Clinical value of folate receptor-positive circulating tumor cells in patients with esophageal squamous cell carcinomas: a retrospective study

Over the past decade, numerous CTC enrichment and detection methods have developed, including density gradient centrifugation, immunomagnetic cell enrichment, flowcytometry, size-based filtration and isolation, PCR-based assays using a variety of chosen markers, and immunoassays against surface antigens [19‐23]. The majority of these procedures are not used on a regular basis in clinical practice due to a lack of standardization, reproducibility, or assay duration, most of these techniques are not implemented in clinical routine. None has emerged as the benchmark for CTC testing [24]. To accurate CTC detection in metastatic breast, colon, and prostatic malignancies, only the CellSearch system has been received approval by the United States (U.S.) Food and Drug Administration (FDA). The CellSearch™ system, which focuses on detecting epithelial marker positive CTCs in ESCC patients, appears to be ineffective at doing so, there was no significant relationship between the presence of CTCs and any of the following patient characters: age, sex, median alcohol consumption, neutrophil/lymphocyte ratio, serum CEA, and WHO PS, even tumor location, size, depth, grade of differentiation, lymph node metastasis, or lymphatic or venous invasion was not significantly [7], which were consistent with our results. Age, sex, albumin, pre-albumin, CRP, ferritin, CRP/Albumin ratio, tumor size, differentiation grade, lymph node metastases, perineural invasion, or vascular invasion were not statistically related with FR ⁺ CTC in our study. The isolation and quantification of mesenchymal CTCs are significant for evaluating the pre-operative condition and treatment efficacy of ESCC patients in comparison to the previous work, which concentrated solely on epithelial marker-positive CTCs. And the findings revealed a statistically significant result between mesenchymal CTC count, ESCC clinical stage, and neoadjuvant chemotherapy effectiveness [8]. Immunomagnetic enrichment for CTCs that express epithelial cell adhesion molecules (EpCAM) using magnetic beads with antibody coatings, followed by fluorescent labeling, is the basis for detection [25]. The major problem of this approach is that it can only detect CTCs that still exhibit epithelial markers, rather than CTCs that undergone the epithelial-mesenchymal transition (EMT) [26]. Because CTCs may have already undergone an EMT process and display stem cell function. Therefore, they can provide false-negative results by upregulating their mesenchymal markers while downregulating their epithelial markers, enabling them to escape hematological detection [27].

The Isolation by Size of Epithelial Tumor cells (ISET)-based CTC assays can detect micro-metastases in ESCC patients that cannot be detected by traditional examination methods and may be used as prognostic indicators of disease progression and clinical outcomes in ESCC. The risk of disease progression was 3.8 fold higher in ESCC patients in whom CTCs > 2, compared with patients CTCs ≤ 2, and CTCs > 0 was an independent prognostic marker for tumor recurrence [9]. CTC-positive ESCC patients typically have a poorer prognosis, which indicates that timely monitoring of the recurrence and metastasis of ESCC may benefit from CTC detection [28]. The CTC quantity was demonstrated to be an independent prognostic factor in patients with unresectable ESCC, before concurrent chemoradiotherapy (CCRT) [29].

The China FDA (CFDA) have approved the use of FR⁺CTCs in clinical lung cancer detection [30]. Numerous earlier investigations have shown a connection between FR⁺CTCs and the prognosis of individuals with lung cancer [14, 15]. In patients with NSCLC, the risk of recurrence/metastasis was 5.49 times higher in the group with serum FR⁺CTC concentration ≥ 11 FU/3 ml in the group with FR⁺CTC concentration < 11 FU/3 ml [14]. Patients with low FR⁺ CTC levels were linked with prolonged OS in a multivariate COX regression analysis; however, the conclusion did not achieve statistical significance. FR⁺ CTC levels (cutoff: 7.9 FU/3 ml) were independent predictive variables of extended RFS [15]. According to a recent study FR is highly expressed in EC patients and serves as an independent prognostic factor in ESCC patients [31]. However, applying FR⁺CTCs in predicting the prognosis of EC requires further clarification. In our study, patients with FR⁺CTCs ≥ 14.79 FU/3ml in pre-operative blood had significantly shorter DFS and OS, suggesting that pre-operative FR⁺CTC may be a useful biomarker for DFS and OS of patients with ESCC, which agreed with the findings of earlier research [28, 29]. Stage IV cancer always indicates an advanced disease, according to our analysis, the stage IV variable has a strong correlation with DFS and OS. Metastasis occurs when tumor cells intrusive into the circulating system and become CTCs. CTC can act as disseminated tumor cells (DTC) to its new metastatic site after leaving the bloodstream. Several key factors determine the fate of DTC, such as the biological characteristics of CTC, and the tumor microenvironment (TME) in the metastatic niche [32]. The activation and proliferation of DTC can be caused by inflammatory stimuli [33], which happened in ESCC TME [34]. As to the biological characteristics, a study has found that the extracted CTCs contain many macrophages adhering to them. Further in vitro research discovered that CTCs obtained better mechanical adaptation phenotypes, with low hardness, adhesion, and deformation [35]. Furthermore, the other study focusing on primary and metastatic tumor ecosystems in ESCC revealed an accumulation of metastatic macrophages in metastatic ESCC lymph nodes [36]. These might be the underpinning mechanisms that could illustrate why esophageal cancer patients with high circulating tumor cell counts have poorer prognoses.

Additionally, in a preliminary study, elevated CRP level was linked to tumor progression and poor survival in patients with ESCC [37, 38]. For the low- and high-CRP groups in the ESCC, The overall 5-year survival rates were 90.6% and 63.6%, respectively, and the results were statistically significant (p = 0.043) [39]. High CRP is a predictive risk factor for DFS and OS in patients with ESCC, and the results of our study in the analysis of DFS in line with the previous finding. Even though the P value of multivariate analyses of CRP in OS is slightly higher than 0.05, which is 0.088, we still consider that it was significant in the real world, as we only have 43 events and could only include four variables in our model. CRP is the most important acute-phase protein. The association between cancer and inflammation is known for years, and the mechanisms behind CRP rise are well understood. Inflammation can be triggered by malignant tumor cells and vice versa. The research on TME of ESCC published on Nature Communication indicates that exhausted T cells, exhausted NK cells, regulatory T (Treg) cells, alternatively activated macrophages (M2), and tolerogenic dendritic cells (tDCs) were enriched in ESCC tissues, suggesting inflammation in TME in ESCC [34]. The hepatocytes’ produced CRP is known to be stimulated by a few proinflammatory cytokines, including interleukin-1, interleukin-6, tumor necrosis factor, interferon, and tumor growth factor. These cytokines also affect the survival, growth, mutation, proliferation, differentiation, and migration of tumor cells [40].Due to the above mechanism, pre-operative serum CRP levels constitute a significant predictive indicator in patients with various carcinomas [41, 42].

There were some inherent limitations in this study. One of them is the limitation of the retrospective nature; hence, a prospective study should be conducted to further explore the relationship between FR⁺CTC level and survival outcome. Furthermore, the predictive value of FR ⁺ CTC in ESCC needs to be explored in studies with larger sample sizes and longer follow-ups.