Abstract
In recent years, the clinical application of circulating tumor cell (CTCs) detection has become one of the research hotspots in the field of precision medicine. CTCs detection is noninvasive, easy to obtain, can be repeatedly collected, and highly repeatable with other advantages. It not only can be a real-time comprehensive monitoring of cancer treatment but also can have a large number of applications, including early diagnosis of tumor, timely evaluation of efficacy, condition monitoring, resistance factor analysis, prognosis judgment, individualized treatment of tumors, drug guidance, and so on. At present, many large-scale clinical studies at home and abroad run through all stages of breast cancer diagnosis and treatment. For different treatment stages of breast cancer, the application value of CTCs detection is different. Compared with traditional detection methods, CTCs have advantages in dynamic monitoring of disease changes and efficacy evaluation in real-time. In the era of breast cancer classificational and individualized treatment, CTCs detection can provide patients with the most timely and optimized treatment plan.
Introduction
In 1869, Australian physician Thomas Ashworth first discovered and proposed the concept of circulating tumor cells (CTCs) by observing the peripheral blood of a metastatic cancer patient. 1 In solid tumor cases, CTCs can reflect the tumor burden, predict disease development, and assist in treatment decision-making. Thus, altogether, CTCs have diagnostic and predictive value, which is crucial and translatable to the clinic. 2 Therefore, CTCs became one of the priorities in oncological research.
According to current statistics, cancer has become the second most common cause of death globally. Breast cancer is the most prevalent malignant tumor and is the leading cause of cancer deaths in women, both in developed and developing countries. 2,3 With the revolutionary development of genetics and molecular pathology, the diagnosis and treatment of breast cancer has entered the era of a targeted approach, based on related specific markers.
From the detection of CTCs in breast cancer patients, disease changes can be inferred in a noninvasive, real-time, and dynamic manner, evaluate the efficacy, and timely adjust the treatment plan for an individualized treatment of the patients. Therefore, this review will mainly focus on CTCs detection and application in diagnosis and treatment of breast tumor.
Circulating Tumor Cells
The dissemination of tumor cells is an important feature in the development of tumors. 4 Tumor cells detach from the primary lesion, infiltrate the basal membrane, and enter the blood circulation through the blood vessel wall to form CTCs. CTCs disperse with blood circulation and colonize at remote organs or replant to the primary lesion, leading to in situ recurrence or formation of distant organ metastases. 5 –10 This explains the fact that CTCs are mostly seen in tumors of high invasiveness and metastatization capacity. 11,12 The CTC peripheral blood count is very low, ∼1 CTCs per millions of other blood cells (e.g., 1 CTC per 106 leukocytes), which makes enrichment techniques necessary for detection. 13,14
CTCs Detection Technology
Thus far, there are several methods validated for CTCs' separation and detection, including techniques based on their biological and on physical characteristics. Enrichment steps are imperative and usually based on the physical (density and size) or immunomagnetic properties, including gradient centrifugation, filtration, and immunomagnetic sorting. Detection then is conducted with immunology, reverse transcription-polymerase chain reaction (RT-PCR), CTCs' chip technology, and flow cytometry (FCM). 5,15,16 Despite great advances, it is still a great challenge to detect, molecularly analyze and characterize those highly rare cells. In addition, CTCs express a high plasticity in relationship to their epithelial–mesenchymal conversion, so that the epithelial markers in the assays become too insensitive for a successful detection. 13 New acoustic separation approaches promise a more intact yield of CTCs with a better preservation of their integrity. 17
Immunology
The principle of immunological techniques is to detect tumor markers expressed by CTCs, including epithelial cell adhesion molecules (EpCAM), cytokeratin (CK), mucin 1, cell surface associated, and tumor-associated glycoprotein-12 (TAG-12). CTCs are thus separated from the blood stream by an antigen-antibody reaction. The most representative immunological technique for detecting CTCs using this method is the CellSearch system, the only FDA-approved CTCs detection technology, a semiautomated CTC counting system, based on immunomagnetic separation. 18 Immunomagnetic bead coated with an EpCAM antibody binds all EpCAM-expressing cells in the peripheral blood by an antigen-antibody reaction. Following, CTCs are separated and captured. CTCs cell corners are then labeled fluorescently so that protein (CKs) antibodies recognize and identify them. The CellSearch system is less affected by errors caused by human factors and the results by other more complex approaches. 19 However, this method mainly focuses on the CTC counting and lacks the depth on the genetic level. As a traditional detection method, it is widely used for a long time, but its drawbacks are increasingly prominent given the rapid development of technology and more in-depth insights of CTC features. For example, studies have shown that CellSearch does not recognize all types of breast cancer cells, especially triple-negative breast cancer cells, because their surface does not express epithelial adhesion molecules (EpCAM). Therefore, the method has a relatively high false negative results rate and does not cover the entire cancer spectrum.
