Vascular endothelial growth factor in pleural effusions of different origin

Patients

A total of 214 patients (94 females, 120 males, mean±sd age 61±18 yrs) were consecutively entered into the study. The diagnosis of malignancy in patients with lung cancer (CA) or lung secondaries with primary tumours elsewhere in the body (TM) was based on either a pleural fluid cytological examination or a pleural biopsy specimen that was positive for malignancy, or if the patient had known metastatic malignancy with no other explanation for the pleural effusion.

In the 58 CA patients, the histological types were adenocarcinoma (n = 28), nonsmall cell carcinoma of undetermined type (n = 19), small cell carcinoma (n = 6) and squamous cell carcinoma (n = 5). The locations of the primary tumours in the 38 TM patients with secondaries to the lung were the breast (n = 6), stomach (n = 2), rectum (n = 2), colon (n = 2), urethra (n = 2), kidney (n = 1), parotic gland (n = 1), leukaemia (n = 3), melanoma (n = 1), and tumours of unknown primary location (n = 18).

The 45 patients with effusions of infectious origin (INF) were mainly intensive care unit patients who suffered from bacterial pneumonia (n = 24), septicaemia in part with multi-organ failure (n = 14), or empyema (n = 7).

The diagnosis of tuberculosis (TB) was based on histological evidence of granulomatous pleuritis in pleural biopsies, microbiological proof in effusion fluid or sputum, and/or on response to anti-TB therapy.

The diagnosis of the 45 patients with congestive heart failure (CHF) was based on clinical symptoms and signs, radiological evidence of cardiomegaly and congested lungs, a transudative effusion in most cases, the exclusion of other aetiologies and the response to appropriate therapy.

The serum of 20 healthy blood donors served as controls.

Collection of pleural fluid and serum samples

Following institutional guidelines, the study was approved by the ethical committees at the University of Leipzig and Robert Koch Hospital, Leipzig, Germany, and the Haven of Hope Hospital and Prince of Wales Hospital, The Chinese University, Hong Kong, China. Informed consent was obtained from each patient. Thoracocentesis was performed for diagnostic or therapeutic purposes, and pleural effusions and serum samples were collected at the time of first tapping. The pleural fluid was collected in tubes containing EDTA (final concentration of 1 mg·mL⁻¹) for determination of the cellular content and in plain tubes for determination of VEGF, lactate dehydrogenase (LDH) and total protein. The total cell, white cell and differential cell counts (May-Grünwald-Giemsa stain) were determined by counting 200 cells using light microscopy. The cell pellet and supernatant were separated by centrifugation (600×g, 30 min, 4°C). Peripheral venous blood was collected in plain tubes for the determination of VEGF, LDH and total protein. The protein and LDH concentrations were determined in effusion fluid and in serum by standardised automated methods. Light's criteria were calculated to differentiate exudative from transudative effusions 11. Serum and effusion fluids were stored at −70°C for later VEGF analysis.

Laboratory analysis

ELISA for human VEGF was performed on all serum and pleural effusion samples, using the commercially available Quantikine^TM kit (R&D Systems GmbH, Wiesbaden, Germany). ELISA plates were coated with specific mouse monoclonal antibodies directed against VEGF. The lower limit of detection, as stated by the supplier, was 5.0 pg·mL⁻¹. After standards and cell-free samples were pipetted into the wells of the microtitre plates, specific horseradish peroxidase-linked polyclonal antibodies were added and immunoreactive levels of VEGF determined. Values below the detection limit were assumed as zero for statistical analyses.

Statistical analysis

Data are presented means±sd. Data were checked on homogeneity (Levene test). Significant differences were identified by ANOVA, followed by Tuckey or Tamhane tests for homogeneously or nonhomogeneously distributed data sets, respectively. Correlations were determined by Pearson's test. All p-values <0.001 were considered statistically significant. ROC curves and the related areas under the curve were calculated using online-provided data analysis tools from Acomed (Acomed, Leipzig, Germany).

Characteristics of study groups

Table 1⇓ depicts the different aetiologies and characteristics of the study patients. No statistical differences between the diagnostic groups were detected for age and sex, even in the comparatively younger control group. The percentage of smokers in the CA patients was higher in comparison with other groups as expected. No difference for all parameters, however, was observed when the smokers and nonsmokers from each study group were compared.

General characteristics of samples collected

Using Light's criteria, transudates and exudates can be differentiated according to their ratios of serum and effusion protein concentrations and respective LDH concentrations 11. The respective mean ratios of the study groups met these criteria (table 2⇓). However, only 35 out of 45 patients (78%) with CHF effusions met the criteria for a transudate, probably due to the different time period between the appearance of effusion until diagnosis and tapping, or due to preceding diuretic therapy (“pseudo-exudate”). Likewise, patients, although comparatively smaller in number, expected to have exudates in cases of malignancy or infection in fact exhibited criteria for a transudate, most likely due to a component of accompanying heart failure or different time periods between the onset of effusion, diagnosis and tapping (table 2⇓).

The classification of patients in this study was, therefore, primarily based on the clinical diagnosis rather than on Light's criteria alone. Following the current authors' observations, it is believed that the VEGF value in such a patient would be closer to clinical reality and, therefore, patients were not excluded from a diagnostic group when the expected criteria by Light were not met.

VEGF in sera and effusions

No dependency of VEGF concentrations from age, sex or smoking history of the patient and control groups investigated could be observed.

In serum, healthy blood donors exhibited only very low VEGF concentrations and were therefore different from patient groups with CA, TM or INF, but not from those with TB or CHF (table 2⇑). Furthermore, INF caused significantly higher VEGF concentrations in serum in comparison with CHF patients. All other groups were not different from one another.

The highest VEGF concentrations in pleural effusions were found in malignancies allowing differentiation from all other entities, but with no significant differences between CA and TM. Except for TB and CHF effusions, all diagnostic groups could be differentiated from one another with adequate accuracy (table 2⇑).

Adjustment of VEGF concentrations to the protein content of effusions negatively influenced significancy levels between tumours (CA as well as TM) and CHF. This was also true for other comparisons. Therefore, the respective ratios were not calculated further (data not shown).

The calculation of ratios between VEGF levels in effusions and sera confirmed differences between malignancies (CA and TM) in comparison with all other patient groups (TB, INF and CHF; table 2⇑).

Of all the investigated cellular parameters, only leukocytes were significantly different between CA and TB, in comparison with CHF but not total cells, lymphocytes, monocytes, or granulocytes (data not shown). Interestingly, the leukocyte counts in effusions correlated with VEGF concentrations.

Receiver-operating characteristic analysis of VEGF

To investigate the potential of VEGF determination for diagnostic use, ROC curves were calculated for the differential diagnosis of malignancies (CA and TM) causing pleural effusions (fig. 1⇓). Although the specificity could be enhanced up to 95% (at 29% sensitivity level), the best sensitivity of an acceptable 80% level had a specificity of only 58%.

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Fig. 1—

Receiver-operating characteristics (ROC) curve (•) for differential diagnosis of tumours (lung cancer and tumours with secondaries to the lung) by vascular endothelial growth factor in pleural effusions. The fitted ROC curve is represented by (––––). The graph also shows the 90% confidence interval ( - - - -) and the hypothetical ROC curve resulting from homogenous distributed data (……). These data indicate 22, 26 or 55% sensitivity at 95, 90 or 80% specificity, respectively.