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Mol Oncol. 2017 Dec;11(12):1687-1697. doi: 10.1002/1878-0261.12113. Epub 2017 Oct 24.

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

Author information

1
Clinical & Experimental Pharmacology Group, CRUK Manchester Institute, University of Manchester, UK.
2
Division of Molecular and Clinical Cancer Sciences, University of Manchester, UK.
3
Molecular Oncology Group, CRUK Manchester Institute, University of Manchester, UK.
4
Institute of Cancer Sciences, University of Manchester, UK.
5
Cancer Research UK Lung Cancer Centre of Excellence, UK.

Abstract

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.

KEYWORDS:

circulating tumour cells; molecular analysis; single cells; stability; storage

PMID:
28741788
PMCID:
PMC5709616
DOI:
10.1002/1878-0261.12113
[Indexed for MEDLINE]
Free PMC Article

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