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Limited genomic heterogeneity of circulating melanoma cells in advanced stage patients.

ABSTRACT: Purpose. Circulating melanoma cells (CMCs) constitute a potentially important representation of time-resolved tumor biology in patients. To date, genomic characterization of CMCs has been limited due to the lack of a robust methodology capable of identifying them in a format suitable for downstream characterization. Here, we have developed a methodology to detect intact CMCs that enables phenotypic, morphometric and genomic analysis at the single cell level. Experimental design. Blood samples from 40 metastatic melanoma patients and 10 normal blood donors were prospectively collected. A panel of 7 chondroitin sulfate proteoglycan 4 (CSPG4)-specific monoclonal antibodies (mAbs) was used to immunocytochemically label CMCs. Detection was performed by automated digital fluorescence microscopy and multi-parametric computational analysis. Individual CMCs were captured by micromanipulation for whole genome amplification and copy number variation (CNV) analysis. Results. Based on CSPG4 expression and nuclear size, 1-250 CMCs were detected in 22 (55%) of 40 metastatic melanoma patients (0.5-371.5 CMCs ml(-1)). Morphometric analysis revealed that CMCs have a broad spectrum of morphologies and sizes but exhibit a relatively homogeneous nuclear size that was on average 1.5-fold larger than that of surrounding PBMCs. CNV analysis of single CMCs identified deletions of CDKN2A and PTEN, and amplification(s) of TERT, BRAF, KRAS and MDM2. Furthermore, novel chromosomal amplifications in chr12, 17 and 19 were also found. Conclusions. Our findings show that CSPG4 expressing CMCs can be found in the majority of advanced melanoma patients. High content analysis of this cell population may contribute to the design of effective personalized therapies in patients with melanoma.

SUBMITTER: Ruiz C

PROVIDER: S-EPMC5023009 | biostudies-literature | 2015 Jan

REPOSITORIES: biostudies-literature

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Limited genomic heterogeneity of circulating melanoma cells in advanced stage patients.

Ruiz Carmen C Li Julia J Luttgen Madelyn S MS Kolatkar Anand A Kendall Jude T JT Flores Edna E Topp Zheng Z Samlowski Wolfram E WE McClay Edward E Bethel Kelly K Ferrone Soldano S Hicks James J Kuhn Peter P

Physical biology 20150109 1

Purpose. Circulating melanoma cells (CMCs) constitute a potentially important representation of time-resolved tumor biology in patients. To date, genomic characterization of CMCs has been limited due to the lack of a robust methodology capable of identifying them in a format suitable for downstream characterization. Here, we have developed a methodology to detect intact CMCs that enables phenotypic, morphometric and genomic analysis at the single cell level. Experimental design. Blood samples fr ...[more]

PMID: 25574741

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Project description:During the process of metastasis, cancer cells dissociate from primary tumors, migrate to distal sites, and finally colonize, eventually leading to the formation of metastatic tumors. These cancer cells, defined circulating tumor cells (CTCs) spreading through the blood stream, may develop metastatic lesions or remain dormant. Some emerging clinical evidence supports that some tumor cells may possess metastatic properties already in the earlier stages of tumorigenesis. Because the initiation and progression of vertical growth in human melanoma is fundamental to the notion of tumor virulence and progression, we decided to immune-magnetic collect and molecularly characterize circulating melanoma cells (CMCs) from melanoma patients AJCC staged = pT1b (i.e., transition from radial to vertical phase). CMCs are phenotypically and molecularly heterogeneous, thus we performed a "home-made Liquid-Biopsy," by targeting the melanoma-associated-antigen, MCAM/MUC18/CD146, and/or the melanoma-initiating marker, ABCB5. We assessed a biomarker qualitative expression panel, contemplating the angiogenic-potential, melanoma-initiating and melanoma-differentiation drivers, cell-cell adhesion molecules, matrix-metallo-proteinases, which was performed on three enriched subpopulations from a total of 61 blood-samples from 21 melanoma patients. At first, a significant differential expression of the specific transcripts was documented between and within the CMC fractions enriched with MCAM-, ABCB5-, and both MCAM/ABCB5-coated beads, when analyzing two distinct groups: early AJCC- (stage I-II) and advanced- staged patients (stage II-IV). Moreover, in the early-AJCC staged-group, we could distinguish "endothelial," CD45-MCAM+ enriched-, "stem" S-CMCs, CD45-ABCB5+ enriched- and a third hybrid bi-phenotypic CD45-MCAM+/ABCB5+ enriched-fractions, due to three distinct gene-expression profiles. In particular, the endothelial-CMCs were characterized by positive expression of genes involved in migration and invasion, whilst the stem CMC-fraction only expressed stem and differentiation markers. The third subpopulation isolated based on concurrent MCAM and ABCB5 protein expression showed an invasive phenotype. All three distinct CMCs sub-populations, exhibited a primitive, "stem-mesenchymal" profile suggesting a highly aggressive and metastasizing phenotype. This study confirms the phenotypic and molecular heterogeneity observed in melanoma and highlights those putative genes involved in early melanoma spreading and disease progression.

Dataset Information

Limited genomic heterogeneity of circulating melanoma cells in advanced stage patients.

Publications

Limited genomic heterogeneity of circulating melanoma cells in advanced stage patients.

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