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Interactive Cardiovascular and Thoracic Surgery 2:201-205(2003)
© 2003 European Association of Cardio-Thoracic Surgery

Negative results - Thoracic general

Lack of prognostic significance of tumor angiogenesis in resected pN2 non-small cell lung cancer

Masaki Tomita*, Yasunori Matsuzaki, Masao Edagawa, Tetsuya Shimizu, Masaki Hara and Toshio Onitsuka

Department of Surgery II, Miyazaki Medical College, Kihara 5200, Kiyotake, Miyazaki 889-1692, Japan

* Corresponding author. Tel.: +81-985-85-2291; fax: +81-985-85-5563
mtomita{at}post.miyazaki-med.ac.jp

Received October 24, 2002; received in revised form January 3, 2003; accepted February 26, 2003


   Abstract
Top
 Abstract
1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 Acknowledgements
 References
 
Objective: Despite extensive studies of tumor angiogenesis in non-small cell lung cancer, only a few studies had concentrated on pN2 disease. Methods: Sixty patients with pN2 non-small cell lung cancer who had undergone a complete resection with a systematic mediastinal lymph node dissection were reviewed retrospectively. Immunohistochemical study, using antibodies against factor VIII, was conducted. We compared between 5-year survivors and the others rather than the cumulative survival rate. Results: There were 13 patients (21.7%) survived longer than 5 years after a surgical resection. The microvessel density in survivor and non-survivor group was 22.9±14.8 and 24.3±21.9, respectively. This data indicated that microvessel density might not be related to the ratio of 5-year survivors (). Multivariate analysis also showed that microvessel density was not independent prognostic factor. Conclusions: We failed to find a prognostic significance of tumor angiogenesis in pN2 disease. Although tumor angiogenesis might be important for the development and maintenance, it was not identified as a prognostic factor of pN2 non-small cell lung cancer.

Key Words: pN2 disease; Tumor angiogenesis


   1. Introduction
Top
 Abstract
 1. Introduction
2. Materials and methods
 3. Results
 4. Discussion
 Acknowledgements
 References
 
The prognosis of surgically resected non-small cell lung cancer with pathologic mediastinal nodal involvement (pN2) is poor in general [1]. However, highly selected patients with pN2 disease survived more than 5 years. The pN2 disease is known to be a heterogeneous subcategory, and several authors have suggested some significant prognostic factors among surgically resected pN2 disease [2–10]. However, controversy still remains.

In addition to a variety of clinical characteristics, tumor angiogenesis, the formation of new blood vessels from the existing vascular bed, had been reported to be associated with a worse prognosis in non-small cell lung cancer [11–15]. Despite extensive studies of angiogenesis, to our knowledge, only one study by Angeletti et al. [11] had concentrated on pN2 non-small cell lung cancer. However the median follow-up of their study was relatively short (median 24 months).

In the present study, therefore, we investigated the prognostic significance of tumor angiogenesis in completely resected pN2 non-small cell lung cancer patients, who were followed for longer than 5 years, using comparison between 5-year survivors and the others rather than the cumulative survival rate.


   2. Materials and methods
Top
 Abstract
 1. Introduction
 2. Materials and methods
3. Results
 4. Discussion
 Acknowledgements
 References
 
2.1. Patients

From 1991 to 1996, 374 patients with primary lung cancer underwent surgical intervention at our institute. The pN2 was present when any mediastinal node was histologically involved. There were 84 among 374 overall patients (22.5%) with pN2 disease. The following patients were excluded: (1) 14 patients who had not received complete resection which consisted of either a lobectomy or a pneumonectomy together with the regional lymph nodes dissection; (2) four patients who died of other diseases within 5 years after surgery; and (3) six patients who were lost to follow-up within 5 years. Because of these reasons, remaining 60 patients with pN2 disease were examined in this study. There were 42 men and 18 women. The ages ranged from 19 to 80 years, with an average of 63.9 years. Preoperative mediastinoscopic examination and induction therapy were not performed during this period, even in patients with clinical N2 (cN2) disease. Tumor-node-metastsis (TNM) staging was recorded for all patients. All surgically resected tumors and lymph nodes were formalin-fixed and paraffin-embedded and routine pathologic studies were performed. When no involved hilar lymph nodes were observed but the mediastinal lymph nodes were involved by cancer, the tumor was regarded as having skip metastasis [16,17]. All patients were followed for longer than 5 years. The follow-up information, including cause of death, was acquired through clinic follow-up notes and direct or family contact. Patients were subdivided into two groups: survivor group, patients surviving more than 5 years; non-survivor group, other patients.

2.2. Immunohistochemical studies

To highlight the microvessels, we used a specific monoclonal antibody against factor VIII (Dako Diagnostika, Hamburg, Germany). Before staining, serial 4 µm-thick sections were pre-treated with microwave heating in 10 mM citric acid buffer for 4x5 min. These sections were immersed in 0.6% H2O2 in methanol for 20 min at room temperature to block endogenous peroxidase activity. After blocking non-specific protein bindings by an overnight incubation with Block Ace (Dainippon, Inc., Osaka, Japan), the sections were incubated with primary antibodies against human factor VIII (1:200) at 4°C overnight. Subsequently, sections were incubated with the secondary antiserum (1:500) for 1 h, followed by an incubation with peroxidase antiperoxidase complex for 30 min at room temperature. The sections were visualized using a Diaminobenzidine/Metal Concentration (10x) and Stable Peroxide Substrate Buffer (1x) system (Pierce, Rockford, IL, USA). The sections were then washed with water and counterstained with hematoxylin. Immunohistological results were assessed semiquantitatively by two authors. Microvessel density (MVD) was determined in the area of the most intense vascularization (hot spot) of each tumor, and the average count was recorded [18].

