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(Chest. 2002;121:1624-1627.)
© 2002 American College of Chest Physicians

Vascular Endothelial Growth Factor Expression in Airways of Patients With Lung Cancer*

A Possible Diagnostic Tool of Responsive Angiogenic Status on the Host Side

Yasuhiko Ohta, MD; Naohiro Ohta, MD; Masaya Tamura, MD; Jian Wu, MD; Yoshio Tsunezuka, MD; Makoto Oda, MD and Go Watanabe, MD

* From the Department of Thoracic Surgery (Dr. Y. Ohta), Ishikawa Prefectural Central Hospital; and Department of Thoracic Surgery (Drs. N. Ohta, Tamura, Wu, Tsunezuka, Oda, and Watanabe), Kanazawa University School of Medicine, Kanazawa, Japan.

Correspondence to: Yasuhiko Ohta, MD, Department of Thoracic Surgery, Ishikawa Prefectural Central Hospital, Minami-Shinbo machi nu 153, Kanazawa 920-8530, Japan; e-mail: yohta{at}ipch.kanazawa.ishikawa.jp


   Abstract
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 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Study objective: We evaluated the expression of vascular endothelial growth factor (VEGF) in airways in patients with lung cancer.

Methods: BAL fluid (BALF) and plasma samples were obtained from 41 patients with primary lung carcinomas and 7 patients with noncancerous diseases, and were analyzed for VEGF by an enzyme-linked immunosorbent assay. After standardization with the albumin protein levels, the relative intensity (VEGF index) was determined as the ratio of VEGF expression on the disease side to that on the healthy side.

Results: In all cases, VEGF concentrations in BALF were greater than those in plasma samples. On the healthy side, the mean value of VEGF in BALF was significantly greater in lung cancer patients than in patients with noncancerous diseases (p = 0.0470). While age, gender, location of cancer (right vs left), histology (adenocarcinoma vs squamous cell carcinoma), and T factor (T1/2 vs T3/4) did not affect the VEGF levels in BALF, the VEGF index was inversely associated with distant metastasis and nodal involvement. The VEGF index of patients in stage I was significantly greater than that of patients in stage IV (p = 0.0308).

Conclusions: The VEGF expression in airways is likely to reflect the response to tumor angiogenesis on the host side. Of direct clinical relevance is that the assessment of VEGF concentrations in airways may provide information concerning the dependency of tumor angiogenesis on VEGF, which is variable according to tumor progression.

Key Words: BAL • BAL fluid • lung cancer • vascular endothelial growth factor


   Introduction
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 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Vascular endothelial growth factor (VEGF) is associated with tumor progression through various biological functions, such as immune reactions mediated by the maturation of dendritic cells, migration of tumor cells, malignant transformation and invasion, tumor survivability, and tumor angiogenesis.1 2 3 4 5 6 Although it has been found that the lung alveoli, including macrophages, are rich in VEGF,7 only a limited number of studies have addressed the clinical significance of VEGF expression in airways. In this study, we investigated the clinical significance of VEGF expression in airways by comparing levels of VEGF in BAL fluid (BALF) on healthy and disease sides.


   Materials and Methods
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 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
In total, 41 lung cancer patients and 7 patients with noncancerous diseases (organizing pneumonia [n = 4], pulmonary sequestration [n = 1], bronchial cyst [n = 1], and pulmonary tuberculosis [n = 1]) were studied. The 41 lung cancer patients included 23 men and 18 women (mean ± SE age, 64.0 ± 2.1 years; range, 39 to 86 years; median, 65 years). The pathologic types included 27 adenocarcinomas, 9 squamous cell carcinomas, 2 adenosquamous carcinomas, 1 large cell carcinoma, and 2 small cell carcinomas. According to the TNM classification, 23 patients had tumors in stage I (19 patients with IA and 4 patients with IB), 6 patients had tumors in stage II (1 patient with IIA and 5 patients with IIB), 7 patients had tumors in stage III (4 patients with IIIA and 3 patients with IIIB), and 5 patients had tumors in stage IV. Table 1 shows the basic clinical features of the patients.


