Review

Progress of Circulating Tumor Cells in Cancer Management

Technology in Cancer Research & Treatment 1–8 ª The Author(s) 2015 Reprints and permission: sagepub.com/journalsPermissions.nav DOI: 10.1177/1533034615583762 tct.sagepub.com

Chufeng Zhang, MD1,2, Lijie Wang, MD1,2, Yan Guan, MD2, Yulan Sun, MD2, Xiuju Liu, MD2, Dongyuan Zhu, MD2, and Qisen Guo, PhD2

Abstract Circulating tumor cells are low-frequency cells that are shed into the peripheral bloodstream from a primary solid tumor and/or metastasis. Although these cells were recognized initially in 1869, it is only in the past 2 decades that they have been isolated for use as a surrogate biomarker to monitor response to therapy, evaluate prognosis, detect tumor mutations, assist in selecting personalized medicine, and enable earlier cancer diagnosis. Keywords circulating tumor cells, enrichment, response to therapy, prognostic evaluation, heterogeneity, personalized treatment, early diagnosis Abbreviations ADT, androgen deprivation therapy; CRPC, castration-resistant prostate cancer; COPD, chronic obstructive pulmonary disease; CTCs, Circulating tumor cells; DEP, dielectrophoresis; EMT, epithelialmesenchymal transition; EpCAM, epithelial cell-adhesion molecule; FDA, Food and Drug Administration; HR, hormone receptor; ISET, isolation by size of epithelial tumor cells; MBC, metastatic breast cancer; mCRC, metastatic colorectal cancer; mHSPC, metastatic hormone-sensitive prostate cancer; MLC, metastatic lung carcinoma; NSCLC, non-small-cell lung cancer; OS, overall survival; PFS, progression-free survival; RFS, relapsefree survival; TKIs, Tyrosine kinase inhibitors Received: November 29, 2014; Revised: March 11, 2015; Accepted: March 30, 2015.

Introduction The occurrence of distant metastases is the cause of nearly all cancer deaths. Circulating tumor cells (CTCs) are more likely to be detected in patients with metastatic cancer, and the number of CTCs significantly increases with tumor progression.1 The number of CTCs also changes in response to anticancer treatment, and therefore, CTCs are a promising marker for evaluating curative effects. So CTCs represent an opportunity for clinicians to identify the best therapy for individuals with cancer over the course of the disease.2 The presence of CTCs has also been associated with overall survival (OS) in several tumor types, such as colorectal, prostate and breast cancer. The CTCs have potential as a biomarker for evaluating prognosis, which is independent of the traditional ‘‘gold-standard’’ pathologic Tumor Node Metastasis (TNM) staging. In the clinic, targeted therapy decisions for cancer are generally guided by the primary tumor genotype. However, tumor genotypes change over time, and we are not able to monitor this with

tumor biopsies or surgical specimens.3 The isolation of CTCs from blood, which is a minimally invasive procedure, is performed to investigate the ability of CTCs to represent the full spectrum of mutations in a primary tumor and metastastic sites as a minimally invasive procedure. Here, we review the progress regarding CTCs as a biomarker and focus on isolation and enrichment methods, response to therapy, prognostic evaluation, anticancer treatment guidance, early diagnostic value, and areas for future improvement.

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School of Medicine and Life Sciences, University of Jinan-Shandong, Academy of Medical Sciences, Jinan, China 2 Academy of Medical Sciences, Shandong Cancer Hospital, Jinan, Shandong, China Corresponding Author: Qisen Guo, PhD, Academy of Medical Sciences, Shandong Cancer Hospital, Ji Yan Road, Jinan, Shandong 250117, China. Email: [email protected]

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Technology in Cancer Research & Treatment

