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Table of Contents
REVIEW ARTICLE
Year : 2018  |  Volume : 29  |  Issue : 4  |  Page : 176-179

Detection of circulating tumor cells and the importance of their measurement in urological cancers


1 Department of Urology, Showa University, Tokyo, Japan
2 Ishiiclinic Kyobashi Edogrand, Tokyo, Japan
3 On-Chip Biotechnologies Co., Ltd., Tokyo, Japan

Date of Web Publication23-Jul-2018

Correspondence Address:
Michio Naoe
Department of Urology, Showa University, Tokyo
Japan
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/UROS.UROS_42_18

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  Abstract 


In recent years, various new drugs such as molecularly targeted drugs and immune checkpoint inhibitors have been developed. Liquid biopsy is becoming increasingly important as a guide for selecting these new drugs and determining their efficacy. In urological cancers, given the lack of serum markers for kidney cancer or urothelial cancers, the development of liquid biopsy is strongly desired. Liquid biopsy is less invasive than conventional tissue biopsy, enabling frequent biopsies, and is therefore considered effective for monitoring of the treatment course. Liquid biopsy is largely divided into three types: circulating tumor cells (CTCs), cell-free DNA, and exosomes, each of which has its own set of advantages and disadvantages with regard to the identification method and utility. In the present article, we focus on CTCs and discuss issues in their identification method as well as recent findings.

Keywords: Circulating tumor cells, liquid biopsy, urological cancer


How to cite this article:
Naoe M, Ohta M, Hasebe Y, Matsui Y, Unoki T, Shimoyama H, Nakasato T, Ogawa Y, Tsukada M, Sunagawa M, Ishii H, Ishige M, Osawa H, Matuzaki M. Detection of circulating tumor cells and the importance of their measurement in urological cancers. Urol Sci 2018;29:176-9

How to cite this URL:
Naoe M, Ohta M, Hasebe Y, Matsui Y, Unoki T, Shimoyama H, Nakasato T, Ogawa Y, Tsukada M, Sunagawa M, Ishii H, Ishige M, Osawa H, Matuzaki M. Detection of circulating tumor cells and the importance of their measurement in urological cancers. Urol Sci [serial online] 2018 [cited 2018 Oct 20];29:176-9. Available from: http://www.e-urol-sci.com/text.asp?2018/29/4/176/237359




  What Is Liquid Biopsy? Top


Liquid biopsy has been the subject of research and development throughout the world in recent years. It differs from conventional tissue biopsy using needles and endoscopies in that it is less burdensome for the patient since it is a diagnostic method which uses bodily fluids such as blood or urine, can be performed frequently, and can be used for screening, treatment selection, treatment monitoring, and recurrence monitoring of cancer. In addition, detailed analysis of genetic mutations in cancer cells by liquid biopsy is thought to allow for sensitivity evaluation of different medications. These diagnoses made through liquid biopsies are called companion diagnostics. The use of molecularly targeted drugs in combination with companion diagnostics has also begun.

Liquid biopsy is largely divided into three types: (1) circulating tumor cells (CTCs), (2) circulating tumor DNA or cell-free DNA, and (3) exosomes. In the present article, we focus on CTCs and discuss challenges in their identification method as well as recent findings.

CTCs are considered to be a type of tumor marker but differ greatly from previous tumor markers in that they are directly captured cancer cells.

One challenge in performing phenotyping or genetic analysis for cancer is the heterogeneity of cells within the cancer lesion in a single patient. Identifying all the heterogeneity is difficult with a tissue biopsy. In contrast, CTC testing uses samples of blood, which circulates throughout the body, making it suitable for the evaluation of heterogeneous cancer cells in primary and metastatic lesions. CTC testing can thus be considered an appropriate evaluation method for cancer profiling. Taken together, CTC offers the potential for further development in the future as a type of liquid biopsy.

Identification method of circulating tumor cell

The CTC tests currently being studied consist of “enrichment” and “detection” (i.e., differentiation between CTCs and leftover peripheral blood mononuclear cells [PBMCs]) of rare CTCs which are mixed with the numerous PBMCs. Specific methods vary and are listed in [Table 1].
Table 1: Devices for CTC enrichment and/or detection

