A versatile method for enumeration and characterization of circulating tumor cells from patients with breast cancer

Abstract

Aim: To establish a standardized protocol for the isolation and enumeration of circulating tumor cells (CTCs) from peripheral blood of patients with metastatic breast cancer.

Methods: The protocol used tumor cells spiked in a lymphoid cell line with detection by flow cytometry and quantitative reverse transcription polymerase chain reaction (QRT-PCR). Cells of the human mammary cancer subtypes were spiked into Jurkat cells, which served as the lymphocyte designate in numbers from 10 to 500 per 10⁵ Jurkat cells. This mixed population was probed for CD45, EpCAM, and pancytokeratin acquired from flow cytometry and characterized by microscopy. QRT-PCR was done for CK-19, MUC-1, EpCAM, and GAPDH. Validation was attained with blood samples from 22 patients with metastatic breast cancer and 20 healthy individuals.

Results: Flow cytometry could detect 1 breast cancer cell per 100,000 Jurkat cells, with similar detection levels in the breast cancer subtypes. Samples from patients with breast cancer showed a range of CTCs from 1-85 per 10 mL of blood. Quantitation of expression for EpCAM, CK-19, Muc-1, and Her2neu confirmed the presence of CTCs in 76% of samples.

Conclusion: Density gradient and immunomagnetic enrichment accomplished isolation of CTCs and quantitation was achieved using flow cytometry. Combined QRT-PCR and imaging further validated these findings, rendering a robust methodology.

Keywords

Circulating tumor cells / cytokeratin-19 / flow cytometry / circulating tumor cell enumeration / quantitative reverse transcription polymerase chain reaction / EpCAM

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