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Clinical Cancer Research Vol. 12, 800-806, February 2006
© 2006 American Association for Cancer Research

Imaging, Diagnosis, Prognosis

Lymphangiogenesis and Angiogenesis in Bladder Cancer: Prognostic Implications and Regulation by Vascular Endothelial Growth Factors-A, -C, and -D

Yasuyoshi Miyata1, Shigeru Kanda2, Kojiro Ohba1, Koichiro Nomata1, Yasushi Hayashida1, Jiro Eguchi1, Tomayoshi Hayashi3 and Hiroshi Kanetake1

Authors' Affiliations: 1 Department of Urology, Nagasaki University School of Medicine, 2 Department of Molecular Microbiology and Immunology, Nagasaki University Graduate School of Biomedical Science, and 3 Department of Pathology, Nagasaki University Hospital, Nagasaki, Japan

Requests for reprints: Yasuyoshi Miyata, Department of Urology, Nagasaki University School of Medicine, 1-7-1 Sakamoto, Nagasaki 852-8501, Japan. Phone: 81-95-849-7340; Fax: 81-95-849-7343; E-mail: int.doc.miya{at}m3.dion.ne.jp.


   Abstract
Top
 Abstract
Materials and Methods
 Results
 Discussion
 References
 
Purpose: Lymph vessel density (LVD) and microvessel density (MVD) correlate with the malignant potential of tumors and patient survival. Vascular endothelial growth factors (VEGF)-A, VEGF-C, and VEGF-D could modulate LVD and MVD. We investigated the clinical and prognostic significance of LVD and MVD on lymphangiogenic and angiogenic function of VEGF-A, VEGF-C, and VEGF-D in human bladder cancer.

Experimental Design: We reviewed tissue samples from patients with nonmetastatic bladder cancer who had undergone transurethral resections (n = 126). The densities of D2-40-positive vessels (LVD) and CD34-positive vessels (MVD) were measured by a computer-aided image analysis system. Expression of VEGF-A, VEGF-C, and VEGF-D was examined by immunohistochemistry; survival analyses and their independent roles were investigated using multivariate analysis models.

Results: LVD was associated with tumor grade but not with pT stage. LVD was associated with metastasis-free survival (log rank P = 0.039), but was not an independent prognostic factor. Although MVD affected survival, the combination of high LVD and high MVD in tumors was an independent predictor of metastasis-free survival. Although VEGF-C expression was positively associated with both LVD and MVD, VEGF-D was associated only with LVD. VEGF-A expression was associated with MVD in univariate analysis, however, it was not an independent factor.

Conclusions: Lymphangiogenesis and angiogenesis influence metastasis-free survival, and are regulated by VEGF-C and/or VEGF-D. Our results suggest that LVD and MVD are useful tools for the selection of postoperative management and treatment strategies in patients with bladder cancer.

Metastatic dissemination of the primary tumor is an important factor that negatively affects the prognosis in most malignancies and neovascularization (angiogenesis) plays a critical role in tumor growth and systemic dissemination of cancer cells (1). As such, much attention has been focused on the pathologic significance and detailed mechanism of the vascular system and angiogenesis in cancers. In addition to dissemination of cancer cells via the bloodstream, the lymphatic system is also thought to play an important role in tumor cell dissemination. Indeed, metastatic spread to regional lymph nodes is an early step in the systemic dissemination of tumors, and lymph node metastasis is generally associated with poor survival (2, 3). However, the clinical significance of the de novo formation of lymphatic capillaries (lymphangiogenesis) and its regulation in cancer remains unclear, largely because specific endothelial markers for lymphatic vessels are unknown and lymphatic vessels cannot be detected in human cancer tissues (4). In recent years, several new specific antibodies for lymphatic endothelial cells have been developed and used to investigate the clinical and pathologic significance of lymphangiogenesis in various cancers (5).

