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Pleural effusion is associated with multiple benign and malignant conditions. Currently no biomarkers differentiate malignant pleural effusion (MPE) and benign pleural effusion (BPE) sensitively and specifically. The present study identified a novel combination of biomarkers in pleural effusion for differentiating MPE from BPE by enrolling 75 patients, 34 with BPE and 41 with MPE. The levels of lactate dehydrogenase, glucose, protein, and total cell, neutrophil, monocyte and lymphocyte counts in the pleural effusion were measured. The concentrations of interleukin (IL)-1β, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12, tumor necrosis factor-α, interferon γ, transforming growth factor-β1, colony stimulating factor 2, monocyte chemoattractant protein-1 and vascular endothelial growth factor (VEGF) were detected using cytometric bead arrays. Protein and VEGF levels differed significantly between patients with BPE and those with MPE. The optimal cutoff value of VEGF and protein was 214 pg/ml and 3.35 g/dl respectively, according to the receiver operating characteristic curve. A combination of VEGF >214 pg/ml and protein >3.35 g/dl in pleural effusion presented a sensitivity of 92.6% and an accuracy of 78.6% for MPE, but was not associated with a decreased survival rate. These results suggested that this novel combination strategy may provide useful biomarkers for predicting MPE and facilitating early diagnosis.
Pleural effusion is a common clinical complication associated with multiple benign and malignant conditions (1). Congestive heart failure, pneumonia and tuberculosis are common causes of benign pleural effusion (BPE) (2). Certain types of malignancy, including lung, breast, ovarian cancer and lymphoma also cause pleural effusion (3–5). Among patients with cancer, ~50% develop malignant pleural effusion (MPE) (6). The median survival time following MPE presentation is 4 months (7).
MPE is induced by the interaction of tumor cells, endothelial cells, myeloid cells and lymphoid cells in the pleural cavity. The concentration of protein and lactate dehydrogenase (LDH) in MPE is a prognostically significant biochemical feature (8). In addition, numerous types of cytokine, including interleukin (IL)-1β, IL-5, IL-6, IL-8, IL-10, IL-12, monocyte chemoattractant protein-1 (MCP-1) and tumor necrosis factor-α (TNF-α) are detected in MPE (9–11). An increased concentration of vascular endothelial growth factor (VEGF), which is mainly secreted from endothelial cells is also detected in MPE (12). Certain biochemical properties of pleural effusion, including glucose and total protein concentration, may predict mortality in patients with MPE (13). Interferon γ (IFNG) and transforming growth factor β (TGFB) 1 are associated with tuberculosis pleural effusion, but not MPE (14,15). However, no cytokines or biochemical features that differentiate MPE and BPE sensitively and specifically have been identified at present.
Since clinical features, biomedical features and numerous cytokines have been reported to be associated with MPE, and a single parameter may not serve as an optimal biomarker for predicting MPE (8–15), the present study assessed whether a combination of biochemical features, clinical features and cytokine levels in pleural effusion may distinguish between BPE and MPE. The clinical and biochemical features of pleural effusion were determined and the concentration of cytokines in collected BPE and MPE samples was evaluated using cytometric bead arrays.
In the present study, 75 patients, including 34 with BPE (22 males and 12 females; median age, 67.59 years) and 41 patients with MPE (19 males and 22 females; median age, 65.68 years) were enrolled between January 2013 and December 2013 at the Kaohsiung Medical University Hospital (Kaohsiung, Taiwan). The Institutional Review Board (IRB) of Kaohsiung Medical University Hospital approved the present study and all patients provided written, informed consent in accordance with the Declaration of Helsinki (IRB no. KMUH-IRB-20120275). Pleural effusion was subsequently collected. Exudative and transudative BPE was classified according to Light's criteria (16). The histology of specimens, obtained using bronchoscopy and lung puncture, or the malignant cells in the pleural effusion were used for malignancy diagnosis (17,18). MPE was collected from patients, including those with malignant tumors.
Aliquots (200 µl) of pleural effusion from the 75 patients were centrifuged for 10 min at 3,000 × g at 4°C and the supernatants were stored at −80°C. The concentrations of IL-1β, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12, IFNG, colony stimulating factor 2, MCP-1, TNF-α, TGFB1 and VEGF were measured using a CBA Flex Set kit (BD Biosciences, Franklin Lakes, NJ, USA) according to the manufacturer's protocol. Each sample was determined once. Data were obtained using a BD Accuri C6 flow cytometer and analyzed using FCAP Array V3.0 software (both from BD Biosciences).
Biochemical features and the concentration of cytokines were compared between BPE and MPE samples using the Kruskal-Wallis or Mann-Whitney U test. The concentrations of cytokines and biochemical features for which these tests revealed a significant difference were used to generate a receiver operating characteristic (ROC) curve. The survival curves were obtained using the Kaplan-Meier method. SPSS version 19 (IBM Corp., Armonk, NY, USA) was used for all statistical analysis and to generate the graphs. P<0.05 was considered to indicate a statistically significant difference.
Of the 75 patients enrolled in the present study, 41 (19 males and 22 females; median age, 65.68 years) exhibited lung cancer (adenocarcinoma, squamous cell or bronchogenic carcinoma, 28 patients) or extrathoracic cancer-induced MPE (including breast and colorectal cancer, 13 patients). The remaining 34 patients (22 males and 12 females; median age, 67.59 years) exhibited transudate (11 patients) or exudate-induced BPE (23 patients). The causes of pleural effusion are presented in Table I.
