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Ann Thorac Surg 2001;71:1635-1639
© 2001 The Society of Thoracic Surgeons

Original article: general thoracic

Expression of vascular endothelial growth factor in thoracic sarcomas

Akira Iyoda, MDa, Kenzo Hiroshima, MDa, Masayuki Baba, MDb, Takehiko Fujisawa, MDb, Toshikazu Yusa, MDc, Hidemi Ohwada, MDa

a Division of Pathology, Institute of Pulmonary Cancer Research, Chiba University School of Medicine, Chiba, Japan
b Department of Surgery, Institute of Pulmonary Cancer Research, Chiba University School of Medicine, Chiba, Japan
c Department of Thoracic Surgery, Chiba Rosai Hospital, Chiba, Japan

Accepted for publication February 1, 2001.

Address reprint requests to Dr Iyoda, Division of Pathology, Institute of Pulmonary Cancer Research, Chiba University School of Medicine, 1-8-1, Inohana, Chuo-ku, Chiba 260-8670, Japan
e-mail: iyoda{at}haibyo1.m.chiba-u.ac.jp


   Abstract
Top
 Abstract
Introduction
 Material and methods
 Results
 Comment
 Acknowledgments
 References
 
Background. A body of data indicates that vascular endothelial growth factor (VEGF) expression by carcinomas is closely related to the prognosis of carcinomas. However, the relationship between VEGF expression and the prognosis of sarcomas is contradictory.

Methods. Tissue from 27 cases of thoracic sarcoma was analyzed immunohistochemically for VEGF expression while tumor vascularity was quantified using an antibody directed against endothelial CD34. The relationship between VEGF expression and the prognosis of patients with sarcomas was then evaluated semiquantitatively.

Results. The microvessel count in sarcomas with strong VEGF expression was significantly higher than that in sarcomas with absent or faint VEGF expression. The disease-free survival rates of sarcomas with strong VEGF expression were significantly lower than those of sarcomas with absent or faint VEGF expression. We found that strong VEGF expression impacted on the disease-free survival in multivariate analyses.

Conclusions. VEGF expression of thoracic sarcomas is directly related to angiogenesis and tumor vascularity, and our findings suggest that strong VEGF expression is an independent prognostic factor in patients with thoracic sarcomas.


   Introduction
Top
 Abstract
 Introduction
Material and methods
 Results
 Comment
 Acknowledgments
 References
 
Tumor growth and metastasis are closely related to angiogenesis. The cytokine vascular endothelial growth factor (VEGF) is a dimeric 45-kDa glycoprotein and acts as an endothelial cell-specific mitogen and vascular permeability factor via the endothelial-specific receptor tyrosine kinase VEGFR-1 (Flt-1) and VEGFR-2 (Flk-1). Four isoforms of VEGF have been described in humans to date and have been designated as VEGF121, VEGF165, VEGF189, and VEGF206 [1–3]. Several lines of evidence suggest that VEGF is a prognostic factor for various malignant tumors. For example, immunohistochemical VEGF expression has been detected in malignant tumors such as renal cell carcinoma [4], gastrointestinal tract tumors [5], glioma [6], and lung cancer [7]. These studies suggest that VEGF plays an important role in the process of tumor angiogenesis. However, at the present time, the relationship between VEGF expression and the prognosis in soft tissue sarcomas is contradictory [8–10]. However, it should be noted that there are few reports documenting VEGF expression in soft tissue sarcomas because soft tissue sarcomas are uncommon tumors. In this study, we have sought to examine the relationship between VEGF expression, tumor vascularity, and the prognosis of soft tissue sarcomas using a semiquantitative analysis.


