(Chest. 2005;128:3382-3387.)
© 2005
American College of Chest Physicians
Prognostic Significance of Vascular Endothelial Growth Factor, Tumor Necrosis, and Mitotic Activity Index in Malignant Pleural Mesothelioma*
Funda Demirag, MD;
Ebru Ünsal, MD;
Aydin Yilmaz, MD and
Atalay Ça
lar
* From the Departments of Pathology (Dr. Demirag) and Chest Diseases (Drs. Ünsal and Yilmaz), Atatürk Chest Diseases and Chest Surgery Education and Research Hospital, Ankara; and Science and Art Faculty (Dr. Çalar), Department of Statistics, Kafkas University, Kars, Turkey.
Correspondence to: Ebru Ünsal, MD, Güleryüz sok. No:16/8 06550 Y.Ayranc
, Ankara, Turkey; e-mail: unsalebru73{at}yahoo.com
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Abstract
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Objectives: Tumor growth and metastasis are angiogenesis-dependent events, and several prognostic factors have been determined in malignant mesothelioma. In this study, we investigated the prognostic significance of vascular endothelial growth factor (VEGF), tumor necrosis (TN), and mitotic activity index (MAI) in malignant mesothelioma. For the first time (to our knowledge), we also demonstrated the association between VEGF staining, TN, and MAI in malignant mesothelioma.
Methods: Decortication and VATS materials of 40 patients were investigated. The routinely processed formalin-fixed, paraffin-embedded, hematoxylin-eosin–stained tumor sections that had been used for the original diagnostic purposes were retrieved. Hematoxylin-eosin sections were selected for VEGF immunostaining, TN, and mitotic count. Clinicopathologic data, stage of disease, and survival of patients were all determined. The correlations between variables were evaluated by Spearman rank correlation test. Both univariate analysis using the log-rank test and multivariate analysis using Cox regression model were performed for survival analysis.
Results: There was a significant correlation between VEGF staining and TN (r = 0.42, p = 0.006). In univariate analysis, both VEGF staining (p = 0.0002) and TN (p = 0.0055) showed significant correlation with short survival. Also, there was a positive correlation between VEGF and tumor stage (p = 0.046). In multivariate analysis, only VEGF was determined as an independent prognostic factor in malignant mesothelioma (p = 0.001). There was no association between MAI and survival (p = 0.504).
Conclusions: VEGF, known as an important angiogenic peptide, is an independent prognostic factor in malignant pleural mesothelioma. TN stimulates angiogenesis, and we observed a significant correlation between VEGF and TN. However, further studies are needed to evaluate the prognostic significance of angiogenic properties in malignant mesothelioma.
Key Words: malignant mesothelioma • mitosis • prognosis • tumor necrosis • vascular endothelial growth factor
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Introduction
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Malignant pleural mesothelioma is an aggressive neoplasm arising from mesothelial cells of the pleura, and it is fatal in most cases. There is a strong association between malignant mesothelioma and asbestos exposure.1 In approximately 80% of malignant mesothelioma cases, an exposure to asbestos is reported.2 Despite advances in chemotherapy or surgery, the mean survival is only approximately 1 year.13
Tumor growth and metastasis are angiogenesis-dependent events; like other tumors, malignant mesothelioma induces a vascular stroma to grow.4 Determination of synthesis and expression of angiogenic factors in tumor tissue shows neovascularization in tumor. There are several factors influencing tumor angiogenesis. Such factors may either stimulate or inhibit growth of the vessels.35 Vascular endothelial growth factor (VEGF) is an important angiogenic peptide secreted by several malignant tumors.6 VEGF expression is associated with poor prognosis in a variety of neoplasms.789 Thus, in malignant mesothelioma, tumor cells synthesize VEGF and its receptors, which contribute to angiogenesis and growth of tumor cells in an autocrine manner.3
Mitotic activity index (MAI) is the most commonly used method of assessing the proliferative activity of a tumor. It is frequently used for classification, grading, and prognosis of solid tumors.101112 MAI has been shown as a prognostic factor in node-negative breast cancer.12 Coagulative necrosis that reflects the level of intratumoral hypoxia is a common feature of solid tumors.13 Hypoxia up-regulates a number of angiogenic factors including VEGF.14 Tumor necrosis (TN) stimulates angiogenesis and correlates with VEGF expression in breast cancer.1415
In this study, we assessed the prognostic significance of VEGF staining, MAI, and TN in malignant pleural mesothelioma specimens and examined the association with tumor stage and histopathologic type. For the first time (to our knowledge), we also demonstrated the association between VEGF staining, TN, and MAI in malignant mesothelioma.
