Endostatin Levels in Exudative Pleural Effusions
- Cite this article as:
- Sumi, M., Kagohashi, K., Satoh, H. et al. Lung (2003) 181: 329. doi:10.1007/s00408-003-1035-9
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Endostatin is an angiogenesis inhibitor that is an endogenously produced proteolytic fragment of type XVIII collagen. Although serum levels of endostatin have extensively been studied in patients with malignant diseases, endostatin in pleural effusion has not been fully evaluated. In order to determine whether endostatin is present in pleural effusion, and to determine whether endostatin levels vary in pleural effusion of different etiology, we measured levels of endostatin in 38 malignant pleural effusion due to lung cancer patients and 29 patients with non-malignant disease using an ELISA kit. Free form of endostaitn was measurable (>11.2 pg/ml) in 26 of 38 malignant and 13 of 29 non-malignant pleural effusion. Endostatin levels in the 38 malignant pleural effusion were significantly higher than those in patients with the 29 patients with non-malignant diseases (p = 0.0131). However, there was not statistically significant difference between the patients with pleuropneumonia and those with tuberculous pleurisy (p = 0.2194). In malignant pleural effusion due to lung cancer, the pleural effusion endostatin levels did not differ when the histological types of lung cancer were considered(p = 0.0674). Endostatin was present in both malignant and non-malignant pleural effusion, and elevated levels of endostatin were observed in malignant pleural effusion. Although the mechanisms are unclear, elevated levels of endostatin in pleural effusion may represent the local productions of endostatin in pleural space.
KeywordsPleural effusionEndostatinLung cancerTuberculosisPleuropneumonia
The formation of pleural effusion is a common problem in patients with advanced lung cancer. The development of malignant effusions is usually a negative prognostic symptom, and is associated with poor quality of life [2, 8]. Several factors that contribute to the formation of pleural effusion have been identified. However, our current understanding of the basic mechanisms by which effusion accumulates within the pleural space is poor. Infiltration of tumor cells into the pleural space may result in production of large amounts of pleural effusion. Additionally, angiogenic factors excreted by the infiltrated tumor cells play an important role in the development of pleural effusion.
Recently, serum levels of endostatin have been extensively investigated to correlate to disease extent or prognosis in patients with malignant diseases, and elevated serum endostatin levels have been observed in patients with cancers originating from several organs [1, 5, 6, 7, 9, 11, 12, 16]. It is well known that microvascular endothelial cells can produce endostatin , whereas elevated serum levels of endostatin were observed in patients with systemic sclerosis, and activated fibroblasts in the lung are speculated to produce endostatin . Therefore, it is likely that endostatin production might be observed in both malignant and non-malignant pleural effusion. This study was undertaken to determine whether endostatin is present in pleural effusion, and to determine whether endostatin levels vary in pleural effusion of different etiology.
Materials and Methods
Between April 2001 and March 2002, we encountered pleural effusion in 67 patients. Diagnosis of lung cancer was histologically or cytologically proven in 38 cases (23 men and 15 women, aged 35–82 years. In all the patients, cancer cells were found in pleural effusion at cytological evaluation. These patients comprised 29 adenocarcinomas, 7 squamous cell carcinomas, 1 small cell carcinoma (SCLC), and 1 large cell carcinoma. A pleural effusion was considered to be pleuropneumonic when there was an acute febrile illness with purulent sputum, and pulmonary infiltration in the absence of malignancy or other disease causing pleurisy [3, 10, 13]. Tuberculous pleurisy was diagnosed with a positive culture for Mycobacterium tuberculosis or a pleural biopsy specimen showing typical epitheloid cell granuloma, and/or clinical and laboratory data suggestive of tuberculosis and response to specific antituberculous therapy [3, 10, 13]. The patients with benign disease comprised 16 with pleuropneumonias and 13 tuberculous pleurisies (21 men and 8 women, 34–84 years of age). Pleural effusion samples were collected at admission by needle puncture and stored at −20°C after centrifugation until assay.
Concentrations of circulating endostatin were measured in duplicate using a commercial enzyme immunoassay kit (CytElisa Human Endostatin, Cytoimmune; College Park, MD) designed to measure the amount of “free” forms of endostatin in tissue cell culture supernatants and biological fluid samples such as serum and pleural effusion.
Data are shown as box plots with median and upper and lower quartiles, if not otherwise indicated. The Kruskal-Wallis test was used for analysis of differences between more than two groups, and the Mann-Whitney test was used for analysis between two specific groups. p less than 0.05 was considered to be statistically significant.
The general characteristics of the 67 patients studied are outlined in Table 1.
Characteristics of study population
Age (median, range), yr.
