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Tumor Biology |
Department of Thoracic Surgery, Faculty of Medicine, Kyoto University, Kyoto, 606-8507 Japan [F. T., S. I., K. Y., R. M., Y. K., M. L., H. W.], and Department of Thoracic Surgery, Seishin-Iryo Center Hospital, Kobe 651-2273, Japan [Y. O.]
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONPATIENTS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONPATIENTS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Ang-1 (4 , 5) and Ang-2 (6) have been identified as ligands for Tie2, which is a receptor tyrosine kinase specifically expressed on ECs, and Angs play critical roles in angiogenesis in concert with VEGF (2) . Ang-1 binds to Tie2, and maintains and stabilizes mature vessels by promoting interaction between ECs and surrounding extracellular matrix. Ang-2 competitively binds to Tie2, and antagonizes the stabilizing action of Ang-1, which results in destabilization of vessels. These destabilized vessels may undergo regression in the absence of angiogenic factors such as VEGF; in the presence of VEGF, however, these destabilized vessels may undergo angiogenic changes. Thus, angiogenesis is controlled by a dynamic balance between vessel regression and growth that are mediated by VEGF, Ang-1, and Ang-2 (2) , which has been proved in many experimental studies (7, 8, 9) . However, little has been reported on the correlation between the status of Angs and angiogenesis in clinical materials (10) , although some clinical studies on Angs expression have been conducted in a variety of malignant tumors, including glioma (10) , glioblastoma (11) , neuroblastoma (12) , colon carcinoma (13) , gastric carcinoma (14) , hepatocellular carcinoma (15) , and NSCLC (16 , 17 , 18) . In addition, only a few clinical studies have documented a correlation between Ang expression and clinical features or prognosis (12 , 14) . We have previously conducted a clinical study on angiogenesis in NSCLC in which IMVD was determined with an antibody against CD34, a pan-endothelial marker (CD34-IMVD), or with an antibody against CD105, a specific marker of activated ECs (CD105-IMVD), and have demonstrated that CD105-IMVD, not CD34-IMVD, was significantly correlated with postoperative survival (19) . In the present study, therefore, we assessed expression of Angs in correlation with angiogenesis and clinical outcome in NSCLC.
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PATIENTS AND METHODS |
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TopABSTRACTINTRODUCTIONPATIENTS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Refs. 19, 20, 21
). Pathological stage was re-evaluated and determined with the present tumor-node-metastasis classification as revised in 1997 (22)
. Histological type and cell differentiation were determined using the current classification by WHO as revised in 2000 (23)
. For analyses according to the differentiation of cancer cells, well-differentiated Sq and Ad were classified as well-differentiated tumors and moderately differentiated Sq and Ad as moderately differentiated tumors. Large cell carcinoma and poorly differentiated Sq and Ad were classified as poorly differentiated tumors, and the other histological types were excluded in the analyses (20)
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All of the primary tumor specimens were immediately fixed in 10% (v/v) formalin, and then embedded in paraffin. Serial 4-µm sections were prepared from each sample and served for H&E staining and IHS. Results of IHS were evaluated by two authors (F. T. and S. I.) independently without knowledge of any clinical data.
Expression of Ang-1, Ang-2, and VEGF.
Expression of Ang-1, Ang-2, or VEGF was evaluated immunohistochemically using a standard streptavidin-biotinylated horseradish-peroxidase complex method (LSAB-2 kit; DAKO, Kyoto, Japan). After retrieval of the antigen by heating the sections in a microwave oven three times for 5 min each, the sections were incubated with an anti-Ang-1 polyclonal antibody (goat IgG, 200 µg/ml; Santa Cruz Biotechnology, Santa Cruz, CA) diluted at 1/50, an anti-Ang-2 polyclonal antibody (goat IgG, 200 µg/ml; Santa Cruz Biotechnology) diluted at 1/50, or an anti-VEGF polyclonal antibody A-20 (rabbit IgG, 200 µg/ml, Santa Cruz Biotechnology) diluted at 1/50. The anti-VEGF antibody recognizes the 165-, 189-, and 121-amino splicing variants of VEGF. As a chromogen, 3,3'-diaminobenzidine (DAB; Sigma Chemical Co., St. Louis, MO) was used, and the sections were counterstained with hematoxylin. For the negative controls, normal goat IgG was used as a substitute for the primary antibody against Ang-1 or Ang-2, and normal rabbit IgG was used for that against VEGF.
