Esophageal cancer (EC) is the sixth leading cause of cancer-related deaths worldwide, and it can be classified into two main histological subtypes: Esophageal adenocarcinoma (EAC) and esophageal squamous cell carcinoma (ESCC). Almost all EACs develop in the lower third of the esophagus and originate from the Barrett mucosa, and ESCC occurs in the upper two-thirds of the esophagus. Approximately 80% of ESCC cases occur in central and southeastern Asia, while the incidence of EAC is high in northern and western Europe, North America, and Oceania [1
]. Overall survival (OS) is similar for ESCC and EAC. Although clinical diagnostic instruments such as endoscopy, computed tomography (CT), and positron emission tomography (PET) have been developed, over sixty percent of patients are diagnosed with EC with advanced progression of the disease because of their poor symptoms in the early stage [2
]. Patients with EC have poorer prognosis than those with other cancers, and even if they receive curative surgical resection, some experience early recurrence or metastasis [3
]. CT and PET are still standard imaging examinations for the diagnosis and monitoring of cancers, while the limitation of low sensitivity for small lesions has been a difficult issue to detect early recurrence sites. Similarly, serum biomarkers, such as carcinoembryonic antigen (CEA) and squamous cell carcinoma antigen (SCC), have low sensitivity and specificity for early diagnosis or the detection of recurrence [4
]. Therefore, a “liquid biopsy”, which is a simple and non-invasive sampling of non-solid biological tissue or DNA/RNAs from peripheral blood, is needed to provide new alternative serum biomarkers for monitoring the malignant behavior of cancer.
Ashworth et al. demonstrated the presence of circulating tumor cells (CTCs) in the peripheral blood of cancer patients in 1869 [5
]. CTCs shed into the peripheral blood stream via primary tumors and extravasate into distant organs to form metastases. In the last two decades, CTCs have been identified as novel biomarkers to elucidate the mechanism of spreading metastasis and the dissemination of cancer. Allard et al. suggested that CTC measurement using the CellSearch system (Menarini-Silicon Biosystem, Bologna, Italy) might have clinical utility for all cancers of an epithelial origin [6
]. To date, many kinds of liquid biopsy technologies have been reported. The most common methods of detecting CTCs are cytometric-based fluorescence immunohistochemical staining (F-IHC) and polymerase chain reaction (PCR) methods.
Quantitative reverse transcription-PCR (qRT-PCR) is a promising technique for quantifying the copy number of mRNA of interest, such as CEA, SCC, and survivin [7
]. Recently, due to the rapid technological advances in molecular biology, CTC analysis has moved to the next stage, which is comprehensive analysis using microarrays or next-generation sequencing (NGS) for genetic variations. These analyses can target hundreds to thousands of microRNAs (miRNAs) or cell-free DNAs/RNAs in peripheral blood with a single procedure.
On the other hand, cytometric detection technologies have been developed that enable the capture of CTCs as a single cell and visual evaluation for phenotype characterization. CTC identification relies on positive or negative selection by leukocyte depletion. The CellSearch system, the only CTC technology cleared by the US Food and Drug Administration (FDA), is among the most representative pieces of equipment for positive selection methods. The clinical utility of CellSearch to predict tumor progression and prognosis has been reported in many cancers, including breast cancer [10
], colorectal cancer [11
], prostate cancer [12
], and ESCC [13
]. Additionally, other technologies have been reported as cytological detection methods: isolation by size of epithelial tumor cells (ISET) [14
], ScreenCell [15
], and MetaCell [16
] as size-based separation systems; magnetic cell separation system (MACS) [17
], CTC-Chip [18
], and IsoFlux [19
] as microfluidic-based, immune-magnetic, positive selection methods; RosetteSep as a density gradient centrifugation method [20
]; and also flow cytometry. Recently, the genomic analysis of CTCs using deep sequencing was reported because of advances in single-cell analysis [21
]. According to these studies, the detection and isolation methods of CTCs or cancer-related genes were called “liquid biopsy” methods. Many studies have attempted to demonstrate the diagnostic or prognostic value of liquid biopsies in EC, and the clinical usefulness of liquid biopsies remains controversial. Herein, we summarize articles published about liquid biopsies in EC and discuss the potential utility of liquid biopsies in clinical use.