Detection and Enrichment Techniques The fundamental challenge in analyzing CTCs is its extremely low concentration in blood, occurring at rates as low as 1 cell per 106 or 107 leukocytes.4 Efficient techniques are required to distinguish CTCs from peripheral blood cells for enrichment. Techniques for CTC enrichment can be classified as positive enrichment (capturing target cells and eluting nontarget cells) or negative enrichment (removing nontarget cells), which are performed based on biological or physical properties. Positive enrichment is the most commonly employed strategy for separating CTCs, and this method is mainly based on an affinity-binding system. This system distinguishes target cells that express certain CTC-specific antigens that are not expressed by blood cells. For example, CTCs can be positively enriched using an anti-epithelial cell-adhesion molecule (EpCAM) antibody combined with magnetic particles, and the mixture is captured in a magnetic field and collected on a glass slide. CellSearch (Veridex; Warren, NJ, USA) is the only platform approved by the Food and Drug Administration (FDA) for detecting CTCs by targeting cell markers, such as EpCAM and cytokeratins 8, 18, and 19, in metastatic breast, colon, and prostate cancer.4 Although positive enrichment methods are able to isolate CTCs at a high purity, they have significant limitations. The EpCAM is not expressed in nonepithelial cancers (eg, ovarian cancer) and certain epithelial cancers (eg, renal cell cancer). Furthermore, circulating epithelial cells have been observed in patients with benign colon diseases.5 In addition, some epithelial antigens on CTCs will be downregulated or lost because of epithelial–mesenchymal transition (EMT).6 A prospective study demonstrated that only 32% (19 of 60) of patients with distant metastatic non-small-cell lung cancer (NSCLC) had positive CTCs at baseline using the CellSearch System.7 To resolve this problem, new biomarkers that cover the complex heterogeneity of tumor cells are required. For example, protein plastin 3 was discovered as a novel CTC marker that is not downregulated during EMT and is not expressed in blood cells.8 It is promising to realize the broad-spectrum enrichment of all CTCs. Negative enrichment is another efficient strategy for capturing CTCs by depleting normal hematopoietic cells based on their physical properties, such as size, density, and deformability. Tumor cells are generally bigger (>8 mm in size) and stiffer than normal blood cells, which can be filtered by a membraneand filtration-based system. Thus, different devices based on cell filtration and centrifugal force have been recently developed.9-11 Membrane-based filter systems, such as isolation by size of epithelial tumor cells (ISET), have demonstrated to be an efficient, inexpensive, and quick method for enriching CTCs.12 A direct comparison of CellSearch and ISET for CTCs isolation demonstrated that ISET performed better than CellSearch in detecting CTCs in patients with metastatic prostate carcinoma and metastatic lung carcinoma.13 However, this method should be further developed to overcome the inherent insufficiency that filter pores are likely to be blocked by CTCs or leukocytes. Ficoll-Hypaque centrifugation is an example of an approach that addresses this problem, but a substantial loss

of CTCs is unavoidable. Considering these issues, a centrifugal force-based size-selective platform was proposed to isolate and enrich CTCs with a high purity. The capture efficiency for whole blood samples varied from 44% to 84%.14 The CTCs can also be captured based on biological characteristics. For example, the characteristics of normal blood cells, such as CD45 expression on leukocytes, were used to isolate heterogenetic CTCs. Using a bead-conjugated anti-CD45þ antibody, leukocytes are negatively depleted in a special magnetic field. The CTCs can also be enriched based on electric charge. Under a particular medium conductivity, different types of cells exhibit different dielectrophoresis (DEP) behavior. A study demonstrated that DEP using a DEP-based microfluidic chip attained enrichment efficiencies as high as 93% with high specificity based on the electric charge of breast cancer CTCs.15 It is controversial whether positive or negative enrichment is more efficient. Positive enrichment can be used to isolate CTCs with high purity, but there are significant limitations; namely, CTCs do not all express the same specific antigens because of heterogeneity. Thus, systems that target different epithelial antigens or novel biomarkers that are expressed by all CTCs might be advantageous. Generally, negative enrichment approaches are easy and highly sensitive but have low specificity. Undoubtedly, the latter is more advantageous in that the target cells remain intact and heterogeneous. This provides researchers with the opportunity for downstream molecular analysis. In addition, positive and negative enrichment can be integrated based on physical and biological characteristics. For example, CTCs can be first selected based on their larger size, which discards the smaller leukocytes; CTCs can be subsequently immunostained with bead-conjugated antibodies against EpCAMs and then captured in a magnetic field.16 Basically, various platforms are being developed to overcome the problem of the sensitivity/specificity trade-off. Recently, a new microfluidics platform called ‘‘CTC-chip’’ was developed rapidly. Current microfluidics technologybased methods for separating CTCs can also be classified as positive enrichment (ie, using anti-EpCAM-coated microfluidic channels or affinity-based magnetophoresis) or negative enrichment (ie, using size-based filtration, size-based hydrophoresis, or normal cells depleting using an anti-CD45 antibody-coated microfluidic channel). Several microfluidics-based platforms have been described.17 This CTC-chip enables the detection of CTCs in 1 mL of blood from almost all patients with metastatic cancer; this method has a high detection efficiency (approximately 100%), even in patients without metastatic disease, and further investigations on assay specificity are therefore warranted.18-20 It also has the advantages of being low cost and high-throughput as well as providing the ability to perform downstream molecular analyses. This microfluidic platform is believed to have laid a solid foundation for clinical practice.