Ang-1 or Ang-2 expression was judged to be positive, when the percentage of cancer cells with positive staining exceeded 5%. VEGF expression was classified according to the following grading system as described previously (19)
. Briefly, a percentage score was defined as follows: score 0 if no VEGF-positive staining cell was documented; score 1 if the percentage of VEGF-positive staining cells was 
25%; score 2 if the percentage was >25% and 50%; and score 3 if the percentage was >50%; an intensity score was defined as follows: score 0 if no staining was documented; score 1 if the staining intensity was weak; score 2 if the intensity was moderate;, and score 3 if the intensity was high. The staining intensity of tumor cells was judged as high (score 3) when the staining intensity was comparable with that of smooth muscle cells of either bronchial wall or blood vessels. Grade of VEGF expression was represented as the sum of the percentage score and the intensity score (VEGF score), and VEGF expression was finally defined as follows: weak expression when the VEGF score is 4; and strong expression when the VEGF score is 5 or 6.
Quantification of Angiogenesis (IMVD).
IHS for CD34 and CD 105 to highlight ECs was performed using a sensitive streptavidin-biotinylated horseradish-peroxidase complex system (TSA-Indirect kit; NEN Life Science Products, Boston, MA) as described previously (19)
. Briefly, dewaxed sections were incubated with an anti-CD34 mAb QBEnd10 (mouse IgG1
, 50 µg/ml; DAKO) diluted at 1/50 or an anti-CD105 mAb SN6 h (mouse IgG1; 366 µg/ml; DAKO Japan) diluted at 1/100. The 10 most vascular areas within a section were selected for evaluation of angiogenesis, and vessels, labeled with the anti-CD34 mAb or the anti-CD105 mAb, were counted under light microscopy at x200. The average counts were recorded as the CD34-IMVD or CD105-IMVD for each case.
Statistical Methods.
The 
2 was used to compare counts. Continuous data were compared using the Student t test if the distribution of samples was normal, or the Mann-Whitney t test if the sample distribution was asymmetrical. The postoperative survival rate was analyzed by the Kaplan-Meier method, and the differences in survival rates were assessed by the log-rank test. Multivariate analysis of prognostic factors was performed using Cox’s regression model. Differences were considered significant when P < 0.05. All of the statistical manipulation was performed using the SPSS for Windows software system (SPSS Inc., Chicago, IL).
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RESULTS |
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TopABSTRACTINTRODUCTIONPATIENTS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. However, the degree and the extent of Angs expression in ECs were extremely heterogeneous, and the staining intensity in ECs was generally not so strong as to be clearly distinguished from negative expression. For these reasons, in the present study, we have focused on Ang expression in tumor cells, and Ang-1 or Ang-2 expression was judged based on the percentage of positive-staining tumor cells as described in the previous section. The staining intensity of IHS for Ang-1 or Ang-2 was heterogeneous even in the same tissue, and we could not evaluate semiquantitatively the degree of Ang-1 or Ang-2 expression.
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. Expression of Ang-2 was positive only in 40 patients (16.9%), and positive Ang-2 expression was significantly less in well-differentiated tumor (Table 1)
. Both Ang-1 expression and Ang-2 expression were positive [Ang-1(+)/Ang-2(+)] in 27 patients (11.4%), and both were negative [Ang-1(-)/Ang-2(-)] in 122 patients (51.7%); the numbers of Ang-1(-)/Ang-2(+) and Ang-1(+)/Ang-2(-) patients were 13 (5.5%) and 74 (31.4%), respectively.
