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

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.

Pulmonary venous circulating tumor cell dissemination before tumor resection and disease relapse

“…Whole genome amplification (WGA) was performed using the Ampli1 WGA kit (Menarini) according to the manufacturer's instructions. The efficacy of WGA was then evaluated by a multiplex quality control PCR (Ampli1 QC kit, Menarini) as previously described 26 followed by visualization of PCR bands on a 1.5% (w/v) agarose gel. This quality control step allowed us to establish a Genome Integrity Index (GII) of 0-4 for each sample and single cells with GII≥2 were considered with good quality DNA and eligible for subsequent downstream analysis.…”

CTC Technologies and Tools

“…Reports on the expression of treatment targets on CTC showed extensive intra and inter patient heterogeneity of expression (49)(50)(51). Therefore, it would be great if tools were available to provide a likelihood that the nucleic acids are of sufficient quality after isolation of the single CTC (58,59). For extensive interrogation of the CTC the individual cells will have to be isolated and for molecular characterization the nucleic acids will have to be amplified.…”

“…As many CTC are undergoing apoptosis, the nucleic acids may not be of a sufficient quality for thorough interrogation. Therefore, it would be great if tools were available to provide a likelihood that the nucleic acids are of sufficient quality after isolation of the single CTC (58,59). This may save effort and cost involved with sequencing, fluorescence in situ hybridization, etc.…”

Circulating tumor cells (CTCs) for the noninvasive monitoring and personalization of non-small cell lung cancer (NSCLC) therapies

“…F o l l o w i n g e n r i c h m e n t a n d i d e n t i f i c a t i o n , t h e molecular characterization of single CTCs requires technology for their isolation, which can be performed by FACS, DEPArray, microfluidic devices or laser capture microdissection. Several overviews and technology articles of the CTC isolation methods used for the genetic analysis have been already published (47)(48)(49)(50)(51)(52).…”