   Material and methods
Top
 Abstract
 Introduction
 Material and methods
Results
 Comment
 Acknowledgments
 References
 
Tissue samples
Twenty-seven patients with a diagnosis of primary thoracic sarcoma were admitted into this study. They had received a complete surgical resection either at the Institute of Pulmonary Cancer Research, Chiba University School of Medicine or at the Chiba Rosai Hospital between 1970 and Mar 2000. Patients with incomplete tumor resection were excluded from the study. The morphological classification of sarcomas was conducted according to the Enzinger and Weiss classification with immunohistochemical analysis [11]. The patients comprised 18 men and 9 women, with a mean age of 44 years (range 3 to 75 years). The histological variants of tumor were eight malignant fibrous histiocytoma, three malignant schwannoma, three leiomyosarcoma, two liposarcoma, two fibrosarcoma, two undifferentiated sarcoma, and seven others. The sites of sarcomas were lung (13 cases), chest wall (9 cases), and mediastinum (5 cases). We classified primary sarcomas based on the histological grade of malignancy and the size of the primary tumor according to the Union Internationale Contre le Cancer (UICC) classification [12]. We used the histological grading system of Coindre and associates [13].

Immunohistochemical staining
Tumor VEGF expression was determined by immunostaining VEGF protein using rabbit polyclonal anti-VEGF antibody (Santa Cruz Biotechnology, Santa Cruz, CA) at 1:400 dilution. The microvasculature of the tumor was identified by immunostaining endothelial cells with a murine monoclonal antibody directed against CD34 (Immunotech, Marseille, France) at 1:10 dilution. Four-micrometer sections from formalin-fixed, paraffin-embedded tissue were used. Endogenous peroxidase was blocked with 3% hydrogen peroxidase in phosphate-buffered saline (0.01 mol/L sodium phosphate buffer, pH 7.2, 0.15 mol/L NaCl), after which the specimens were incubated with normal goat serum (VEGF) or normal rabbit serum (CD34) to block nonspecific binding sites. Sections were then incubated overnight at 4°C with the primary antibody. After washing, slides were incubated with biotinylated anti-rabbit (VEGF) or anti-mouse (CD34) IgG for 10 minutes at room temperature. After washing, peroxidated streptavidin was added for 5 minutes and the peroxidase activity subsequently visualized with 3,3'-diaminobenzidine. Hematoxylin was used as a counterstain.

VEGF expression and tumor vascularity were analyzed semiquantitatively [14]. The number of VEGF-positive tumor cells was calculated using the Cell Analysis System (Becton Dickinson & Co, San Jose, CA) and was graded as follows: absent (no positive cells), faint (< 10% positive cells), moderate (10% to 50% positive cells), and strong (>50% positive cells). The density of tumor microvessels was determined as described by Weidner and associates [15]. Individual microvessel counts were performed on 10 high-power fields at a magnification of 400x (0.1885 mm2). The mean count was used as the final score.

Statistical analysis
Fisher’s exact test was used to compare binomial proportions. The {chi}2 test was used to compare the difference in the proportion of VEGF-positive tumors between each grade. An unpaired t test was used to determine whether or not there was a significant difference in microvessel density between VEGF-positive tumors and VEGF-negative tumors. The overall survival and disease-free survival were determined from the date of surgery until the date of death or the date of first local or distant recurrence, respectively, and were evaluated using the method of Kaplan and Meier. The curves obtained were compared by the log-rank test. The prognostic impact of the following variables was investigated using Cox’s proportional hazards multivariable regression model: VEGF expression (absent or faint vs moderate vs strong), gender, age, tumor site (lung vs chest wall vs mediastinum), tumor size, grade (low grade [grade 1] vs high grade [grade 2 or 3]), mitotic rate, and adjuvant therapy (performed vs not performed). Multivariate analyses was performed to factors in which univariate analyses showed that p values were less than 0.2. A p value of less than 0.05 was considered to be statistically significant.


   Results
Top
 Abstract
 Introduction
 Material and methods
 Results
Comment
 Acknowledgments
 References
 
VEGF expression
Tumor cells with a predominantly cytoplasmic pattern of VEGF immunoreactivity are illustrated in Figure 1. The results of VEGF expression were four absent, three faint, six moderate, and 14 strong. Of grade 1 sarcomas, the results of VEGF expression were two absent (15.4%), three faint (23.1%), four moderate (30.8%), and four strong (30.8%). Of grade 2 sarcomas, the results of VEGF expression were one absent (12.5%), two moderate (25.0%), and five strong (62.5%). Of grade 3 sarcomas, the results of VEGF expression were one absent (16.7%) and five strong (83.3%). The degree of strong VEGF expression in grade 3 sarcomas was higher than that in grade 1 sarcomas, although the result did not quite reach significant difference (p = 0.096) (Table 1).



