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.