disease progression and early-stage brain metastasis in breast cancer and potentiate further molecular analysis to elucidate the biology and significance of tumor cells in the CSF.

Figure 1. Detection by the CellSearch system of breast cancer cells but not glioblastoma cells present in human blood

(A) Western blot analysis of EpCAM expression in SF767 human glioblastoma cells and SKBR3 human breast cancer cells. Ran shown as a loading control. (B) Number of CTCs enumerated by CellSearch criteria in normal human blood spiked with 1,000 SF767 and SKBR3 cells from cell culture. (C) Exemplary image of an SKBR3 cell isolated by the CellSearch system.

Figure 2. Schematic demonstrating use of the CellSearch system to detect breast cancer cerebrospinal fluid tumor cells

Figure 3. Recovery rate of breast cancer cells present in cerebrospinal fluid

(A) Enumeration and (B) recovery rate of cultured SKBR3 cells spiked into normal blood (n>3).

Figure 4. Detection of CSFTCs in breast cancer patients with CNS metastasis and correlation with Karnofsky performance status

(A) Exemplary images of species detected in CSF samples. (B) Correlation of Karnofsky performance status with CSF cytology versus CSFTC number in subjects 1,2, 3, and 5 throughout treatment. (C) Correlation of CSFTCs with CSF results that report positive (+), equivocal (?), or negative (-). (D) MRI of subject 5 on day of CSF withdrawal.

Figure 5. CSFTC counts dynamically change with chemotherapy

(A) CSFTC count over time and (B) MRI of subject 1 at baseline and days post-treatment initiation. CSFTC count over time in (C) subject 2 and (D) subject 3. (E) MRI of subject 3 at baseline (left panel), 42 (middle panel), and 68 days (right panel) post-treatment initiation. (F) MRI of subject 4 at baseline (left panel) and 57 days post-treatment initiation (right panel).