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  		Fig 1. Immunohistochemical staining of an undifferentiated sarcoma with anti-VEGF antibody. The expression of VEGF was mainly detected in the cytoplasm of tumor cells (x120).

 

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  		Table 1. The Relationship Between VEGF Expression and Grading

 
Tumor vascularity
An example of microvessel staining using anti-CD34 antibody is shown in Figure 2 (same case as Fig 1). The mean microvessel count (mean ± SD/mm2) of all sarcomas was 220.3 ± 130.7 (range 37 to 536). The mean microvessel count in sarcomas with strong VEGF expression (276.2 ± 134.7, range 96 to 536) was significantly higher than that in sarcomas with absent or faint VEGF expression (137.9 ± 112.0, range 37 to 313) (p < 0.05, unpaired t test; Table 2). There was no significant difference between moderate VEGF expression and absent or faint VEGF expression, and between moderate VEGF expression and strong VEGF expression.



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  		Fig 2. Immunohistochemical staining of endothelial cells using anti-CD34 antibody (x120).

 

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  		Table 2. The Relationship Between VEGF Expression and Microvessel Density

 
We showed clinicopathological features of thoracic sarcomas in each decade. There was no significant difference in the extent of VEGF expression or microvessel density (Table 3).


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  		Table 3. Clinicopathological Features in Each Decade

 
Survival rates
The 5-year overall survival rates in sarcomas with absent or faint VEGF expression, moderate VEGF expression, and strong VEGF expression were 83.3%, 75.0%, and 15.3%, respectively. The overall survival for patients with strong VEGF expression was considerably lower than that for patients with absent or faint VEGF expression, though it did not reach statistical significance (p = 0.061; Fig 3). The 5-year disease-free survival rates in sarcomas with absent or faint VEGF expression, moderate VEGF expression, and strong VEGF expression were 83.3%, 41.7%, and 13.2%, respectively. The disease-free survival for patients with strong VEGF expression was significantly lower than that for patients with absent or faint VEGF expression (p < 0.05; Fig 4).



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  		Fig 3. Analysis of the overall survival for thoracic sarcomas. The overall survival for sarcomas exhibiting strong VEGF expression was lower than that for sarcomas with absent or faint VEGF expression.

 


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  		Fig 4. Analysis of the disease-free survival for thoracic sarcomas. The disease-free survival for sarcomas exhibiting strong VEGF expression was significantly lower than that for sarcomas with absent or faint VEGF expression (p < 0.05).

 
Multivariate analyses for survival were performed to look for differences in the disease-free survival rates (Table 4). The presence of tumors with strong VEGF expression impacted on the disease-free survival rates in multivariate analyses.


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  		Table 4. Univariate and Multivariate Analyses for Disease-free Survival

 

   Comment
Top
 Abstract
 Introduction
 Material and methods
 Results
 Comment
Acknowledgments
 References
 
VEGF expression by tumors is related to tumor growth and metastasis, and many authors suggest that VEGF is a useful prognostic parameter in malignant tumors.

However, the prognostic value of VEGF in soft tissue sarcomas is contradictory. Kawauchi and associates [8] examined VEGF expression in synovial sarcomas and found that VEGF expression did not have any prognostic value on the overall survival, concluding that angiogenesis did not play an important role in metastasis and the overall survival. However, they did not quantitate VEGF expression by tumor cells. Furthermore, their results are in contrast to other studies [16], which have indicated that angiogenesis is closely linked to the metastatic behavior of tumors. It may be the case that Kawauchi and associates [8] results are restricted to synovial sarcoma alone, but because our study did not include any patients with synovial sarcoma, we are unable to clarify this point further.

