|Ahead of print
S100A2: A potential biomarker to differentiate malignant from tuberculous pleural effusion
Ting Wang1, Ning Wang1, Linpei Zhang2, Yuchun Liu3, Asmitananda Thakur4
1 Department of Respiratory Medicine, Xi'an No. 4 Hospital, Xi'an 710004, People's Republic of China
2 Department of Scientific Research, Xi'an No. 4 Hospital, Xi'an 710004, People's Republic of China
3 Department of Cardiovascular Medicine, Xi'an No. 4 Hospital, Xi'an 710004, People's Republic of China
4 Department of Respiratory and Critical Care Medicine, The first affiliated hospital of Xi'an Jiaotong University, People's Republic of China
|Date of Submission||16-Feb-2019|
|Date of Decision||28-Apr-2019|
|Date of Acceptance||02-May-2019|
Department of Internal and Critical Care Medicine, Life Guard Hospital and Department of Chest Diseases, Nepal Anti Tuberculosis Association, Morang, Biratnagar
People's Republic of China
Source of Support: None, Conflict of Interest: None
Background: S100 calcium binding protein A2 (S100A2)—which has been testified to have an abnormal expression in non-small cell lung cancer (NSCLC)—is considered as an effective biomarker in the diagnosis and prognosis of this malignancy. In this study, we detected the S100A2 levels in pleural effusion, aiming to evaluate its potential value in differentiating malignant pleural effusion (MPE) from tuberculous pleural effusion (TPE).
Methods: We collected pleural effusion from 104 NSCLC patients with MPE and 96 tubercular pleurisy cases. Enzyme-linked immunosorbent assay (ELISA) was performed to measure the levels of S100A2 in these samples. Meanwhile, the serum S100A2 levels were also examined in same subjects. The data concerning the expression of those commonly-used markers, including CEA, CYFRA211 and NSE, were obtained from medical records.
Results: Like other classified biomarkers, S100A2 had an over-expression in both pleural effusion and sera of the NSCLC patients compared with controls (P = 0.000), though having a lower P value. Receiver operating characteristic (ROC) analysis showed that the levels of S100A2 in pleural effusion (PE) could distinguish MPE from tuberculous pleurisy (Area Under the Receiver Operating Characteristic Curve (AUC) = 0.887), and its diagnostic value in hydrothorax was obviously higher than in serum (AUC = 0.709).
Conclusion: Our results indicate that levels of S100A2 are significantly elevated in MPE, and that S100A2 may serve as a diagnostic biomarker for NSCLC patients with MPE. In further studies, we will validate our findings with a larger sample population.
Keywords: Biomarker, NSCLC, pleural effusion, S100A2
Key Message: S100A2 is significantly elevated in pleural effusion of patient's with NSCLC, it may serve as a diagnostic biomarker for NSCLC patients with malignant pleural effusion.
| » Introduction|| |
Lung cancer has come to be an elevated occurrence in recent years due to the trend of population aging, as well as an increasing prevalence of risk factors such as smoking, air pollution, etc. According to the global cancer statistic in 2018, lung cancer—which has been the leading cause of cancer-related death for both genders in developed countries—is placing a tremendous burden on the society. In the last few years, even though there has been obvious development in the techniques of diagnosing and treating this ailment, including molecule-targeted and immunization therapy, lung malignancy still kills more than 1.5 million people every year.
Among all the lung cancer patients, those having pleural effusion (PE) at the time of initial diagnosis is approximately 15%. Detecting tumor biomarkers in the samples of PE has been rendered into an area of interest in screening and diagnosing lung cancer due to its relatively small trauma and high acceptability.,, However, relevant researches show that a tumor marker panel, rather than a single pleural fluid marker, represents a helpful adjunct to cytology in order to rule in malignancy as a probable diagnosis., Therefore, searching for new indices, especially those with adequate sensitivity and/or specificity, is essential for clinical practice.
