Clinical significance of circulating tumor cells in blood from patients with gastric cancer

Abstract Circulating tumor cells (CTC) have been focused on as a target for detecting occult tumors, predicting therapeutic responses and prognoses, and monitoring postoperative recurrence in the clinical management of patients with various malignancies, including gastric cancer. Recent advances in molecular diagnostic tools have contributed to high sensitivity and specificity for the detection of CTC. A conspicuous disparity exists in the incidence of CTC among studies. However, a close relationship has been reported between positivity for CTC and well‐known prognostic clinicopathological factors including depth of tumor invasion, lymph node metastasis, stage, and lymphatic and venous invasion in patients with gastric cancer. According to most studies published on the clinical impact of CTC, the presence of CTC negatively affects the prognosis of patients with gastric cancer. Moreover, the study of CTC based on a meta‐analysis demonstrated their importance as a poor prognostic indicator. In clinical management, pre‐ and post‐therapeutic monitoring of CTC using liquid biopsy may be useful for early detection of subclinical patients or disease recurrence, prediction of tumor progression, and administrative control of adjuvant chemotherapy. Although their functional properties remain unclear, molecular profiling of CTC may contribute to the development of personalized treatment that effectively inhibits tumor progression in patients with advanced gastric cancer. We herein review the clinical significance of CTC as a promising blood marker and therapeutic target in patients with gastric cancer.

patients with early gastric tumors and free of lymph node metastasis.
However, there are clinical limits for accurate tumor detection and diagnoses using preoperative examinations such as endoscopy, endoscopic ultrasonography (EUS), computed tomography (CT), and positron emission tomography-computed tomography. [2][3][4] Carcinoembryonic antigen (CEA) and carbohydrate antigen 19-9 (CA19-9) are now commonly used as established serum markers in the clinical management of patients with gastric cancer. Nevertheless, the sensitivity and specificity of detecting patients with early gastric cancer are clinically insufficient and few candidate blood markers have clinical utility for overcoming these key problems. 5 Five-year survival rates of patients with International Union Against Cancer stages IIIA, IIIB, and IV gastric cancers are 30.8-54.0%, 16.1-36.5%, and 9.2-23.9%, respectively. 6,7 Furthermore, advances in chemotherapy have contributed to improvements in the prognosis of patients with advanced gastric cancer. 8 However, difficulties are associated with predicting tumor responses to chemotherapy and disease recurrence after surgery in patients with advanced stage cancer. Although blood monitoring using serum CEA and CA19-9 has been conventionally introduced for the managements of patients with gastric cancer, serum levels of conventional blood markers do not necessarily coincide with tumor behavior. 9 Therefore, surrogate blood markers are needed clinically to monitor tumor aggressiveness in real time. Moreover, liquid biopsy using blood specimens has the clinical benefit of being a simple and repeatable sampling tool.
In 1869, the presence of circulating tumor cells (CTC) in peripheral blood was proposed by Ashworth. 10 CTC are generally isolated from primary tumors or metastatic sites and these cells flow in the bloodstream of patients with malignancies. 11 To date, CTC have been focused on as a target for detecting occult tumors, predicting therapeutic responses and prognoses, and monitoring postoperative recurrence in the clinical management of patients with various malignancies, including gastric cancer. 12,13 Non-invasive liquid biopsy has enabled CTC to be characterized and their numbers assessed. Therefore, the assessment of CTC using liquid biopsy may support new perspectives for the diagnosis and treatment of patients with gastric cancer.
The present review will focus on the clinical significance of CTC as an important therapeutic target in gastric cancer, including recent topics.

