In the present study, we evaluated CTCs and TWIST expression in CTCs using FAST in 31 patients with metastatic GC; CTCs and TWIST (+) CTCs were detected in 25 (80.6%) and 24 (77.4%) patients, respectively. Patients with CTCs > 7.5/7.5 mL of blood showed shorter overall survival than those with CTCs ≤ 7.5/7.5 mL of blood. In addition, patients with TWIST (+) CTCs > 2.5/7.5 mL of blood tended to show shorter overall survival than those with TWIST (+) CTCs ≤ 2.5/7.5 mL of blood. These results suggest the potential role of CTCs as a prognostic biomarker in patients with metastatic GC.
Recently, CTCs have been suggested to be promising biomarkers for early diagnosis and predictor of survival and prognosis in GC patients [3, 11]. In our previous study, we measured CTCs using FAST in 116 GC patients and 31 healthy individuals [11]. When CTCs ≥ 2/7.5 mL of blood was set as the cut-off value, the sensitivity and specificity of differentiating GC patients from healthy controls were 85.3% and 90.3%, respectively. Although the CTCs were not associated with any clinicopathologic feature including histopathologic type, mucin phenotype, or staging, we suggested the potential of CTCs as an early diagnostic biomarker for GC. In another recent study, using a wedge-shaped microfluidic chip, CTCs were associated with tumor differentiation, lymphovascular invasion, and staging [22]. In the present study, we only included patients with metastatic GC; there was no difference in CTC counts according to histopathologic type, peritoneal dissemination, and hematogenous metastasis. These differences in the association between CTCs and clinicopathologic parameters may be due to heterogeneity in the baseline clinicopathologic background.
In a prospective study that evaluated CTCs in 251 patients with advanced GC using the CellSearch™ system, CTCs were detected in 62 patients (62/103, 60.2%) in the non-resectable group and the overall survival rate was significantly lower in patients with CTCs than in those without CTCs in the non-resectable group [6]. In a meta-analysis that included 26 studies comprising 2,566 GC patients, the frequency of CTC detection was significantly related to disease-free and overall survival of patients [23]. Similarly, in the present study, CTCs were detected in 80.6% of patients with metastatic GC, and high CTC number (> 7.5/7.5 mL of blood) was associated with poor overall survival. Our results are also consistent with the results of a previous study in which the overall survival rate of patients with CTCs > 5/7.5 mL of blood tended to be lower than that of the patients with CTCs ≤ 5/7.5 mL of blood [24].
CellSearch™ system is the most commonly used and the only United States Food and Drug Administration-approved CTC detection test applicable in patients with various cancers. This test system requires the enumeration of epithelial cells, which are separated from the blood using EpCAM antibody-coated magnetic beads and identified with fluorescently labeled antibodies against CKs and a fluorescent nuclear stain. However, EpCAM-based enrichment cannot detect CTC subpopulations that have already or partially undergone EMT [25, 26]. In contrast, FAST is a size-based isolation kit, with several advantages compared to the other CTC detection methods, including user-friendly, cost-effective, and efficient CTC capture technique [9, 10]. Furthermore, FAST enables further downstream molecular analysis for detection of TWIST (+) CTCs using immunostaining. Therefore, we could investigate TWIST expression in the CTCs using FAST in patients with metastatic GC. EMT is a multi-step process that plays a key role in metastasis, cancer progression, and therapeutic resistance. Therefore, we predicted that TWIST expression in CTCs would be strongly associated with poor prognosis and response to chemotherapy in patients with metastatic GC. In the present study, although the difference was not statistically significant, TWIST (+) CTCs tended to be associated with shorter survival. However, TWIST (+) CTCs were not associated with poor response to chemotherapy. We believe that these results may be caused by the relatively small sample size of our study and the use of various chemotherapy regimens, and that examination of larger cohorts may reveal a more significant impact of TWIST (+) CTCs on these clinical parameters.
CTCs can also be used to monitor response to chemotherapy [4]. The measurement of CTCs using peripheral blood is non-invasive and can be performed repeatedly, and the periodic monitoring of CTCs may be useful to predict the efficacy of chemotherapy. Although carcinoembryonic antigen (CEA) and cancer antigen 19 − 9 (CA19-9) are frequently used as markers in GC patients, these markers are elevated in less than 40% of patients with advanced GC and they temporary increase following chemotherapy; therefore, the sensitivity and specificity of assays to detect these markers are insufficient for prognosis [27]. In the present study, CEA and CA19-9 were elevated in 13 (41.9%) and 18 (58.0%) patients with metastatic GC, respectively. In a study that measured CTC levels serially in 52 patients with advanced GC, patients with ≥ 4 CTCs/7.5 mL of blood at 2- and 4-weeks post-chemotherapy showed shorter median progression free survival and overall survival [28]. In another study CTC levels were measured in 136 patients with advanced GC, and patients with ≥ 3 CTCs/7.5 mL of blood after chemotherapy had shorter progression free survival and overall survival, and elevated levels of CTCs after chemotherapy was associated with ineffective therapeutic response [26]. We also found that CTC and TWIST (+) CTC counts decreased following chemotherapy compared to before chemotherapy in some patients (data not shown). We intend to evaluate the potential of CTCs and TWIST (+) CTCs as useful supplementary markers to monitor the response to chemotherapy in GC patients in the future.
Our study has several limitations that need to be addressed. First, the number of patients included in the study was relatively small. Second, other mesenchymal markers such as vimentin or stem cell markers (such as CD44) were not studied along with TWIST in the CTCs. Third, we detected CTCs based on an EpCAM-based enrichment technique, and evaluated TWIST expression in these CTCs. Based on the expression of epithelial and mesenchymal markers, CTCs is classified into three subpopulations; epithelial CTCs, biphenotypic epithelial/mesenchymal CTCs, and mesenchymal CTCs [29]. We did not evaluate mesenchymal CTCs without EpCAM expression, which are reported to be associated with metastasis and disease progression [29]. Finally, CTCs detected in patients with metastatic GC are unlikely to be organ specific. Although we excluded other current malignancies through the examinations mentioned earlier, it is possible that undetected malignancies may be present in other organs and these could be a source of the CTCs.
In conclusion, CTCs and TWIST (+) CTCs were detected in 4/5 of patients with metastatic GC, and high level of CTCs and TWIST (+) CTCs was associated with worse overall survival. Our study provides promising results for the use of CTCs and TWIST (+) CTCs as prognostic biomarkers in patients with metastatic GC. The additional role of CTCs and TWIST (+) CTCs as biomarkers for prediction of response to chemotherapy should be investigated in large, prospective, long-term follow-up studies.