Circulating Melanoma Cell Subpopulations: Their Heterogeneity and Differential Responses to Treatment

Abstract

Metastatic melanoma is a highly heterogeneous tumor; thus, methods to analyze tumor-derived cells circulating in blood should address this diversity. Taking this into account, we analyzed, using multiparametric flow cytometry, the co-expression of the melanoma markers melanoma cell adhesion molecule and melanoma-associated chondroitin sulphate proteoglycan and the tumor-initiating markers ATP-binding cassette sub-family B member 5 (ABCB5), CD271, and receptor activator of NF-κβ (RANK) in individual circulating tumor cells (CTCs) from 40 late-stage (III–IV) and 16 early-stage (I–II) melanoma patients. CTCs were heterogeneous within and between patients, with limited co-expression between the five markers analyzed. Analysis of patient matched blood and metastatic tumors revealed that ABCB5 and RANK subpopulations are more common among CTCs than in the solid tumors, suggesting a preferential selection for these cells in circulation. Pairwise comparison of CTC subpopulations longitudinally before and 6–13 weeks after treatment initiation showed that the percentage of RANK+ CTCs significantly increased in the patients undergoing targeted therapy (N=16, P<0.01). Moreover, the presence of ⩾5 RANK+ CTCs in the blood of patients undergoing targeted therapies was prognostic of shorter progression-free survival (hazards ratio 8.73, 95% confidence interval 1.82–41.75, P<0.01). Taken together, our results provide evidence of the heterogeneity among CTC subpopulations in melanoma and the differential response of these subpopulations to targeted therapy.

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

“…Using this approach we provided for the first time, a detailed insight into the diversity of MelCTCs within each patient, and showed that the prognostic utility of MelCTCs may not rely on the total count of CTCs but on the CTC subpopulations circulating within an individual. This study indicated that a high number of MelCTCs express melanomainitiating or stem cell markers (ABCB5 and RANK) while only very low numbers of CTCs express melanoma markers MCSP and MCAM [25]. Importantly, the common expression of these melanomainitiating markers by MelCTCs did not correlate with the expression of these markers in patient-matched tumors, where a low frequency of melanoma tumor cells positive for these markers was observed.…”

“…Based on our previous identification of heterogeneous MelCTCs [21], we recently developed a flow-cytometry multimarker approach to detect and analyze CTCs for the presence of melanomaassociated markers, such as MCSP and MCAM, in combination with melanoma stem cell markers, such as ABCB5, RANK (receptor activator of NF-kb) and CD271 [25]. Using this approach we provided for the first time, a detailed insight into the diversity of MelCTCs within each patient, and showed that the prognostic utility of MelCTCs may not rely on the total count of CTCs but on the CTC subpopulations circulating within an individual.…”

“…In these techniques a cocktail of antibodies against cell surface or intracellular markers associated with melanocyte biology or melanoma pathogenesis [21,25,30,55] are used to recognize the cells. Alternately, molecular approaches that detect RNA or DNA from enriched MelCTCs, by quantitative real-time PCR (qRT-PCR) [4,39] or droplet digital PCR (ddPCR) [45], respectively, have been used for CTC detection and characterization.…”

“…Despite MelCTC heterogeneity [25,29,53], methods for their capture and enrichment have relied predominantly on the expression of one or two known cell surface markers (Fig. 1).…”

“…For melanoma, the difficulties are magnified because common CTC markers, such as EpCAM, used in CTC enrichment of epithelial cancers including breast and prostate cancers, are not commonly expressed by MelCTCs, since melanocytes originate from the neural crest and not the epithelium [19]. In addition, MelCTCs are a very heterogeneous population of cells [21,25,29], yet current techniques used to enrich melanoma cells from blood do not commonly consider this factor, as their principle for CTC detection relies on the expression of only one or two markers. The isolation of MelCTCs usually follows two common steps: first, CTCs are enriched from the background of millions of blood cells and second, CTCs are detected and characterized in the enriched fraction.…”