Patients with BPE were divided into transudate and exudate groups, while patients with MPE were divided into lung and extrathoracic cancer groups. The levels of LDH, glucose and protein and the number of total cells, neutrophils, lymphocytes and monocytes among four groups (transudate, exudate, lung cancer and extrathoracic groups) are presented in Table II. A significant difference was demonstrated in protein concentration and lymphocyte number among the four groups (P=0.0001 and P=0.040, respectively). However, protein concentration was the only factor for which a significant difference between the BPE and MPE groups was demonstrated (P=0.007). No significant difference was observed between the level of LDH, glucose, count of total cell, neutrophil, lymphocytes and monocytes between the entire BPE and entire MPE groups (P=0.310, 0.117, 0.699, 0.840, 0.589 and 0.333, respectively). This result suggested that protein concentration but not lymphocyte number, may serve as a predictor for distinguishing between BPE and MPE.
The concentration of cytokines was analyzed using a CBA Flex Set kit (Table III). The concentration of TNF-α (P=0.035), VEGF (P=0.002) and IFNG (P=0.020) differed significantly across the four groups. The highest concentration of IFNG was detected in the exudate group and the highest concentration of TNF-α was detected in the extrathoracic cancer group. However, neither IFNG nor TNF-α distinguished BPE and MPE; there was no significant difference between the BPE and MPE groups in the concentration of TGFB1 (P=0.865), TNF-α (P=0.589), CSF-2 (P=0.814), IFNG (P=0.321), IL-1B (P=0.594), IL-4 (P=0.783), IL-5 (P=0.449), IL-6 (P=0.568), IL-8 (P=0.530), IL-10 (P=0.827), IL-12 (P=0.371) and MCP-1 (P=0.489). The results of the present study revealed that VEGF concentration in MPE was increased compared with that in BPE (P=0.001). Therefore, the present study suggests that VEGF may potentially distinguish MPE and BPE.
The ROC curve of protein and VEGF concentration was used to generate the optimal cutoff point for MPE and BPE. The protein concentration cutoff point [area under the curve (AUC): 0.708] was 3.35 g/dl and the VEGF cutoff point (AUC: 0.728) was 214 pg/ml for predicting MPE (Fig. 1). In accordance with the cutoff value of VEGF and protein, the sensitivity, specificity and accuracy of VEGF (>214 pg/ml; sensitivity, 70.6%; specificity, 82.4%; accuracy, 76.0%), protein (>3.35 g/dl; sensitivity, 75.6%; specificity, 70.6%; accuracy, 73.3%), and VEGF and protein (VEGF, >214 pg/ml; protein, >3.35 g/dl; sensitivity, 92.6%; specificity, 61.7%; accuracy, 78.6%) were presented in Table IV. However, the concentration of VEGF and protein was not associated with a poor survival rate (Fig. 2).
The clinical and biochemical features of pleural effusion, including the level of procalcitonin, adenosine deaminase, C-reactive protein, carcinoembryonic antigen, and LDH have been demonstrated to represent diagnostic markers in differentiating MPE from tuberculosis pleural effusion (19,20). However, the present study demonstrated no significant difference in the level of LDH between BPE and MPE groups. BPE samples were collected in the present study from patients with different diseases, including 8 patients with tuberculosis, and this may have decreased the sensitivity of LDH. A previous study suggested that the ratio of serum LDH to adenosine deaminase in pleural fluid enhanced the sensitivity and specificity for identifying MPE (21), a result that requires further study. Furthermore, protein, glucose concentration, total cell, neutrophil, monocyte and lymphocyte counts represent MPE-associated features (22,23). However, protein concentration was the only parameter to reveal a significant difference between BPE and MPE groups in the present study. Therefore, protein concentration may potentially serve to distinguish between MPE and BPE.
Although numerous types of cytokine may be detected in BPE and MPE, the pattern of cytokines may not differentiate MPE and BPE (24,25). The present study demonstrated no significant difference in the concentration of cytokines between the MPE and BPE groups, except for TNF-α, IFNG and VEGF. However, while increased concentrations of TNF-α and IFNG were observed in the extrathoracic cancer and exudates groups, respectively, there was no significant difference between MPE and BPE overall. VEGF was used as a biomarker in the present study (optimal cutoff value=214 pg/ml). Duysinx et al (26) suggested that the optimal value of VEGF for differentiating MPE from BPE is 382 pg/ml and Fiorelli et al (27) demonstrated that sensitivity and specificity were 63 and 83%, respectively, when VEGF is >652 pg/ml. These cutoff values may differ from that of the present study due to the use of different experimental designs and sample sizes.
VEGF induces vascular permeability and is a critical mediator of pleural effusion formation (28), suggesting that blocking VEGF potentially represents a strategy for suppressing the formation of pleural effusion (29). A previous study demonstrated that the level of VEGF in pleural effusion was associated with lymph node and distant metastasis and that the IL-8 level in pleural effusion was associated with lymph node metastasis (30). Due to the limitation of a small sample size, patients with MPE were not divided into patients with primary and metastatic tumors in the present study. The association between VEGF and IL-8 and metastasis as described by this previous study was not observed in MPE samples in the present study.
The combination of VEGF and protein for differentiating BPE and MPE increased the sensitivity and accuracy but decreased the specificity compared with using a single parameter. In addition, a poor survival rate was not significantly associated with VEGF, protein or the combination of the two. To the best of our knowledge, the present study is the first to use a combination of pleural effusion VEGF and protein levels to predict whether pleural effusion from patients was malignant. To conclude, this novel combination may represent a tool for predicting MPE and facilitating early diagnosis, but not for predicting the survival rate of patients with MPE and BPE.
The present study was supported by the Ministry of Science and Technology (grant no. MOST 104-2320-B-037-014-MY3) and the Kaohsiung Medical University Hospital Research Foundation (grant nos. KMUH104-4R08 and KMUH101-1M65).