title: Efficiency of whole genome amplification of single circulating tumor cells enriched by CellSearch and sorted by FACS.
abstract: BACKGROUND
  Tumor cells in the blood of patients with metastatic carcinomas are associated with poor survival. Knowledge of the cells' genetic make-up can help to guide targeted therapy. We evaluated the efficiency and quality of isolation and amplification of DNA from single circulating tumor cells (CTC).
  
  
  METHODS
  The efficiency of the procedure was determined by spiking blood with SKBR-3 cells, enrichment with the CellSearch system, followed by single cell sorting by fluorescence-activated cell sorting (FACS) and whole genome amplification. A selection of single cell DNA from fixed and unfixed SKBR-3 cells was exome sequenced and the DNA quality analyzed. Single CTC from patients with lung cancer were used to demonstrate the potential of single CTC molecular characterization.
  
  
  RESULTS
  The overall efficiency of the procedure from spiked cell to amplified DNA was approximately 20%. Losses attributed to the CellSearch system were around 20%, transfer to FACS around 25%, sorting around 5% and DNA amplification around 25%. Exome sequencing revealed that the quality of the DNA was affected by the fixation of the cells, amplification, and the low starting quantity of DNA. A single fixed cell had an average coverage at 20× depth of 30% when sequencing to an average of 40× depth, whereas a single unfixed cell had 45% coverage. GenomiPhi-amplified genomic DNA had a coverage of 72% versus a coverage of 87% of genomic DNA. Twenty-one percent of the CTC from patients with lung cancer identified by the CellSearch system could be isolated individually and amplified.
  
  
  CONCLUSIONS
  CTC enriched by the CellSearch system were sorted by FACS, and DNA retrieved and amplified with an overall efficiency of 20%. Analysis of the sequencing data showed that this DNA could 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 were successfully sequenced to 20× depth making it possible to call 72% of the variants. The overall coverage was reduced to 30% at 20× depth, making it possible to call 56% of the variants in CellSave-fixed cells.
authors.affiliation: Department of Medical Cell BioPhysics, MIRA Institute, University of Twente, Carre Room C4437, Hallenweg 23, 7522 NH Enschede, The Netherlands.
authors.forename: Joost F
authors.initials: JF
authors.lastname: Swennenhuis
authors.affiliation: Janssen Research & Development, Janssen Pharmaceutical Companies of Johnson & Johnson, Beerse, Belgium.
authors.forename: Joke
authors.initials: J
authors.lastname: Reumers
authors.affiliation: Janssen Research & Development, Janssen Pharmaceutical Companies of Johnson & Johnson, Beerse, Belgium.
authors.forename: Kim
authors.initials: K
authors.lastname: Thys
authors.affiliation: Janssen Research & Development, Janssen Pharmaceutical Companies of Johnson & Johnson, Beerse, Belgium.
authors.forename: Jeroen
authors.initials: J
authors.lastname: Aerssens
authors.affiliation: Department of Medical Cell BioPhysics, MIRA Institute, University of Twente, Carre Room C4437, Hallenweg 23, 7522 NH Enschede, The Netherlands.
authors.forename: Leon Wmm
authors.initials: LW
authors.lastname: Terstappen