Angs Expression and IMVD.
The average CD34-IMVD for Ang-1-negative tumor and that for Ang-1-positive tumor were 179.3 and 180.3, respectively, showing no correlation between Ang-1 status and CD34-IMVD. There was no significant correlation between Ang-1 status and CD105-IMVD, and the average CD105-IMVD for Ang-1-positive tumor was somewhat higher than that for Ang-1-negative tumor (48.0 versus 36.9; P = 0.093). The average CD105-IMVD for Ang-2-positive tumor was significantly higher than that for Ang-2-negative tumor (56.7 versus 38.5; P = 0.032), suggesting that Ag-2 plays an important role in angiogenesis. The average CD34-IMVD for Ang-2-positive tumor was somewhat higher than that for Ang-2-negative tumor (199.3 versus 177.0), but the difference was not statistically significant (P = 0.347). When angiogenesis was analyzed in combination with Ang-1 status and Ang-2 status, Ang-1-negative and Ang-2-positive tumor or Ang-1-positive and Ang-2-positive tumor showed higher IMVD, suggesting that Ang-2, not Ang-1, played major roles in tumor angiogenesis (Table 2)
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Next, correlation between IMVD and Angs status in combination with VEGF status was examined, because many experimental studies showed that angiogenesis was promoted by Angs in concert with VEGF. The average CD34-IMVD for Ang-1-negative and VEGF-low tumor and that for Ang-1-positive and VEGF-low tumor were 166.7 and 168.8, respectively (P = 0.843); the average CD34-IMVD for Ang-1-negative and VEGF-high tumor and that for Ang-1-positive and VEGF-high tumor were 214.1 and 196.4, respectively (P = 0.446). In addition, the average CD105-IMVD for Ang-1-negative and VEGF-low tumor and that for Ang-1-positive and VEGF-low tumor were 25.1 and 29.1, respectively (P = 0.543); the average CD105-IMVD for Ang-1-negative and VEGF-high tumor and that for Ang-1-positive and VEGF-high tumor were 65.3 and 73.6, respectively (P = 0.513). These results showed that Ang-1 did not affect angiogenesis induced by VEGF. In contrast, Ang-2 status affected CD105-IMVD when VEGF expression was high as follows: when VEGF expression was high, the average CD105-IMVD for Ang-2-positive tumor was significantly higher than that for Ang-2-negative tumor (89.1 versus 63.6; P = 0.045); when VEGF expression was low, the average CD105-IMVD for Ang-2-positive tumor and that for Ang-2-negative tumor were almost the same (27.4 and 27.1, respectively; P = 0.982; Fig. 2
). When VEGF expression was high, the average CD34-IMVD for Ang-2-positive tumor was somewhat higher than that for Ang-2-negative tumor (216.7 versus 201.1), but the difference was not statistically significant (P = 0.166). When VEGF expression was low, there was no difference between CD34-IMVD for Ang-2-positive tumor and that for Ang-2-negative tumor (171.8 and 166.1, respectively; P = 0.797).
