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 Table of Contents  
ORIGINAL ARTICLE
Year : 2018  |  Volume : 3  |  Issue : 2  |  Page : 23-27

Role of Angiopoietin-2 in the control of malignant pleural effusion and survival in patients with primary lung adenocarcinoma


Department of Respiratory and Critical Care Medicine, The First Affiliated Hospital of Nanjing Medical University, Nanjing, Jiangsu, China

Date of Submission22-Dec-2017
Date of Acceptance01-Mar-2018
Date of Web Publication27-Jun-2018

Correspondence Address:
Dr. Wenkui Sun
Department of Respiratory and Critical Care Medicine, The First Affiliated Hospital of Nanjing Medical University, 300 Guangzhou Road, Nanjing 210029, Jiangsu
China
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/ts.ts_25_17

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  Abstract 


Aim: This study aimed to investigate the role of angiopoietin-2 (Ang-2) in the local control of malignant pleural effusion (MPE) and survival in patients with primary lung adenocarcinoma. Methods: Pleural effusion and blood samples were obtained from 85 patients with lung adenocarcinoma and MPE. Enzyme-linked immunosorbent assay was performed to investigate the expression levels of Ang-2 in pleural effusions and serums. SPSS software (Version 18.0) was used to analyze the data. Results: We found that the expression levels of Ang-2 in pleural effusion and serum were 26.82 ± 8.82 pg/mL and 358.78 ± 95.93 pg/mL, respectively. The expression level of Ang-2 in pleural effusion was found to be correlated with the control of pleural effusion. Based on receiver operating characteristic curve analysis, we found that when the cutoff value was 25.57 pg/mL, the efficacy of pleural effusion Ang-2 level in evaluating the control of pleural effusion could get the best result. Both the pleural effusion Ang-2 level and the local control of pleural effusion are independent factors affecting the prognosis of patients with lung cancer and MPE. However, this study also found that serum Ang-2 levels had no significant correlation with MPE local control or prognosis. Conclusion: Pleural effusion Ang-2 levels can be used as a molecular biomarker to predict both the MPE local control and prognosis of the MPE patients, which has significant clinical value.

Keywords: Angiopoietin-2, lung adenocarcinoma, malignant pleural effusion, prognosis, therapeutic efficacy evaluation


How to cite this article:
Li X, Sun W. Role of Angiopoietin-2 in the control of malignant pleural effusion and survival in patients with primary lung adenocarcinoma. Transl Surg 2018;3:23-7

How to cite this URL:
Li X, Sun W. Role of Angiopoietin-2 in the control of malignant pleural effusion and survival in patients with primary lung adenocarcinoma. Transl Surg [serial online] 2018 [cited 2018 Jul 22];3:23-7. Available from: http://www.translsurg.com/text.asp?2018/3/2/23/235392




  Introduction Top


Malignant pleural effusion (MPE) is caused by either the direct or indirect invasion of pleural or pleural primary tumors. MPE is the first clinical manifestation among over 25% of tumor patients, of which lung cancer is the most common one.[1] MPE in patients with lung cancer indicates tumor metastasis, which seriously affects the patient quality of life, and is associated with a poor prognosis (<6-month survival period).[2] At present, little is known about the mechanism of MPE formation. The common causes of MPE formation are lymphatic drainage disorders, pleural metastatic disease, and increased capillary permeability. Palliative treatment of MPE is associated with certain shortcomings, deficiencies, and high recurrence rates.[2],[3] Therefore, a thorough understanding of the mechanism of MPE formation can help to better prevent and control MPE.

Angiopoietin-2 (Ang-2) is a member of the angiogenin family and was first isolated from cell culture supernatant in 1985. By binding to the tyrosine kinase receptor (Tie-2) on the cell membrane of endothelial cells, Ang-2 participates in both the regeneration and function regulation of blood vessels. In malignant disease, Ang-2 regulates vascular stability and promotes neovascularization. Further, the Ang-2 expression level in nonsmall cell lung cancer patients has been shown to be correlated with tumor burden.[4] In addition, significantly increased level of Ang-2 has been observed in patients with exudative pleural effusions.[5] By measuring the Ang-2 level in MPE, this study investigated the role of Ang-2 in therapeutic efficacy evaluation and prognosis of patients.


