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
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Abstract |
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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
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Introduction |
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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) markersanti-CD31, -CD34, -factor VIII and -endoglin antibodieswere used in an attempt to identify high-risk patients who might benefit from anti-angiogenic therapeutic approaches.
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Patients and methods |
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Immunohistochemistry
The avidinbiotinperoxidase 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 avidinbiotinperoxidase complexes (VectaStain ABC Kit) for 30 min. Reaction products were visualised with diaminobenzidine as the chromogen and sections were counterstained with Harriss 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 ECswith at least two of the CD31, CD34, endoglin or factor VIII markersdistinct 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 Fishers 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 KaplanMeier 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 Coxs 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.
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Results |
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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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Discussion |
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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 and -2
, 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.
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Acknowledgements |
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Footnotes |
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References |
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2. Heney NM, Ahmed S, Flannagan MJ et al. Superficial bladder cancer: progression and recurrence. J Urol 1983; 130: 10831086.[ISI][Medline]
3. Malkowicz SB. Intravesical therapy for superficial bladder cancer. Semin Urol Oncol 2000; 4: 280288.
4. Cookson MS, Herr HW, Zhang ZF et al. The treated natural history of high risk superficial bladder cancer: 15-year outcome. J Urol 1997; 158: 6267.[ISI][Medline]
5. Streeter EH, Harris AL. Angiogenesis in bladder cancerprognostic marker and target for future therapy. Surg Oncol 2002; 11: 85100.[CrossRef][ISI][Medline]
6. Inoue K, Slaton JW, Karashima T et al. The prognostic value of angiogenesis factor expression for predicting recurrence and metastasis of bladder cancer after neoadjuvant chemotherapy and radical cystectomy. Clin Cancer Res 2000; 6: 48664873.
7. OBrien T, Cranston D, Fuggle S et al. Different angiogenic pathways characterise superficial and invasive bladder cancer. Cancer Res 1995; 55: 510513.[Abstract]
8. Crew JP, OBrien T, Bradburn M et al. Vascular endothelial growth factor is a predictor factor of relapse and stage progression in superficial bladder cancer. Cancer Res 1997; 57: 52815285.[Abstract]
9. Jaeger T, Weidner N, Chew K et al. Tumor angiogenesis correlates with lymph node metastases in invasive bladder cancer. J Urol 1995; 154: 6971.[ISI][Medline]
10. Reiher F, Ozer O, Pins M et al. p53 and microvessel density in primary resection specimens of superficial bladder cancer. J Urol 2002; 167: 14691474.[ISI][Medline]
11. WHO. Histological typing of urinary bladder tumours. International Histological Classification of Tumours, no. 10. Geneva, Switzerland: World Health Organization 1973.
12. American Joint Committee on Cancer. AJCC Cancer Staging Manual, 5th edition. Philadelphia, PA: Lippincott-Raven 1997; 241246.
13. Pow-Sang JM, Seigne JD. Contemporary management of superficial bladder cancer. Cancer Control 2000; 7: 335339.[Medline]
14. Stein JP. Indications for early cystectomy. Semin Urol Oncol 2000; 4: 289295.
15. Lamm DL, Rigs DR, Traynelis CL et al. Apparent failure of current intravesical chemotherapy prophylaxis to influence the long-term course of superficial TCC of the bladder. J Urol 1995; 153: 14441450.[ISI][Medline]
16. Pawinski A, Sylvester R, Kurth KH et al. A combined analysis of European Organization for Research and Treatment of Cancer and Medical Research Council randomized clinical trials for the prophylactic treatment of stage Ta, T1 bladder cancer. J Urol 1996; 156: 19301940.
17. Kerbel RS, Klement G, Pritchard KI, Kamen B. Continuous low-dose anti-angiogenic/metronomic chemotherapy: from the research laboratory into the oncology clinic. Ann Oncol 2002; 13: 1215.
