Circulating Melanoma Cells (CMCs), End of a Stalemate

Circulating Melanoma Cells (CMCs), End of a StalemateKhoja L1,2,3, Booth J1, Aung K1,2,3, Ward T 1,3, Clack G4, Hughes A4, Lorigan P2,3, Dive C1,31Clinical and Experimental Pharmacology Group, Paterson Institute for Cancer Research, Manchester, UK; 2The Christie NHS Foundation Trust, Manchester, UK; 3School of Cancer and Enabling Sciences, University of Manchester, Manchester Cancer Research Centre and Manchester Academic Health Sciences Centre, Manchester, UK; 4AstraZeneca Pharmaceuticals, Alderley Park, SK10 4TG, UKBackground: Melanoma is the fastest growing cancer worldwide and whilst 80% of cases are cured surgically, 20% develop metastatic disease. Outcome is poor with a median survival of 9 months. The definition of melanoma subtypes by genetic alterations ( BRAF mutations in 50%, NRAS in 20% and c-Kit aberrations in accral and mucosal melanomas) [1, 2] has lead to new targeted therapies such as Vemurafenib (a BRAF inhibitor). CMCs are a potential biomarker to assess response to treatment, monitor disease and relapse and provide new targets for drug development. Methods: The semi automated CellSearch platform enriches for CMCs using melanoma specific antibodies (Melcam antibody for CMC capture and High Molecular Weight Melanoma associated Antigen (HMW-MAA) antibody for CMC detection)[3]. An additional marker of choice can be added to the standard kit. The Isolation by Size of Epithelial Tumour Cells (ISET) platform enriches for CMCs based on cell size (filtration through 8µm pores onto a polycarbonate membrane) and allows downstream analysis of CMCs. The two platforms were evaluated for CMC enumeration (recovery, morphology, marker expression).Results: Cell line western blot analysis showed marker expression heterogeneity which determines CMC recovery with CellSearch in cell spiking experiments. MART-1 could be used as a 4th channel marker with further characterisation of CMCs. The ISET platform enriches for CMCs independently of marker expression. Downstream analysis by immunohistochemistry revealed heterogeneity of melanoma antigens within and between cell lines.Conclusion: Both platforms are valid methods for CMC detection and provide the potential to detect different populations of CMCs. The CellSearch platform is reproducible and provides a robust CMC count whilst the ISET platform provides a flexible platform for CMC analysis and a possible role for CMCs as a ‘virtual biopsy.’ Clinical studies are now underway to explore the potential utility of these platforms.1. Curtin, J.A., et al., Distinct sets of genetic alterations in melanoma. N Engl J Med, 2005. 353(20): p. 2135-47.2. Curtin, J.A., et al., Somatic activation of KIT in distinct subtypes of melanoma. J Clin Oncol, 2006. 24(26): p. 4340-6.3. Rao, C., et al., Circulating melanoma cells and survival in metastatic melanoma. Int J Oncol, 2011. 38(3): p. 755-60.