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  		Table 1.. Clinical Background Factors of the Lung Cancer Patients

 
BALF and Blood Samples
Informed consent was obtained from all patients for the use of both BALF and blood samples. At the time of bronchoscopy, BALF was collected from both sides, ie, from the lobar bronchus on the disease side and from the corresponding lobar bronchus on the healthy side. After lavage with 20 mL of saline solution, the fluid was collected by syringe suction. The lavage fluid was initially collected from the healthy side in all cases in order to avoid the possible contamination of cancer cells. Venous blood samples were also collected in a vacutainer containing anticoagulant sodium ethylenediamine tetra-acetic acid for plasma before bronchoscopic examination. Both BALF and blood samples were immediately centrifuged at 2,000 revolutions per minute for 10 min, and supernatant aliquots were frozen at - 80°C for later analysis. Cytologic examination of BALF was also performed.

VEGF and Albumin Immunoassays
The BALF and plasma samples were analyzed for VEGF using enzyme-linked immunosorbent assay kits (R&D Systems; Minneapolis, MN). The limit of sensitivity of the assay was 9.0 pg/mL, and the coefficient of variation was < 5.0%. Albumin in BALF was measured on a Cobas Integra (Roche Diagnostics; Basel, Switzerland). The interassay coefficient of variation was < 4%, and the intra-assay coefficient of variation was < 2%. The limit of sensitivity of the assay was 6 µg/mL. Since the retrievable volume of the lavage fluid was different in cases, the VEGF levels in BALF were standardized with the corresponding albumin protein levels; that is, the VEGF expression in BALF was determined as the ratios of VEGF levels (picograms per milliliter) to corresponding albumin levels (milligrams per milliliter). The relative intensity of VEGF expression (VEGF index) was calculated as the ratio of VEGF expression on the disease side to that on the healthy side in each patient.

Statistics
Differences in the intensities of VEGF expression in BALF were analyzed using the Mann-Whitney U test. Spearman rank correlation coefficient test was used to examine the association between different variables. The criterion for statistical significance was p < 0.05. Mean values are shown ± SEs.


   Results
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 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
The results of cytologic examination of BALF were negative in all cases. VEGF concentrations in BALF samples were greater than those in plasma samples. While the mean VEGF concentration in plasma was 38.3 ± 8.3 pg/mL, the mean values in BALF were 469.7 ± 57.6 pg/mL on the disease side and 553.3 ± 67.7 pg/mL on the healthy side. If standardized by albumin, the mean values of VEGF expression on the disease and healthy sides were 17.7 ± 2.3 and 22.5 ± 3.9, respectively, in the patients with lung cancer. However, in the patients with noncancerous diseases, the values were 9.1 ± 6.4 on the disease side and 10.8 ± 8.8 on the healthy side. On the healthy side, the mean value of VEGF expression was significantly greater in patients with lung cancer than in patients with noncancerous diseases (p = 0.0470), but there was no difference on the disease side (Table 2 ). The mean plasma VEGF level was 68.1 ± 12.7 pg/mL in the patients with lung cancer and 30.5 ± 7.9 pg/mL in the patients with noncancerous diseases (p = 0.1374). No significant association was found between BALF and plasma VEGF levels (p = 0.8078 on the cancerous side and p = 0.7934 on the healthy side). In the lung cancer patients, age < 65 years vs >= 65 years, gender, location of cancer (right vs left), histology (adenocarcinoma vs squamous cell carcinoma), and T factor (T1/2 vs T3/4) did not affect the VEGF index in BALF samples (Table 3 ). The VEGF index of patients without distant metastasis was significantly greater than that of patients with distant metastasis (1.06 ± 0.11 vs 0.48 ± 0.12, p = 0.045). Although the difference did not reach statistical significance, the VEGF index in patients without nodal metastasis tended to be greater than that of patients with nodal involvement (1.13 ± 0.13 vs 0.63 ± 0.10, p = 0.0639). According to stage, the VEGF indexes were 1.27 ± 0.16 in stage I (n = 23), 0.62 ± 0.10 in stage II (n = 6), 0.76 ± 0.38 in stage III (n = 7), and 0.48 ± 0.12 in stage IV (n = 5). The VEGF index of patients in stage I was significantly greater than that of patients in stage IV (p = 0.0308).


