Challenges and limitations in CTC analysis

The reward of information gained from the isolation and analysis of circulating tumor cells is great, but challenges in the process are numerous. CTC characteristics currently being analyzed in cancer patients include phenotypic and genomic heterogeneity, EMT-like properties, resistance to anoikis in circulation(self-destruction upon loss of ECM-adhesion), metastatic potential, and single-cell or clustering properties (Figure1.3) [93].

General hurdles to obtaining this information include the detection of such rare cells, overcoming bias in the methods, and translation into a clinical setting. The methods that struggle in one areas, such as with detection of rare cells (negative depletion of leukocytes), excel in other areas like selection bias, and vice versa with positive selection.

However, the methods as a whole are limited by their lack of standardization. Further

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Figure 1.3: CTC characteristics as currently described. Reprinted with permission from Macmillan Publishers Ltd: Nature, copyright 2014 [93]

challenge lies in interpreting the meaning of CTCs once detected. Analysis of single cells to understand CTC populations and subsequent assays to ascertain function could be solutions to the problem.

1.2.5.1 | Rarity of cells

A small number of CTCs (commonly between 1-10) are found in the majority of cancer patients [93]. This could be due to the nature of the location of the sampling, the nature of the tumor, or the systemic environment. Portal veins have been considered as an option and found to contain much higher number of cells [102], however this is clearly more invasive than a typical venipuncture. The main reason liquid biopsies are sought after is the ease of sample retrieval. Another option that is less invasive than arterial sampling is leukaphoresis. Several liters of blood can be filtered and collected at one time. In a comparison with peripheral blood and the CellSearch workflow, this method was found to collect a much higher number of CTCs and while revealing significant associations with TNM stage and metastasis-free survival [53].

1.2.5.2 | Capture Bias

All methods are based on assumptions on the cell populations being collected or removed.

There is no method that is 100% effective or precise due to basic biological variation.

This is further complicated by the heterogeneity of individual tumors between and within patients which is further reflected in the CTC populations. It is difficult to define CTCs

by morphology or molecular and genetic properties. If we would point to a characteristic present in all tumor cells, we would be solving a much larger problem in the cancer treatment.

All of the methods beyond pure enumeration showcase this heterogeneity of CTCs.

This is not surprising given the variability of cells in the primary tumor, and the innate ability for cancer cells to adapt to their environment. The best methods going forward will be the ones that allow for capture and detection across a wide variety of cellular characteristics. The more details known, perhaps the better we can understand the cancer and provide more personalized and effective treatments.

There are ways to control for this in both the enrichment and characterization steps to the best of our ability. This can be done by first not selecting CTCs based on EpCAM, as this is known to be a overly-selective property and excludes many cells that may be the most predictive [85, 103]. The selective nature of different methods is made clear in many comparison studies [56,61,77, 81]. In addition, the characterization methods should also be inclusive enough to analyze and gain information from as many cells as possible.

1.2.5.3 | Functional Characteristics

Despite the evidence demonstrating the clinical relevance of CTC numbers, the func-tional characteristics of CTCs are not as intensely investigated. Surface receptors present on the cells, along with gene expression, can give some idea of what is happening within the cell on a molecular level, but how that effects the function of the cell is unknown.

The CTCs with the most clinical value are those that survive circulation, dissemination, and go on to form metastasis. Functional assays are needed to find the specific charac-teristics that support these actions. Some studies have been done that investigate these features, such as metastatic initiating cells (MICs) in xenografts [46], and growing and monitoring cell cultures from CTCs [80,90].

1.2.5.4 | Lack of standardization and translational medicine

Medical decisions can hopefully be enhanced with the input from CTC science, but many challenges and limitations remain for their translation to the clinic [104]. The methods presented here present only a snapshot of the hundreds of publications every year in CTC analysis. With so many methods and techniques being used, it makes comparison and standardization in the field more difficult. Biologics is a complicated medical field, but to be used in the clinic a CTC method must be rigorously proven and validated and for this, a standard and routine set of methods must be developed. Unfortunately, we are still trying to arrive at what the best methods may be for the most clinical value. The

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best method will ultimately be easy, effective, and minimize inaccuracies, with function being more important than novelty.