Palmirotta Raffaele, Lovero Domenica, Silvestris Erica, Felici Claudia, Quaresmini Davide, Cafforio Paola, Silvestris Franco
Department of Biomedical Sciences and Human Oncology, University of Bari 'Aldo Moro', Bari, Italy
Department of Biomedical Sciences and Human Oncology, University of Bari 'Aldo Moro', Bari, Italy.
Cancer Genomics Proteomics. 2017 May-Jun;14(3):173-179. doi: 10.21873/cgp.20029.
Isolation and genotyping of circulating tumor cells (CTCs) is gaining an increasing interest by clinical researchers in oncology not only for investigative purposes, but also for concrete application in clinical practice in terms of diagnosis, prognosis and decision treatment with targeted therapies. For the mutational analysis of single CTCs, the most advanced biotechnology methodology currently available includes the combination of whole genome amplification (WGA) followed by next-generation sequencing (NGS). However, the sequence of these molecular techniques is time-consuming and may also favor operator-dependent errors, related to the procedures themselves that, as in the case of the WGA technique, might affect downstream molecular analyses.
A preliminary approach of molecular analysis by NGS on a model of CTCs without previous WGA procedural step was performed. We set-up an artificial sample obtained by spiking the SK-MEL-28 melanoma cell line in normal donor peripheral whole blood. Melanoma cells were first enriched using an AutoMACS® (Miltenyi) cell separator and then isolated as single and pooled CTCs by DEPArray™ System (Silicon Biosystems). NGS analysis, using the Ion AmpliSeq™ Cancer Hotspot Panel v2 (Life Technologies) with the Ion Torrent PGM™ system (Life Technologies), was performed on the SK-MEL-28 cell pellet, a single CTC previously processed with WGA and on 1, 2, 4 and 8 recovered CTCs without WGA pre-amplification.
NGS directly carried out on CTCs without WGA showed the same mutations identified in SK-MEL-28 cell line pellet, with a considerable efficiency and avoiding the errors induced by the WGA procedure.
We identified a cost-effective, time-saving and reliable methodological approach that could improve the analytical accuracy of the liquid biopsy and appears promising in studying CTCs from cancer patients for both research and clinical purposes.
循环肿瘤细胞（CTC）的分离和基因分型不仅因其研究目的，而且因其在肿瘤学临床研究中在诊断、预后和靶向治疗决策等临床实践中的具体应用而越来越受到临床研究人员的关注。对于单个CTC的突变分析，目前可用的最先进生物技术方法包括全基因组扩增（WGA）后接下一代测序（NGS）。然而，这些分子技术的操作流程耗时，并且可能还会出现与操作本身相关的人为误差，就像WGA技术那样，这可能会影响下游的分子分析。
对未经过WGA程序步骤的CTC模型进行了NGS分子分析的初步方法。我们构建了一个人工样本，通过将SK-MEL-28黑色素瘤细胞系加入正常供体的外周全血中获得。首先使用AutoMACS®（美天旎）细胞分选仪富集黑色素瘤细胞，然后通过DEPArray™系统（硅生物系统公司）将其分离为单个和汇集的CTC。使用Ion AmpliSeq™癌症热点区域panel v2（赛默飞世尔科技）和Ion Torrent PGM™系统（赛默飞世尔科技）对SK-MEL-28细胞沉淀、一个先前经过WGA处理的单个CTC以及1、2、4和8个未进行WGA预扩增的回收CTC进行NGS分析。
对未进行WGA的CTC直接进行NGS分析显示出与SK-MEL-28细胞系沉淀中相同的突变，效率相当高，并且避免了WGA程序引起的误差。
我们确定了一种经济高效、节省时间且可靠的方法，该方法可以提高液体活检的分析准确性，并在研究癌症患者的CTC用于研究和临床目的方面显示出前景。