Bladder cancer is the second most common malignant tumor of the urogenital region. This tumor is associated with frequent muscle invasion and metastasis at the time of diagnosis and after initial treatment (6). Bladder cancer, clinically diagnosed as a superficial tumor, is generally treated with a transurethral resection due to its relatively indolent nature and low malignant potential. However, this superficial carcinoma often recurs and invades the muscle after the initial treatment; such progression is associated with a high risk of subsequent metastasis and poor survival (7, 8). Various growth factors and molecules have been reported to be associated with tumor growth, progression, and survival in bladder cancer (9, 10). Among these factors, tumor microvessel density (MVD) is thought to be one of the most useful prognostic markers for disease development, grade, recurrence-free survival, and overall survival (11–13). In contrast, the clinical and pathologic significance of lymphangiogenesis (lymph vessel density, LVD) in human bladder cancer tissues are yet to be investigated.

The mechanisms of angiogenesis and lymphangiogenesis are complex. Vascular endothelial growth factor (VEGF)-A is a representative proangiogenic factor and plays an important role in neovascularization and tumor progression (14–16). Traditionally, VEGF-A is largely associated with angiogenesis in cancer, although recent studies showed that VEGF-A could also stimulate lymphangiogenesis in vivo (17, 18). In this context, there is little or no information on the relationship between VEGF-A and lymphangiogenesis in human cancer, although recent research showed that VEGF-C and VEGF-D are major factors associated with the growth of lymphatic endothelial cells (19, 20). Furthermore, several investigators found that inhibition of VEGF-C and VEGF-D activity diminished the level of lymphangiogenesis and lymph node metastasis (21, 22). Thus, it is currently accepted that VEGF-C and VEGF-D are important regulators of lymphangiogenesis in a variety of cancers. In addition to their prolymphangiogenic function, VEGF-C and VEGF-D also play important roles in various aspects of angiogenesis under physiologic and pathologic condition (23–26). Thus, it is possible that VEGF-A, VEGF-B, and/or VEGF-C could potentially stimulate angiogenesis and/or lymphangiogenesis independently or cooperatively. In the present retrospective study, we investigated the relationship between various clinicopathologic features, prognosis, and survival and angiogenesis/lymphangiogenesis in patients with transitional cell carcinoma of the urinary bladder. Furthermore, we also used multivariate analysis to assess the independent effects of VEGF-A, VEGF-C, and VEGF-D on LVD and MVD.


   Materials and Methods
Top
 Abstract
 Materials and Methods
Results
 Discussion
 References
 
Patients and tumor samples. Data from patients who were clinically diagnosed with noninvasive and nonmetastatic transitional cell carcinoma of the urinary bladder between January 1993 and December 2003, were reviewed retrospectively. For tumor staging, all patients underwent ultrasonography of the urinary bladder, computed tomography of the abdomen and urinary bladder, drip infusion pyelography, bone scanning, lung X-ray photography, and cystoscopy. Patients with a history of malignancy, including a prior history of urinary tract malignancies were excluded from this study, as were patients who had previously received neoadjuvant therapy. All patients were treated with a transurethral resection, and all samples were evaluated by one pathologist. Tumors were staged according to the American Joint Committee on Cancer (27) and graded according to the WHO and International Society for Urological Pathology classification system (28). In the present study, tumors were grouped for statistical analysis into the following groups: low-stage (pTa and pT1) and high-stage (pT2-4) or low-grade (grades 1 and 2) and high-grade (grade 3). Furthermore, we also examined 21 normal tissue samples of the urinary bladder obtained from apparently normal areas of the bladder of patients with transitional cell carcinoma of the upper urinary tract. All of them did not recur for 5 to 12 years. The study protocol met the ethical standard of the Human Ethics Review Committee of the Nagasaki University School of Medicine.

