Neovascularisation is a prognostic factor of early recurrence in T1/G2 urothelial bladder tumours

L. Santos1,+, C. Costa2, S. Pereira2, M. Koch2, T. Amaro2, F. Cardoso3, T. Guimarães3, M. J. Bento4, F. Lobo1, S. Pinto3 and C. Lopes2

Departments of 1 Surgical Oncology, 2 Pathology, 3 Medical Oncology and 4 Epidemiology, Portuguese Institute of Oncology, Porto, Portugal

Received 19 December 2002; revised 24 April 2003; accepted 3 June 2003


    Abstract
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
Background:

Of patients with superficial bladder cancer, a group are still at risk of disease recurrence, progression and death from their cancer after curative treatment. Angiogenesis is a crucial pathogenic mechanism for this type of urothelial cell carcinoma (UCC), and is a potential therapeutic target. However, the selection of the appropriate patients remains a dilemma.

Patients and methods:

Vascular endothelial growth factor (VEGF) expression and the presence of angiogenesis and occurrence of CD31, CD34, endoglin and factor VIII immunoexpression, were evaluated in 66 superficial papillary UCCs of the bladder and were correlated with classical histopathological factors and disease outcome.

Results:

VEGF immunoreactivity was observed in 100% of cases, and more intensely in the luminal surface. The presence of microvessel clusters independently of a fibrovascular core was observed in 22.7% of cases. Of these, the T1/G2 subgroup had an independent and significantly lower recurrence-free survival (P = 0.0002).

Conclusions:

These results indicate that the presence of angiogenesis in tumour urothelium is a potential prognostic factor in superficial UCC, particularly in T1/G2 tumours, and may be used to select patients for anti-angiogenic treatments.

Key words: angiogenesis, bladder cancer, prognosis, vascular endothelial growth factor


    Introduction
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
Superficial bladder cancer is an important genito-urinary malignancy. Approximately 65–70% of initial bladder tumours are superficial lesions, and approximately 70% of these are stage Ta, 20% T1 and 10% Tis [1]. Sixty to seventy per cent of the superficial lesions recur after therapy and one-quarter to one-third of these show progression to higher grade and/or stage [2]. Overall, 10–20% of superficial lesions will progress to muscle-invasive bladder cancer [3]. The treatment goal for superficial bladder cancer is reducing tumour recurrence and preventing tumour progression, which would require additional aggressive therapies. The administration of intravesical chemotherapy (IVT) or immunotherapy, following transurethral resection (TUR), has become the main therapeutic approach to accomplish these goals. However, despite this therapy, a group of patients with superficial bladder cancer still remain at risk of disease progression, metastasis and death from their cancer [4].

Angiogenesis is the process by which tumours induce a blood supply, crucial for growth and progression [5]. Taking this into account, potential anti-angiogenic therapies may have an important role in the treatment procedure. The increased expression of angiogenic factors, such as vascular endothelial growth factor (VEGF), basic fibroblast growth factor and microvessel density (MVD), identifies patients with muscle-invasive urothelial cell carcinoma (UCC) of the bladder who are at high risk of developing metastasis after aggressive systemic chemotherapy [6]. VEGF, representing an essential factor for endothelial growth, has been evaluated in superficial UCC, but no definitive conclusion can yet be reached regarding its prognostic value [7, 8]. High MVD, a histological surrogate for angiogenesis, has been shown to be correlated with aggressive clinical behaviour for a number of different neoplasms, including invasive bladder cancer [9]. However, its relationship with disease progression in patients with superficial bladder tumours is not yet accepted [10].

In the present study we evaluated the prognostic significance of VEGF expression and the presence of microvessels in the neoplastic urothelium of superficial UCC of the bladder. Classical endothelial cell (EC) markers—anti-CD31, -CD34, -factor VIII and -endoglin antibodies—were used in an attempt to identify high-risk patients who might benefit from anti-angiogenic therapeutic approaches.