  Methods Top


General information

A total of 85 inpatients in the First Affiliated Hospital of Nanjing Medical University and Jinling Hospital from January 2013 to December 2014 were identified with adenocarcinoma and MPE. Basic information of patients is shown in [Table 1]. Inclusion criteria were based on the following: lung adenocarcinoma confirmed by histology or cytology during hospitalization; no systemic antitumor therapy (systemic chemotherapy, radiotherapy, or targeted therapy) or intrathoracic local treatment; Eastern Cooperative Oncology Group Performance Status score of 0–2; first-line treatment included at least two cycles of platinum-based two-drug combination chemotherapy; and ability to accept regular follow-up and therapeutic efficacy evaluation. Exclusion criteria were based on the following: combined with other primary tumors; unable to accept follow-up. All patients have signed the informed consent. This study was approved by the Ethics Committee of the Jinling Hospital.
Table 1: Basic clinical features of 85 cases of patients

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Research methods

Pleural effusion and blood samples of patients were collected before pleurodesis and chemotherapy. Collect 100–200 mL pleural effusion after closed thoracic drainage, place that sample in a sterilized centrifuge tube, and draw 3 mL peripheral venous blood in the early morning on an empty stomach. The samples were centrifuged (1000 rpm, 4°C, and 15 min) within 30 min after being collected. The supernatant was aspirated and stored in a sterilized centrifuge tube. It was then marked and stored in a refrigerator (−80°C) to avoid thawing and refreezing repeatedly. Pleural effusion and serum Ang-2 levels were measured with the commercially available Ang-2 enzyme-linked immunosorbent assay systems obtained from R and D Systems (Minneapolis, MN, USA). The assay for each marker was performed according to the manufacturer's instructions. At the same time, clinical data and follow-up data were collected for each patient.

According to the changes of pleural effusion and the WHO standard, we evaluated the objective response rate. Specifically, the evaluation criteria of pleural effusion were defined as follows: Complete remission (CR): effusion disappeared completely for >4 weeks; Partial response (PR): effusion reduced by >50% (compared with that before treatment) for 4 weeks; Stability of disease (SD): effusion reduced by <50% or increased by no >25% (compared with that before treatment); Progression of disease (PD): effusion increased by >25%; and Unknown: no progress of disease was documented.[6] Both CR and PR were considered an effective treatment endpoint. The first evaluation of efficacy was performed after the first treatment course as well as 3 days before the second treatment course. After the second treatment course, therapeutic efficacy evaluation was performed using imaging examination. Therapeutic efficacy evaluation was then performed once every 3 weeks until PD. Follow-ups were performed by telephone or outpatient visit, which ended on June 1, 2016. Progression-free survival (PFS) was defined as the time from diagnosis of MPE to the progression of disease. Overall survival (OS) was defined as the time from diagnosis of MPE to death.

Statistical analysis

Statistical analysis was performed using SPSS 18.0 statistical software (SPSS, Chicago, IL, USA). Differences in the values among the groups were determined by analysis of variance. Correlation analysis of different covariates was used with the Spearman's rank-order correlation. The Kaplan–Meier method was used to estimate PFS distributions. The Cox proportional-hazard regression model was used as a multivariate analysis to assess the prognostic potential of covariates adjusted for the possible confounding effects of all other factors. To find the cutoff value and to evaluate and compare the performance of factors in predicting response to therapy, receiver operating characteristic curves and Youden indices were constructed. A value of P < 0.05 was considered as statistically significant.


  Results Top


General information

According to the criteria for inclusion and exclusion established in this study, 85 patients with lung adenocarcinoma complicated with MPE were enrolled in this study. Basic clinical information of the patients is shown in [Table 1]. We found the expression levels of Ang-2 in pleural effusion and serum are 26.67 ± 8.82 pg/mL and 361.18 ± 97.58 pg/mL, respectively. The expression levels of Ang-2 in pleural effusion and serum have no significant difference in gender, age, smoking status, PS score, or disease stage (P > 0.05). Similarly, the expression levels of Ang-2 in pleural effusion and serum showed no statistically significant correlation (P = 0.453).

The correlation between the Ang-2 expression level and the MPE control of pleural effusion

According to the local control of pleural effusion, patients were divided into two groups as follows: (1) the MPE local control group (CR + PR) and (2) the MPE difficult local control group (SD + PD). Here, there were 59 (69%) patients in the MPE local control group and 26 (31%) patients in the MPE difficult local control group. The expression level of Ang-2 in pleural effusion of the MPE local control group was 23.97 ± 7.58 pg/mL, and that of the MPE difficult local control group was 33.29 ± 8.08 pg/mL. The difference between the two groups was statistically significant (P < 0.01). However, there was no significant difference between two groups of patients in serum Ang-2 expression level, gender, age, smoking status, PS score, or disease stage (P > 0.05).