Immunohistochemistry. Paraffin sections (5 µm thick) were deparaffinized and rehydrated. Antigen retrieval treatment was done at 95°C for 40 minutes in 0.01 mol/L sodium citrate buffer (pH 6.0), and endogenous peroxidases were blocked using 3% hydrogen peroxide for 30 minutes. The primary antibodies were purchased from DakoCytomation (Glostrup, Denmark; D2-40 at 1:50 dilution, and CD34 at 1:50 dilution), Santa Cruz Biotechnology, Inc. (Santa Cruz, CA; VEGF-A, at 1:120 dilution), Zymed Laboratories, Inc. (San Francisco, CA; VEGF-C, at 1:70 dilution), and R&D Systems, Inc. (Abingdon, United Kingdom; VEGF-D, at 1:100 dilution). All sections were incubated overnight with the primary antibody at 4°C. The sections were then treated with peroxidase using the labeled polymer method with DAKO EnVision+ Peroxidase (Dako Corp., Carpinteria, CA) for 30 minutes. The peroxidase reaction was visualized with liquid 3, 3'-diaminobenzine substrate kit (Zymed Laboratories). Sections were then counterstained in hematoxylin. Tonsil, breast cancer, and colon cancer specimens that were prepared, and then confirmed to be immunoreactive for the relevant antigens in preliminary studies, were used as positive controls for D2-40 and CD34, VEGF-A, VEGF-C, and VEGF-D. Consecutive sections from each sample processed without the primary antibody were used as negative controls. Furthermore, we also stained the 21 control tissue sections of the urinary bladder for D2-40 and CD34.

Evaluation. To analyze the LVD and MVD, tumor sections stained with anti-D2-40 antibody and anti-CD34 antibody were examined using a Nikon E-400 and digital images were captured using a digital camera (Nikon DU100, Japan) at x200 magnification. D2-40 antibody, which reacts with an O-linked sialoglycoprotein expressed by the lymphatic endothelium, was used previously to detect the lymph vessels and measure LVD (29, 30). For each tumor section, three to five fields with the highest lymphatic vascular density and blood vessel density (hotspots) were evaluated. To determine the MVD and LVD, defined as the number of vessels per square millimeter, we used a computer-aided image analysis system (Win ROOF, version 5.0, Mitani Corp., Japan). To determine the relationships between these variables and clinical outcome, we divided the tissue samples into two groups according to MVD levels; those with values higher than the median value for the entire group, and those with lower than the group median value. The same was applied based on the LVD values.

The expression levels of VEGF-A, VEGF-C, and VEGF-D were assessed semiquantitatively, taking into account the percentage of expressing carcinoma cells (at least 500 carcinoma cells were examined). The staining intensity was classified into four grades: none, weak, moderate, and strong. Specimens were considered positive when >25% of carcinoma cells were clearly (moderately or strongly) stained for statistical evaluation. This cutoff level was selected based on previous studies (31). Two investigators, blinded to the patients' clinical characteristics and survival data, independently did the semiquantitative analysis and staining interpretations.

Statistical analysis. Data were expressed as the median (interquartile range). The Mann-Whitney U test was done for continuous variables. Survival analysis was evaluated by Kaplan-Meier analysis and the log-rank test, and variables that achieved statistical significance (P < 0.05) in the univariate analysis were subsequently entered into a multivariate analysis using a cyclooxygenase proportional hazards analysis [described as odds ratios (OR) with 95% confidence intervals (95% CI), together with the P values]. The crude and adjusted effects on immunohistochemical staining, as well as other risk factors, were estimated by logistic regression analysis. All statistical analyses were two-sided and significance was defined as P < 0.05.


   Results
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 Abstract
 Materials and Methods
 Results
Discussion
 References
 
Patient characteristics and the clinical significance of lymphatic and blood vessels. Table 1 lists the characteristics and clinical outcome of 126 patients. After tumor resection, 28 patients (22.2%) did not receive any adjuvant therapy. In contrast, 89 patients (70.6%) and 9 patients (7.1%) received intravesical installation of chemical or Bacillus Calmette-Guerin and systemic adjuvant therapy (chemotherapy and radiation therapy), respectively. The mean (± SD) follow-up period after operation was 60.2 ± 39.0 months (median, 47 months; range, 5-146 months).