S100 protein family, comprising a total of 25 known human members, is the largest subfamily of EF-hand type calcium-binding proteins. Among all these genes, 22 are described to be clustered at chromosome locus 1q21, which is predisposed to chromosomal rearrangements.,, Through the inter-relationship with the target proteins, such as cell cycle's regulator genes (e.g., p53), enzymes, the S100 proteins fulfill a wide range of crucial intracellular/extracellular functions and participate in tumor genesis., S100A2—a member playing a significant and unique role in this family—has been found to be closely related to a number of tumors, including lung cancer.,,,, It was initially reported as a tumor suppressor in breast cancer by Lee et al. However, subsequent studies in the following decades have brought out conflicting findings about the role S100A2 played in neoplasia. In gastric cancer, for instance, down-regulated S100 gene was confirmed to have an association with advanced invasion, poor differentiation and distant metastasis. Similarly, contradictory results also appeared in researches with respect to S100A2 in NSCLC. Consequently, some specialists proposed the dual concept, that S100A2 has a peculiarly dominant expression at the early stage of NSCLC, and then decreases as the carcinomas gets invasive. Our previous researches confirmed that S100A2 experienced an increased expression in the sera and tissues of NSCLC patients., In this study, the levels of S100A2 were examined for the first time in PEs, and its clinical diagnostic value in NSCLC patients with MPE was evaluated.
| » Methods|| |
A total of 200 patients with PE who received repeated thoracocentesis and were newly diagnosed at the department of respiratory medicine, Xi'an No. 4 hospital, were recruited in this study. This included 104 NSCLC patients with MPE and 96 tubercular pleurisy cases. Through diverse inspection methods, such as pleural biopsy, endoscopic examination or percutaneous biopsy, all patients were histologically confirmed, having definitive etiology documented. Remarkably, the major pathologic types of the MPE patients were adenocarcinoma (65.4%) and squamous cell carcinoma (29.8%), and all cases were diagnosed by histopathological biopsy on small specimen. Patients with metastatic lung cancer or other malignant tumors were excluded from the study.
Histology reports were issued on the basis of World Health Organization (WHO) criteria. Apropos of the control group, the diagnostic criteria for tubercular pleural effusion (TPE) were as follows: caseous granulomas in a pleural biopsy specimen, positive acid-fast bacilli in pleural fluid, or an obviously high level of pleural fluid adenosine deaminase (>45 U/L). The control subjects were age and gender matched, free from malignancy and family history of tumor. Clinical data such as age, gender, family history of cancer, location of tumor, as well as laboratory characteristics of PE in two groups, were collected from the patients' medical records. Additionally, we obtained the information of the levels of lung cancer biomarkers carcinoembryonic antigen (CEA), neuron-specific enolase (NSE), and cytokeratin 19 (CYFRA21-1), in sera and PEs of these 200 patients, making a further comparison with S100A2.
The study was approved by the Human Research Committee for research involving human subjects at the Xi'an No. 4 hospital; all the participants were informed of the purpose and the exact experimental procedures of our study.
PE sample from each subject was immediately centrifuged at 1500 × g for 10 min at 4°C after the collection. The supernatants were divided into 100uL aliquots in cryotubes, stored at −80°C, and then examined for the expression of S100A2 using enzyme linked immunosorbent assay (ELISA) kits (USCN Life Sciences Inc., Hubei, China). All experimental steps were in strict accordance with the manufacturer's instructions of the ELISA kits with a catalog number of SEC009Hu. All assays were run in duplicate, having a dilution of 1:5, the test sensitivity and assay range for S100A2 are 0.124 ng/mL and 0.312–20 ng/mL, respectively.
Microsoft Excel and the SPSS 22.0 statistical package were used to collect, tabulate and analyze data. Distribution of demographic characteristics and biomarker analysis were compared between two groups. We used the analytic method of covariance significance of the mean to evaluate the age variables between cases and controls, and the χ2 test was performed in comparison between qualitative variables, such as gender, differentiation, etc.
The Fisher's Exact test was adopted if a cell in the table had few expected cases (i.e., <1). For all the quantitative data, normally distributed variables were compared in two groups using Student's t-tests; for those did not conform to normal distribution, nonparametric Mann–Whitney U test was applied. A two-tailed P < 0.05 was considered to indicate statistical significance. Receiver operating characteristic (ROC) curves was constructed to assess the accuracy of S100A2 to distinguish MPE from TPE by plotting sensitivity versus 1-specificity for every possible cut-off score, and area under the ROC curve (AUC) was calculated.