Reverse transcription polymerase chain reaction
The principle of nucleic acid-based detection technology is used to detect free DNA in the blood. It is generally believed that there are four sources of free DNA in peripheral blood: (1) spontaneous DNA release by proliferating tumor cells into the blood circulation; (2) release of necrotic tumor cells; and (3) lysis of CTCs and micrometastases—tumor cell apoptosis. Therefore, the presence of CTC can be confirmed by detecting free DNA by PCR and RT-PCR. RT-PCR has been used since the 1990s to detect tumor-specific nucleotides in peripheral blood. 19 –21 Compared with the detection method based on antibody binding, its sensitivity is better and applicability wider. However, due to the nonspecific expression of target RNA, contamination of specimens and PCR products, improper control of conditions during PCR amplification, and low level of undesirable transcription of material from nonspecific cells, and the results of RT-PCR method are prone to be false positives. Also, this technique cannot be used to observe the morphological characteristics of tumor cells, and many drawbacks limit the clinical application of this method.
CTCs chip technology
CTCs chip technology combines bioseparation microbead technology with microfluidic chip technology to build a fast and efficient magnetic bead (MB)-based CTC separation chip system. MBs are coated with CTCs-specific biomolecules to bind those CTCs that contain the corresponding target material to form a new complex. This complex can be retained by the magnetic field and separated from other components. The MBs hold the characteristics of paramagnetic and high-molecular particles in the magnetic field. The MBs contain small particles, their capacity is large and the suspension stability good. Overall, their paramagnetism makes the solid–liquid separation easier. Thus, the complicated traditional operations such as filtration become unnecessary. The microfluidic chip is formed by binding a substrate integrated with a micromagnetic field component and a substrate containing a microflow path, further exchange of fluid with the external environment through the macro–micro interface. This is based on the fact that microelements have good magnetic properties. By applying an external magnetic field to the micro-MB system, the soft magnetic microcomponent is strongly magnetized, and a magnetic field is locally generated in a microfluidic channel. When the MB solution flows through the microchannel at a certain flow rate, the magnetic field is applied to the paramagnetic microcomponents to achieve the purpose of CTCs separation. 22 –24
In the recent years, with the development of microelectromechanical technology, processing technology and the biomedical engineering, chip-level MB manipulation, and reaction devices using microfluidic chip technology and MB detection technology have emerged. Focusing on the construction of an efficient, fast, and integrated MB-based biological sample separation and detection with microfluidic chip system is becoming a priority in that research area. 25
Flow cytometry
FCM is a single-cell quantitative analysis and sorting technique. Since most of the breast cancer originates from epithelial cells, in the study of breast cancer CTCs, CK is fluorescently labeled for FCM. However, this method is least favorable to be used alone, since measurement of CK only does not distinguish tumor cells from normal tissue cells. 26,27
Application of CTC in Breast Cancer Diagnostic, Therapy, and Follow-Up Processes
In recent years, the incidence of breast cancer has been increasing and research on breast cancer diseases has become one of the major oncological fields. Once breast cancer is diagnosed, it is considered to be a systemic disease. The detection of CTCs in breast cancer patients is important in assisting diagnosis, early detection of tumor micrometastases, guiding individualized treatment, evaluating treatment effects, and prognosis. Thus, the clinical significance is manifold and undeniable.