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  		Table 2.. Mean VEGF Expression Levels in BALF From Patients With Lung Cancer and Noncancerous Diseases

 

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  		Table 3.. Correlation of Clinicopathologic Features and VEGF Index in BALF From Patients With Lung Cancer*

 

   Discussion
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Although high levels of VEGF have been found in BALF,8 9 10 the clinical relevance of such high levels in lung cancer patients has been investigated in only a few studies. Only one previous study8 has shown that VEGF expression levels in BALF were affected by anticancer therapies such as radiation therapy and chemotherapy. In our study, all BALF samples were collected before the initiation of any anticancer therapy.

In a study on prostate tumors, VEGF overexpression in the tumor samples was found to be more frequent in early stages than in advanced stages.11 However, in lung cancer, some researchers have demonstrated that high tumoral VEGF gene expression was associated with advanced stage, and concentrations of VEGF in circulation have also been found to increase according to the stage progression of lung tumors.12 13 We previously reported that VEGF gene and protein expression in cancer cells of lung cancer patients in early stages have a strong prognostic impact and a close association with nodal and bone marrow micrometastases and with recurrence after complete resection of the tumors.14 15 16 17 One point that is not clear is to what degree the angiogenic circumstances on the host side are affected by the tumor angiogenesis. The findings regarding VEGF expression in BALF in the present study appear to have some implications on this question. Firstly, the VEGF concentration in airways on the healthy side was significantly greater in lung cancer patients than in patients with noncancerous disease, suggesting that the potency of angiogenic capacity in the host lung of patients with lung cancer is elevated. Secondly, the expression levels on the disease side showed an inverse relationship with stage or progression of tumors. The VEGF expression level was higher in early stage tumors, particularly stage I tumors, than in tumors of more advanced stages. Patients with distant metastasis had a significantly smaller VEGF index in airways than did patients without distant metastasis. The VEGF expression in the lavage of patients with nodal involvement was also smaller than that of patients without nodal metastasis. In addition, although the value only trends toward significance due to the small sample size, the lungs on the cancerous side showed smaller levels of VEGF expression than did those on the healthy side. These results suggest that the dependency of tumor angiogenesis on VEGF, which is variable during the process of tumor progression, can be determined by evaluation of VEGF concentrations in BALF.

In summary, we assessed the VEGF expression in airways of patients with lung cancer using BALF samples. Compared with its expression levels in circulation, the expression levels of VEGF in BALF were high. In airways on the contralateral healthy side, the VEGF expression levels in lung cancer patients were higher than those with noncancerous diseases. On the disease side, however, the VEGF expression in airways appeared to be inversely associated with tumor progression. If the VEGF expression in BALF represents the responsive status on the host side to tumor angiogenesis, the assessment of VEGF expression in airways may provide information concerning the dependency of tumor angiogenesis on VEGF.


   Footnotes
 
Abbreviations: BALF = BAL fluid; VEGF = vascular endothelial growth factor

Received for publication May 29, 2001. Accepted for publication November 14, 2001.


