Abstract
Circulating melanoma cells (CMCs) are responsible for the hematogenous spread of melanoma and, ultimately, metastasis. However, their study has been limited by the low abundance in patient blood and the heterogeneous expression of surface markers. The FDA-approved CellSearch platform enriches CD146-positive CMCs, whose number correlates with progression-free survival and overall survival. However, a single marker may not be sufficient to identify them all. The Parsortix system allows enrichment of CMCs based on their size and deformability, keeping them viable and suitable for downstream molecular analyses. In this study, we tested the strengths, weaknesses and potential convergences of both platforms to integrate the counting of CMCs with a protocol for their genetic analysis. Samples run on Parsortix were labeled with a customized melanoma antibody cocktail, which efficiently labeled and distinguished CMCs from endothelial cells/leukocytes. The capture rate of CellSearch and Parsortix was comparable for cell lines, but Parsortix had a higher capture rate in real-life samples. Moreover, double enrichment with both CellSearch and Parsortix succeeded in removing most of the leukocyte contamination, resulting in an almost pure CMC sample suitable for genetic analysis. In this regard, a proof-of-concept analysis of CMCs from a paradigmatic case of a metastatic uveal melanoma patient led to the identification of multiple genetic alterations. In particular, the GNAQ p.Q209L was identified as homozygous, while a deletion in BAP1 exon 9 was found hemizygous. Moreover, an isochromosome 8 and a homozygous deletion of the CDKN2A gene were detected. In conclusion, we have optimized an approach to successfully enrich and retrieve viable CMCs from metastatic melanoma patients. Moreover, this study provides proof-of-principle for the feasibility of a marker-agnostic CMC enrichment followed by CMC phenotypic identification and genetic analysis.Kindly check and confirm the processed contributed equally is correctly identify We confirm.