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Before discussing the details of each method, however, we will reflect on the history of CTC identification. Various CTC identification methods have been explored. Many of the CTC identification methods used over 20 years ago were based on polymerase chain reaction (PCR) and were problematic in their sensitivity and reproducibility. One of the many CTC identification methods developed after the PCR-based methods, the CellSearch System ® (Veridex, USA) established by Cristofanilli et al.[1] using automated immunostaining, was based on direct observation of CTCs under a fluorescent microscope and was anticipated to be a new cancer biomarker. The CellSearch System ® obtained the United States Food and Drug Administration (FDA) approval for prediction of progression-free survival (PFS) and overall survival (OS) in metastatic breast cancer (approved 2004), monitoring treatment effect of metastatic breast cancer (approved 2006), prediction of PFS and OS in metastatic colon cancer (approved 2007), and prediction of PFS and OS in metastatic prostate cancer (approved 2008). However, as will be discussed below, several challenges have been reported since then. Here, we discuss the principles of CTC identification used in the CellSearch System ® and their problems. Only several to several tens of CTCs are present in 1 mL of blood, which contains roughly 5 billion mostly red blood cells and PBMCs, and identification and isolation of CTCs are extremely difficult. Currently, various techniques are being developed to improve CTC detection rate. These varied methods include separation using antibodies against epithelial cell surface antigens with immunomagnetic beads, flow cytometry and cell sorting, size-based filtration, density gradient centrifugation, and high-throughput imaging. In the present article, we introduce the CTC identification methods as divided into three categories: (1) immunologic methods, (2) methods using differences in cell size and cell deformability, and (3) density gradient centrifugation method.

The first category, as the name suggests, differentiates CTCs and PBMCs based on “immunologic methods.” The CellSearch System ® and On-chip Sort ® (On-chip Biotechnologies) are representative methods in this category, as will be discussed later. We will first introduce the GILUPI CellCollector™ (GILUPI, Potsdam, Germany), an interesting, recently developed medical device for CTC identification. This system involves placing a wire coated with anti-epithelial cell adhesion molecule (EpCAM) antibodies in a blood vessel and capturing CTCs in the large amount of blood that comes in contact with the wire, thereby improving the CTC detection rate. Contact of the wire with about 5 L of blood reportedly results in a 96%–99% detection rate of one or more CTCs.[2] However, this method depends on the EpCAM antigen expressed on the CTC surface, as will be discussed later, and may not capture CTCs that do not express the EpCAM antigen, so concerns remain in its use for the quantitative evaluation of CTCs.

Going back to the previous discussion, we will now discuss the principles behind CTC identification in the CellSearch System ® and their problems. Ten milliliters of blood contains many blood cell components, such as red blood cells (40–50 billion cells), white blood cells, and platelets. Red blood cells can be differentiated from cancer cells due to their lack of nuclei and can also be removed through hemolysis; however, the differentiation between nucleated white blood cells (30–90 million cells) and rare CTCs is difficult. The CellSearch System ® was the first to overcome this challenge. The principle behind the CTC identification method of the CellSearch System ®, in brief, involves capturing cells that express EpCAM, which is a cell adhesion molecule on epithelial cells and cytokeratin as “cancer cells” and removing coexisting white blood cells using the blood cell marker cluster of differentiation (CD) 45 and removing anti-CD45 antibody-positive cells. These immunostaining processes are performed by an automated device. The captured cancer cell candidates are ultimately visualized under a fluorescent microscope. Cancer cells were previously thought to have the characteristics of epithelial cells, which are their origin, and to express the epithelial cell marker EpCAM antigen. However, highly malignant cancer cells have recently been reported to undergo epithelial–mesenchymal transition (EMT), lose the characteristics of epithelial cells, and have reduced expression of the EpCAM antigen. Therefore, CTC identification methods that rely on the EpCAM antigen, including the above-mentioned CellSearch System ®, have been reported to possibly be missing CTCs that have little or no EpCAM antigen expression (EMT-CTC).

Another immunologic CTC identification method that we are developing combines On-chip Sort ® and the “CD45 depletion method” and aims to improve detection of EMT-CTCs. The “CD45 depletion method” utilizes Dynabeads ® CD45, which is an anti-CD45 antibody with a covalent bond to a magnetic bead that binds to the CD45 antigen (leukocyte common antigen) expressed on PBMCs. When a sample containing CTCs and PBMCs bound to Dynabeads ® CD45 is passed through a magnetic separator (DynaMag ®), only PBMCs are captured by DynaMag ®, and by collecting the anti-CD45 antibody-negative cells (i.e., negative selection), CTCs can be isolated. Indeed, most PBMCs can be removed with Dynabeads ® CD45; however, their removal is not perfect, and thus, we then use anti-G250 antibodies, which are specific for renal cancer cells, and anti-CD45 antibodies to stain CTCs and remaining PBMCs and differentiate them using On-chip Sort ®. The significance of this CTC identification method, as mentioned before, is that it isolates CTCs independent of EpCAM and therefore independent of EMT. The results of renal cancer CTC identification using this method will be discussed later.

