Elevated serum eotaxin and IP‐10 levels as potential biomarkers for the detection of esophageal squamous cell carcinoma

Abstract Background and Aims Esophageal squamous cell cancer (ESCC) is one of the leading malignant cancers with a high incidence and mortality. Exploring novel serum biomarkers will help improve the management and monitoring of ESCC. Methods In the present study, we first used a ProcartaPlex Array to screen for serum proteins that were increased in 40 ESCC patients compared with matched normal controls; we found that eight proteins (IL‐2, IL‐5, IP‐10, IL‐8, eotaxin, TNF‐α, HGF, and MIP‐1b) had higher serum levels in ESCC patients than in normal controls. We further verified the clinical relevance of the candidate biomarkers with a larger sample of sera. Results In the 174 tested ESCC patients and 189 normal controls, the serum levels of eotaxin and IP‐10 were significantly higher in patients than in normal controls (p = 0.0038, 0.0031). In particular, these two proteins were also elevated in the sera of patients with early‐stage (0‐IIA) ESCC (p = 0.0041, 0.0412). When combining CEA and CYFRA21‐1 (in use clinically) with eotaxin or IP‐10, the effectiveness of detecting ESCC was superior to that of CEA and/or CYFRA21‐1 alone. Moreover, the serum level of eotaxin dropped significantly after surgical resection of primary tumors compared with that in preoperative ESCC samples (p < 0.001). Conclusions The data suggest that serum eotaxin and IP‐10 might be potential biomarkers for the detection of ESCC.


| INTRODUC TI ON
Esophageal squamous cell cancer (ESCC) is one of the leading malignant cancers in the world. China is one of the regions with the highest incidence rates, and over 90% of cases are esophageal squamous cell carcinomas. Most patients with ESCC have unresectable tumors or radiographically visible metastases at the time of diagnosis, and the overall 5-year survival rate of this disease has been low. 1,2 Thus, it is urgent and important to develop new molecular approaches for improving the diagnosis and treatment of ESCC.
Serum proteins are the most commonly used biomarkers in clinical diagnosis and treatment monitoring, with characteristics of being rapid, less invasive, and easy to accept by patients. As reported, αfetoprotein (AFP) is the preferred serum marker for the diagnosis and monitoring of hepatocellular carcinoma (HCC). 3 Cancer testis antigen sperm-associated antigen 9 (SPAG9) could distinguish lung cancer from normal controls (p < 0.001), and the level of the SPAG9 autoantibody in the sera of untreated patients was significantly higher than that in treated patients. 4 An increase in serum carcinoembryonic antigen (CEA) and carbohydrate antigen  has been associated with tumor progression and decreased overall survival of colorectal cancer. 5 Insulin-like growth factor binding protein 7 (IGFBP7) has been suggested as a serum marker for ESCC with an area under the curve (AUC) of 0.794, and a sensitivity of 40.6% and specificity of 90.7% based on a cutoff of 2.993 ng/ml. 6 L1 cell adhesion molecule (L1CAM) has diagnostic performance in ESCC with an AUC of 0.644. 7 CEA, cytokeratin 19 fragment 21-1 (CYFRA21-1), and squamous cell carcinoma antigen (SCC) were also evaluated for the diagnosis of ESCC. However, the sensitivity and specificity of these biomarkers are very limited in ESCC. 8,9 In the present study, we first used a ProcartaPlex Array to screen for serum proteins increased in 40 ESCC patients compared with the normal controls. By further verification of candidate serum markers with a larger sample of sera (174 ESCC patients and 189 normal controls), we found that serum eotaxin and IP-10 might be potential biomarkers in the diagnosis and therapeutic monitoring of ESCC patients.

| Study participants
In the biomarker discovery stage, we selected 80 blood samples

| Collection of blood samples
Fasting blood samples from cancer patients and normal controls were processed in an identical manner, collected into anticoagulant-free tubes, and centrifuged at 2500 g for 5 min. The supernatant was then subpacked and frozen at -80°C until the day before the experiment.

| Assessment of serum proteins using multiplex immunoassays
Serum samples were detected with ProcartaPlex multiplex immunoassay kits from the eBioscience division of Affymetrix. Briefly, 50 µl of serum was used for cytokine quantification using a MAGPIX® MILLIPLEX® system (Merck) with xPONENT software (Luminex).
The data were analyzed with MILLIPLEX® analyst software (Merck) using a cubic spline curve and background subtraction to convert the mean fluorescence intensity to pg/ml values. The kit detected up to 40 protein targets in a single sample.

| Measurement of candidate biomarkers by ELISA
ProcartaPlex Mix&Match Human 8-plex kits (Thermo Fisher) were used to measure the serum levels of the candidate biomarkers IL-2, Samples of patients and normal controls were assayed together in the same batch. Quality control (QC) was carried out as described in previous literature. 12

| Statistical analysis
The obtained data were statistically analyzed with Microsoft Excel, SPSS (version 22.0), and GraphPad Prism 7.0 software. The differences between groups were tested by the Mann-Whitney U test.
Comparison of positive rates was performed using chi-squared tests.
Receiver operating characteristic (ROC) curves and the area under the ROC curve (AUC) were used to evaluate the predictive performance of candidate biomarkers. p < 0.05 was considered statistically significant.