CTCs are unfavorable cells that are shed into the peripheral blood stream from primary tumors that can develop via metastasis. CTCs have been identified in many cancers, and their malignant behavior has been extensively demonstrated. Several investigators have reported the clinical importance of CTC in managing treatment strategies and predicting prognosis in many solid cancers, including EC. However, compared to other cancers, the clinical impact of CTCs in EC is still unclear due to the lower number of published studies. In the early 2000s, circulating mRNAs in peripheral blood were focused on as a new biomarker for the early diagnosis and prognostic prediction of various cancers. The PCR-based detection of cancer-related genes, such as CEA mRNA, CK mRNA, SCC mRNA, and survivin mRNA, presents good sensitivity in terms of predicting poor prognosis; however, some investigators have noticed that the false positive results from normal epithelial cells might constitute contamination. Since the late 2000s, cytometric methods that could morphologically identify CTCs and count the number of CTCs have been developed and have improved detection specificity. Moreover, we analyzed the genetic and mutational characteristics of each CTC. This has provided a strong contribution to clarifying the metastatic mechanisms of cancer. Although the CellSearch system is the only CTC platform that has been cleared by the FDA, several studies, including our previous study, have demonstrated the clinical utility of CellSearch as a prognostic predictor; however, the detection rate of EC, ranging from 18.0% to 50.0%, was not as high as we expected. Among the reasons for this result was that the isolation procedures of CellSearch depended on EpCAM expression. Therefore, the other non-EpCAM-dependent platforms have demonstrated a higher detection rate than CellSearch, ranging from 25.6% to 79.7%. In addition, several investigators have suggested that the measurement of the DNA methylation of cancer-related specific genes and exosomes may be used to detect early cancer or predict prognostic and therapeutic responses for several types of cancer. However, few studies have reported on DNA methylation and exosomes in esophageal cancer, and DNA methylation-based epigenetic signatures are considered to be valuable cancer biomarkers [98
]. Nevertheless, both cytometric and non-cytometric methods could isolate and analyze cancer-related molecules or cancer cells in the peripheral blood, and these procedures have been called “liquid biopsies”, which can be performed repetitively with usual blood sampling.
In the 2010s, owing to the development of scientific technology, comprehensive gene analysis with microarrays was applied to identify circulating cancer-related non-coding RNAs, such as miRNAs and lncRNAs, and NGS was applied to sequence ctDNA. Numerous studies have been reported for many cancers, including EC, and this has become the next standard method for liquid biopsies.
In this meta-analysis, we demonstrated the pooled hazard ratio of the OS and PFS for both cytometric and non-cytometric assays. For OS, the pooled HR of the cytometric assay was relatively higher than that of the non-cytometric assay. For PFS, the pooled HR of the cytometric assay was relatively higher than that of the non-cytometric assay. It seems that the cytometric assay may be a more useful prognostic method than the non-cytometric assay; however, these results depended on the difference of each detection theory and its sensitivity. For both the cytometric and non-cytometric assay, the median CTC detection rates in stages I–II were lower than III–IV. On the other hand, the detection rate for the non-cytometric assay was relatively higher than that of the cytometric assay. These results led the prognostic value of the non-cytometric assay to be relatively lower.