The second category of methods physically separates and collects CTCs based on “the difference in cell size and cell deformability.” Representative examples include ClearCell FX ® and Celsee . The ClearCell FX ® method uses a disposable chip called CTChip ® FR1 to enrich and collect CTCs. By passing the sample suspension (which includes PBMCs and CTCs, but not red blood cells which are removed in a before step through hemolysis) through the spiral microfluidic biochip CTChip ® FR1, the cells get distributed into inner and outer components based on cell size, and the inner component is collected as the CTC-enriched component. Of note, a 7.5-mL sample takes about 1 h to process. CTCs collected by this method differ from those collected through immunologic methods in that they have not undergone fixation or been marked by magnetic beads, and thus, they can be easily used in various types of research such as sequencing, PCR, or fluorescence in situ hybridization analysis.[2] However, this method depends on the size difference between CTCs and PBMCs, and thus according to our examination, although the CTC collection rate when the cell size difference is large is relatively good at about 70%, the detection rate is only about 30% when the cell size difference is small. Therefore, we believe that this method is effective for monitoring the clinical course in a single patient, such as determining treatment efficacy, or PCR analysis of specific substances in CTCs enriched by this method but is not appropriate for comparison of CTC number between different patients.

The Celsee system is another representative CTC-enrichment system that uses the difference in cell size and deformability of CTCs and PBMCs. It uses a microfluidic chip with 56,320 individual wells in an intricate tunnel. The entrance of each well is 25 μm and the exit is 8 μm. A blood sample containing CTCs enters through the central part of the chip and flows through the microfluidic chip divided into four blocks at the same pressure as a capillary blood vessel (about 1 PSI). Most red blood cells and PBMCs, which are smaller in size and higher in deformability than CTCs, pass through the 8 μm hole, but CTCs, which are unable to pass through the hole, get captured in the wells at a rate >85%. Some PBMCs do remain in the wells but can be differentiated from CTCs by staining with anti-CD45 antibodies in a later step.

The third group of methods is the “density gradient centrifugation” method, which uses the specific gravity difference between CTC and blood cells and is used in the Ficoll ® (GE Healthcare, Buckinghamshire, UK) and OncoQuick ® (Greiner Bio-One, Kremsmünster, Austria) systems. The authors have tried these methods in the past; however, several concerns, including the large number of remaining PBMCs and loss of CTCs during pipetting, limit the utility of these methods.

Significance of circulating tumor cell detection in urological cancers

In the realm of urological cancers, we believe that CTC testing will play a significant role. With no serum markers currently available for renal cell carcinoma or urothelial cancers (renal pelvis cancer, ureteral cancer, and bladder cancer), determination of disease state and treatment efficacy relies on radiologic testing, and thus, the development of diagnostic methods equivalent to serum markers is desired. The authors have reported that CTCs can be identified in metastatic urothelial cancer patients using the CellSearch System ® in 2006[3] and reported the relationship between the number of CTCs and computed tomography (CT) findings in urothelial cancer patients undergoing treatment in 2007.[4] However, as discussed above, it was later discovered that some CTCs may not be detected by the CellSearch System ®, which depends on EpCAM expression of CTCs, potentially resulting in false-negative results for some patients. Therefore, the authors began examination of a CTC identification method that does not rely on EpCAM, such as the above-mentioned CD45 depletion method, or those based on nonimmunologic methods, such as the ClearCell FX ® and Celsee systems.

In Japan, many molecularly targeted drugs for “renal cell carcinoma that is incurable with resection or metastatic” have been approved since obtaining insurance coverage in 2008, as well as Opdivo, which is an anti-PD-1 antibody that obtained insurance approval in 2016. Determination of responders to these medications and monitoring of cancer cell status during treatment will become important. In addition, given the lack of serum markers in renal cell carcinoma, similar to urothelial cancer, evaluation of treatment effect relies on diagnostic imaging, and thus, this type of cancer is another one that would benefit from liquid biopsy.[5]

The authors are engaged in basic research on identification of CTCs specific to renal cancer, which target the G250 antigen, expressed specifically in renal cell carcinoma. This antigen is expressed in approximately 95% of renal cell carcinoma primary lesions and in approximately 75% of metastatic lesions and is not expressed in normal tissue including the kidney. We have confirmed that highly sensitive identification of renal cell carcinoma-specific CTCs targeting the G250 antigen is possible, and publication of this finding has been planned. In addition, regarding the above-mentioned anti-PD-L1 antibody therapy, we believe that measuring PD-L1 expression in renal cancer CTCs before or during treatment will play a significant role in anti-PD-L1 antibody treatment in the future and thus are engaging in the development of a quantitative detection method.