    Patients and methods
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
Tissue samples
From 1989 to 1996, 66 patients (23 women and 43 men) with superficial low-grade papillary UCC of the bladder were retrospectively studied. These patients were admitted consecutively, and treated with curative intention by TUR, at the Portuguese Institute of Oncology, Porto. The carcinomas were selected from the Department of Pathology archival files based on available paraffin blocks and adequate follow-up. Two independent pathologists reviewed all the tumours. For each one, histopathological criteria from the World Health Organization classification were used [11] and staging was made according to the American Joint Committee on Cancer system [12]. Twenty-one tumours were pTa and 45 were pT1; 26 cases were GI and 40 were GII. Surgical therapy (TUR) alone was done in 34 patients, while 32 received adjuvant IVT (BCG in nine patients and intravesical chemotherapy in 23), according to the standard management of superficial bladder cancer [13]. Median age was 66.5 years (range 48–87). Median follow-up was 74.3 months (range 6.6–105). Recurrence was defined as the reappearance of a lesion in the bladder more than 3 months after intended curative treatment. Disease progression was defined as a local recurrence resulting in an increase of stage. Recurrence-free survival was defined as the time from the beginning of the treatment until the first observed recurrence in the bladder, or until the last clinical assessment without recurrence. A group of seven normal urothelium samples, obtained from autopsy donors with a median age <45 years and with traumatic cause of death, were used as the control group.

Immunohistochemistry
The avidin–biotin–peroxidase complex method was used for immunohistochemical detection. Briefly, 3-µm sections were cut from formalin-fixed, paraffin-embedded primary bladder cancers and normal urothelium specimens. A microwave oven was used for antigen extraction; endogenous peroxidase was blocked by incubation with 3% hydrogen peroxide. The slides were incubated with horse normal serum (VectaStain ABC kit; Vector Laboratories®, Burlingame, CA) for 20 min at room temperature. The presence of VEGF was evaluated using a monoclonal antibody (1:50 AB-5; Neomarkers®, Fremont, CA) reacting with the 121, 165, 189 and 206 isoforms. Anti-CD31 (1:30, Dako®, Glostrup, Denmark), -CD34 (1:25, Novocastra®, Newcastle, UK), -endoglin (1:10, Novocastra®) and -factor VIII (1:50, Dako®) antibodies were also used. After incubation with each primary antibody, sections were incubated with the secondary biotinylated antibody (VectaStain ABC Kit) and avidin–biotin–peroxidase complexes (VectaStain ABC Kit) for 30 min. Reaction products were visualised with diaminobenzidine as the chromogen and sections were counterstained with Harris’s haematoxylin.

Sections from previously studied tumours of breast cancer, known to express VEGF, CD31, CD34 endoglin and factor VIII were used as positive controls. Negative controls were carried out by replacing the primary antibody with 2.5% bovine serum albumin in phosphate-buffered saline, pH 7.

Immunohistochemical evaluation
Immunohistochemical evaluation was done by two observers in independent readings (T.A. and L.S.). The readers were ‘blinded’ to clinical outcomes and to the results obtained by the other reader. Cases that varied significantly between readers were re-evaluated in order to determine a consensus.

VEGF positivity was indicated by the presence of cytoplasmic or membrane brown staining. VEGF-positive cases were defined when the whole tumour or extended tumoural areas (>50% of cells) were stained. The staining intensity was also recorded. Angiogenesis was evaluated by immunohistochemical staining of tumour microvessels for CD31, CD34, endoglin and factor VIII. The presence of microvessels was evaluated qualitatively. Thus, the entire section was screened to find a region with microvessels. Cases with no tumour clusters of stained cells and where only the fibrovascular core was stained for CD31, CD34, endoglin and factor VIII, were classified as negative for angiogenesis, while tumours with clusters of stained ECs—with at least two of the CD31, CD34, endoglin or factor VIII markers—distinct from the main papillary vessels (fibrovascular core), surrounded by tumour cells and independent of stromal cells, were considered to have angiogenesis.

Statistical analysis
A descriptive study was carried out for all variables included in the study. Chi-square and Fisher’s exact tests were used to compare categorical variables with the presence of angiogenesis. The dependent variables of interest were recurrence-free and progression-free survival. To determine the effect of the different variables in prognosis, we conducted a survival analysis using Kaplan–Meier methodology and the differences between categories of each variable were evaluated by a log-rank test. To determine the way in which recurrence and progression were affected by other covariables and to control their confounding effect, a Cox’s proportional hazards analysis was carried out. The hazard ratios were estimated with their 95% confidence intervals. All statistical analyses were carried out with SPSS 8.0 software (SPSS Inc.). P <0.05 was accepted as statistically significant.


