Diagnostic value of calprotectin in differentiation between benign and malignant pleural effusion

Background Pleural effusion can arise as a result of more than 50 recognized causes and the differentiation between benign and malignant origin of the fluid is still a diagnostic challenge. The ability of tumor markers and other biological markers to make better diagnosis of malignant pleural effusion (MPE) remains questionable. Out of these, the calcium-related proteins S100-A8 and S100-A9 (the noncovalent heterodimer calprotectin) were demonstrated in a small amount in malignant not in benign pleural effusion. Objectives This research aimed to assess the diagnostic value of calprotectin in the differentiation between infectious or benign and MPE. Patients and methods Sixty patients were divided into group I: malignant and group II: infectious pleural effusions (which were further divided into group IIA: parapneumonic effusion and group IIB: tuberculous effusion) Quantitative measurement of calprotectin was done using the enzyme-linked immunosorbent assay technique in pleural effusion. Results Pleural calprotectin level in MPEs (229.2±168.6 ng/ml) was significantly lower than its level of infectious pleural effusions (3202.2±1304.8 ng/ml; P<0.001). The cutoff value of calprotectin level for the diagnosis of MPE was less than or equal to 730.5 ng/ml, with 95% confidence interval and the area under the curve was 0.999, the corresponding sensitivity was 96.7 and the specificity was 100% (P<0.001). Conclusion Calprotectin is a valuable biomarker in differentiating malignant from infectious pleural effusion.


Introduction
Diagnosis of pleural effusion remains a challenge. The list of the diseases they cause is big as well as it is heterogeneous. The more important difficulty in the diagnosis of exudative effusions is to differentiate benign from malignant effusion [1]. Most of malignant pleural effusions (MPEs) (90-97%) are exudative; they result from increased filtration from pleural vessels [2]. The initial semi-invasive method to diagnose exudative pleural effusion (thoracocentesis) allows cytological, microbiological, and biochemical analyses of the fluid [3]. More invasive procedures are required to diagnose exudative effusion with negative cytology particularly if malignancy is suspected. Closed pleural biopsy has a little diagnostic value and because of high diagnostic yield of thoracoscopy (≥90%), it is the method of choice [4].
Many studies have assessed the capability of tumor markers and other biological markers to make a better diagnosisofMPE [5].Severalproteins weredemonstrated in malignant versus benign pleural effusion (BPE). Out of these, the calcium-related proteins S100-A8 and S100-A9 (noncovalent heterodimer calprotectin) were demonstrated in a small amount in MPE [6].
Calprotectin is a calcium-and zinc-binding protein of the S100 group heterodimeric complex [7]. Magne Fagerhol and colleagues first described calprotectin in 1980 [8], it is a 36 kDa protein with two 14 kDa and one 8 kDa chain of amino acid, these proteins were named so because they were 100% soluble in ammonium sulfate solution [9]. Calprotectin is heat resistant and resistant to proteolysis in the presence of calcium [10]. When neutrophil activation or endothelial adhesion of monocytes occurred, calprotectin begins to be secreted by a microtubulemediated alternative pathway, thus acting as a marker for the influx of phagocytes into the site of inflammation [11], leading to increase its concentration in the plasma, serum, spinal fluid, synovial fluid, pleural fluid, urine, saliva, and stool during bacterial infection or inflammation in the relevant organs [12]. It has bacteriostatic and fungistatic properties that arise from its ability to sequester manganese and zinc [13]. It causes inhibition of metalloproteinases and chelation with zinc and manganese ions to inhibit microbial proliferation, as these metals are of vital importance for bacterial growth [14]. Calprotectin induces the apoptosis both in malignant and nonmalignant cell lines [15].
The function of calprotectin in cancer biology is conflicting to some extent. It is a strong apoptotic factor when produced by immune cells. On the contrary, expression in cancer cells is related to tumor development, cancer invasion, and metastasis [9]. Its relationship with inflammation is obvious with an established proinflammatory role in various inflammatory states [16]. A high calprotectin level could be expected in a patient with inflammatory disorder [15]. It could be reasonably expected to find high quantities of this protein in MPE because it is also concerned with inflammation-associated carcinogenesis [9].
The higher levels of calprotectin found in BPE could attribute to the antimicrobial role of this protein.
When neutrophil dies as a policy to suppress the growth of various fungal and bacterial pathogens, a massive amount of calprotectin is released [17]. Accordingly, calprotectin levels were high in parapneumonic and tuberculous pleural effusion and low in additional benign noninfectious etiologies [18].

Aim of the work
The objective of this study is to clarify the diagnostic value of calprotectin in the differentiation between infectious or benign and MPE.