| Biomarker discovery by serum-based multiplex immunoassays
In the biomarker discovery stage, we first analyzed serum samples of 40 ESCC patients and 40 normal controls using ProcartaPlex multiplex kits to initially screen for candidate biomarkers. The results showed that eight proteins (IL-2, IL-5, IP-10, IL-8, eotaxin, TNFα, HGF, and MIP-1b) had higher serum levels in ESCC patients than in normal controls ( Figure 1).

| Serum levels of candidate biomarkers in ESCC patients and normal controls
We then expanded the serum sampling to 174 ESCC patients and 189 normal controls to verify the clinical value of the eight candidate F I G U R E 1 Results of multiplex immunoassay arrays to measure serum proteins in 40 ESCC patients. Differences in serum protein levels between patients and normal controls (p < 0.05) biomarkers, IL-2, IL-5, IP-10, IL-8, eotaxin, TNFα, HGF, and MIP-1b.
The results showed that among the above candidate biomarkers, eotaxin was the most effective for detecting ESCC. Serum levels of eotaxin and IP-10 were significantly higher in patients than in controls (p = 0.0038, 0.0031) ( Figure 2; Figure S1). In particular, elevated serum eotaxin and IP-10 were also observed in patients with earlystage (0-IIA) tumors (p = 0.0041, 0.0412; Table 2). In addition, high eotaxin was correlated with N staging (p = 0.042). However, there was no statistically significant association between the other six serum proteins and clinicopathological parameters (all p > 0.05) ( Table 3).
We simultaneously measured the classical tumor biomarkers CEA, CYFRA21-1, and SCC and found that serum CEA, CFYRA21-1, and SCC levels were also higher in ESCC patients than in healthy controls ( Figure 2).

| Performance of serum biomarkers for detecting advanced and early-stage ESCC
We further analyzed the ROC curves of candidate serum biomarkers to assess their diagnostic value ( Figure 3A). We found that serum levels  (Table 4). In patients with early-stage tumors, the sensitivity of eotaxin was 32.88% (Table 5).

| Sensitivity and specificity of serum protein panels in detecting ESCC
We further analyzed the combinations of different serum proteins and found that eotaxin and/or IP-10 combined with CEA and CYFRA21-1 were superior to CEA and/or CYFRA21-1 alone. When for the combination of the proteins was higher than that for each single protein ( Figure 3B; Table 6). In particular, the three combinations also showed higher sensitivity and/or accuracies for patients with early-stage (0-IIA) ESCC (Table 7).

| Difference in serum eotaxin levels between preoperative and postoperative patients
We carried out a comparative analysis of serum samples from 40 ESCC patients before and after surgery, in which postoperative samples were taken at 1 week after tumor resection. The results showed that the level of serum eotaxin in postoperative samples decreased significantly compared with that preoperatively (p < 0.0001). A similar postoperative decrease was also observed for CEA and SCC ( Figure 4; Figure S2).   TA B L E 6 The sensitivities, specificities, and accuracies of protein panels for detecting ESCC HPV in cervical carcinoma. 18 In addition, IP-10 is a major natural killer (NK) cell recruiting chemokine, and it exerts antitumor effects by inhibiting angiogenesis, 25 making IP-10 a potent antitumor factor. 26 Regarding cancer studies, overexpression of IP-10 in human cancer is mediated through the Raf, PI3K, p38/MAPK, JNK/MAPK, and NF-kB signaling cascades, which promote cell proliferation and contribute to the development of tumors. 27,28 Moreover, IP-10 induces antitumor and antimetastatic activities in different ways, including through immunological and antiangiogenic mechanisms. 29 Therefore, IP-10 immunotherapy is considered a promising strategy for breast cancer treatment. 19 In the present study, we found that serum levels of IP-10 in ESCC patients were significantly higher than those in normal controls. In particular, this protein was also elevated in the sera of patients with early-stage (0-IIA) ESCC, suggesting that serum IP-10 is a potential early biomarker of the disease.

| DISCUSS ION
Eotaxins are C-C motif chemokines that were first identified as potent eosinophil chemoattractants. Chemokines are responsible for promoting leukocyte attraction to sites of inflammation and cancer.
Additionally, some chemokines may promote and regulate metastasis and angiogenesis. 30,31 Chemokines are a family of secreted proteins that act through autocrine or paracrine mechanisms, and they are thought to influence tumor development. 32 The eotaxin family currently includes three members: eotaxin-1 (CCL11), eotaxin-2 (CCL24), and eotaxin-3 (CCL26). 33 Eotaxin-1 is customarily referred

| CON CLUS ION
In summary, we evaluated the clinical relevance of serum eotaxin and IP-10 for detecting ESCC for the first time. Our data demonstrate that serum eotaxin and IP-10 are potential biomarkers for early diagnosis and that eotaxin can also be used to monitor treatment progress.

CO N FLI C T O F I NTE R E S T
The authors declare that this research is not related to any commercial or financial interests.

DATA AVA I L A B I L I T Y S TAT E M E N T
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.