Prevalence and heterogeneity of circulating tumour cells in metastatic cutaneous melanoma

Abstract

We previously demonstrated that circulating tumour cells (CTCs) are detectable by the MelCAM and high molecular weight melanoma-associated antigen (HMW-MAA)-dependent CellSearch platform. However, CTCs which do not express these capture and detection markers are not detectable by CellSearch. Consequently, we explored the use of isolation by size of epithelial tumour cells (ISET), a marker independent, filtration-based device to determine the prevalence and heterogeneity of CTCs in metastatic cutaneous melanoma patients. Ninety patients were prospectively recruited and blood samples taken before treatment. Patients' blood was filtered using the ISET platform. CTCs were enumerated using dual immunohistochemistry with positive selection by S100 expression and exclusion of leucocytes and endothelial cells expressing CD45 or CD144, respectively. A panel of markers (Melan-A, MITF, MelCAM, high molecular melanoma-associated antigen, CD271 and MAGEC) was also examined. Fifty-one patients (57%) had CTCs (range 1-44 CTCs/4 ml blood) and 12 patients also had circulating tumour microemboli. Seven patients had S100- CTCs, 11 patients' CTCs were S100+ and 33 patients had S100+ and S100- CTCs. Substantial intrapatient and interpatient heterogeneity was observed for all other melanoma-associated markers. CTCs in metastatic cutaneous melanoma are detectable using the flexible marker-independent ISET platform. CTCs display significant marker expression heterogeneity implying that marker-dependent platforms would not detect all CTCs and multimarker assays are now required to reveal the biological significance of this CTC heterogeneity.

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

“…However, this approach also detected benign circulating nevus cells [15], suggesting the inability of this assay to distinguish between benign nevus cells and melanoma cells. Alternatively, when using the same ISET enrichment technique, with CTCs defined by positive immunohistochemistry expression of S100 and negative expression for CD45 or CD144 (leukocyte and endothelial cell markers, respectively), 51/90 (57%) metastatic melanoma patients had detectable CTCs (1e44 CTCs/mL of blood) [29]. The low percentage of metastatic patients with high-burden disease found with CTCs, shows the unsuitability of this method for detecting all CTCs present in patients.…”

“…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.…”

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

“…Importantly, a methodology has been previously described to isolate live CTCs and has been effective for melanoma, glioma, bladder cancer, and nonsmall cell lung cancer [27,[95][96][97].The continued development of this technique, along with other CTP analysis methods, enables better understanding of tumor changes that accumulate over the course of treatment. Marker dependent: HMW-MAA via CellSearch Captured cells were first confirmed as CTCs using qRT-PCR, then a second sample was used to detect BRAF mutation in 81% of patients [21] Marker dependent: Negative selection (CD45 and RBC depletion), cytospin, and IHC Performed microRNA analysis of CTCs using qRT-PCR, which enriched microRNA 106a, 20a, and 21 [85] Marker independent: ISET plus IHC No genetic testing done, but molecular phenotype assessed through IHC, which found intrapatient and interpatient heterogeneity in S100, Melan-A, MITF, MCAM, HMW-MAA, CD271, and MAGEC1 expression [25] Marker independent: Oncoquick plus telomerase assay…”

“…Size-based isolation techniques, such as isolation by size of epithelial tumor cells (ISET), use a porous filter to trap large cells (larger than 8 mm) regardless of surface marker expression. The cells can be evaluated for mRNA or DNA mutations or transferred onto a slide for immunohistochemistry (IHC) evaluation [23][24][25]. Densitybased techniques use Ficoll-hypaque or Oncoquick centrifugation separation media to enrich for a layer of cells containing CTCs, suitable for further isolation [15,26,27].…”