A139
Characterisation of circulating melanoma cells in patients with advanced disease
Roger Ramcharan1, Ioannis Karydis1, Glenn Clack2, Chandra Rao3, Michael Malone2, Mark Middleton1
1University of Oxford, UK, 2Astrazeneca, Alderley Park, UK, 3Immunicon, Huntington Valley, USA
Background
Advanced melanoma is heterogenous, but associated with treatment resistance and a poor prognosis. The enumeration and characterisation of circulating tumour cells in patients with metastatic breast or colorectal cancer has been shown to provide prognostic and predictive information that can be used to monitor patient management. We describe the development of an automated assay for capturing and detecting circulating melanoma cells (CMC) in the blood of patients with melanoma, and the results of that assay on blood from healthy donors and melanoma patients.
Method
The CellTracks AutoPrep System and CellTracks Analyzer II were used to capture and detecy CMCs. The CMC assay was developed by modifying an existing circulating endothelial cell assay. In the CMC assay, magnetic particles conjugated to antibody specific for the melanoma cell adhesion molecule (CD146) are used to capture melanoma cells from blood. Enriched CMCs are then stained with the nucleic acid dye DAPI, and a monoclonal antibody specific for the High Molecular Weight Melanoma Associated Antigen. The assay also contains monoclonal antibodies to CD45 and CD34 to exclude co-purified leukocytes and circulating endothelial cells, respectively. Ki67 staining was used to determine the proportion of CMCs in cell cycle (G1, S, G2 or M phase). Enriched, stained cells were mounted magnetically and scored as CMCs, based on fluorescence and cell morphology. The assay was validated using blood from healthy donors (n=60) and patients with metastatic melanoma (n=71).
Results
The assay consistently recovered >65% of SK-MEL28 melanoma cells when spiked into 7.5 mL of blood from healthy donors. The assay was linear over the tested range of from 1 to 1200 melanoma cells/7.5mL. In 7.5 mL blood from normal donors 0 CMC were detected in 57/60 (95%). One cell was stained as a CMC in 3/60 (5%) normal donors and all were Ki67 negative. In melanoma patients CMCs ranged from 0 to 8000 /7.5mL blood. One or more CMCs were detected in 39% of the patients, > 2 in 25%, > 5 in 8%, > 10 in 4% and > 100 in 3%. Interestingly, 30-100% (mean 84%) of the CMCs were Ki67 positive suggesting a high proportion of melanoma cells shed into blood are actively dividing. Multiple samples from the patients during the course of treatment failed to establish a correlation between CMC numbers and treatment outcome.
Conclusion
The assay may prove useful as a tool for evaluating biomarkers, targets, and potential treatments in melanoma. The high proportion of samples with no CMCs limits the potential of the assay as a tool for monitoring treatment.
