eISSN: 2084-9869
ISSN: 1233-9687
Polish Journal of Pathology
Current issue Archive Manuscripts accepted About the journal Supplements Editorial board Abstracting and indexing Subscription Contact Instructions for authors Ethical standards and procedures
SCImago Journal & Country Rank
1/2013
vol. 64
 
Share:
Share:
more
 
 

Microvessel density and expression of vascular endothelial growth factor in clinically localized prostate cancer

Elżbieta Łuczyńska
,
Anna Gasińska
,
Wacław Wilk

Pol J Pathol 2013; 1: 33-38
Online publish date: 2013/04/25
Article file
- Microvessel density.pdf  [0.28 MB]
Get citation
ENW
EndNote
BIB
JabRef, Mendeley
RIS
Papers, Reference Manager, RefWorks, Zotero
AMA
APA
Chicago
Harvard
MLA
Vancouver
 
 

Introduction

Late prostate cancer (PC) recognition is the main cause of treatment failure. Earlier recognition based on biological markers could be helpful in identifying biological differences in benign and malignant lesions, which could further facilitate precise indication for more aggressive post-operative treatment (chemo/radiotherapy). Tumour growth and metastasis are dependent upon tumour angiogenesis, which relies on growth of new vessels toward and within a tumour [1]. Such vascularization may be stimulated by factors released from the tumour cells, tumour-associated inflammatory cells, and/or from the extracellular matrix. In prostate tumour, vascular endothelial growth factor (VEGF) is one of the most potent facilitators in angiogenesis and takes a direct role in oncogenesis as an important hypoxia-inducible pro-angiogenic protein [2, 3]. The proteins by auto- and paracrine mechanisms also induce proliferation, growth and dissemination of tumour cells [4]. In the case of prostatic cancer it was shown that VEGF is connected with cell malignancy and has been linked with an adverse survival outcome [5, 6]. It was also indicated that microvessel density (MVD) has been associated with tumour aggressiveness, PSA recurrence, metastatic capability after radical prostatectomy and patient survival [7-9]. The results on VEGF expression and microvessel density in PC are inconsistent; nevertheless, control of the angiogenic switch and regulation by these soluble factors is of great importance for the rational design of cancer treatment strategies. Therefore, the aim of the study was to establish whether VEGF and MVD in localized PC are associated with pathological tumour stage and grade, which could be helpful in patients’ selection for more aggressive treatment (e.g. adjuvant anti-angiogenic therapy).

Material and methods

Patients



The study involved evaluation of 140 consecutive radical prostatectomy specimens obtained from patients who underwent radical prostatectomy for localized PC between 2007 and 2011. Clinical stage and tumour grade were analysed earlier [10] as this is the same group of patients. The protocol was approved by the Ethical Committee of the Centre of Oncology, and each patient submitted written consent.



Immunohistochemical assessment of tumour markers



Following rehydration, blocking the endogenous peroxidase, in 5 m sections heat-based antigen retrieval was carried out (20-60 min or 1 h at 98°C with 10 mM citric acid buffer, pH 6.0). After 20 minutes, the sections were washed, flooded with 10% normal goat serum for 20 min and incubated with the appropriate antibody overnight at 4°C in a humidified chamber. For CD34 it was a mouse anti-human monoclonal antibody (DAKO, 1 : 200), and for VEGF a mouse anti-VEGF monoclonal antibody (DAKO, 1 : 25) in Tris-buffered saline (TBS, pH 7.4). After washing, slides were incubated at 37°C for 1 h with the DAKO En Vision visualisation system containing goat anti-mouse (CD34) or VECTOR ImmPRESS Reagent Kit (VEGF). The sections were stained with diaminobenzidine (DAB), counterstained with haematoxylin, dehydrated and mounted. Negative control slides omitting the primary antibody were included in each run of stains. The intensity of staining was evaluated by light microscopy at 400× magnification. Cytoplasmic VEGF expression was presented as a percentage of positively immunostained tumour cells (brown) in several (5-7) malignant areas of the tissue section. High-grade prostatic intraepithelial neoplasia foci and non-hyperplastic benign acini were not evaluated; however, they were stained heterogeneously.

For vascular density assessment 7-10 high power (400×) tumour fields, where one field is equivalent to 0.292 mm2, were counted for each patient. We counted highlighted endothelial cells or cell clusters clearly separated from adjacent microvessels, tumour cells and other connective tissue elements regarded as a distinct countable microvessel. The mean vessel count (MVD) per 1 mm2 of tumour volume was used in the analysis.