   References
TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Gabrilovich, DI, Chen, HL, Girgis, KR, et al (1996) Production of vascular endothelial growth factor by human tumors inhibits the functional maturation of dendritic cells. Nat Med 2,1096-1103[CrossRef][ISI][Medline]
  2. Skobe, M, Rockwell, P, Goldstein, N, et al (1997) Holting angiogenesis suppresses carcinoma cell invasion. Nat Med 3,1222-1227[CrossRef][ISI][Medline]
  3. Herold-Mende, C, Steiner, H, Addl, T, et al (1999) Expression and functional significance of vascular endothelial growth factor receptors in human tumor cells. Lab Invest 79,1573-1582[ISI][Medline]
  4. Arbiser, JL, Larsson, H, Claesson-Welsh, L, et al (2000) Overexpression of VEGF 121 in immortalized endothelial cells causes conversion to slowly growing angiosarcoma and high level expression of the VEGF receptors VEGFR-1 and VEGFR-2 in vivo. Am J Pathol 156,1469-1476[Abstract/Free Full Text]
  5. Baek, JH, Jang, J, Kang, C, et al (2000) Hypoxia-induced VEGF enhances tumor survivability via suppression of serum deprivation-induced apoptosis. Oncogene 19,4621-4631[CrossRef][ISI][Medline]
  6. Bruns, CJ, Liu, W, Davis, DW, et al (2000) Vascular endothelial growth factor is an in vivo survival factor for tumor endothelium in a murine model of colorectal carcinoma liver metastases. Cancer 89,488-499[CrossRef][ISI][Medline]
  7. Berse, B, Brown, LF, Water, LVD, et al (1992) Vascular permeability factor (vascular endothelial growth factor) gene is expressed differentially in normal tissues, macrophages, and tumors. Mol Biol Cell 3,221-233[Abstract]
  8. Beinert, T, Binder, D, Oehm, C, et al (1999) Increased levels of vascular endothelial growth factor in bronchoalveolar lavage of patients with bronchial carcinoma effect of tumor activity and oxidative stress due to radio-chemotherapy? Eur J Med Res 4,328-334[Medline]
  9. Demoly, P, Maly, FE, Mautino, G, et al (1999) VEGF levels in asthmatic airways do not correlate with plasma extravasation. Clin Exp Allergy 29,1390-1394[CrossRef][ISI][Medline]
  10. Meyer, KC, Cardoni, A, Xiang, ZZ (2000) Vascular endothelial growth factor in bronchoalveolar lavage from normal subjects and patients with diffuse parenchymal lung disease. J Lab Clin Med 135,332-338[CrossRef][ISI][Medline]
  11. Latil, A, Biéche, I, Pesche, S, et al (2000) VEGF overexpression in clinically localized prostate tumors and neuropilin-1 overexpression in metastatic forms. Int J Cancer 89,167-171[CrossRef][ISI][Medline]
  12. Matsuyama, W, Hashiguchi, T, Mizoguchi, A, et al (2000) Serum levels of vascular endothelial growth factor dependent on the stage progression of lung cancer. Chest 118,948-951[Abstract/Free Full Text]
  13. Yuan, A, Yu, CJ, Chen, WJ, et al (2000) Correlation of total VEGF mRNA and protein expression with histologic type, tumor angiogenesis, patient survival and timing of relapse in non-small-cell lung cancer. Int J Cancer 89,475-483[CrossRef][ISI][Medline]
  14. Ohta, Y, Endo, Y, Tanaka, M, et al (1996) Significance of vascular endothelial growth factor messenger RNA expression in primary lung cancer. Clin Cancer Res 2,1411-1416[Abstract]
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  16. Ohta, Y, Nozawa, H, Tanaka, Y, et al (2000) Increased vascular endothelial growth factor and vascular endothelial growth factor-c and decreased nm23 expression associated with microdissemination in the lymph nodes in stage I non-small cell lung cancer. J Thorac Cardiovasc Surg 119,804-813[Abstract/Free Full Text]
  17. Ohta, Y, Nozaki, Z, Nozawa, H, et al (2001) The predictive value of vascular endothelial growth factor and nm23 for the diagnosis of occult metastasis in non-small cell lung cancer. Jpn J Cancer Res 92,361-366[ISI][Medline]



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