Patients and pleural fluid collection
Among 112 consecutive patients with exudative pleural effusion who underwent thoracentesis or pleural biopsy between September 2008 and November 2014, 81 were included in this study after excluding 29 who did not provide consent to this study and two who were transferred out after initial evaluation. All 81 patients were clinically suspected of MPE. Patients with MPE had not received any kind of systemic chemotherapy before pleural effusion analysis. Clinical and pathology data, including tumour type, were acquired for all patients, with approval from the Institutional Review Board at Hallym University, and written informed consent was obtained from all patients (application no. 2014-18). Pleural fluid and serum were collected at the same time as a standard procedure for all individuals. Obtained pleural fluid and blood samples were immediately centrifuged at 2000 g for 10 min, and the supernatants were stored at –80oC until assayed.
MPE was primarily diagnosed through observation of the malignant cells using either cytologic analysis of the pleural fluid or histologic examination of the pleural tissue . Because pleural fluid cytological examination has a variable yield (range 62–90%) , the following criteria were also used to diagnose MPE: 1) confirmed histology obtained from the origin of malignancy; and 2) a clinical course compatible with MPE (treatment modality and survival time).
BPE was diagnosed when the following criteria were satisfied: 1) no evidence of MPE; and 2) a clinical course compatible with BPE for a six-month follow-up period at minimum. Among the BPE patients, pleural TB was diagnosed based on the following criteria: 1) a positive acid-fast bacilli smear, growth of Mycobacterium tuberculosis in culture, or detection of Mycobacterium tuberculosis by polymerase chain reaction, using pleural fluid as the source specimen; 2) a pleural biopsy revealing granuloma, with or without caseous necrosis; 3) a positive sputum culture for TB with improvement of the pleural effusion after anti-TB treatment; or 4) a lymphocytic exudate with adenosine deaminase ≥40 IU/L and improvement of the pleural effusion [14, 15]. Diagnosis of parapneumonic effusion was based on the evidence of an infection (a fever, an elevated white blood cell count, and an elevated serum level of C-reactive protein) as well as a compatible clinical course, which was assessed by the attending physicians.
Analysis of Pro-Cathepsin D
For analysis, 96-well microtiter plates were coated by applying 100 ul/well of anti-cathepsin D monoclonal antibody clone 6410, Abcam, Cambridge, UK) at 5 ug/ml in 100 mM sodium carbonate, pH 9.6 incubated overnight at room temperature (RT). Plates were washed with PBS and blocked with 2% BSA and 10% lactose in PBS prior to use. Next, 100 ul of standard or sample diluted in PBS with 4% BSA or in PBS with 4% BSA and 0.7% NP40 was added to each well and incubated overnight at RT. Plates were washed 6 times with wash buffer (10 mM phosphate, pH 7.5, 150 mM NaCl, 0.05% Tween-20), and 100 ul of anti-pro-cathepsin D rabbit polyclonal detector antibody (4 ug/ml) was added and incubated for 1 hr at RT. Plates were washed 6 times as before, followed by addition of 100 ul of goat anti-rabbit HRP conjugate (KPL) at 0.25 ug/ml. After 30 min at RT, the plates were again washed 6 times, and 100 ul of O-phenylenediamine substrate (Dako, 1 mg/ml in 100 mM citrate buffer, 0.03% hydrogen peroxide) was added. Development proceeded for 1 hr at RT in the dark and was stopped by addition of 100 ul of 4N N2SO4. Absorbance was measured at 490 nm using a Biotek EL 309 autoreader.
The data are presented as median and IQR (interquartile range) for continuous variables, and as numbers and percentages for categorical variables. Data were compared using the Mann–Whitney U test for continuous variables and Pearson’s chi-square test or Fisher’s exact test for categorical variables. Spearman's test was used to assess correlations between variables. To determine the accuracy of plasma and pleural fluid pro-cathepsin D in discriminating MPE from BPE, the sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), positive likelihood ratio (LR+), and negative likelihood ratio (LR−) were calculated. The receiver operating characteristic (ROC) curves were analysed to determine the optimal cut-off value, calculated using the highest sum of sensitivity and specificity, and to compare the diagnostic accuracies of pro-cathepsin D. To evaluate the association between pleural fluid pro-cathepsin D and the diagnosis of MPE, both univariable and multivariable logistic regression analyses were performed. We adjusted the age, sex, and pleural fluid glucose, adenosine deaminase, and pro-cathepsin D levels (cases suggested as malignant pleural effusion by the cut-off value of pleural fluid pro-cathepsin D versus those suggested as benign pleural effusion). All tests were two-sided, and a P-value <0.05 was considered significant. Data were analysed using IBM SPSS Statistics version 24 (IBM Corp., Armonk, NY, USA) and STATA (version 16; Stata Corp., College Station, TX, USA).