Statistical analysis



Statistical analysis was performed with STATISTICA vs. 9 (StatSoft Inc.). Intergroup differences in the original data were tested with ANOVA test or Student’s t test. P values of less than 0.05 were considered to indicate statistical significance.

The correlation between the immunohistochemical expression of VEGF and MVD or PSA was determined using Spearman’s test.

Results

We examined human histological specimens from 140 PC patients treated with radical surgery. The mean age of the patients was 62.9 ±6.2 (range 49.0-77.0) years. There were 13 (9.3%) at pTNM stage 1, 78 (55.7%) at stage 2, 40 (28.6%) at stage 3 and 9 (6.4%) at stage 4. In the analysed group there were 75 (53.6%) well-differentiated, 53 (37.8%) moderately differentiated and 12 (8.6%) poorly differentiated tumours.

Pre-operative PSA revealed a mean PSA of 9.9 ±0.5 ng/ml. Concentration of serum PSA was significantly increased with pTNM stage (p = 0.011), Gleason score (p = 0.011) and tumour grade (p = 0.003) (Table I). In 100 (75.7%) tumours positive VEGF expression was shown. Staining was entirely cytoplasmic (Fig. 1) and restricted mainly to secretory cells. Uniform stain intensity was seen within the cells of selected tumour acini; however, heterogeneous staining patterns were observed between different tumour foci. Very occasional positive staining was also visible in endothelial and muscle cells, macrophages and neutrophils as these cells are able to synthesize and secrete VEGF. In the analysed group of tumours the mean percentage of positive VEGF cells was 14.8 ±1.4%. The presence of positively stained cells was not correlated with tumour grade (p = 0.649) and Gleason scores (p = 0.697). However, growth of VEGF expression was observed with increase of pTNM stages. Significantly higher values for the protein were observed in pTNM 3 (p = 0.035) and 4 (p = 0.037) than in pTNM stage 1 (Fig. 2).

In the whole series of tumours the mean MVD was 97.5 ±2.4/mm2. Single cell sprouts as well as larger vessels are included in the counts (Fig. 3). A non-significant decrease in the number of microvessels was observed in the highest pathological tumour volume (P = 0.631), Gleason score (p = 0.368) and tumour grade (p = 0.233) (Table I). Preoperative serum PSA level was not associated statistically with either MVD (p = 0.466) or VEGF expression (p = 0.188). There was also no correlation between the immunohistochemical expression of VEGF and MVD (p = 0.925).

Discussion

We did not observe a significant correlation between VEGF expression, Gleason scores and tumour grade. However, we noted a statistically significant increase of VEGF expression between stages pTNM 1 and pTNM 3 or pTNM 4.

In our study, positive staining for VEGF was seen in 75.7% of cases, which is in accord with previous findings [5, 11]. However, the published data regarding VEGF association with clinico-pathological variables in prostate cancer are conflicting. We did not observe a significant association between VEGF expression and nuclear grade, and in this respect our findings contradict some earlier studies [5, 6, 11]. Although the number of negative VEGF cases decreased with tumour grade – from 19 (25.3%) in G1 to 1 (8.3%) in G3 – this had no influence on increase of percentages of VEGF positive cells. Similarly to the findings of Ferrer et al. [12] we observed more intense VEGF staining in well-differentiated than in poorly differentiated tumours. Therefore, our data are in agreement with those authors who noted no difference in the intensity or distribution of VEGF immunoreactivity between well-, moderately and poorly differentiated tumours [4, 12]. This may indicate that VEGF expression might not be related to malignancy, which could confirm no difference in VEGF immunoreactivity between malignant and benign prostatic epithelium shown by some studies [7, 11]. However, similarly as in earlier studies [11], we observed increased VEGF immunoreactivity with pathological stage. We noted elevation of VEGF expression in stages pTNM 3 and pTNM 4, in which there was seen a subsequent decrease of vascular density.

The mean value of MVD obtained in our study (97.5/mm2) is within the range given by other authors [8, 9, 13-15]. The lowest microvessel density was observed in the highest Gleason scores, tumour grade and pT classification. There was no significant relationship between MVD and pTNM. Lack of correlation between tumour volume and MVD has also been reported by other authors [13, 14, 16, 17]. However, some authors found that MVD is associated with tumour stage [8, 13, 18-20]. Gleason score was associated with MVD in some studies [9, 17, 19-21], but in others, similarly to our results, such a correlation was not indicated [8, 16]. Several key studies concerning MVD in PC are summarized in Table II.