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  		Table 1. Clinicopathologic characteristics

 
In normal urinary bladder, D2-40-positive lymphatic vessels were detected only in stromal tissue but not in the epithelium (Fig. 1A). D2-40-positive lymphatic vessels were also detected in stromal tissues of all cases of transitional cell carcinoma (Fig. 1B). However, within the tumor, they were detected only in 16% of the tumors and almost all of the vessels had collapsed (Fig. 1C and D). Cancer cells did not stain with anti-D2-40 antibody, and the lumen of vessels lined with D2-40-positive cells did not contain any erythrocytes, suggesting that they were lymphatic vessels rather than blood capillaries (Fig. 1E). Furthermore, these vessels were confirmed to be lymphatic vessels by immunohistochemical staining with CD34 antibody (Fig. 1F).


Figure 1
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  		Fig. 1. D2-40-positive vessels in normal tissues (A, magnification x200). Stromal area (B, magnification x100) and intratumoral area of tumor tissues (C, magnification x100; D, magnification, x400). Intratumoral lymph vessels were small in number and had collapsed. In serial section of the same patients, D2-40-positive vessels (E, magnification x400) were clearly distinguished from CD34-positive vessels (F, magnification x400). Examples of positively stained tumor cells for VEGF-A (G), VEGF-C (H), or VEGF-D (I, magnification, x200). The expression of these proteins was located mainly in the cytoplasm of tumor cells.

 
The median (IR) LVD was significantly higher in stromal tissue of tumors (26.3/mm2, 17.9-34.7) than in that of normal tissues (8.1/mm2, 3.2-14.2; P < 0.001). On the other hand, the median LVD (IR and range) inside the tumor was 0/mm2 (0-0 and 0-5.2). Based on these findings, we examined the clinical significance of stromal LVD in this study. Table 2 shows the relationship between clinicopathologic features and LVD. LVD was significantly higher in high-grade tumors than that in low-grade tumors (P = 0.002). However, LVD was not associated with other variables including pT stage (P = 0.130).


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  		Table 2. Relationships between clinicopathologic features and LVD and MVD

 
The median (IR) MVD was significantly higher in tumor tissue (71.2 /mm2, 56.8-83.2) than in normal tissues (32.3/mm2, 22.4-43.2, P < 0.001). The MVD in tumor tissues correlated with pT stage (P < 0.001) and grade (P = 0.036; Table 2). On the other hand, both LVD and MVD did not correlate with gender, age at diagnosis, or multicentricity.

Significance of LVD and MVD on survival. Kaplan-Meier curves of urinary tract recurrence-free survival, metastasis-free survival, and cause-specific survival according to the LVD status are presented in Fig. 2A, B, and C, respectively. The 5-year metastasis-free survival rate in patients with low stromal LVD (96.5%) was significantly higher (P = 0.039) than that of patients with high LVD (80.2%, Fig. 2B). However, LVD was of no significant value for other variables. In contrast, MVD did not predict urinary tract recurrence-free survival (log rank P = 0.638), metastasis-free survival (log rank P = 0.156), or cause-specific survival (log rank P = 0.499). Analysis of the independent predictive value of LVD for metastasis-free survival using a multivariate analysis model including pT stage, grade, and adjuvant therapy, identified pT stage as the only independent and significant predictive factor (OR, 4.05; 95% CI, 1.13-12.22; P = 0.013), whereas LVD was not (OR, 2.27; 95% CI, 0.68-7.53; P = 0.181; Table 3, model A). When we investigated the combined effect of LVD and MVD, patients with high LVD and high MVD had shorter metastasis-free survival than those with other patterns in Kaplan-Meier curve (log rank P = 0.002, Fig. 2D) and multivariate analysis model (OR, 3.19, 1.03-9.86; P = 0.044; Table 3, model B). However, this variable did not significantly influence survival for the recurrence of urinary tract carcinoma (log rank P = 0.888) or cause-specific survival (log rank P = 0.865) in Kaplan-Meier curves.


Figure 2
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  		Fig. 2. Kaplan-Meier recurrence to urinary tract-free (A), metastasis-free (B), and cause-specific survival (C) curves according to the LVD status. Kaplan-Meier curve of metastasis-free survival according to LVD and MVD (D).