Monitoring Therapeutic Response Changes in the number or genotype of CTCs can provide a reliable marker of clinical response to anticancer treatment

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by sensitive detection methods such as CTC-chip.20,21 Various studies have been published supporting this as a powerful tool for monitoring therapeutic response, predicting clinical outcome, and realizing accurate, early decision making. Genotypic changes may occur during disease progression and may predict a lack of response to targeted therapy. Tyrosine kinase inhibitors (TKIs) are effective in patients with NSCLC with an EGFR mutation but are limited by mutations that confer resistance to anti-EGFR therapies, such as the T790M mutation.22 Maheswaran et al used CTCs as a noninvasive strategy to monitor tumor genotypic changes during personalized targeted treatment.23 The CTCs were enriched from 27 patients with NSCLC, and an Epidermal Growth Factor Receptor (EGFR) mutation analysis was performed on DNA from CTCs and tumor-biopsy specimens using polymerase chain reaction; T790M mutation analysis was also performed on CTCs from patients with EGFR mutations who had received TKIs. The results showed that EGFR mutations were detected in 92% of patients and that the presence of T790M in CTCs correlated with worse progression-free survival (PFS). These data suggested the possibility of monitoring genotypic changes during the course of treatment and investigating the reasons for drug resistance. Furthermore, serial analysis of CTCs demonstrated that decreasing CTC numbers were associated with a radiographic tumor response; increasing CTC numbers were associated with tumor progression. These data suggested that the CTC number is promising as a predictor of tumor progression and treatment response. Similarly, the Kirsten rat sarcoma viral oncogene (KRAS) mutation has been successfully detected in CTCs from patients with metastatic colorectal cancer (mCRC), and the KRAS mutation status in CTCs may differ from that of the corresponding primary tumor over the course of targeted therapy,24 which supports the use of CTCs in monitoring mutation status and predicting the response to targeted therapy in real time over the course of treatment. The number of CTCs, which is influenced by chemotherapy, is a well-developed indicator of therapeutic response and provides valuable information regarding the effect of anticancer treatment. Changes in the number of CTCs at baseline and after treatment are significant in terms of clinical outcomes. In the clinical trial registration number (IMMC38) study,25 serial monitoring of CTC count demonstrated that patients with castration-resistant prostate cancer (CRPC) had significantly better outcomes when an unfavorable baseline CTC count converted to a favorable CTC count after treatment and worse outcomes when favorable baseline counts converted to unfavorable counts after treatment. This finding was confirmed by other studies in CRPC.26,27 The value of CTC count as an indicator of treatment response is also well established in metastatic and local breast cancer,28,29 mCRC,30 advanced lung cancer,31,32 metastatic melanoma,33 and other tumor types.

Prognosis Evaluation The CTC count has been shown to be affected by systemic chemotherapy and to be an independent prognostic factor (pretreatment or posttreatment) for survival in patients with

metastatic carcinoma and in several other tumor types, such as breast, prostate, and colorectal cancer. In a prospective, multicenter study, the CTC count in 177 patients with measurable metastatic breast cancer (MBC) was determined before a new line of treatment and at the first follow-up visit. Compared to the group with 18 months, P < .001). This difference between the groups persisted at the first follow-up visit (PFS, 2.1 months vs 7.0 months; P < 0.001; OS, 8.2 months vs >18 months; P < 0.001). A multivariate Cox regression analysis that included treatment methods, Eastern Cooperative Oncology Group (ECOG) score, and estrogen receptor/progesterone receptor (ER/PR) status demonstrated that the number of CTCs was the strongest predictor of PFS and OS.34 In a retrospective study, the individual CTC data for 1944 patients with MBC also confirmed this finding.35 In a prospective multicenter study by Cohen et al, the CTC count at baseline or during treatment was confirmed to be an independent predictor of PFS and OS in patients with mCRC. In this study, 430 patients were divided into 2 groups based on CTC count (3 CTCs or