The serum level of CC chemokine ligand 18 correlates with the prognosis of non-small cell lung cancer
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Hui Huang, Jing Li, Wen-jia Hu, ...
First Published May 3, 2019 Research Article 
https://doi.org/10.1177/1724600819829758
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 Article has an altmetric score of 1   Open Access Creative Commons Attribution, Non Commercial 4.0 License
Abstract
Background:
CC chemokine ligand 18 (CCL18) is a chemotactic cytokine involved in the pathogenesis and progression of various cancers. Our previous research showed that the expression of CCL18 is obviously higher in non-small cell lung cancer (NSCLC) than in the adjacent normal tissues, suggesting its role in NSCLC.

Methods:
We further examined the serum level of CCL18 in 80 NSCLC patients with enzyme-linked immunosorbent assay and simultaneously analyzed the survival curve of these patients by the Kaplan–Meier method, and then utilized a log-rank test to evaluate the correlation of CCL18 expression with the malignant progression of NSCLC.

Results:
Our results showed that the median serum concentration of CCL18 was significantly elevated to 436.11 ng/mL in NSCLC patients compared to 41.97 ng/ml in healthy people (P<0.01), which was also positively related to the expression of lung cancer biomarkers carcinoma–embryonic antigen and cytokeratin fragment antigen 21-1. Moreover, correlation analysis showed that an increased level of serum CCL18 was associated with a worse survival time in NSCLC patients.

Conclusion:
Our findings suggest that the serum CCL18 level of NSCLC patients was negatively correlated with the prognosis, thus suggesting that CCL18 may serve as a potential circulating biomarker for NSCLC diagnosis.

Keywords NSCLC, CCL 18, prognosis
Introduction
Lung and bronchus cancer, the most common type in the respiratory system, is one of the leading causes of cancer-related death worldwide.1 It is generally accepted that the poor prognosis of lung cancer mainly results from its high aggressiveness and its early invasion and metastasis. A large number of clinical studies have shown that patients with NSCLC (the most common form of lung cancer) show a low general 5-year survival rate and a high recurrence rate even in the early phase of the disease.2,3 Even though several clinical parameters, such as serum levels of carcinoma–embryonic antigen (CEA) and cytokeratin fragment antigen 21-1 (CYFRA21-1), may be helpful (to certain degrees) to the diagnosis and prognosis of lung cancer, there are currently no definitive biomarkers that can be reliably used to predict the responses of NSCLC patients to clinical therapies. Therefore, identification of novel and effective biomarkers becomes urgent for the early diagnosis and prognosis of lung cancer.

CC-Chemokine ligand 18 (CCL18) is a kind of CC chemokine family that usually has been found to play a critical role in the progression of malignant tumors,4 which is mainly expressed in monocytes, macrophages, and immature dendritic cells.5 It is known that tumor-associated macrophages (TAMs) are one of the main subgroups of immune cells in the tumor microenvironment and represent up to 50% of neoplastic mass.6 CCL18 is one of the most plentiful factors expressed in M2-like TAMs polarized by such stimulators as interleukin (IL)-4, AMP, and IL-13.7 Excessive production of CCL18 from M2-like TAMs has been demonstrated under various chronic inflammatory settings.8 In addition, numerous studies have shown that overexpression of CCL18 played an important role in the malignant progression and was closely related to a poor prognosis in various malignancies, including breast, ovarian, bladder, pancreatic, and colorectal cancers.9-13 Furthermore, our previous study revealed that expression of CCL18 was significantly elevated in NSCLC tissues compared with that in adjacent normal lung tissues, whereby the high expression of CCL18 was correlated with tumor stage and lymph node metastasis.14 In the present study, we further determined the serum level of CCL18 in NSCLC patients and evaluated its potential use as a biomarker for the prognosis of these patients.