 

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  		Table 3. Independent predictive values of LVD (model A) and interaction of LVD and MVD (model B) for metastasis-free survival by Cox proportional hazard regression analysis

 
Correlation between VEGF-A, -C, and -D expression and clinicopathologic features, LVD, or MVD. Figure 1G, H, and I show representative examples of positive staining for VEGF-A, -C, and -D, respectively. The median (IR) of VEGF-A-, VEGF-C-, and VEGF-D–positively stained cells was 37.9% (23.6-44.9), 30.3% (18.4-42.9), and 28.1% (19.4-41.2), respectively. The relationship between clinicopathologic features and the expression of these proteins is summarized in Table 4. In all cases, positive staining for VEGF-A, VEGF-C, and VEGF-D in high pT stage and high-grade tumors was higher than in low pT stage (P = 0.023, P = 0.043, and P = 0.030, respectively) and low-grade tumors (P < 0.001, P < 0.001, and P = 0.020, respectively).


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  		Table 4. Relationships between clinicopathologic features and expressions of VEGF-A, VEGF-C, and VEGF-D

 
Univariate logistic regression analysis was done to investigate the influence of VEGF-A, VEGF-C, or VEGF-D on lymphangiogenesis and angiogenesis (Table 5). Univariate analysis showed that VEGF-C and VEGF-D were significantly associated with LVD. Furthermore, both proteins were also identified as independent factors in a multivariate analysis model including grade (OR, 3.32; 95% CI, 1.45-7.55; P = 0.005 and OR, 2.36; 95% CI, 1.08-5.13; P = 0.031; Table 5, model A). However, univariate analysis showed no significant correlation between VEGF-A and LVD. In contrast, the same analysis showed that VEGF-A and VEGF-D were associated with MVD, whereas VEGF-C was not (Table 5, model B). On the other hand, multivariate analysis identified only VEGF-C expression as a significant and independent factor for high MVD (OR, 4.18; 95% CI, 1.93-9.05; P = 0.001), but not VEGF-A (OR, 1.88, 95% CI = 0.85-4.14; P = 0.121).


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  		Table 5. Multiple logistic regression analysis for prediction of LVD (model A) and MVD (model B)

 

   Discussion
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 Abstract
 Materials and Methods
 Results
 Discussion
References
 
To our knowledge, the present study is the first to investigate the clinical significance and prognostic value of LVD in patients with transitional cell carcinoma of the urinary bladder. Our results showed that LVD correlates positively with tumor grade. However, there was no significant correlation between LVD in the low pT stage tumors and in high stage tumors. With respect to the relationship between LVD and T classification, several investigators reported that for several cancer types, the LVD was higher in invasive tumors than in noninvasive tumors (32, 33). In contrast, other investigators reported that LVD was not associated with T stage and tumor invasion in head and neck cancer (30, 34) and cutaneous melanoma (35). Based on our results, we speculate that lymphangiogenesis does not directly influence tumor invasion in human bladder cancer at least.

The finding that LVD is a significant predictive factor for metastasis-free survival is particularly interesting. Conflicting views exist regarding the predictive value of LVD for disease progression and survival. Previous reports showed that a high LVD was a useful predictor of poor disease-free survival and overall survival in patients with cutaneous malignant melanoma (36). In contrast, Franchi et al. (30) reported that high LVD was associated with increased risk of lymph node metastasis, but not with disease-free or overall survival in head and neck squamous cell carcinoma. Another study reported that increased LVD had no effect on tumor progression (37). Furthermore, Nakamura et al. (34) reported that high LVD was associated with shorter duration of disease-free survival in Kaplan-Meier analysis; it was not identified as an independent factor by multivariate analysis. Our data is in line with the findings of Nakamura and colleagues.