Patients and methods
This study was conducted on 60 patients with pleural effusion admitted at the Chest Department, Benha University Hospital during the period from December 2016 till January 2018. Ethical research approval from Benha University Hospitals ethics committee and informed consent from the patient were obtained.
The patients were classified according to their final diagnosis into two groups. (1) Under treatment with anticancer chemotherapy.
All patients were subjected to the following: (1) Thorough medical history: age, sex, residence, occupation, smoking, and other particular habits of medical importance. Statistical analysis [21] The collected data were tabulated and analyzed using SPSS, version 16 software (SPSS Inc. Released 2007, SPSS for Windows, version 16.0; SPSS Inc., Chicago, Illinois, USA). Categorical data were presented as number and percentages, using χ 2 test to analyze them. Quantitative data were expressed as a mean ±SD, median, interquartile range. They tested for normality using the Shapiro-Wilk test, assuming normality at a P value of more than 0.05, Student's t test, and analysis of variance for normally distributed variables. Nonparametric variables were analyzed using the Mann-Whitney, Wilcoxon test, and Kruskal-Wallis test. Receiver operating characteristic curve was used to detect the cutoff value of calprotectin in the prediction of MPE.
The degree of significance in this work started below 0.05 (P<0.05 was considered significant). A P value of more than 0.05 is nonsignificant (NS), P value less than 0.05 is significant (S), and a P value less than or equal to 0.001 is highly significant (HS).
The ages of the studied patients ranged from 19 to 76 years: group I (malignant effusions) with a mean age of 57.6±13 while group II (infectious effusions) was subdivided into group IIA, parapneumonic pleural effusion (their ages ranged 29-75 years with a mean 52±14) and group IIB, tuberculous pleural effusion (their ages ranged 13-65 years with a mean 44.8 ±14.5) with a significant difference between tuberculous and MPE.
The pleural calprotectin in group I MPE (229.2 ±168.6 ng/ml) was significantly lower than its level in group II; infectious pleural effusion was (3202.2 ±1304.8 ng/ml) with highly significant difference between two groups ( Table 1).
The patients suffered from parapneumonic pleural effusions, group IIA demonstrated the highest levels of calprotectin (3333 ng/ml) and its level in patients with tuberculous pleural effusions group IIB (3071.3 ng/ml) were significantly higher than those of MPE group I (229.2 ng/ml), P value less than 0.001 (HS) ( Table 2). However, the pleural calprotectin level in parapneumonic pleural effusion group IIA (3333±1151.8 ng/ml) was not substantially different from those in the tuberculous pleural effusion group IIB (3071.3 ng/ml) (P=0.92) ( Table 3 and Fig. 1).     increased in nonmalignant pleural fluid compared with malignant pleural fluid, which supported the measurement of both biomarkers in pleural effusion as a possible noninvasive strategy for the differential diagnosis of MPE. Also Lou et al. [22] found that the median level of calprotectin and CXCL12 in MPE were 447.15 and 4.12 ng/ml, and both were significantly lower than that of the BPE (P=0.003 and 0.020, respectively) and that of tuberculous pleural effusion (P=0.002 and 0.003, respectively). The higher calprotectin levels found in BPE could attribute to the antimicrobial role of this protein [17].
Calprotectin level in patients with parapneumonic and tuberculous pleural effusions were significantly higher than those with MPE [3333, 3071.3, and 229.2 ng/ml, respectively with P<0.001 (HS)] (Table 2). However, the pleural calprotectin level in parapneumonic pleural effusion was not significantly different from those in the tuberculous pleural effusion (P=0.92) ( Table 3). Sanchez-Otero et al. [18] found that patients with pneumonia showed the highest calprotectin level (3517.9 ng/ml) and were significantly higher than those of the MPF group (257.2 ng/ml), but these levels were not considerably different from those in the tuberculous group (2982.3±1573.0 ng/ml).
In the present work, the cutoff value of calprotectin level for the diagnosis of MPE was less than or equal to 730.5 ng/ml with 95% confidence interval and the AUC was 0.999; the corresponding sensitivity was 96.7% and specificity was 100% (P<0.001) ( Table 4). Sanchez-Otero and colleagues detected the ability of calprotectin to differentiate between MPE and BPE by initiating two cutoff points. The first one (≤545 ng/ml) was determined as it gave the highest accuracy level (92.31%), and a sensitivity of 97.01% and specificity of 88.76%, while the second one (≤736.4 ng/ml) represented a sensitivity of 100%, specificity of 83.1%, accuracy of 90.4%, and AUC was found to be 0.963 (95% confidence interval, 0.932-0.994). Besides, they analyzed the calprotectin capability to differentiate pleural effusion subtypes. It showed the highest accuracy in differentiating MPE from pleural effusion of tuberculous and parapneumonic origin (97.94 and 95.83%, respectively) [18]. Luo et al. [22] stated that calprotectin showed high specificity in discriminating MPE from BPE and tuberculous pleural effusion (71.43 and 84.09%, respectively). On the contrary, Su and colleagues found a strong expression of S100-A8, S100-A9 in lung adenocarcinoma and end-stage lung cancer tissue. Also, Blanco-Prieto and colleagues found a similar level of calprotectin in the sera of lung cancer patients (221.21 ng/ml) but a lower serum level was found in benign inflammatory conditions (141.93 ng/ ml). They explained that finding by the different nature of the fluids (pleural vs. serum) and their higher expression in lung tissues may be explained by the combined effect of inflammation on cancer progression [24,25].

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Conflicts of interest
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