One of the main challenges with isolating CTCs is their low abundance relative to the normal circulating blood cells. Even in metastatic settings, patients generally have fewer than 10 CTCs/mL of blood. Hence, most isolation techniques start with an enrichment step in which the concentration of the CTCs is increased by several log units to enable or facilitate their detection. Numerous CTC detection methods have emerged over the years. Nonetheless, they all take advantage of the physical properties of the CTCs such as their large size in comparison to circulating leukocytes (median diameter 15 µm vs. 10 µm), or the unique expression of cell surface markers such as epithelial cell adhesion molecule (EpCAM) and cytokeratins that are heavily expressed on the surface of epithelial cancer cells. Of note, CTCs can undergo epithelial to mesenchymal transformation, and thereby lose their expression of EpCAM as well as other surface markers via this process.
Detection of cancer by monitoring cfDNA has also garnered much enthusiasm because tumor specific alterations in cfDNA are unique to the tumor and not present in normal or noncancerous cells. Hence, cfDNA potentially offers a sensitive and specific strategy for cancer detection. Similar to CTCs, one of the greatest technical challenges is the identification of very low amounts of tumor cfDNA where it accounts for less than 1% of total circulating free DNA in the blood. Thus, standard sequencing techniques, such as Sanger sequencing or pyrosequencing, can detect cfDNA only among patients with heavy tumor burden. However, the introduction of digital polymerase chain reaction; beads, emulsification, amplification and magnetics; or pyrophosphorolysis-activated polymerization have enabled the detection of cfDNA derived from tumors in a considerably consistent manner.
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