Saenz and associates [17] studied soft tissue sarcomas of the extremity and suggested that neovascularity had no prognostic significance in mesenchymal tumors. Their study included patients with a positive microscopic margin of sarcoma tissue and suggested that positive microscopic margin had prognostic significance but neovascularity did not. However, it is apparent that patients with a positive microscopic margin have a poor prognosis and that the relationship between neovascularity and prognosis should be evaluated in patients with complete tumor resection. In addition, they stratified sarcoma patients to groups above and below the median vessel count for the entire group (146 microvessels/field) and compared the proportion of patients free of distant metastasis between the two groups. However, it was unclear from their studies whether or not there were differences between the small microvessel count group (for example, < 50) and the large vessel count group (> 400). Therefore, although the authors stated that there was no correlation between neovascularity and the prognosis, the relationships between very high and low microvessel density were not actually studied in their report.

On the other hand, Kim and associates reported that the growth of rhabdomyosarcoma, glioblastoma, and leiomyosarcoma cell lines injected into nude mice was significantly inhibited by a function blocking monoclonal antibody specific for VEGF [9]. Furthermore, Angelov and associates reported that the growth of neurogenic sarcoma xenografts was reduced by a small molecule inhibitor of VEGF receptor 2 [3]. There have also been reports of elevated serum VEGF levels in the majority of patients with sarcomas [18]. Linder and associates [10] suggested that VEGF levels increased with tumor progression and might therefore be a useful marker for clinical monitoring of sarcoma.

Our results revealed that patients with sarcomas exhibiting strong VEGF expression have lower survival rates than patients with sarcomas exhibiting absent or faint VEGF expression. In particular, the disease-free survival rates were significantly different. In multivariate analysis, strong VEGF expression impacted on the disease-free survival. These data support the proposition that strong VEGF expression by the tumor is a significant prognostic factor of sarcomas. In addition, we found that the overall and disease-free survival rates and microvessel density of patients with moderate VEGF expression were intermediate between patients with strong VEGF expression and patients with absent or faint VEGF expression. This result suggests that the angiogenic, growth-promoting effect of VEGF and consequently the metastatic propensity of the tumor depends upon the quantity of available VEGF protein.

Our findings are limited to thoracic sarcomas because the prognosis of thoracic sarcomas may be different from the prognosis of sarcomas originating at other sites. Potter and associates [19] showed that the 5-year overall survival rates of high-grade soft tissue sarcomas of arm/shoulder, forearm/hand, leg/foot, and thigh/buttock were about 85%, 83%, 80%, and 70%, respectively. Gordon and associates [20] revealed that the 5-year overall survival rate of high-grade soft tissue sarcomas of the chest wall was 49%. These results indicated that it was possible that soft tissue sarcomas of the chest wall had a poorer prognosis than sarcomas of the extremity. However, most reports on VEGF expression of sarcomas contained tumors originating at many kinds of sites because it is very difficult to collect sufficient sarcomas originating from only one site. We deliberately limited our study to thoracic sarcomas and found a significant difference of prognosis and microvessel density depending on the pattern of expression of VEGF. It may be the case that sarcomas originating in organs with both large vessels and hypervascularity, such as lung, are more readily influenced by promoters of angiogenesis such as VEGF, ie, the prognosis of sarcomas may be influenced by the site of origin of the tumor.

In conclusion, strong VEGF expression in thoracic sarcomas is related to angiogenesis and the prognosis of the tumor.


   Acknowledgments
Top
 Abstract
 Introduction
 Material and methods
 Results
 Comment
 Acknowledgments
References
 
The authors thank Ayaka Sato, Michiko Hanazono, and Kazuko Abe for their technical assistance.


   References
Top
 Abstract
 Introduction
 Material and methods
 Results
 Comment
 Acknowledgments
 References
 

  1. Dvorak H.F., Brown L.F., Detmar M., Dvorak A.M. Vascular permeability factor/vascular endothelial growth factor, microvascular hyperpermeability, and angiogenesis. Am J Pathol 1995;146:1029-1039.[Abstract]
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Related Article

Invited commentary
David R. Jones
Ann. Thorac. Surg. 2001 71: 1639. [Extract] [Full Text] [PDF]




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