Detection of micrometastases for early detection of tumors
Studies have shown that as a systemic disease, breast cancer can spread throughout the body at an early stage and about 15% of breast cancer patients, even after axillary lymph node biopsy, will experience recurrence and metastases. This suggests that these events are related to micrometastases of breast cancer that are present in patients, but cannot be found by clinical routine examination or pathological examinations. Identifying these patients with high risk of recurrence and metastases is critical to improving their prognosis. Biggers et al. performed CTC testing on 41 patients with early breast cancer, of which 10 (24.4%) were positive for CTC before surgery. At present, most patients' lesions are discovered by mammography when they are in advanced stages. Compared with traditional imaging studies, detection of CTCs in blood detects micrometastases earlier. 28 –30 Therefore, CTC testing is conducive to early diagnosis of breast cancer. 31
Monitoring tumor progression
CTCs count measurement can also be used as an early indicator of tumor progression. CTC detection has less interobserver variability and a higher specificity than imaging evaluations. 14 Schwarzenbach et al. 15 conducted a prospective clinical study to investigate the value of CTC counts and imaging findings in the monitoring of therapeutic efficacy. The results showed that CTC counts were correlated with the degree of disease progression, as shown by imaging, but at a much earlier stage. Disease progression was monitored from 7 to 9 weeks. CTC counts ≥5 after chemotherapy were highly suggestive of disease progression. The study concluded that continuous CTC count monitoring combined with imaging examination could improve the accuracy of patient's follow-up and assist in the development of optimal management efficacy 32 and individualized treatment plan. Blood sampling at different time points during treatment can help clinicians to more accurately predict the clinical benefit, reduce the time for patients of receiving ineffective treatment, and guide the development of the most beneficial treatment options.
Guidance for individualized treatment
Currently, endocrine and molecular targeted therapies are based on the estrogen receptor, progesterone receptor, and human epidermal growth factor receptor status of primary mammary tumors. Choosing to study the biological characteristics of CTCs will provide a new entry point for clinical treatment of breast cancer. 33 For example, studies have shown that CTC in metastatic breast cancer are similar to EMT and stem cells, thus a potential markers of an inferior prognosis and possible drug modeling. 16,34
Evaluation of therapeutic effect and prognosis
Studies have shown that CTCs in breast cancer lead to creation of distant metastases through blood dissemination, so the evaluation of peripheral blood CTCs can give a solid feedback on the therapeutic effect of a current treatment. 35 –37 This evaluation might be much more sensitive and conducted earlier than conventional imaging examination, which would then allow clinicians to adjust the treatments timely, as to avoid excessive and ineffective ones. 38 Reports showed that CTCs are an independent predictor of breast cancer prognosis in patients with primary breast cancer and associated with poor prognosis in metastasized breast cancer. 11,37,39 –42 Lucci et al. also confirmed that CTCs were detected in peripheral blood of patients with nonmetastatic breast cancer, directing to a poor prognosis. 43
Conclusion and Outlook
CTCs play an important role in formation of distant tumor metastases. CTC detection has obvious advantages compared with traditional imaging and serological evaluation methods and is of great significance for the diagnosis and treatment of cancer patients. So far, there has been a rapid progress in the research on real-time monitoring and prognostic predictions in metastatic breast cancer. For patients with early breast cancer, due to the relatively low detection rate of CTCs and the high costs of a quite complex technology, the CTC-related methods have not found clinical application. At present, epithelial-specific antigens are often used in place of tumor cell-specific antigens, but no absolute specificity of markers for breast cancer has been found. The detection and analysis of CTCs will contribute to the diagnosis of early metastases in breast cancer patients, assist in accurate clinical staging, real-time monitoring of tumor recurrence and metastatic spread in postoperative patients, assessment of prognosis, and rational selection of individualized treatment strategies. On January 1, 2018, AJCC (American Joint Committee on Cancer) listed CTCs in peripheral blood of breast cancer as prognostic factors in the eighth edition of the cancer staging system. The latest NCCN (Nation Comprehensive Cancer Network) included CTCs in the TN (tumor node metastasis) staging system, stating that “there is no clinical or radiographic evidence of distant metastases, but in patients with no signs of symptoms or metastases, peripheral blood, and/or bone marrow, and/or circulating tumor cells were found by microscopy or molecular deposition in nonregional lymph node tissues with swelling <0.2 mm, which is cM0(+) phase.” 44 With the continued development of biotechnology, CTCs will hopefully be more widely used in clinical settings.
Footnotes
Author Disclosure Statement
No competing financial interests exist.