| DETECTION OF CTC
Many investigators have reported several approaches for the detection of CTC in patients with gastric cancer. Representative detection methods have been classified into two categories: polymerase chain reaction (PCR)-based methods and cytometric-based methods.
Reverse transcription-polymerase chain reaction (RT-PCR) is one of the PCR-based methods. A RT-PCR assay permits the molecular detection of CTC by assessing the mRNA expression of tumor-associated markers. Moreover, quantitative RT-PCR (qRT-PCR) is a promising tool for quantifying mRNA copy numbers. The greatest advantage of the RT-PCR assay is its high sensitivity for the molecular detection of CTC. We previously investigated its sensitivity using an in vitro model system with serially diluted gastric tumor cells mixed with peripheral blood cells from healthy donors. 14 The findings of this cell spiking study showed that the RT-PCR assay detected 10 tumor cells/10 7 donor-derived peripheral blood cells.
Additionally, a recent RT-PCR system has the ability to assess multiple gene expressions for the detection of CTC in one run. However, several investigators identified some limitations in the clinical application of RT-PCR assays to the detection of CTC. 15,16 False-positive results associated with RT-PCR may be yielded as a result of the illegitimate expression of targeted genes by normal cells and epidermal contamination in blood collecting or processing. 15 Furthermore, false-negative results may be obtained as a result of the heterogeneous expression of the targeted markers. 16 Further studies are needed in order to resolve the problems associated with the detection of CTC using RT-PCR-based methods. Table 1 summarizes studies reported since 2001 on CTC assessed using PCR-based methods in blood specimens from patients with gastric cancer. [16][17][18][19][20][21][22][23][24][25][26][27][28][29][30][31][32][33][34] In RT-PCR assays, cytokeratin (CK) and CEA are commonly selected as gene target markers for CTC.
Both genes are epithelial-specific antigens that are expressed in the normal cells of gastrointestinal tissues or most tumor cells, including gastric cancer. 35,36 Recent studies reported the clinical utility of new molecular markers for RT-PCR assays to detect CTC in the peripheral blood of patients with gastric cancer. 37 Survivin has been attracting attention as a promising blood marker for CTC in gastric cancer. 25,26,32 Survivin is a member of the inhibitor of apoptosis gene family and plays an important role in tumor progression. 38 It has been shown to control tumor apoptosis, promote proliferation, and enhance angiogenesis by a vascular endothelial growth factor signaling pathway. 39,40 Furthermore, survivin is overexpressed in the tumor cells of various malignant neoplasms, including gastric cancer. 41 Liu et al., in a meta-analysis of 1365 patients with gastric cancer from 16 eligible studies, demonstrated a close relationship between strong survivin expression in primary tumor sites and a poor prognosis. 42 Consequently, survivin has potential as an indicator for monitoring CTC in patients with gastric cancer. In contrast, we recently reported the clinical availability of B7-H3 and B7-H4 as blood biomarkers of CTC in patients with gastric cancer. 28,31 These molecules are members of the B7 family and regulate T-cellmediated immune responses. 43,44 The signaling pathway between B7 family members and their CD28 receptors on activated T cells has a marked impact on the immune surveillance system. 43,44 Although B7-H3 is considered to have two opposing characteristics as a coinhibitory or co-stimulatory mediator in T-cell-mediated immunity, B7-H4 is known to function as a negative modulator of immune responses. 43 Immunohistochemical studies showed that B7-H3 and B7-H4 were abundantly expressed in the primary tumor cells of patients with gastric cancer. 45 56 In the CellSearch system, CTC are captured based on enrichment using antibody-coated magnetic beads with epithelial-cell adhesion molecules and discrimination using fluorescently labeled antibodies against CK and CD45.
We investigated the presence or absence of CTC in peripheral blood cells from patients with gastric cancer using the CellSearch system. 51 The findings obtained showed that CTC were morphologically detected using the CellSearch system, particularly in patients with unresectable advanced or recurrent gastric cancers. Recently, a new size-based separation system has been developed for enrichment and cultivation of CTC. 55 The greatest appeal of this system is that it can easily separate viable CTC from peripheral blood. Moreover, we can assess functional properties by culture of enriched viable CTC. Accordingly, the size-based filtration system may be focused as a novel tool for isolating viable CTC.