Methods
Patients and sample collection
This study included 80 NSCLC patients who were recruited from the affiliated hospital of Guangdong Medical University from January 2014 to June 2018. Criteria included patients who were diagnosed with NSCLC, but excluded patients who received any preoperative anticancer therapies. Also, we recruited 50 people as the healthy control group, whose serum tumor marker diagnoses were negative. The clinicopathologic features of each patient was carefully collected from the clinical database, including sex, age, pathological type, tumor node metastasis (TNM) stage and smoking history. When the NSCLC patients were included, their blood was drawn and their overall condition was followed-up every 2 months. This study was approved by the research ethics committee of the affiliated hospital of Guangdong Medical University, and informed consents were obtained from all participants.

Enzyme-linked immunosorbent assays for serum CCL18, CEA, and CYFRA21-1 detection
The serum levels of CCL18, CEA, CYFRA21-1, and IL-4 in blood samples were detected by using enzyme-linked immunosorbent assay (ELISA) kits (Tong Wei, China). Briefly, venous blood samples were withdrawn by a traditional procedure and kept at room temperature for 30 minutes, then centrifuged with 1500 rpm/min for 30 minutes to separate serum and blood cells. The serum was transferred to a clean EP tube, and stored at −80℃ before analysis. The ELISA assays were conducted according to the manufacturer’s instructions, and calibration curves were prepared using purified standards for each protein assessed.

Statistical analysis
All statistical analyses were performed using SPSS 19.0 software (IBM Inc., US). The differences in concentration of CCL18 between NSCLC patients and healthy controls were evaluated using an independent t-test. Correlation analyses of concentration of CCL18 with the various clinicopathological features were performed using the chi-square test. The relationship of CCL18 and CEA,CCL18 and CYFRA21-1 were evaluated using the Pearson correlation coefficient analysis. The diagnostic value of CCL18 and the optimal CCL18 level cutoff value for diagnosis were determined using a receiver operating characteristic (ROC) curve. Survival rates were estimated using the Kaplan–Meier method. Univariate and multivariate analysis of the prognostic factors was performed using Cox regression analysis. Differences were considered significant at values of P < 0.05.

Results
The characteristics of NSCLC patients
This study included 80 NSCLC patients, 52 males and 28 females with a mean age of 64±5 years, and 50 healthy people, 27 males and 23 females with a mean age of 52±5 years. Among the 80 NSCLC patients, 42 had cancer diagnosed as adenocarcinoma and 38 as squamous carcinoma according to their pathological types. A total of 32 cases were classified as TNM stages I/II, and 48 were classified as TNM stages III/IV (Table 1).

Table
Table 1. The correlation between CCL18 expression and clinicopathological factors of non-small cell lung cancer.

Table 1. The correlation between CCL18 expression and clinicopathological factors of non-small cell lung cancer.


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Serum Levels of CCL18 in NSCLC Patients and Healthy Subjects
As shown in Figure 1, the concentration of CCL18 was present as median (25%, 75%) on account of their non-normal distribution. According to the results from Figure 1(a), the mean serum level of CCL18 in the NSCLC patients (n=80) was 39.69 (28.74, 150.69) ng/mL, which was significantly higher than that of healthy subjects (n=50), 14.19 (7.67, 25.54) ng/mL (P<0.001). Additionally, further analysis revealed that the median serum level of CCL18 in patients with lung squamous cell carcinoma, 78.35 (30.34, 258.1) ng/mL, was obviously elevated compared to that in patients with adenocarcinoma, 34.46 (27.97, 90.9) ng/mL (P<0.05), even though the serum levels of both were obviously higher than that in healthy subjects (P<0.01, Figure 1(b)).


                        figure
                   
Figure 1. The expression of serum CCL18. (a) CCL18 level in serum of NSCLC patients. (b) CCL18 level in patients with squamous carcinoma or adenocarcinoma.

CCL18: CC-chemokine ligand 18; NSCLC: non-small cell lung cancer.