18. Miller KD, Sweeney CJ, Sledge GW. Redefining the target: chemotherapeutics as anti-angiogenics. J Clin Oncol 2001; 19: 11951206.
19. Bello L, Carrabba G, Giussani C et al. Low-dose chemotherapy combined with antiangiogenic drugs reduces human glioma growth in vivo. Cancer Res 2001; 61: 75017506.
20. Sato K, Sasaki R, Ogura Y et al. Expression of vascular endothelial growth factor gene and its receptor (flt-1) gene in urinary bladder cancer. Tohoku J Exp Med 1998; 185: 173184.[ISI][Medline]
21. Campbell SC, Volpert OV, Ivanovich M, Bouck NP. Molecular mediators of angiogenesis in bladder cancer. Cancer Res 1998; 58: 12981304.[Abstract]
22. Izawa JI, Dinney CP. The role of angiogenesis in prostate and other urologic cancer: a review. CMAJ 2001; 164: 662670.
23. Schweiki D, Itin A, Soffer D, Keshet E. Vascular endothelial growth factor induced by hypoxia initiated angiogenesis. Nature 1992; 359: 843845.[CrossRef][ISI][Medline]
24. Jones A, Fujiyama C, Blanche C et al. Relation of vascular endothelial growth factor production to expression and regulation of hypoxia-inducible factor-1 and hypoxia-inducible factor-2
in human bladder tumors and cell lines. Clin Cancer Res 2001; 7: 12631272.
25. Reiher FK, Ivanovich M, Huang H et al. The role of hypoxia and p53 in the regulation of angiogenesis in bladder cancer. J Urol 2001; 165: 20752081.[ISI][Medline]
26. Turner KJ, Crew JP, Wykoff CC et al. The hypoxia-inducible genes VEGF and CA9 are differentially regulated in superficial vs invasive bladder cancer. Br J Cancer 2002; 86: 12761282.[CrossRef][ISI][Medline]
27. Chaudhary R, Bromley M, Clarke NW et al. Prognostic relevance of micro-vessel density in cancer of the urinary bladder. Anticancer Res 1999; 19: 34793484.[ISI][Medline]
28. Bochner B, Esrig D, Groshen S et al. Relationship of tumor angiogenesis and nuclear p53 accumulation in invasive bladder cancer. Clin Cancer Res 1997; 3: 16151622.[Abstract]
29. Sapino A, Bongiovanni M, Cassioni P et al. Expression of CD31 by cells of extensive ductal in situ and invasive carcinomas of the breast. J Pathol 2001; 194: 254261.[CrossRef][ISI][Medline]
30. McKenney J, Weiss S, Folpe A. CD31 expression in intratumoral macrophages: a potential diagnostic pitfall. Am J Surg Pathol 2001; 25: 11671173.[CrossRef][ISI][Medline]
31. Sagol O, Yorukoglu K, Sis B et al. Does angiogenesis predict recurrence in superficial transitional cell carcinoma of the bladder? Urology 2001; 57: 895899.[CrossRef][ISI][Medline]
32. Dinney CP, Babkowski RC, Antelo M et al. Relationship among cystectomy, microvessel density and prognosis in stage T1 transitional cell carcinoma of the bladder. J Urol 1998; 160: 12851290.[ISI][Medline]
33. Korkolopoulou P, Konstantinidou AE, Kavantzas N et al. Morphometric microvascular characteristics predict prognosis in superficial and invasive bladder cancer. Virchows Arch 2001; 438: 603611.[CrossRef][ISI][Medline]
34. Dickinson AJ, Fox SB, Persad RA et al. Quantification of angiogenesis as an independent predictor of prognosis in invasive bladder carcinomas. Br J Urol 1994; 74: 762766.[ISI][Medline]
35. Philp EA, Stephenson TJ, Reed MW. Prognostic significance of angiogenesis in transitional cell carcinoma of the human urinary bladder. Br J Urol 1996; 77: 352357.[ISI][Medline]