| » Results|| |
A total of 200 patients were recruited in our study, including 104 MPE patients (52.0%) and 96 tubercular pleurisy cases with TPE (48.0%); the demographic, pathologic, and clinical details of the study population are listed in [Table 1]. The mean age of two groups, which was displayed by mean and standard deviation (SD) values was 62.37 ± 0.76 years (95% confidence interval (CI) 53.38, 71.36, MPE) and 58.56 ± 0.52 years (95% CI 49.51, 67.61, TPE), respectively, and the difference was not significant (P > 0.05). Men accounted for 76.9% of the MPE patients, which had no significant difference from the controls (69.8%) (P > 0.05), as shown in [Table 1]. Besides that, most of the population studied had a history of smoking, taking up 67.3% in the case group and 61.5% in the control group, and there was no statistical significance, with P > 0.05. Specifically, for the NSCLC patients with MPE, the leading pathology was adenocarcinoma, accounting for 65.4%, followed by squamous cell carcinoma (29.8%), and 4.8% uncertain pathologic type. Regrading the pathologic grade, the predominant was poorly differentiated neoplasm, with a proportion of 64.4%, and only 2.9% were well differentiated.
According to the information illustrated in [Table 2], MPE patients had a tendency to share similar symptoms with their counterparts; frequent clinical symptoms included cough, expectoration, chest tightness, shortness of breath, etc., However, compared to the MPE patients, a greater proportion in TPE cases were more likely to have signs of fever (P = 0.044) and night sweats (P = 0.038).
[Table 3] and [Table 4] gives us information about measurements for routine and biochemistry tests of PE.
As shown in the table, the level of lactate dehydrogenase (LDH) (presented as mean ± SD values) were higher in the TPE group 872.6 ± 72.1 U/L (95% CI 685.4, 1059.8) than the MPE group 460.9 ± 64.9 U/L (95% CI 396.8, 525.0), respectively (P = 0.00). These were contrary to the glucose values [(6.1 ± 0.1 in MPE, 95% CI 4.8, 7.3 vs. 4.6 ± 0.4 in TPE, 95% CI 3.8, 5.5), with a P value of 0.002]. There was no statistical significance in other laboratory indexes – including leukocyte, differential cell counts (lymphocytes, neutrophils), total protein, and the positive rate of Rivalta test [Table 4] (all P > 0.05).
Levels of S100A2 and common clinical biomarkers in sera/PEs
[Table 5] demonstrates S100A2's expression in sera and PEs in the two groups, as well as the levels of those biomarkers frequently used in clinical practice, including CEA, CYFRA211 and NSE. As shown, the mean serum S100A2 level in the patients with MPE was 16.7 ± 0.9 ng/mL (95% CI 14.3, 19.1), while the mean level of serum S100A2 in the control subjects was 11.6 ± 1.0 ng/mL (95% CI 9.6, 13.5). The S100A2 serum levels were significantly higher in MPE patients than in TPE controls (P = 0.000). This upward trend was more conspicuous in PE of the case group, with an average concentration of 34.4 ± 1.6 ng/mL (95% CI 24.8, 43.9), compared with only 11.4 ± 0.9 ng/mL (95% CI 9.2, 13.6) in TPE, and the P value was 0.000. Meanwhile, statistically significant differences in the serum/PE levels of CEA were also observed between two groups, while P values were relatively higher (0.001 and 0.002). With regard to CYFRA211 and NSE, their levels in hydrothorax—rather than serum—had great value for accessory diagnosis of PE caused by primary lung neoplasm, with P = 0.001 and P = 0.002, respectively.
Furthermore, we investigated the correlation between the levels of S100A2 in MPE and clinical features. In our study, S100A2's levels in MPE were confirmed to have no statistically significant correlation with age, gender, smoking status, differentiation, and tumor location (all P > 0.05, data not shown).
ROC curve analysis was carried out to assess the value of S100A2 concentrations in sera/PE as a potential biomarker to differentiate MPE from TPE [Figure 1]. The serum level of S100A2, represented by the blue line, resulted in an AUC of 0.709 with a specificity of 78.1% at a sensitivity of 63.5% (the cut-off was 13.807 pg/ml). By comparison, as the green line indicates, the levels of S100A2 in PE had an apparently higher diagnostic value, having an AUC of 0.887 with a specificity of 85.4% at a sensitivity of 76.9% for distinguishing MPE from the controls (the cut-off was 17.646 pg/ml).
|Figure 1: ROC analyses for S100A2 in sera/PEs to differentiate MPE from TPE|
Click here to view
| » Discussion|| |
In this study, we focused on the diagnostic potency of S100A2 for NSCLC patients with MPE. To the best of our knowledge, our study is the first on a relatively lager sample of PE in which S100A2 levels were detected. Our results presented and verified that the pleural fluid concentration of S100A2 was extremely elevated in MPE, compared to benign pleural effusion. In particular, from the aspect of either sensitivity or specificity, the diagnostic potency of S100A2 in PE was much greater than in serum, for patients with MPE. In addition to that, by comparison with those popularly-used biomarkers (CEA, CYFRA211 and NSE), the sera/PE levels of S100A2 also had a diagnostic capacity to distinguishing NSCLC cases form the control group, however, having a lower P value.