Our results showed that patients with high MVD and LVD tumors had the worst metastasis-free survival, and high MVD/LVD was identified as an independent factor in a multivariate analysis model. Our results showed that MVD did not influence prognosis in any survival analyses. However, angiogenesis is expected to play a significant role in malignant aggressiveness because of its association with tumor grade and pT stage. In fact, our results showed that the combined function of angiogenesis and lymphangiogenesis was important for cancer metastasis in patients with bladder cancer. Contrary to our expectations, LVD and MVD were not useful predictors for cause-specific survival in our study population. The pathways into the lymphatic or vascular system were expected to be promoted by stimulation of lymphangiogenesis or angiogenesis. However, various other processes, including migration, invasion, adhesion, and growth at metastatic sites, are necessary to form the metastatic tumor and influence survival, and these processes are regulated by numerous factors (9, 10). Therefore, we speculate that the predictive values of LVD and MVD for clinical outcome are dependent on the malignant potential of cancer cells, factors produced by cancer cells or released by stromal tissues, and organ microenvironment at metastatic sites.

The main objective of the present study was to investigate the relationship between VEGF-A, VEGF-C, and VEGF-D expression and LVD or MVD in human bladder cancer tissues. VEGF-A acts as a proangiogenic factor under normal physiologic and pathologic conditions including cancer (15, 16, 38). In contrast, divergent opinions exist on the proangiogenic function and clinical significance of VEGF-A in bladder cancer. Several investigators showed no correlation between the expression of VEGF-A and MVD in bladder cancer patients (39, 40). Our univariate analysis showed that overexpression of VEGF-A correlated with high MVD, however, VEGF-A was not identified as an independent and significant factor in multivariate analysis. We speculate that although VEGF-A plays a pathologic role, it is not an important or essential determinant of angiogenesis in our study population. We did not find a significant correlation between VEGF-A expression and LVD. A similar trend was reported in pancreatic cancer (41) and cutaneous melanoma (35). Recently, Hirakawa et al. (42) reported that VEGF-A induced lymphangiogenesis and promoted lymphatic metastasis in an animal model with chemical skin cancer. However, a significant correlation between VEGF-A expression and LVD in human cancer has not been reported.

One of the most interesting findings in our study was that high expression of VEGF-D was associated with only high LVD, although VEGF-C expression also showed a positive correlation with both LVD and MVD. Findings reported by various investigators have supported the hypothesis that VEGF-C and VEGF-D are stimulators of lymphangiogenesis and lymph node metastasis in human cancers (19–22, 33). Other investigators reported that VEGF-C and VEGF-D were positively associated with MVD in cancer tissues (43, 44). However, VEGF-C and VEGF-D do not always stimulate lymphangiogenesis and angiogenesis. Several reports indicated that VEGF-C expression in cancer cells did not correlate with MVD (45, 46). White and colleagues reported that increased VEGF-D expression was associated with lymphatic involvement, but not with MVD in human colorectal cancer tissues and suggested that VEGF-D does not have angiogenic function, but may stimulate the development and/or function of lymphatic vessels (47). In addition, Choi et al. (48) reported that the expression of VEGF-C and VEGF-D correlated with LVD, but not with MVD, in breast cancer. An explanation for such different functions of VEGF-C and VEGF-D was not provided by data from the present study. However, several investigators speculated that the properties of VEGF-C and VEGF-D in lymphangiogenesis or angiogenesis might depend on the degree of their proteolytic processing. Briefly, proteolytic processing alters their binding affinity to their receptor on the surface of lymphatic endothelium and vascular endothelium (49, 50). Although we cannot discuss such phenomena with respect to our study population because proteolysis of VEGF-A, VEGF-C, and VEGF-D was not examined, we speculate that the different influences of VEGF-C and VEGF-D on MVD may be partly explained by such phenomena.

Our findings indicate that targeting lymphangiogenesis and angiogenesis could be a potential powerful tool to predict and control progression of bladder cancer. Furthermore, we speculate that the regulation of VEGF-C and VEGF-D may present a useful method for inhibition of lymphangiogenesis and angiogenesis.


   Acknowledgments
 
We thank Takumi Shimogama and Yoshikazu Tsuji for their excellent assistance.


   Footnotes
 
Grant support: Japanese Society for the Promotion of Science Grant-in-Aid no. 17791080 (Y. Miyata).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 6/15/05; revised 10/25/05; accepted 11/10/05.


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 Discussion
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