ROC curve analysis was used to analyze the pleural effusion Ang-2 level and predict the performance of pleural effusion local control. The results showed that the area under the curve was 0.85. When the cutoff value was 25.57 pg/mL, the sensitivity was 90.40% and the specificity was 81% (this was the best prediction performance) [Figure 1]. Logistic regression analysis further confirmed that pleural effusion Ang-2 level may be an independent factor of pleural effusion local control (odds ratio = 5.65; 95% confidence interval [CI], 2.40–16.78; and P < 0.001). Local control of pleural effusion had no statistically significant correlation with serum Ang-2 expression level, gender, age, smoking status, PS score, or disease stage (P > 0.05).
Figure 1: Receiver operating characteristic curve predicting the malignant pleural effusion local control by using pleural effusion angiopoietin-2 level

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The connection between pleural effusion Ang-2 level and prognosis of patients

In this study, the median PFS of the 85 patients with pulmonary adenocarcinoma and MPE was 3 months, and the median OS was 9 months. According to the ROC curve analysis, 25.57 pg/mL was selected as the cutoff value of pleural effusion Ang-2 expression level. The patients were divided into low-level and high-level groups. When performing the univariate survival analysis, we found that the pleural effusion Ang-2 expression level and the pleural effusion local control were correlated with the OS prognosis of patients. The survival time of the low-level group was significantly longer than that of the high-level group (11.82 months vs. 8.54 months, P < 0.001), while the survival time of the pleural effusion local control group was significantly longer than that of the pleural effusion difficult local control group (10.96 months vs. 7.96 months, P < 0.001). The survival curves for MPE Ang-2 cutoff value and local control are shown in [Figure 2].
Figure 2: Kaplan–Meier survival curve of pleural effusion angiopoietin-2 and malignant pleural effusion local control. (a) Patients with a pleural effusion angiopoietin-2 level <25.57 pg/mL survived longer than those who had pleural effusion angiopoietin-2 level ≥ 25.57 pg/mL. (b) Malignant pleural effusion local control group survived longer than the malignant pleural effusion difficult local control group

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Multivariate Cox regression analysis showed that pleural effusion Ang-2 expression level (hazard ratio [HR], 1.15; 95% CI, and 1.01–1.32) and pleural effusion local control (HR, 0.42; 95% CI, and 0.19–0.89) were independent prognostic factors for total survival [Table 2]. No correlations were observed in patient prognosis and serum Ang-2 expression level, gender, age, smoking status, PS score, or disease stage.
Table 2: Multivariate Cox regression analysis of prognosis of patients with lung adenocarcinoma and malignant pleural effusion

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  Discussion Top


MPE is the first symptom of lung cancer, and it can also be a symptom of lung cancer recurrence. MPE patients generally have a short survival time. Previous studies have shown that incidences of pleural effusion in the first diagnosis of lung cancer are 11%–32%.[6] However, even the presence of a very small amount of pleural effusion (<10 mm) may suggest poor prognosis of nonsmall cell lung cancer.[7] The basic treatment of MPE is targeted at the primary disease, such as lung adenocarcinoma. The palliative treatment is usually used to control the pleural effusion and relieve clinical symptoms. However, MPE may persist or relapse. Updated knowledge regarding the biological factors that influence the efficacy and prognosis of MPE would contribute to improved treatment regiments, based on individual clinical features. This study found that the pleural effusion Ang-2 expression level of MPE patients was correlated with pleural effusion local control and prognosis.

The emergence of MPE indicates that tumor cells have broken through the protective mechanism of the pleura. Although the pathophysiological process is still not fully known, it is generally considered to include reduced adhesion, migration of primary tumors, pleural metastases, blood vessels and lymphatic wall adhesion and exudation, the generation of vascular growth factor, and angiogenesis and lymphangiogenesis.[8] In the formation of MPE, tumor angiogenesis and lymphangiogenesis have similar formation processes, and both play important roles in the formation of MPE. In the development of pleural metastases, tumor cells secrete vascular endothelial growth factors (VEGFs), which stimulate neovascularization, tumor-associated lymphangiogenesis, and regional lymph node metastasis. Such newborn vessels have the features of large lumen, thin wall, crisp wall, discontinuous basement membrane, and high permeability. At the same time, tumor cell metastasis occurs through blood vessels and lymphatic vessels, where tumor cells gather and block the lymphatic vessels. This is an important cause of MPE.[9]