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Materials and Methods
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Patients
Forty patients with a diagnosis of malignant mesothelioma were selected from the archives of the Department of Pathology in Atatürk Chest Diseases and Chest Surgery Education and Research Hospital between 1996 and 2003. Decortication and VATS materials were investigated retrospectively, and diagnostic slides were reviewed by a pathologist to confirm the diagnosis. The routinely processed formalin-fixed, paraffin-embedded, hematoxylin-eosin–stained tumor sections that had been used for the original diagnosis were retrieved. For each case, hematoxylin-eosin-stained sections were reviewed from all the diagnostic tissue blocks, and the sections were selected for immunostaining and mitosis counting. Demographic and clinicopathologic data, stage of the disease, and survival were determined. Survival was calculated from the date of the diagnostic biopsy to the date of death. The tumors were subclassified into epithelioid, sarcomatoid, and biphasic subtypes according to the criteria given by the World Health Organization.16
Assessment of TN and MAI
Sections were inspected at low power (x 40) using light microscopy by an observer blinded to clinicopathologic data and outcome for necrosis. Mitotic figures were counted in areas selected on the basis of the following criteria: (1) presence of good cellularity and fields with no necrosis, inflammation, or calcifications; (2) high density of mitotic figures. Counting was carried out in 10 consecutive fields of 0.196 mm2 (x 400). Only cells with clear morphologic features of metaphase, anaphase, and telophase were counted, avoiding apoptotic and hyperchromatic nuclei. In clinical analysis in which mitotic counts were analyzed as categorized variables, we used the cutoff values of 
9 (score 1), 10 to 19 (score 2), and > 19 (score 3) for mitotic figures per 10 fields (MAI).
Immunohistochemical Staining for VEGF
Immunostaining of VEGF expression was performed using the streptavidin-biotin method with a rabbit polyclonal antibody (Zymed Labs; San Francisco, CA). Sections of 6 µm were cut from formalin-fixed, paraffin-embedded tissue specimens and mounted on ply-l-lysme coated slides—paraffin sections were dewaxed by xylene, rehydrated, and finally washed in phosphate buffer (pH 7.6) for 10 min. VEGF required boiling in 0.01 mol/L citrate buffer for 20 min in a microwave oven, and sections were incubated with primary antibody solution for VEGF at a dilution of 1:150 for 1 h at room temperature. Immunostaining was performed with the streptavidin-biotin complex kit (Dako Corporation; Copenhagen, Denmark). After incubation, the chromogen specimens were counterstained with Harris hematoxylin and cover-slipped. The intensity of VEGF immunostaining was evaluated by light microscopy (Labophot 2; Nikon, Tokyo, Japan). The immunostaining results were classified as VEGF negative (VEGF –), VEGF slight (VEGF +), VEGF moderate (VEGF ++), or VEGF strong (VEGF +++).
Statistical Analysis
Statistical analysis was performed using statistical software (SPSS for Windows, release 10.0; SPSS; Chicago, IL). The correlations among VEGF, MAI, and TN were evaluated by Spearman rank correlation test. Univariate analysis was performed, and the significance of differences in survival between the groups was determined using log-rank test. Survival curves were computed according to the Kaplan-Meier method. To evaluate the independent prognostic relevance of VEGF, MAI, and TN, multivariate analysis using Cox regression model was performed. A value of p < 0.05 was accepted as statistically significant.
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Results
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There were 23 male (57.5%) and 17 female (42.5%) patients (mean age, 58.9 ± 11.6 years [± SD]; range, 33 to 74 years). The mean smoking history of patients was 14 ± 19.3 pack-years (range, 0 to 80 pack-years). The mean asbestos exposure was 28 ± 11.7 years (range, 0 to 40 pack-years). In our study, 30 specimens were classified as epithelioid type and 10 specimens were biphasic type. According to the new international TNM staging system,17 4 patients had stage I, 11 patients had stage II, 12 patients had stage III, and 13 patients had stage IV.
In 32 of 40 tumors (80%), VEGF immunostaining was found (Fig 1 , 2
). VEGF staining was slight in 13 specimens (32.5%), moderate in 12 specimens (30%), and strong in 7 specimens (17.5%). Also, in eight specimens (20%), VEGF staining findings were negative. TN was identified in 10 specimens (25%), and it was absent in 30 specimens (75%). MAI was score I in 29 specimens (72.5%), score II in 9 specimens (22.5%), and score III in 2 specimens (5%), respectively (Table 1
).
In our study, we found a statistically significant correlation between VEGF staining and TN (r = 0.42, p = 0.006). There was no significant correlation between VEGF and MAI (p > 0.05), TN, and MAI (p > 0.05).
Correlation With Histopathologic Type and Tumor Stage
VEGF staining, TN, and MAI did not show any significant correlation with histopathologic type (p > 0.05). There was a significant correlation between VEGF staining and tumor stage (p = 0.046). However, TN and MAI did not show correlation with tumor stage (p > 0.05).
Correlation With Survival
The median survival of patients was determined as 15.3 ± 10.5 months. At the end of the study, 25 patients (62.5%) had died and 15 patients (37.5%) were still alive. VEGF staining showed a significant correlation with short survival (p = 0.0002; Table 2
). Median survival was 24 months (95% confidence interval [CI], 17.5 to 30.6 months) in patients with negative VEGF staining, whereas it was 28 months (95% CI, 9.9 to 46 months) in VEGF (+), 7 months (95% CI, 4.2 to 9.7 months) in VEGF (++), and 5 months (95% CI, 0 to 10 months) in patients having VEGF (+++) staining (Fig 3
). TN showed a significant correlation with poor survival (p = 0.0055). The median survival was 20 months (95% CI, 6.8 to 33.1 month) in TN-absent patients, whereas median survival was 9 months (95% CI, 6.3 to 11.6 months) in patients with TN (Fig 4
). However, there was no association between MAI and survival (p = 0.504; Fig 5
).
