Molecular analysis of single circulating tumour cells following long-term storage of clinical samples

The CellSearch^® semiautomated CTC enrichment and staining system has been established as the 'gold standard' for CTC enumeration with CellSearch^® CTC counts recognized by the FDA as prognostic for a number of cancers. We and others have gone on to show that molecular analysis of CellSearch^® CTCs isolated shortly after CellSearch^® enrichment provides another valuable layer of information that has potential clinical utility including predicting response to treatment. Although CellSearch^® CTCs can be readily isolated after enrichment, the process of analysing a single CellSearch^® patient sample, which may contain many CTCs, is both time-consuming and costly. Here, we describe a simple process that will allow storage of all CellSearch^® -enriched cells in glycerol at -20 °C for up to 2 years without any measurable loss in the ability to retrieve single cells or in the genome integrity of the isolated cells. To establish the suitability of long-term glycerol storage for single-cell molecular analysis, we isolated individual CellSearch^® -enriched cells by DEPArray™ either shortly after CellSearch^® enrichment or following storage of matched enriched cells in glycerol at -20 °C. All isolated cells were subjected to whole-genome amplification (WGA), and the efficacy of single-cell WGA was evaluated by multiplex PCR to generate a Genome Integrity Index (GII). The GII results from 409 single cells obtained from both 'spike-in' controls and clinical samples showed no statistical difference between values obtained pre- and postglycerol storage and that there is no further loss in integrity when DEPArray™-isolated cells are then stored at -80 °C for up to 2 years. In summary, we have established simple yet effective 'stop-off' points along the CTC workflow enabling CTC banking and facilitating selection of suitable samples for intensive analysis once patient outcomes are known.