Among the challenging tasks of liquid biopsies is their application to the early diagnosis of cancers. Several investigators have examined the diagnostic value of liquid biopsies for differential diagnosis between pre-cancerous diseases and early EC. Although the pooled AUC for the early diagnosis of EC was 0.79 (0.75–0.83) in this meta-analysis, it was slightly lower than other cancers; for example, the pooled AUC for hepatocellular carcinoma was 0.87 (0.83–0.89) [102
], 0.89 for ovarian cancer [103
], and 0.88 for colorectal cancer [104
]. There were also two main reasons for this, among which was the presence of heterogeneity for the meta-analysis, owing to the small number of patients in this cohort. The other was that most studies in this review used single molecules as diagnostic predictors. Another cancer meta-analysis, as described above, applied a combination of multiple molecules and the copy-number of ctDNA as a comprehensive marker (AUC = 0.99, 95% CI = 0.98–1.00), as well as a combination of miR-30a-5p, miR-205-5p, and miR-574-3p (AUC = 0.95, 95% CI = 0.90–1.00) [64
], demonstrating a favorable diagnostic value. These results suggest that it might be difficult to determine a single definitive biomarker for cancer diagnosis and that exhaustive analysis will be required.
There were some limitations to this study. First, although the meta-analysis required detailed extracted data from as many publications as possible, the number of published studies according to EC and liquid biopsies was fewer than for other cancers. Due to the small number of studies, the heterogeneity became slightly larger than expected. Second, for the non-cytometric assays, differential microarray techniques were used to determine the potential of new biomarkers, and the background of the patient in each study was different. These factors may affect the variability of the prognostic and diagnostic values, even if the same molecules came from other studies. Third, this study did not deal with the pathological differences between ESCC and EAC because of the relatively small number of EAC patients. Therefore, large-scale multicenter studies with matched-pair patients are needed to more accurately estimate the diagnostic and prognostic values of liquid biopsies.
For future perspectives on liquid biopsies, a strong and simple combination of circulating molecules will be anticipated for the clinical management of patients with EC. The early diagnosis of EC using non-invasive liquid biopsies will improve the clinical outcomes of EC, and this will provide a great contribution to reducing healthcare costs. In this meta-analysis, we did not deal with CTM because of lack of sufficient published reports for EC. Umer et al. found that CTM has higher metastatic potential and resistance to apoptosis when compared to their single cell counterparts [105
]. As Umer mentioned, several investigators reported the malignant behavior of CTM in other cancers. The analysis of gene mutations, not only in CTCs but also CTM, will be a new candidate for molecular targeted therapy. Alix-Panabieres et al. advocated that CTC-derived cell lines and xenograft models are promising tools for identifying new therapeutic targets and for the development of new medicines [106
4. Materials and Methods
4.1. Literature Search Strategy
We searched relevant articles using PubMed and Embase with the keywords “esophageal cancer”, “liquid biopsy”, and “circulating tumor cells”. An additional search with Google Scholar was performed to check for other relevant publications.
4.2. Inclusion and Exclusion Criteria
The following inclusion criteria were used: (1) studies were written in English; (2) studies demonstrated prognostic or diagnostic value of CTC in EC; and (3) at least 15 cases were enrolled. The exclusion criteria were the following: (1) meta-analysis, review, commentary, and laboratory articles; or (2) duplicated data reported in other studies.
4.3. Data Extraction
Data were retrieved from the included studies by two reviewers (M.D. and A.T.). The extracted data included the following: the first author, publication year, country, number of controls, amount of blood samples, and positive rate of CTCs in each stage. For further analysis, the hazard ratio (HR) and 95% confidence intervals (CIs) for prognosis, sensitivity, and specificity for diagnosis were retrieved. Two reviewers performed literature selection independently, and any discrepancies were resolved by discussion.
4.4. Statistical Methods
Prognostic meta-analysis was performed to evaluate HRs and 95% CIs by forest plot analysis using the free downloaded software EZR [107
]. For diagnostic meta-analysis, the forest plot for the AUC and the summary sensitivity and specificity point with summary ROC were estimated using the JMP®
In the forest plot, the error bars indicate the 95% confidence interval (CI), the heterogeneity is indicated by I2 (intuitive statistic), and P-values less than 0.05 were considered statistically significant. The random effects model was applied to estimate the pooled HR. A funnel plot was used to evaluate publication bias.