Ang-1 and Ang-2 participate in vessel regeneration and function regulation by binding to Tie-2 on the cell membrane of endothelial cells. Tie-2 is widely expressed in the endothelial cell system, and it regulates the function of endothelial cells in recruiting stromal cells. It also stabilizes the tubular structure formed by the monolayer of endothelial cells and promotes vessel regeneration. Ang-1 and Ang-2 play important roles in the development of pleural effusion. Ang-1 has the function of anti-inflammation and anti-exudation.[10] Ang-2 can antagonize Ang-1 and increase the permeability of blood vessels in epithelial cells. Recent studies have confirmed that Ang-2 plays an important role in the occurrence and development of lymphatic vessels by regulating the functional connection of lymphatic endothelial cells. Studies have shown that inhibition of Ang-2 can inhibit tumor angiogenesis and lymphangiogenesis.[11] Ang-2 may affect the generation of MPE, the local control, and survival time by regulating the thoracic local angiogenesis and lymphangiogenesis. At the same time, this study found that the local control of pleural effusion is correlated with patient prognosis. Patients whose pleural effusion can be locally controlled generally have longer survival times than those whose pleural effusion cannot be locally controlled. This may be associated with the heavy tumor burden caused by the uncontrolled tumor pleural metastasis. Currently, more efforts are required to clarify the mechanism of the refractory pleural effusion.

We also found no significant correlation between serum Ang-2 expression level and the survival of MPE patients. It is well known that malignant tumor survival and metastasis rely on angiogenesis. VEGF is the most representative regulation factor of angiogenesis, due to its specific role in promoting endothelial cell proliferation.[12] Recent clinical studies have found that serum VEGF levels in patients with lung cancer increased significantly with the stage of development, which was an independent risk factor affecting prognoses.[13] However, the role of VEGF in promoting endothelial cell proliferation still needs the help of the leading factor, Ang-2. There is a key synergistic connection between VEGF and Ang-2 during the process of angiogenesis. It has been reported that Ang-2 induces the coopted vessels for regression through an apoptotic mechanism. Moreover, VEGF upregulation is consistent with Ang-2 expression at the tumor periphery and correlated with robust angiogenesis.[14]

At first, Ang-2 mediates mature blood vessel to make them weak.[15] Then, VEGF promotes the formation of new blood vessels.[14],[16] During angiogenesis, Ang-2 plays the earliest start-up and stimulation role with its start-up increase, while VEGF plays a subsequent promoting role with its delayed increase.[17] A comparison of nonsmall cell lung cancer patients at different stages revealed that, although serum Ang-2 levels had an increasing trend with the staging progress, there was no statistically significant difference among different stages.[18] Park et al.[19] researched the Ang-2 played a role in the prognosis of patients and performed a univariate survival analysis. The study concluded that the expression level of Ang-2 was correlated with the prognosis of nonsmall cell lung cancer patients. However, after a Cox regression analysis that considered the development stage as a correction factor, it was found that Ang-2 level had no correlation with patient prognosis. This is consistent with the results of our study. Currently, there are a few clinical studies on the expression of Ang-2 and VEGF, in particular, their roles contributing to the prognosis of nonsmall cell lung cancer patients. Large-scale, multicenter studies should be performed to further clarify the clinical significance of early occurrence Ang-2, as well as the involvement in the development and prognosis of nonsmall cell lung cancer.

In conclusion, this study has revealed that the expression level of Ang-2 in pleural effusion is correlated with local control of pleural effusion and prognosis of patients with lung cancer complicated with MPE. The expression levels of Ang-2 in pleural effusion and the local control of pleural effusion are two independent factors, which can affect the prognosis of patients with lung cancer complicated with MPE. No significant correlation was demonstrated between serum Ang-2 expression levels and local control of pleural effusion and prognosis of patients. Currently, researches regarding anti-Ang-2 drugs are being developed.[20] It is worth further studying whether the topical use of anti-Ang-2 drugs in the thoracic cavity of MPE patients can inhibit the growth of MPE more effectively. A deeper understanding of lung cancer complicated with MPE may result in improved treatment options in the future.

Financial support and sponsorship

This study was supported by the National Natural Science Foundation of China (Grant No. 81770009).

Conflicts of interest

There are no conflicts of interest.



 
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