    Results
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 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
VEGF, CD31, CD34 endoglin and factor VIII immunostaining was done successfully in all tumours.

Immunohistochemistry for VEGF
Immunostaining for VEGF was clearly positive in all tumours, being similar in both normal and malignant bladder urothelial cells. Its immunoexpression was mainly localised in the cytoplasm and was weakly positive in some ECs of the fibrovascular core. Several tumours (n = 48) had a stronger VEGF staining intensity in the luminal surface (Figure 1A). No significant relationship was found between these cases and tumours with microvessel clusters.



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Figure 1. (A) Strong vascular endothelial growth factor staining intensity in the luminal surface. (B) Normal urothelium specimen; no angiogenesis was observed (CD31). (C) Angiogenesis in the tumoural urothelium (CD34). (D) CD31 expression limited to the neoplastic cell membrane.

 
Immunohistochemistry for CD31, CD34, endoglin and factor VIII
All stromal and papillary fibrovascular cores were stained with all the studied EC markers. The intensity of factor VIII immunoexpression was weaker compared with the other studied markers. In normal urothelium specimens, no angiogenesis was observed (Figure 1B). With respect to the vessel density, a significant heterogeneity was noted in all cases. Angiogenesis was observed in the urothelium of 15 tumours (22.7%) (Figure 1C). The microvessel clusters were concomitantly stained for CD31, CD34 and endoglin. Seven cases in 15 were factor VIII-negative. CD31 immunoreactivity was also observed in the interface between the urothelium tumour cells and the papillary fibrovascular core of all the angiogenic cases. In some, the expression was limited to the neoplastic cell membrane (Figure 1D). This CD31 immunoreactivity was not superimposed on the immunoreactivity for the other studied EC markers. The majority of the tumours with angiogenic clusters had a solid pattern and were G2 tumours (Table 1).


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Table 1. Relationship between angiogenesis and clinicopathological variables
 
Outcome analysis
The rate of recurrence and progression was not significantly different when the patients treated with surgery alone were compared with those treated with adjuvant IVT. Progression was observed in six cases with primary pTa tumours and in two with primary pT1 tumours, being three of those submitted to adjuvant intravesical chemotherapy. In the univariate analysis, multifocality was associated with progression (P = 0.047; Table 2). Regarding the rate of recurrence, only the presence of microvessel clusters (angiogenesis) achieved statistical significance (P = 0.0001), being an independent prognostic factor (Table 3). These survival differences were maintained when a stratified analysis for gender, multifocality and treatment was carried out. Fifty per cent of the tumours recurred within the first 24 months after treatment (Figure 2A). In the pT1/G2 subgroup (n = 36), tumours with angiogenesis had a significantly reduced recurrence-free survival (P = 0.0002; Figure 2B), independent of gender status and treatment. All pT1/G2 tumours with angiogenesis were unifocal (n = 12).


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Table 2. Recurrence and progression: univariate analysis as a function of clinicopathology, angiogenesis and treatment
 

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Table 3. Multivariate recurrence-free survival analysis
 


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Figure 2. (A) Recurrence-free survival according to the angiogenesis status in all cases (P = 0.0001). (B) Recurrence-free survival according to the angiogenesis status in the pT1/G2 sub-group (P = 0.0002).

 

    Discussion
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
The appropriate treatment for superficial bladder cancer is not straightforward, and the risk of tumour recurrence/progression must be balanced against the toxicity of IVT. It must be emphasised that bladder preservation with effective TUR and IVT is clearly preferable to exanterative surgery only if equivalent efficacy is proven [14]. Nevertheless, as pointed out by Lamm et al. [15] and Pawinski et al. [16], long-term analyses regarding the effectiveness of intravesical chemotherapy are still scarce. A rational development for using IVT in order to achieve constant and durable results must be tested using as a target the recurrence of neovascularisation.

Anti-angiogenic effects, mediated by conventional cytotoxic anticancer drugs, have been reported [17]. In this sense Miller et al. [18] redefined the chemotherapeutic drugs with more anti-angiogenic effects. It is known that chemotherapy regimens combined with anti-angiogenic endothelial-specific drugs can induce remarkable responses [19].