S100A2 gene—also known as CaN19 and S100L—belongs to the S100 protein family, a multigenic group of cytoplasmic EF-hand Ca2+-binding proteins., The S100A2 protein has been reported to be closely related to a number of tumors – because its gene is located on chromosome 1q21, an area which is frequently rearranged. It has been confirmed that S100A2 gets involved in cytoskeletal components, protein phosphorylation, and calcium homeostasis both outside and inside of cells. This calcium-binding protein was initially thought to have special properties as it has a downregulated expression in a variety of cancers, in contradiction to most members in S100 protein family. Besides that, lower S100A2 expression is validated to have a relationship with poor prognosis and high risk for metastasis. For instance, in gastric carcinoma, Liu YF et al. found that downregulation of S100A2 was associated with poor differentiation, lymph node metastasis, advanced depth of invasion as well as a decline in overall survival. In recent decades, the close relationship between S100A2 and tumor genesis becomes increasingly clear along with its rising attraction to researchers, and S100A2 was found to have opposite characteristics. In epithelial tumors, overexpressed S100A2 could promote epithelial-mesenchymal transition (EMT) followed by increased invasion, loose colony morphology, and enhanced Akt phosphorylation, resulting in increased tumor growth.
In lung cancer, Feng et al. used a cDNA array to screen for genes that have different expressions in normal human bronchial epithelial (NHBE) cells and a tumorigenic cell line (1190-I), which was derived from immortalized human bronchial epithelial (HBE) cells, identifying that S100A2 had a down regulation in the 1170-I cells. Their further studies showed that the levels of S100A2 transcription and translation remained undetectable in transformed (1198) and tumorigenic (1170-I) HBE cells, compared to their previously high expressions in NHBE. However, S100A2 expression in a majority of NSCLC cells partially restored after 5-aza-2-deoxycytidine use. Thus, their results suggested that S100A2 acted as a suppression factor during lung carcinogenesis. Conflicting results appeared one year later, as Heighway et al. found that, at both mRNA and protein levels, the gene of S100A2 was highly over-represented in multiple lung tumor samples, which was parallel with other studies.,,,
Based on this discrepancy, some researchers proposed the dual role concept, that is, S100A2 expression is particularly obvious in the early and advanced stages of lung cancer and decreased in the middle stages. Evaluating the role S100A2 played in the genesis, development and metastasis of lung cancer, Naz S et al. demonstrated that higher expression of S100A2 induced epithelial-mesenchymal transition (EMT) in experimental cells (A549), through which it got involved in protumorigenic actions, such as increased invasion, loose colony morphology in soft agar, and enhanced Akt phosphorylation. Taken together, all these researches have provided some valuable information, but the concrete function of S100A2 is still unclear in lung cancer.
To explore the clinical value of S100A2, we have conducted a series of researches in recent years. Our previous studies identified that S100A2 experienced a significantly high expression in NSCLC at both serum and tissue levels, compared with healthy controls.,
In this study, the S100A2 concentrations in PEs were detected, and its ability was evaluated in differentiating malignant from nonmalignant pleural effusion, a common and complicated clinical problem., As data shown, S100A2 is up-regulated in MPEs, which is consistent with the dual role concept – this protein has an apparent expression in the advanced stage of lung cancer. Cumulatively, our results suggest that S100A2 could be used as a tumor marker for the diagnosis of MPE. With a relatively high sensitivities (specificity 85.4%, sensitivity 76.9%), the PE level of S100A2 can be considered to be a reliable detection method. In future, we will conduct experiments with larger samples to determine the potential efficacy of this marker and elucidate its precise biological role in NSCLC development.
Informed consent was obtained from all individual participants included in the study.
This study was supported by Key Research and Development Program of Shaanxi Province (Grant No. 2018SF-219), Xi'an Municipal Science and Technology Project (Grant No. 201805093YX1SF27(4)).
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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[Table 1], [Table 2], [Table 3], [Table 4], [Table 5]