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Figure 3.. Survival analysis of patients with malignant mesothelioma based on VEGF staining. There is a significant correlation between VEGF staining and short survival (p = 0.0002).
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Multivariate survival analysis was performed using Cox regression model. Other prognostic factors such as age, sex, histopathology, and stage were all included in the multivariate analysis. We found that women have better survival than men (p = 0.047), and that VEGF staining was an independent prognostic factor (p = 0.001; Table 3
).
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Discussion
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Angiogenesis is involved in a variety of pathologic processes, such as tumor growth, metastasis, and inflammation.6 Tumor growth and metastasis are angiogenesis-dependent events. Tumor growth is known to be dependent on neovascularization, and it may be caused by an impaired balance between angiogenic and antiangiogenic factors.1819 In vitro studies1820 have shown that malignant mesothelioma cells stimulate their growth in autocrine, paracrine fashion by releasing numerous growth factors, including the proangiogenic VEGF. Edwards et al19 assessed microvessel density and found angiogenesis as an independent prognostic factor in malignant mesothelioma. In this study, we assessed the prognostic significance of VEGF, TN, and MAI in malignant mesothelioma. VEGF staining in tumor tissue was determined as an independent prognostic factor in malignant mesothelioma. This may be explained by the key role of VEGF in regulating multiple mechanisms of tumor progression, such as formation of new blood vessels and proliferation of neoplastic cells.5 We also found significant correlation between VEGF staining and tumor stage. VEGF expression is widespread in most tumors and is predictive of more aggressive and metastatic forms and poor prognosis in a variety of neoplasms.1
Malignant mesothelioma, like other tumors, must induce a vascular stroma to grow beyond a minimal size.6 It is known that VEGF acts on endothelial cells to increase microvascular permeability in a variety of tumors. VEGF not only acts on vascular endothelium and microvessel growth but moreover induces tumor growth by an autocrine-acting loop mechanism.36 Also, VEGF secreted by tumor cells stimulates capillary permeability and fluid accumulation. Zebrowski et al21 found VEGF levels significantly higher in pleural effusions of patients with malignant mesothelioma.
TN has been reported as an indicator of poor prognosis in a number of solid tumors, including non-small cell lung cancer (NSCLC), GI system tumors, and breast cancer.142223 Edwards et al13 demonstrated the presence of association between TN and poor prognosis in malignant mesothelioma. In our study, we found TN correlated with poor prognosis. Also, for the first time (to our knowledge), we showed the significant correlation between VEGF staining and TN in malignant mesothelioma. In breast cancer TN has been shown to correlate with increased tumor size, high vascular density, and macrophage infiltration that express VEGF.15 These findings suggest that in rapidly growing tumors, TN stimulates angiogenesis due to the release of angiogenic growth factors from infiltrating macrophages.13
MAI is a well-known method for the assessment of primary tumor proliferation rate. The main advantage of MAI over other prognostic factors is that MAI is both an easy and inexpensive technique.24 In breast cancer, it has been shown as a poor prognostic factor.12 However, Daniels et al24 did not find MAI as a predictor of prognosis in stage IA NSCLC. They suggested that metastatic patterns of NSCLC differ from breast cancer and proliferation markers less valuable for predicting survival in NSCLC. Also, for the first time (to our knowledge), we assessed the prognostic significance of MAI in malignant mesothelioma. However, we did not find MAI to be a useful prognostic factor in malignant mesothelioma. This might be due to the low proliferation rate of tumor in malignant mesothelioma.
In conclusion, we have shown that the angiogenic peptide VEGF is an independent prognostic factor in malignant mesothelioma, We also found that the proliferation marker MAI does not predict prognosis and clinical behavior of tumor. Evaluating VEGF and TN together in tumor tissues might show the degree of tumor growth and invasiveness. Both VEGF and TN might play an important role in angiogenesis and poor prognosis in malignant mesothelioma. In the future, it may be useful to evaluate the degree of angiogenic properties to determine the prognosis of disease, and it may be advantageous to combine standard cytotoxic drugs with an antiangiogenic therapy.
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Acknowledgements
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The authors thank William D. Travis for his support in the revision of the manuscript.
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Footnotes
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Abbreviations: CI = confidence interval; MAI = mitotic activity index; NSCLC = non-small cell lung cancer; TN = tumor necrosis; VEGF = vascular endothelial growth factor; VEGF (–) = vascular endothelial growth factor negative; VEGF (+) = vascular endothelial growth factor slight; VEGF (++) = vascular endothelial growth factor moderate; VEGF (+++) = vascular endothelial growth factor strong
Received for publication December 28, 2004.
Accepted for publication May 5, 2005.
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References
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Abstract
Introduction