CCL18 correlates with clinicopathological characteristics of NSCLC patients
The relationship between various clinicopathological features of the NSCLC patients and serum levels of CCL18 was investigated and the results are shown in Table 1. NSCLC patients were divided into two groups according to how their serum levels of CCL18 compared with the median CCL18 value (39.69 ng/mL): the low-level group had a concentration of CCL18 <39.69 ng/mL; the high-level group had a concentration of CCL18 ⩾39.69 ng/mL. There was no significant correlation serum level of CCL18 and sex, age, pathological type, or smoking history (P>0.05). In contrast, the serum level of CCL18 significantly correlated with TNM stage and lymphatic metastasis (P<0.05).

Correlation between CCL18 expression and tumor biomarker in NSCLC Patients
To further clarify the correlation between CCL18 and NSCLC tumorigenesis, we also detected the serum levels of lung cancer associated tumor markers, such as CEA and CYFRA21-1, in NSCLC patients by ELISA assay, and then evaluated their correlations with serum CCL18 levels by Pearson correlation coefficient analysis using SPSS 19.0 software. Our results showed that CCL18 expression was significantly correlated with both CEA and CYFRA21-1 (Table 2).

Table
Table 2. The correlation analysis between CCL18 and tumor biomarkers.

Table 2. The correlation analysis between CCL18 and tumor biomarkers.


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Diagnostic and prognostic value of CCL18 for NSCLC
ROC analyses of serum CCL18 levels were performed to distinguish between healthy persons and patients. As seen from Figure 2a, our results showed, our results showed the value of the cutoff point is 23.8 ng/mL, which revealed an area under the curve of 0.867(P<0.001,), indicating a high sensitivity and specificity to discriminate between healthy controls and NSCLC.

According to the cutoff point of serum CCL18 levels defined above, we stratified the NSCLC patients with the serum CCL18 concentration at 23.8 ng/mL as the point of equal sensitivity and specificity. Our results showed that NSCLC patients with the serum CCL18 level higher than 23.8 ng/mL had a mean survival time of 555.49 days, which was significantly shorter than 864.97 survival days of those patients with the serum CCL18 level below 23.8 ng/mL (P<0.01) (Figure 2(b)). In addition, further classification analysis found that the survival time of squamous cell carcinoma patients with negative serum CCL18 (857.36 days) was obviously longer than 676.29 days of those patients with positive serum CCL18 (P<0.05) (Figure 2(c)). Consistently, adenocarcinoma patients with positive serum CCL18 had a mean survival time of 497.27 days, which was markedly shorter than 1001.38 days of those adenocarcinoma patients with negative serum CCL18 (P<0.05) (Figure 2(d)).


                        figure
                   
Figure 2. CCL18 serum level and survival time of NSCLC patients. (a) Receiver operating characteristic (ROC) curve. (b) NSCLC patients. (c) Squamous cell carcinoma patients. (d) Adenocarcinoma patients. CCL18: CC-chemokine ligand 18; NSCLC: non-small cell lung cancer.

In addition, we performed univariate and multivariate analysis to determine whether the serum level of CCL18 and the clinicopathological parameters of Table 1 were independent prognostic indicators of NSCLC patient outcomes. The results are shown in Table 3. The results indicated that TNM stage, lymphatic metastasis, smoking history and CCL18 expression were independent predictors of a poor prognosis for NSCLC patients.

Table
Table 3. Univariate and multivariate analyses for overall survival in NSCLC patients.

Table 3. Univariate and multivariate analyses for overall survival in NSCLC patients.


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Discussion
CCL18 is a member of the serum-based cytokine family of secreted proteins involved in immune-regulatory and inflammatory process, which is considered to be involved in the pathogenesis and progression of various disorders, including cancer. We have previously found that the level of CCL18 expression was significantly elevated in NSCLC tissues, which correlated with tumor stage and lymph node metastasis.14 Similarly, numerous studies have shown that increased expression of CCL18 was observed in a variety of cancers and is associated with their clinical pathological features, including oral squamous cell carcinoma,15 colorectal cancer,13 ovarian cancer,16 breast cancer,9 bladder cancer,17 and so on. Recently, Eriksson Hogling et al.18 showed that circulating and WAT-secreted CCL18 was correlated with such metabolic risk factors as insulin resistance and metabolic risk score in women. Taken together, CCL18 is thought to be involved in the progress of multiple types of cancers by regulating the tumor microenvironment.

