Although circulating tumor cells (CTCs) hold a wealth of information, the only clinically approved use of CTCs is enumeration for the prediction of progression-free survival and overall survival. The use of CTCs for prognosis is groundbreaking, but specifically enriching and counting tumor cells does not fully exploit the information available in CTCs. A real‑world analogy would be visiting the library and carefully selecting a stack of books, counting them, looking at the titles, but never reading the text inside. Research is intensely focused on reading the whole “book” of molecular information provided by CTCs. Several techniques exist to take advantage of CTC content. The techniques can be subdivided into three general categories: DNA, RNA and protein analysis.
Characterization of CTC DNA requires whole-genome amplification (WGA) using PCR-based or multiple displacement amplification (MDA). Although both techniques are powerful enough to amplify DNA from a single cell, they are also prone to method-specific drawbacks. PCR-based WGA introduces errors and amplification bias, while MDA is subject to allelic dropout, the random failure of one allele to amplify. Another technique for DNA analysis is fluorescence in situ hybridization (FISH) with probes for specific oncogenes, such as HER2, EGFR and PTEN (1). FISH analysis requires CTC enrichment, which may be accomplished by multiple techniques, such as the CellSearch® system, microfluidic chips or isolation by size of epithelial tumor cells (ISET). ISET utilizes specialized filters to take advantage of the fact that solid tumor CTCs are larger than blood cells, and recovers intact CTCs (1). Using ISET, Pailler et al. identified c-ros oncogene 1 (ROS1) rearrangement in CTCs of patients with biopsy-confirmed ROS1 rearrangements (2). This group also detected a decrease in ROS1 copy number in patients who responded to a ROS1 inhibitor, and an increase in ROS1 copy number in patients who progressed despite treatment.
RNA analysis of CTCs can be performed using expression arrays, single-molecule RNA sequencing or single CTC RNA sequencing (3). Transcriptional analysis of single CTCs requires an amplification step. Using quantitative PCR, Powell et al. examined the expression of a selected panel of 87 cancer-associated genes (4). They identified expression differences when CTCs from breast cancer patients were compared to breast cancer cell lines and demonstrated the feasibility of CTC expression analysis from a single cell.
Historically, protein detection has been the primary technique used for isolation and analysis of CTCs. Immunomagnetic separation with EpCAM-specific antibodies or other cell surface markers forms the basis for many CTC enrichment and separation techniques. CTCs have also been characterized using immunofluorescence with specific antibodies to EGFR, HER2, prostate-specific antigen and estrogen receptor (1). Newer enrichment technology uses marker-independent isolation, but antibody-based techniques remain essential for verifying the identity of CTCs.
- 1. Lowes, L.E. and Allan, A.L. (2014) Recent advances in the molecular characterization of circulating tumor cells. Cancers (Basel) 6, 595.
- 2. Pailler, E. et al. (2015) High level of chromosomal instability in circulating tumor cells of ROS1-rearranged non-small-cell lung cancer. Ann. Oncol. 26, 1408.
- 3. Pantel, K. and Speicher, M.R. (2015) The biology of circulating tumor cells. Oncogene, doi:10.1038/onc.2015.192.
- 4. Powell, A.A. et al. (2012) Single cell profiling of circulating tumor cells: transcriptional heterogeneity and diversity from breast cancer cell lines. PLoS One 7, e33788.