Our findings of equal VEGF expression in tumour and normal epithelium are in agreement with those of Sato et al. [20], who observed the presence of VEGF mRNA in all bladder tumours and normal mucosae. Campbell et al. [21] also observed that, in bladder tumours and despite the higher angiogenesis rate, VEGF expression was similar to that found in normal urothelium. On the other hand, VEGF expression in human UCCs was also evaluated for its prognostic value and no definitive conclusion could be reached [22].

Tumour hypoxia regulates genes that enhance vascularity and oxygen delivery, such as VEGF [23]. Jones et al. [24], studying cell lines and bladder tumours, revealed that the components of the hypoxia response pathway, including hypoxia-inducible factor-1{alpha} and -2{alpha}, are important cofactors in the regulation of VEGF in bladder cancer. Reiher et al. [25] observed that hypoxia is also crucial in bladder cancer angiogenesis. The distribution of VEGF mRNA and the presence of microvessels were compared with the expression of a hypoxia marker, carbonic anhydrase 9 (CA IX), in superficial bladder cancer. There was an overlap in expression of VEGF and CA IX mainly in the luminal surface of tumours and in areas within 80 µm of a microvessel [26]. Therefore, angiogenesis is an important event in superficial bladder cancer, being related to the hypoxic environment. In our sample we also observed an increase of VEGF staining intensity in the luminal surface of several tumours. However, no relationship between these cases and tumours with microvessel clusters was found. Upregulation of VEGF may have been a previous event.

Papillary superficial bladder cancer is a pathological entity with a well-developed branching fibrovascular core. In fact, it has been speculated that angiogenesis is the initial pathogenic mechanism for this type of urothelial carcinoma [27]. This architectural pattern in papillary superficial UCCs increases the difficulty of MVD assessment; therefore, the discrimination between prior microvessels (fibrovascular core) and new ones (neovascularisation) is a hard task. Based on our observation, anti-CD31, -CD34 and -endoglin antibodies recognise small-calibre vessels that are associated with angiogenesis in bladder cancer more efficiently than anti-factor VIII antibody. Similar results were observed by others [28]. The CD31 expression limited to the neoplastic cell membrane without co-expression of other EC markers was observed in our results and also by Sapino et al. [29] in aggressive breast carcinomas. These authors concluded that CD31 expression was associated with other functions, distinct from angiogenesis, such as cell-to-cell adhesion and migration. Recently, others have pointed out that misinterpretation of CD31-positive macrophages as tumour cells may result in erroneous diagnosis of neoangiogenesis [30]. Sagol et al. [31] used a stereological method to evaluate the number of CD31-positive vessels/mm2 in urothelial superficial bladder carcinoma. They showed that this angiogenic assessment did not predict recurrence and/or progression. Dinney et al. [32], studying T1 bladder cancer, did not find MVD as a prognostic marker. However, another study of stereological parameters in superficial and invasive bladder cancer pointed out that the presence of microvessel clusters was an independent predictor of adverse prognosis in T1 bladder cancer [33]. In invasive urothelial bladder tumours, MVD was found to be an independent prognostic marker [27, 34, 35]. Our study showed that superficial bladder carcinomas with tumour microvessel clusters (angiogenesis) have an earlier and significantly greater recurrence rate, particularly in pT1/G2 tumours. The results of our method of assessing microvessels could be translated into an angiogenic phenotype and seems to be a functional and useful approach in the clarification of angiogenic profiles and their impact on prognosis in superficial bladder tumours.

We have shown different intensities of VEGF expression, which may reflect the tumour angiogenesis process. A qualitative tumour microvessel assessment was useful and identified an angiogenic tumour phenotype. The presence of microvessel tumour clusters, in superficial papillary UCCs, added prognostic information and identified high-risk patients who could benefit from anti-angiogenic therapeutic regimens.


    Acknowledgements
 
We thank F. Schmitt from the Instituto de Patologia & Imunologia Molecular da Universidade do Porto (IPATIMUP) for a critical review of the manuscript.


    Footnotes
 
+ Correspondence to: Dr L. Santos, Department of Surgical Oncology, Instituto Português de Oncologia, Rua Dr António Bernardino de Almeida, 4200-072 Porto, Portugal. Tel: +351-22-5026489; Fax: +351-22-5026489; E-mail: ucib{at}ipoporto.min-saude.pt Back


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 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
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