The tumor microenvironment is a complex ecology of cells and contains a milieu of cellular and non-cellular factors, such inflammatory cytokines and TAMs, which influence the tumor’s status and promote its development and progression.19,20 Among these factors, TAMs—also known as tumor-infiltrating macrophages—are the major players in accelerating tumor progression by altering the tumor microenvironment.21 Basically, CCL18 is the most abundant and specific chemokine released by TAM in the tumor microenvironment,22 suggesting that CCL18 is a possible marker of TAMs. Therefore, we hypothesized that CCL18 is directly involved in pathogenic processes in cancer and might serve as a serum biomarker for cancer patients.

In the present study, we first tested the serum level of CCL18 from a cohort of 130 subjects by ELISA analysis and found that serum CCL18 was significantly elevated in the NSCLC patients, which confirmed our previous findings.14 Further, we demonstrated that the median serum CCL18 in patients with squamous cell carcinoma was obviously elevated compared to patients with adenocarcinoma. In addition, we also found that the serum level of CCL18 was significantly correlated with TNM stage and lymphatic metastasis, but not with sex, age, pathological type, and smoking history. Our research results were in agreement with other people’s research. For example, Wu et al.23 found that the serum level of CCL18 in patients with gastric cancer was obviously higher than that of the control group; Sun and his coauthors revealed that the elevated serum level of CCL18 was associated with worse prognosis in female patients with breast cancer.9 Likewise, Plönes et al.24 also indicated that the level of serum CCL18 was increased in NSCLC patients and predicted a diminished survival time in adenocarcinomas. However, Yuan et al.13 found that a high CCL18 level was significantly correlated with a better overall survival in colorectal cancer patients, which we consider may be related to the tissue specificity of CCL18 expression.

Since CCL18 is produced by TAMs and the latter are associated with poor prognosis,25 we then investigated whether CCL18 could predict the poor prognosis of NSCLC. Our ROC analyses revealed NSCLC patients with a CCL18 concentration of 23.8 ng/mL or above had a significantly shorter mean survival time than those patients with a CCL18 concentration below 23.8 ng/mL. However, this effect seems to be differential in the NSCLC subgroup, whereby the survival time of squamous cell carcinoma patients with negative serum CCL18 was obviously longer than those with positive serum CCL18; that is, from 857.36 days to 676.29 days (P<0.05). However, adenocarcinoma patients with positive serum CCL18 had a mean survival time of 497.27 days, which was markedly lower than that of negative serum CCL18 adenocarcinoma patients with 1001.38 days. Moreover, the Pearson correlation coefficient analysis showed that serum CCL18 levels were significantly correlated with serum levels of the lung-cancer-associated biomarkers CEA and CYFRA21-1. Taken together, these data indicated that an increased serum level of CCL18 could be associated with a poor prognosis in NSCLC patients.

Conclusion
The present study has provided convincing evidence that the serum CCL18 level in NSCLC patients was positively related with the expression of the lung cancer biomarkers, CEA and CYFRA21-1, but negatively correlated with their survival time. Therefore, serum CCL18 may serve as a potential circulating biomarker for the diagnosis and prognosis of NSCLC patients.

Author contributions
Hui Huang and Jing Li contributed equally to this work.

Declaration of conflicting interest
The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding
The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This study was supported by Zhanjiang City Finance Competition Grant (2015A01017, 2016A01024), the Open Program of Guangdong Provincial Key Laboratory of Medical Molecular Diagnostics (FZZD201605), Doctor Funding of the Affiliated Hospital of Guangdong Medical College (BJ201504) and special funds for Science and Technology Innovation Strategy of Guangdong Province (2018A030310155).

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