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 (NBD) were prospectively collected. A panel of 7 chondroitin sulfate proteoglycan 4 (CSPG4)-specific monoclonal antibodies (mAb) 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 (WGA) and copy number variation (CNV) analysis. Results Based on CSPG4 expression and nuclear size, 1 to 250 CMCs were detected in 22 (55%) of 40 metastatic melanoma patients (0.5 to 371.5 CMCs/ml). 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 population may contribute to develop effective therapeutic strategies.

Melanoma circulating tumor cells: Benefits and challenges required for clinical application

“…Detection of MelCTCs without a previous enrichment step has been reported by Ruiz and colleagues [51], where the Epic Sciences platform detected MelCTCs from whole blood, using a panel of seven anti-MCSP monoclonal antibodies. MCSP is a cell surface protein involved in melanoma proliferation, spreading and migration of cells and it is overexpressed in more than 90% of melanoma tumor tissue samples [6,13].…”

“…Using this marker for detection, 1e250 CTCs were detected in 8 mL of blood (0.5e371.5 CTCs/mL of blood) from 22/40 metastatic melanoma patients (55%). Interestingly, this method also enabled whole genome amplification and copy number variation (CNV) analyses of single MelCTCs, which revealed deletions of CDKN2A and PTEN, and amplifications of melanoma related genes, TERT, BRAF, KRAS and MDM2 amongst others [51].…”

Biophysical technologies for understanding circulating tumor cell biology and metastasis

“…This is now possible on a single cell basis at the level of whole genome copy number analysis (63)(64)(65)(66)(67). Combined with morphologic analysis of CTCs, the addition of genomic analysis provides a capacity to study cancer populations as a collection of individual cells, often revealing cancers to be oligoclonal proliferations of cancer cells, rather than truly clonal populations possessing a singular genome (68). The unique nature of CTC analysis, permitting serial analysis has been successfully performed on patients, creating a method for the identification of new tumor lineages to be identified as they evolve in real time (69).…”

Circulating Tumor Cells, DNA, and mRNA: Potential for Clinical Utility in Patients With Melanoma

“…Marker-dependent strategies use melanomaspecific surface antigens and immunomagnetic beads to positively select for melanoma CTCs in blood. Surface markers such as high molecular weight melanoma-associated antigen (HMW-MAA), also known as melanoma-associated chondroitin sulfate proteoglycan (MCSP), CD146 or melanoma cell adhesion molecule (MCAM), and ATP-binding cassette subfamily B member 5 (ABCB5) are used either in isolation or combination to facilitate CTC capture [16][17][18][19][20][21][22]. In contrast, marker-independent strategies capitalize on CTCs' physical properties of size and density.…”

“…This may be motivated by ease of harvesting genetic material, but it also compromises the assurance that the detected mutations are derived from intact circulating cells. Currently, the only mechanism of isolating intact melanoma CTCs for DNA extraction and analysis is through microcapillary dissection [19,27]. The identification of melanoma-specific genetic and molecular mutations has primarily served as proof-of-concept findings in many CTC and ctDNA studies, but these changes may also have potential prognostic and predictive significance [77,78,[82][83][84].…”