2.3. Statistical analysis

A chi-square test was used to compare factors examined between 5-year survivors and the others. The data of MVD was analyzed using the Mann–Whitney U-test. Factors related to prognosis were analyzed by multivariate analyzes according to the Cox proportional hazards model. Differences were considered significant when the p value was less than 0.05.


   3. Results
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
4. Discussion
 Acknowledgements
 References
 
3.1. Clinicopathologic factors

The 5-year survival rates were 21.7% and 13 patients have survived longer than 5 years. As shown in Table 1, gender, age, side of operated on, location of the tumor, surgical procedure, adjuvant therapy and cN0 disease were not different between these groups. In contrast, survivor group was characterized by significantly higher proportions of T1 disease (), squamous cell histology (), skip metastasis without hilar node metastasis () and metastasis of a single mediastinal station () in comparison to the non-survivor group (Table 1).


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  		Table 1 Clinical characteristics

 
3.2. Immunohistochemical results

At a magnification of 200x, microvessels appeared as brown linear fragments; occasionally a nucleus was visible. Tiny lumens within these microvessels often resembled small circular or fusiform structures (Fig. 1). Microvessels were present in the tumor parenchyma in addition to those observed at the margin. The MVD (per x200 field) in survivor and non-survivor group was 22.9±14.8 and 24.3±21.9, respectively. As shown in Fig. 2, this data demonstrates no difference in MVD between survivor and non-survivor group (). Since this result was contrary to our expectations, the survival rates according to MVD (MVD<20 versus >20) were also assessed by Kaplan–Meier method. The 5-year survival rate of the patients with MVD<20 and >20 were 28.4 and 16.8%, respectively. There was also no significant difference in patients’ survival (). This result was also found in patients with adenocarcinoma () as well as in patients with squamous cell carcinoma ().



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  		Fig. 1 Immunohistochemical staining of microvessels. Magnification x100.

 


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  		Fig. 2 Microvessel density between 5-year survivor and non-survivor group ().

 
Variables found as prognostic factors in Table 1 and immunohistochemical results were entered into the Cox proportional hazards model by a forward stepwise procedure. Our result of multivariate analysis showed that single N2 station () had independent prognostic importance (Table 2). The other variables, including MVD, were not independent.


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  		Table 2 Multivariate analysis

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
Acknowledgements
 References
 
Many investigators reported the prognosis of pN2 non-small cell lung cancer patients using a calculated cumulative survival rate. However, the cumulative survival rate was occasionally confounded due patients with short follow-up period. In the present study, non-small cell lung cancer patients diagnosed after 1997 were omitted, and follow-up period of all patients was more than 5 years. We investigated prognostic significance of MVD in pN2 non-small cell lung cancer using the actual number of survivors.

In our results, we failed to find prognostic significance of tumor angiogenesis in pN2 disease. Several studies have associated tumor angiogenesis with a poor prognosis in non-small cell lung cancer [11–15], but not others [19–22]. Despite extensive studies for angiogenesis, only a few studies had concentrated on pN2 non-small cell lung cancer [11]. Before this investigation, we hypothesized that tumor angiogenesis might be a prognostic factor of pN2 non-small cell lung cancer. However our results showed that the ratio of 5-year survivors was not related to tumor angiogenesis. It was demonstrated that the prognostic value of MVD in non-small cell lung cancer was strictly associated to nodal status [23]. Pastorino et al. [20] also showed that MVD did not represent an independent prognostic factor in stage I (pN0) non-small cell ling cancer. Decaussin et al. [22] also failed to demonstrate a prognostic significance of tumor angiogenesis, and they suggested that MVD is more strongly correlated with TNM stage than predictive of survival at any given stage. Thus it might be suggested that tumor angiogenesis is important and related to patients’ survival but not an independent prognostic factor in patients with identical nodal status. Furthermore most of previous studies examined patients with resectable early stage or all stage (I–III), while we investigated pN2 disease (late stage). Therefore it is also suggested that tumor angiogenesis might be related to patients’ survival in early stage of the disease but not in late stage. In the present study, we used an antibody against factor VIII (von Willebrand factor). In addition to factor VIII, immunohistochemical detection of MVD has been also performed using antibodies against CD31 (platelet/endothelial cell adhesion molecule) and CD34 (progenitor cell antigen). Yano et al. [24] reported that staining for CD34 significantly correlated with survival in lung adenocarcinoma but staining for factor VIII did not. Duarte et al. [25] demonstrated that factor VIII staining was a significant prognostic predictor in non-small cell lung cancer, but CD31 staining was not. Therefore, the possibility remains that there might be another different findings when we use other antibodies.

This study is a retrospective study and the number of patients is relatively small. However we believe that the result in this study is interesting. From our result, there is a possibility that anti-angiogenic agents may not be a treatment for patients with pN2 non-small cell lung cancer. The role of tumor angiogenesis remains unclear, mainly because the process of tumor angiogenesis itself is still under examination. Further study is warranted.

In conclusion, we failed to find prognostic significance of tumor angiogenesis in pN2 disease. Although tumor angiogenesis might be important for tumor development and maintenance, it was not identified as a prognostic factor of pN2 non-small cell lung cancer.


   Acknowledgements
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 Acknowledgements
References
 
The authors wish to thank Mrs Yasuko Tobayashi for her skillful technical assistance.

doi:10.1016/S1569-9293(03)00051-3


   References
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 Acknowledgements
 References
 

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