| PROGNOSTIC IMPACT OF CTC
A large number of studies have investigated the clinical significance of CTC in patients with various malignancies, such as esophageal cancer, colorectal cancer, and pancreatic cancer. [57][58][59] Similarly, many investigators have assessed the prognostic impact of CTC in patients with gastric cancer, and most studies have suggested a close relationship between the presence of CTC and a poor prognosis. [16][17][18][19][20][21][22][23][24][25][26][27][28][29][30][31][32][33][34][47][48][49][50][51][52][53][54][55] In a qRT-PCR study of 123 gastric cancer patients with stages I-IV, Qiu et al. 29 reported that 3-year disease-free survival (DFS) rates in patients who were positive or negative for CEA mRNA were 43.9% and 74.1%, respectively (P = 0.001). A multivariate analysis identified CEA mRNA positivity as an independent prognostic factor (P = 0.02). 29 Moreover, the sensitivity and specificity of CEA mRNA expression for predicting disease recurrence were 56.8% and 74.7%, respectively. 29 However, the sensitivity and specificity of the serum CEA status were 31.8% and 79.7%, respectively. 29 They concluded that the presence or absence of CTC by qRT-PCR detection for CEA mRNA was a promising predictor for disease recurrence in patients with gastric cancer. 29 In contrast, in a qRT-PCR study on 59 gastric cancer patients with stages I-IV, Ikeguchi and Kaibara reported that there were no significant differences in overall survival (OS) rates among patients with or without CEA mRNA expression (P = 0.744). 19 In that study, CTC were assessed using a qRT-PCR assay on blood specimens after gastrectomy. 19 The findings obtained indicated that CTC were destroyed shortly after gastrectomy. 19 They hypothesized host-related immunological defense mechanisms as one of the reasons for these findings. 19 Cao et al. 32 focused on survivin as a novel blood marker of CTC in a qRT-PCR study on 98 gastric cancer patients with stages I-IV. 32 They reported that 3-year DFS rates in patients who were positive or negative for survivin mRNA were 53.1% and 84.3%, respectively (P < 0.001). Furthermore, a multivariate analysis identified the status of survivin mRNA as an independent prognostic factor (P < 0.001). 32 In a study on 55 gastric cancer patients with stages I-IV, Yie et al.
showed that the specificity, sensitivity, and accuracy of survivinexpressing CTC for predicting disease recurrence were 100%, 100%, and 84.6%, respectively. 25  In recent years, immunotherapy has begun to attract attention as a drug treatment for patients with several malignant neoplasms. 60 According to the findings of a phase 1b trial on immunotherapy for patients with advanced gastric cancer, the anti-programmed cell death protein 1 (PD-1) antibody pembrolizumab was found to be safe and exerted antitumor effects. 61 Although PD-1 is one of the representative molecules for immune checkpoints, we focused on other immune checkpoint molecules, such as B7-H3 and B7-H4. 28,31 We investigated the prognostic impact of B7-H3 and B7-H4 in the peripheral blood of patients with stages I-IV gastric cancer. 28,31 In a qRT-PCRbased study on 95 patients with gastric cancer, 5-year OS rates in patients who strongly or weakly expressed B7-H3 were 57.1% and 76.4%, respectively (P = 0.02). 31 Additionally, multivariate analyses selected the status of B7-H3 expression as an independent prognostic factor (P = 0.046). 31 In a B7-H4 study on 94 patients with gastric cancer, 5-year OS rates in patients who were positive or negative for mRNA expression were 60.4% and 87.2%, respectively (P = 0.04). 28 Our findings propose that the evaluation of B7-H3 and B7-H4 mRNA expression in blood specimens is useful as a CTC-associated tool for predicting the prognosis of patients with gastric cancer.

CONF LICTS OF INTEREST
Authors declare no conflicts of interest for this article.