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and Table 3
). In contrast, there was no difference in the survival between Ang-1-positive patients and Ang-1-negative patients (Fig. 3A
; Table 3
). When analyzed in combination with Ang-1 status and Ang-2 status, Ang-1-positive and Ang-2-positive tumor showed the poorest prognosis (Table 2)
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. For pathological stage-I disease, the 5-year survival rate for Ang-2-positive patients was significant lower than that for Ang-2-negative patients (61.8% versus 81.1%; P = 0.022; Table 3
). A multivariate analysis confirmed that positive Ang-2 expression was a significant factor to predict a poor prognosis (Table 4)
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, whereas VEGF expression was the most potent angiogenic factor. Because angiogenesis was highly promoted by Ang-2 in the presence of high VEGF expression (Fig. 2)
, postoperative survival was analyzed according to the status of Ang-2 expression in combination with the status of VEGF expression (Fig. 4)
. The 5-year survival of Ang-2-positive and VEGF-high patients was extremely low, suggesting that aggressive angiogenesis caused by Ang-2 in concert with VEGF might bring the poorest postoperative survival.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONPATIENTS AND METHODSRESULTSDISCUSSIONREFERENCES |
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With respect to a correlation between Angs expression and tumor progression or prognosis, only two clinical studies have been reported (12 , 14) . Eggert et al. (12) reported that increased Ang-2 gene expression, not Ang-1 gene expression, was significantly correlated with advanced tumor stages in neuroblastoma, but they failed to demonstrate a significant correlation between Ang-2 gene expression and postoperative survival. Etoh et al. (14) documented that increased Ang-2 gene expression was significantly correlated not only with an advanced tumor-stage but also with a poor prognosis in gastric carcinoma; this was the only clinical study that showed a significant correlation between Angs expression and a prognosis. Thus, the present study added clinical evidence that Ang-2 played important roles in tumor progression through tumor angiogenesis and that Ang-2 could be a new prognostic marker. In addition, we demonstrated that the postoperative survival of patients with Ang-2-positive and VEGF-high tumor was extremely poor, which might be attributable to active angiogenesis in tumor tissues. Although Ang-2 significantly affected angiogenesis only when VEGF expression was high, a multivariate analysis of prognostic factors showed that Ang-2 proved to be an independent factor to predict a poor postoperative survival. The reason why Ang-2 status was an independent prognostic factor regardless of the VEGF status should be examined in a future study for a larger number of patients with more homogeneous characteristics.
In the present study, Angs expression was judged based on Angs expression in tumor cells, not in ECs, whereas Angs were expressed in ECs as well as in tumor cells. Although it had been initially reported that Angs were expressed in ECs, many experimental and clinical studies have revealed that Angs were expressed not only in ECs but also in tumor cells (9, 10, 11, 12, 13, 14 , 18 , 26) . In the present study, we did not assess Angs expression in ECs, not only because the degree and the extent of Angs expression in ECs were very heterogeneous, but also because the intensity of Angs expression was not so strong as to be clearly distinguished from negative expression. In fact, Ang-2 expression levels in whole tumor tissues were evaluated in most clinical studies, and detailed analyses of Ang-2 expression in tumor cells and that in ECs have not been reported. Only one clinical study has been reported on the quantitative evaluation of Ang-2 expression in ECs; that study used in situ hybridization to evaluate Ang-2 gene expression and demonstrated only that a correlation existed between the number of Ang-2-expressing vessels and VEGF expression (17) . These results suggest that it may be difficult to assess Ang-2 expression quantitatively in ECs in clinical materials.
In conclusion, Ang-2 expression in NSCLC was significantly correlated with active angiogenesis, and can be a significant prognostic factor. In future studies, expression of Tie2 along with expression of the Ang ligands should be studied.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported by Grant-in-Aid 14370410 (to F. T. ) for Scientific Research (B) from the Ministry of Education, Culture, Sports, Science and Technology of Japan. 
2 To whom requests for reprints should be addressed, at Department of Thoracic Surgery, Faculty of Medicine, Kyoto University, Shogoin-kawahara-cho 54, Sakyo-ku, Kyoto, 606-8507, JAPAN. Phone: 81-75-751-4975; Fax: 81-75-751-4974; E-mail: ftanaka{at}kuhp.kyoto-u.ac.jp
3 The abbreviations used are: NSCLC, non-small cell lung cancer; Ang, angiopoietin; VEGF, vascular endothelial growth factor; IMVD, intratumoral microvessel density; EC, endothelial cell; Sq, squamous cell carcinoma; Ad, adenocarcinoma; IHC, immunohistochemical staining; mAb, monoclonal antibody.
Received 6/17/02. Accepted 10/ 4/02.
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TopABSTRACTINTRODUCTIONPATIENTS AND METHODSRESULTSDISCUSSIONREFERENCES |
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