*3.2. Expression Levels of Biomarkers in Malignant Pleural Mesothelioma and Benign Pleural E*ff*usion*

β In order to describe the diagnostic power of pleural effusion derived Angiopoietin-1, HGF, MMP-7, Osteopontin, TIMP-1, Galectin, Mesothelin, NRG1-b1, Syndecan-1 and VEGF, we compared the expression level of these 10 angiogenesis related biomarkers in pleural effusion from malignant pleural mesothelioma patients (*n* = 42) and benign samples (*n* = 40). The expression levels of Galectin-1, Mesothelin, Osteopontin, shed SDC-1, VEGF, MMP-7, HGF and TIMP-1 were significantly higher, and NRG1-β1 was significantly lower in malignant mesothelioma effusions, compared with the benign effusions (Figure 2). Expression level of these 10 angiogenesis related biomarkers in pleural effusion from metastatic lung adenocarcinoma patients (*n* = 38) showed in Supplementary Figure S1. Differences

between malignant mesothelioma cases and benign cases were statistically significant for these nine biomarkers (*p* < 0.05).

**Figure 2.** *Cont*.

**Figure 2.** Biomarker levels in pleural effusion of malignant pleural mesothelioma (MPM) patients compared to benign effusions (BE). (**A**–**H**) Levels of Galectin-1, Mesothelin, Osteopontin, shed SDC-1, VEGF, MMP-7, HGF and TIMP-1 are significantly higher in pleural effusion from malignant pleural mesothelioma (MPM) patients (*n* = 42) comparing with benign (BE) patients (*n* = 40). (**I**) Level of NRG1-β1 is significantly lower Significance was assessed by two-tailed *t*-test at *p* ≤ 0.05.

### β ≤ *3.3. Diagnostic Biomarkers for Distinguishing Malignant Mesothelioma from Metastatic Adenocarcinoma*

In order to distinguishing malignant mesothelioma from other adenocarcinoma, the expression levels of these 10 angiogenic related biomarkers in pleural effusion were compared between malignant pleural mesothelioma patients and metastatic adenocarcinomas. Of these, expression level of Mesothelin and Galectin-1 were significantly higher in malignant mesothelioma effusions, compared to adenocarcinoma effusions whereas the expression level of shed Syndecan-1 and MMP-7 were significantly lower in malignant mesothelioma effusions compared to the adenocarcinoma effusions (Figure 3). Differences between malignant mesothelioma and adenocarcinoma were statistically significant for these four biomarkers (*p* < 0.05). β ≤

Stepwise logistic regression based on biomarker levels showed that MMP-7, Mesothelin and Osteopontin are three variables with a higher predictive value for distinguishing malignant mesothelioma from metastatic adenocarcinoma (Table 1).

**Table 1.** Parameter estimates of logistic regression model. MMP-7 has higher predictive value for distinguishing malignant pleural mesothelioma from adenocarcinoma, based on estimate and *p*-value.


**Figure 3.** *Cont*.

β ≤

**Figure 3.** Different biomarkers for distinguishing malignant mesothelioma from metastatic adenocarcinoma patients. (**A**,**B**) Levels of Galectin-1 and Mesothelin are significantly higher in pleural effusion from malignant pleural mesothelioma (MPM) patients (*n* = 42) compared with adenocarcinoma (AD) patients (*n* = 36). (**C**,**D**) Levels of MMP-7 and shed SDC-1 are significantly lower in malignant pleural mesothelioma patients (*n* = 42) compared with adenocarcinoma patients (*n* = 36). Significance was assessed by two-tailed *t*-test at *p* ≤ 0.05. **˃** − − − − − −