Squamous cell carcinoma
Small cell carcinoma
Large cell carcinoma
Free form of endostatin was measurable (>11.2 pg/ml) in 26 of 38 malignant and 13 of 29 non-malignant pleural effusions. In the 38 patients with lung cancer, pleural effusion endostatin levels (median: 23.4 pg/ml; range: 2.2–933.3 pg/ml) were significantly higher than those in the 29 patients with non-malignant diseases (median: 8.5 pg/ml; range: 3.1–47.2 pg/ml) (p = 0.0131, Mann-Whitney U test) (Fig. 1). We observed 8 patients who had endostatin levels in pleural effusion of more than 50 pg/ml. All 8 patients had malignant pleural effusion due to lung cancer (4 with adenocarcinomas, 3 with squamous cell carcinomas, and 1 with large cell carcinoma). The median pleural effusion endostatin levels of 16 patients with pleuropneumonia and 13 patients with tuberculous pleurisy was 17.1 pg/ml (range: 3.1–37.2 pg/ml) and 6.8 pg/ml (range: 3.4–47.2 pg/ml), respectively. There was no statistically significant difference between the patients with pleuropneumonia and those with tuberculous pleurisy (p = 0.2194).
We next evaluated pleural effusion endostatin levels and the histological type of lung cancer. The median (and range) pleural effusion endostatin levels for adenocarcinoma and non-adenocarcinoma were pg/ml 18.3 (4.2–933.3) and 43.9 (2.2–498.9), respectively. No statistical difference was found between these two groups (p = 0.2229, Mann-Whitney U test). The pleural effusion endostatin levels did not differ when the 4 histological types of lung cancer were considered (p = 0.0674, Kruskal-Wallis test).
There have been at least two different types of kits measuring human endostatin. One, ACCUCYTE human endostatin, is a competitive ELISA designed for the measurement of endostatin in biological samples, and the dynamic range in this assay ranges from 2.0 ng/ml to 500 ng/ml. There have been a number of articles referring to this kit [1, 4, 12]. On the other hand, the CytElisa kit is the more sensitive of the two types, which is designed to measure the amount of “free” form of endostatin (sensitivity >11.2 pg/m, dynamic range: 15.6 pg/ml to 4000 pg/ml). In the present study, we measured serum levels of endostatin using the CytElisa kit, and revealed significantly elevated endostatin levels in malignant pleural effusion due to lung cancer compared with non-malignant pleural effusions. The levels were variable according to the histology, and the pleural effusion endostatin levels did not differ when the histological type of lung cancer was considered. The present study also demonstrates that there is much overlap between the endostatin levels in the various groups.
Accordingly, endostatin levels are unlikely to be useful diagnostically. The clinical use of endostatin measurement as a diagnostic tool warrants further validation in a prospective clinical trial. From this retrospective patient series, however, it was evident that endostatin levels above 50.0 pg/ml were suggestive of malignancy. Therefore, the elevated levels of endostatin in some of the pleural effusion samples are noteworthy and raise the possibility that the accumulation of pleural effusion, in at least some patients, may be related to endostatin.
The origin of endostatin is unknown and many cells, such as cancer cells and fibroblasts, are potential sources. van Hensbergen et al.  showed that there were higher concentrations of endostatin in intratumoral fluid, which was a fluid phase present in the extracelullar space of a tumor, from various malignant diseases than those with non-malignant disease. Their finding was a support for the local production of endostatin by tumor cells in pleural or peritoneal space rather than the transport from the circulation. We speculated that the origin of endostatin in the pleural effusion might be the cells in the pleural effusion, and that endostatin in pleural effusions of lung cancer patients might be largely derived from cancer cells. In addition, however, it is likely that endothelial cells and fibroblasts, which are activated by cancer cells, are also responsible for endostatin in pleural effusion. These results are in higher levels of endostatin in malignant effusions.
In this study, although the number of patients with pleuropneumonic and tuberculous pleural effusions was small, we also found that endostatin was detectable in non-malignant pleural effusion. Therefore, elevations in pleural endostatin levels were not unique to patients with lung cancer. The mechanisms of elevated pleural effusion endostatin levels in patients with non-malignant diseases might be different from those in patients with lung cancer. However, we believe that endostatin could also participate in the formation of non-malignant pleural effusion. Additional studies are necessary to delineate whether endostatin has an important role in the pathogenesis of pleural effusions in patients with pleuropneumonia or tuberculous pleurisy.
In summary, this study has demonstrated increased levels of endostatin in the majority of malignant pleural effusions due to lung cancer. The specific cells responsible for the release of endostatin in lung cancer patients have to be identified. Additional research is needed to identify the mechanism of endostatin excretion and to define its possible physiological function in pleural effusion development.