Efficiency of whole genome amplification of Single Circulating Tumor Cells enriched by CellSearch and sorted by FACS. Efficiency of whole genome amplification of Single Circulating Tumor Cells enriched by CellSearch and sorted by FACS

Background, Tumor cells in the blood of patients suffering from metastatic carcinomas are associated with poor survival. Knowledge of the cells’ genetic make-up can help to guide targeted therapy. In this study we evaluate the efficiency and quality, of isolation and amplification, of DNA from single CTC; Methods, The efficiency of the procedure is determined by spiking blood with SKBR-3 cells, enrichment with the CellSearch system followed by single cell sorting by FACS and Whole Genome Amplification. A selection of single cell DNA from fixed and unfixed SKBR-3 cells is exome sequenced and the DNA quality is analyzed. Single CTC’s from lung cancer patients are used to demonstrate the potential of single CTC molecular characterization; Results, The overall efficiency of the procedure from spiked cell to amplified DNA was ~20%. Losses attributed to the CellSearch system were about 20%, transfer to FACS ~25%, sorting ~5% and DNA amplification ~25%. Exome sequencing revealed that the quality of the DNA is affected by the fixation of the cells, the amplification and the low starting quantity of DNA. A single fixed cell has an average coverage at 20xdepth of 30% when sequencing to an average of 40xdepth, whereas a single unfixed cell has a 45% coverage. Genomiphi-amplified genomic DNA has a coverage of 72% versus a coverage of 87% of genomic DNA. 21% of the CTC from lung cancer patients identified by the CellSearch system could be isolated individually and amplified; Conclusions, CTC’s enriched by the CellSearch system can be sorted by FACS, and DNA retrieved and amplified with an overall efficiency of 20%. Analysis of the sequencing data shows that this DNA can be used for variant calling, but not for quantitative measurements such as copy number detection. Close to 55% of the exome of single SKBR-3 cells can be successfully sequenced to 20x depth making it possible to call 72% of the variants. The overall coverage is reduced to 30% at 20x depth making it possible to call 56% of the variants in cellsave fixed cells.

背景：转移性癌患者血液中的肿瘤细胞与不良预后相关。明确此类细胞的遗传组成，可为靶向治疗提供指导。本研究旨在评估从单个循环肿瘤细胞（Circulating Tumor Cell, CTC）中分离并扩增DNA的效率与质量。方法：本研究通过向血液中加入SKBR-3细胞开展加标实验，采用CellSearch系统完成细胞富集，随后通过荧光激活细胞分选（Fluorescence-Activated Cell Sorting, FACS）进行单细胞分选，并实施全基因组扩增（Whole Genome Amplification）以评估实验流程的整体效率。对固定与未固定的SKBR-3单细胞DNA进行外显子测序，并分析DNA质量；此外，本研究使用肺癌患者的单个CTC，以验证单细胞CTC分子表征技术的应用潜力。结果：从加标细胞到获得扩增DNA的整体实验效率约为20%。其中，CellSearch系统导致的细胞损失约20%，转移至FACS分选环节损失约25%，分选环节损失约5%，DNA扩增环节损失约25%。外显子测序结果显示，细胞固定方式、扩增过程及初始DNA模板量偏低，均会对DNA测序质量产生影响。当测序平均深度达40×时，单个固定细胞的平均覆盖度为30%；而单个未固定细胞的覆盖度可达45%。采用Genomiphi扩增的基因组DNA覆盖度为72%，未扩增的基因组DNA覆盖度则为87%。经CellSearch系统鉴定的肺癌患者CTC中，有21%可被单独分离并完成扩增。结论：经CellSearch系统富集的CTC可通过FACS进行分选，获取并扩增DNA的整体效率可达20%。测序数据分析表明，此类DNA可用于变异位点检出，但不适用于拷贝数检测等定量分析。单个SKBR-3细胞的外显子组中，近55%可在20×测序深度下成功被覆盖，从而可检出72%的变异位点。在20×测序深度下，经细胞保存液固定的细胞整体覆盖度降至30%，此时可检出56%的变异位点。