In prostate cancer as well, with the development of various new androgen receptor (AR) antagonists and chemotherapeutic agents, we believe that CTC testing will play a significant role. CTC testing can enable analysis of changes to AR on CTCs, thereby guiding selection of AR antagonists or chemotherapeutic agents. Indeed, detection of AR-splicing variant 7 (AR-V7) in CTCs in a castration-resistant prostate cancer (CRPC) patient has been reported to potentially be related to resistance to new AR antagonist agents.[6]

We would now like to focus on the AR-V7 identification method by Antonarakis et al. They performed PCR analysis of AR-V7 after CTC enrichment by AndraTest, with results reported qualitatively as either AR-V7 positive or negative, rather than quantitatively. However, CTCs are thought to be heterogeneous among CRPC patients, and thus, we believe that measuring expression rate of AR-V7-positive CTC in total CTCs will be important in determining resistance to new AR antagonist agents. Therefore, we have been involved in studies on the quantitative identification method for AR-V7; however, given several challenges such as unstable responsiveness of preexisting anti-AR-V7 antibodies, we have not yet developed a stable quantitative method.

Management of negative companion diagnostic cases

The FDA defines biomarkers including companion diagnostics as “a characteristic that is objectively measured and evaluated as an indicator of normal biological processes, pathogenic processes, or pharmacologic responses to treatment.”[7] With such a large variety of newly developed agents to choose from, such as molecularly targeted drugs and immune checkpoint inhibitors, research and development of liquid biopsy using companion diagnostics is becoming increasingly popular throughout the world. The global market of liquid biopsy is estimated to be 580 million dollars in 2016 and is predicted to continue growing.

On the other hand, along with the development and spread of companion diagnostics, its use requires caution. For instance, there is currently no evidence that a negative companion diagnostic result for a particular drug corresponds to lack of responsiveness to that drug. Theoretical reasons that the drug may still be effective in negative companion diagnostic cases include (1) the possibility that a molecule different from the therapeutic agent's targeted molecule is involved in its efficacy, (2) a false-negative result due to a problem in the test kit, and (3) a potential decrease in sample quality during processing.

From these standpoints, the FDA urges data collection on whether drugs have lower efficacy in cases of negative companion diagnostic results or no efficacy at all. The guidelines point out that to accept removal of the treatment of negative cases, there must be pathophysiological evidence of lack of efficacy in negative cases and early phase clinical trial results that indicate a large difference in effect between positive and negative cases.[8]


  Conclusions Top


As introduced in the present article, various methods have been developed for CTC identification. While we anticipate improvements in identification rates, various challenges remain in the identification of all CTCs at the present time, with room for methodological improvements as well. In addition, one must proceed with caution when using CTC testing (a type of liquid biopsy) as a companion diagnostic tool. Further investigation is needed concerning the management of negative results in companion diagnostics.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
  References Top

1.
Cristofanilli M, Hayes DF, Budd GT, Ellis MJ, Stopeck A, Reuben JM, et al. Circulating tumor cells: A novel prognostic factor for newly diagnosed metastatic breast cancer. J Clin Oncol 2005;23:1420-30.  Back to cited text no. 1
    
2.
Coumans FA, Ligthart ST, Uhr JW, Terstappen LW. Challenges in the enumeration and phenotyping of CTC. Clin Cancer Res 2012;18:5711-8.  Back to cited text no. 2
    
3.
Naoe M, Ogawa Y, Morita J, Omori K, Takeshita K, Shichijyo T, et al. Detection of circulating urothelial cancer cells in the blood using the cell search system. Cancer 2007;109:1439-45.  Back to cited text no. 3
    
4.
Naoe M, Ogawa Y, Takeshita K, Iwamoto S, Miyazaki A. Use of the cellSearch circulating tumor cell test for monitoring urothelial cancer: Two case reports of metastatic urothelial cancer. South Med J 2008;101:439-41.  Back to cited text no. 4
    
5.
Naoe M, Hasebe Y, Ogawa Y. Radiologic diagnoses and tumor markers useful for follow up in urological cancers: Circulating tumor marker testing in urological cancers. Urol Surg 2012;25:29-39.  Back to cited text no. 5
    
6.
Antonarakis ES, Lu C, Wang H, Luber B, Nakazawa M, Roeser JC, et al. AR-V7 and resistance to enzalutamide and abiraterone in prostate cancer. N Engl J Med 2014;371:1028-38.  Back to cited text no. 6
    
7.
Mizuno N, Yokoyama S, Togashi I, Sugatani N, Sato K, Sugibayashi Y, et al. Circulating tumor cells: Detection method and clinical application. Appl Ther 2015;7:49-62.  Back to cited text no. 7
    
8.
Guidance for Industry Enrichment Strategies for Clinical Trials to Support Approval of Human Drugs and Biological Products. U.S. Department of Health and Human Services Food and Drug Administration Center for Drug Evaluation and Research (CDER) Center for Biologics Evaluation and Research (CBER) Center for Devices and Radiological Health (CDRH) December, 2012 Clinical Medical December, 2012 Clinical Medical; 2012.  Back to cited text no. 8
    



 
 
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