CTC: Circulating tumor cell. Read about what they are and why they’re important here.
Leukocyte: White blood cell.
Erythrocyte: Red blood cell.
I’ve previously discussed how to sort CTCs, and the standards used to characterize device performance. Today, I’ll explain what some of the most common evaluation metrics are, and place them in context of eventual clinical/industrial application.
What are common performance criteria?
Capture Efficiency is number of target cells captured divided by total number of target cells introduced into the cell isolation system. This metric can only be assessed using cancer cell lines spiked into solution at known concentrations, as the number of actual CTCs in a patient sample is always unknown.
Enrichment is closely related to capture efficiency. It is the factor increase of the target cells per unit volume, post cell-isolation/sorting. As in the case of capture efficiency, this can only be assessed using model CTCs at controlled concentrations.
Capture Purity is the number of target cells captured divided by the total number of nucleated cells captured by the device. As I’ve mentioned before, the major contaminating cell type is usually leukocytes. Nucleated blood cells are an issue for genetic analyses, where their DNA can swamp out the signal from CTCs. Non-nucleated blood cells (e.g. erythrocytes), do not carry any DNA, and are less of a concern.
Here’s simple example to understand how these criteria are calculated: presume you have a 1mL blood sample that contains 4 cancer cells and 1e9 leukocytes. After processing the blood in your device of choice, you have captured 2 cancer cells and 100 leukocytes. Then the performance stats would be: 5e6-fold enrichment, 50% capture efficiency and 2% capture purity.
Capture efficiency, enrichment, and capture purity are the values you will see reported most commonly in the literature. However, there are two more metrics that are gaining increasing attention as the field moves forward:
Cell Viability is percentage of target cells that are still alive after the cell sorting/isolation process. It can also be defined as percentage of cells alive after a certain number of days in cell culture. The first definition can only be assessed using model CTCs, while the second can be calculated from actual patient samples. This is important for assays that require live cells to investigate how CTCs respond to different biomechanical and biochemical cues.
Release Efficiency is the percentage of captured target cells that can be successfully removed from a device in a viable state. The material and optical properties of CTC isolation devices are often suboptimal for traditional biological assays, requiring researchers to create new protocols for each new type of CTC isolation device. Releasing viable cells is gaining increasing focus as scientists and engineers try to streamline and standardize this technology for use in hospitals, drug development, and pharma companies.
Criteria are a baseline, not the end goal
Characterizing device performance is important for optimization, as well as for comparison to the industrial and academic state-of-the-art. However, the area of greatest impact is no longer optimizing every performance metric, but what you can do with CTCs once you’ve captured them. CTCs are a platform for a variety of tests, ranging from morphological analyses1 and molecular profiling2 to chemotherapeutic testing3. Depending on the test that will be applied to the CTCs, the performance criteria that are prioritized will be different. For example, if the end-goal is to enable sensitive genetics testing, you might build a more discriminating device and trade capture efficiency (sensitivity) for capture purity (specificity). If the goal is to culture CTCs ex-vivo for other types of assays, then cell viability and release efficiency become paramount.
CTC enumeration is the traditiaonal tool to inform patient prognosis. However, there is increasing work using CTCs to infer information about the primary tumor or metastasis. These analyses range from [L-R] looking for novel gene fusions correlated with patient outcomes, to using patient CTCs to evaluate chemotherapeutic efficacy, to culturing CTCs outside the body.
At the World CTC Summit
, this was termed fit-to-purpose
CTC isolation technology. While it would be ideal to have a “magic bullet” tech that has perfect performance metrics and is facile for every time of biological and pharmacological test, in reality most CTC isolation technologies will have to be designed, and selected, based on their end-goal application.
1. Marrinucci D., Bethel K., Lazar D., Fisher J., Huynh E., Clark P., Bruce R., Nieva J. & Kuhn P. (2010). Cytomorphology of circulating colorectal tumor cells:a small case series., Journal of oncology, PMID: 20111743
2. Stott S.L., Lee R.J., Nagrath S., Yu M., Miyamoto D.T., Ulkus L., Inserra E.J., Ulman M., Springer S. & Nakamura Z. & Isolation and characterization of circulating tumor cells from patients with localized and metastatic prostate cancer., Science translational medicine, PMID: 20424012
3. Kirby B.J., Jodari M., Loftus M.S., Gakhar G., Pratt E.D., Chanel-Vos C., Gleghorn J.P., Santana S.M., Liu H. & Smith J.P. & (2012). Functional characterization of circulating tumor cells with a prostate-cancer-specific microfluidic device., PloS one, PMID: 22558290