A model workflow for microfluidic enrichment and genetic analysis of circulating melanoma cells

Circulating melanoma cells (CMCs) are responsible for the hematogenous spread of melanoma and ultimately metastasis. However, their study has been limited by their low abundance in patient blood and their heterogeneous expression of surface markers. The FDA-approved CellSearch platform enriches CD146-positive CMCs, the number of which 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 analysis. 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. CellSearch and Parsortix were used to enrich CMCs from samples of a paradigmatic uveal melanoma patient. Enriched CMCs and cfDNA were subjected to custom NGS analysis and results were confirmed by ddPCR or MLPA assays.Sequencing libraries, from lysed-only CMCs, WGA-CMCs, and cfDNA (for a total of 7 samples) were prepared using the Magnis SureSelect XT HS Rev B Reagent Kit on the Magnis NGS Prep System (Agilent Technologies). The following genes were sequenced on a NextSeq 550 instrument (Illumina) using a 300 cycle NextSeq 500/550 Mid Output v2 kit: ABL1, ADAMTS18, ALK, ARID2, ATM, BAP1, BRAF, CDK4, CDKN2A, CTNNB1, CYSLTR2, EGFR, EIF1AX, ERBB2, ERBB4, FBXW7, FGFR1, FGFR2, FGFR3, GNA11, GNAQ, GRIN2A, HOXD8, HRAS, IDH1, KDR, KIT, KRAS, MAP2K1, MAP2K2, MC1R, MET, MITF, MLH1, NF1, NRAS, PDGFRA, PIK3CA, PLCB4, PPP6C, PREX2, PTEN, PTPN11, RAC1, RB1, RET, ROS1 SF3B1, SRC, SRSF2, STK11, STK19, TERT, TP53, U2AF1 for SNP/INDEL analysis and BRAF, CDC42, CDK4, ERBB2, FGFR1, FGFR3, NEDD9, NFAT5, PIK3CA, PPARG, PTK2, TUSC3 for CNV assessment.