Background Circulating tumor cells (CTCs) have recently emerged as a new dynamic soluble marker for several malignancies including cutaneous melanoma (CM) and are suitable for prognostic evaluations and treatment monitoring. However, to date many limitations still hamper the wide-scale application of CTCs in CM setting, including the lack of standardized methods as well as both low levels and heterogeneity of these cells. Methods We developed a protocol for CTC detection in CM based on immune-magnetic sorting to deplete CD45-, CD31- or CD34-positive cells, followed by dielectrophoretic DEPArray separation according to cell morphology and immunophenotype. To this end, we explored the expression of melanoma stem cell antigens (CD271, ABCB5, and RANK) and the epithelial-to-mesenchymal transition markers (N-Cad, -CD44, and -MCAM/CD146) on CTCs from 17 stage IV CM patients, and investigated their BRAF mutational status by droplet digital PCR. Results The number of CTCs isolated from CM patients ranged from 2 to 91 cells (38 ± 6.4) with respect to healthy donors (p < 0.0002). To confirm the melanoma origin of isolated cells, we observed an 80% agreement between their BRAFV600 mutational status and matched primary tumors. The characterization of the immune phenotype of isolated cells revealed high interindividual and intraindividual heterogeneity that was found to correlate with the clinical outcome. Conclusions The dual-step protocol of immune-magnetic sorting and subsequent dielectrophoretic DEPArray separation, turned out to be a suitable method to isolate viable CTCs from stage IV melanoma patients and enabled quantitative and qualitative analyses on these cells, which may deserve prospective evaluation for potential use in the clinical practice.

中文翻译：

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皮肤黑色素瘤中 CTC 的双程序分离为分子诊断和预后提供了有用的信息。

背景 循环肿瘤细胞 (CTC) 最近已成为包括皮肤黑色素瘤 (CM) 在内的多种恶性肿瘤的一种新的动态可溶性标志物，适用于预后评估和治疗监测。然而，迄今为止，许多限制仍然阻碍 CTC 在 CM 环境中的广泛应用，包括缺乏标准化方法以及这些细胞的低水平和异质性。方法 我们开发了一种基于免疫磁性分选的 CM 中 CTC 检测方案，以消耗 CD45、CD31 或 CD34 阳性细胞，然后根据细胞形态和免疫表型进行介电泳 DEPArray 分离。为此，我们探索了黑色素瘤干细胞抗原（CD271、ABCB5 和 RANK）和上皮间质转化标志物（N-Cad、-CD44、和-MCAM/CD146）对来自 17 名 IV 期 CM 患者的 CTC 进行研究，并通过液滴数字 PCR 研究其 BRAF 突变状态。结果 从 CM 患者中分离出的 CTC 数量范围为 2 至 91 个细胞（38 ± 6.4），相对于健康供体（p < 0.0002）。为了确认分离细胞的黑色素瘤起源，我们观察到它们的 BRAFV600 突变状态与匹配的原发性肿瘤之间有 80% 的一致性。分离细胞的免疫表型特征揭示了个体间和个体间的高度异质性，这与临床结果相关。结论 免疫磁性分选和随后的介电泳 DEPArray 分离的双步骤方案，