This indicates that data are inconclusive and the differences may depend upon several parameters, such as the kind of endothelial marker (CD31, CD34, factor VIII antibody) used, the way in which the microvessels are analysed [Chalkley point eyepiece graticule, “hot spot” automated digital image analysis, tissue microarray (TMA)] or size of tumour sample analysed (surgical or tumour biopsy) [8, 20, 24], and the small biopsy specimens (TMA) seem to be critical. Counting MVD on large sections appears to be superior to the counting of all microvessels in each TMA spot [20].

In our study, the lack of correlation between MVD and tumour volume is not surprising. In a higher tumour volume with lower oxygen concentrations tumour cells can remain viable, and they can exist at greater distances from the vasculature because they acquire the ability to take up glucose and perform glycolysis. In the same series of tumours we found a significantly larger number of GLUT-1 positive cells in higher than in lower pTNM stages [10]. This may suggest that in pT3 and pT4 tumours the lower vascular number was compensated by glucose uptake for cell metabolism. It is known that in hypoxic conditions, the cells produce hypoxia-inducible factor (HIF), a transcription factor which stimulates the release of VEGF. Circulating VEGF then binds to VEGF receptors on endothelial cells, triggering a tyrosine kinase pathway leading to angiogenesis. In higher tumour stages we observed significant increase of VEGF expression; therefore expression of this protein seems to reflect better tumour progression in PC than MVD.

Recently it has been considered that microvessel density is not a measure of the angiogenic dependence of a tumour. It rather reflects the metabolic burden of the supported tumour cells [25]. Contrary to common belief, microvessel density does not reflect the angiogenic activity but the intercapillary distance. Oxygen and nutrients consumption sets a limit on how far away from the vasculature tumour cells can remain viable. It is approximately 150 µm and viable cells form a viable cuff around a vessel. However, cuff size tends to vary with the tumour metabolic demand [25]. Tumours with low metabolic demand have relatively large cuff sizes with many cell layers and relatively low vascular density. As the metabolic needs of cancer cells vary with the tissue of origin and change with tumour progression [25], MVD may not be an indicator of antiangiogenic efficacy. Current opinion is that a proportion of primary PCs may progress via a non-angiogenic pathway without neo-vascularisation, and be clinically more aggressive than angiogenic tumours [26].

We did not observe a significant correlation between VEGF and PSA level, although such a correlation was found by some authors [11, 27]. In our series, patients who had higher PSA levels manifest lower VEGF expression. This might confirm earlier studies showing that VEGF expression may be controlled by androgens [2, 28]. Also PSA was not related to MVD, similarly as in other authors’ studies [16, 23]. Additionally, we did not observe a correlation between MVD and VEGF expression. However, contradictory results were obtained in other studies [6], where VEGF expression was correlated with MVD.

The assessment of the examined proteins’ expression may be important not only for prediction of biological tumour behaviour but also for development of therapeutic strategies incorporating antiangiogenic drugs with chemotherapy. Perhaps it should be remembered that MVD is not a measure of the angiogenic dependence of a tumour, and may not be an indicator of angiogenic treatment efficacy [20, 25]. Low microvessel density within tumours might not be a sufficient criterion to exclude patients from treatment with angiogenesis inhibitors [26]. Furthermore, rapid tumour growth may not imply high MVD [24, 25].





The authors would like to thank Anna Cichocka M.Sc. for her valuable assistance. This work was supported by

the Polish Ministry of Science and Higher Education (grant no. N403 240837).

The authors report no conflict of interest.

References

 1. Folkman J. What is the evidence that tumors are angiogenesis dependent? J Natl Cancer Inst 1990; 82: 4-6.

 2. Boddy JL, Fox SB, Han C, et al. The androgen receptor is significantly associated with vascular endothelial growth factor and hypoxia sensing via hypoxia-inducible factors HIF-1a, HIF-2a, and the prolyl hydroxylases in human prostate cancer. Clin Cancer Res 2005; 11: 7658-7663.

 3. Gordan JD, Simon MC. Hypoxia-inducible factors: central regulators of the tumor phenotype. Curr Opin Genet Dev 2007; 17: 71-77.

 4. Jackson MW, Roberts JS, Heckford SE, et al. A potential autocrine role for vascular endothelial growth factor in prostate cancer. Cancer Res 2002; 62: 854-859.

 5. Green MML, Hiley CT, Shanks JH, et al. Expression of vascular endothelial growth factor (VEGF) in locally invasive prostate cancer is prognostic for radiotherapy outcome. Int J Radiat Oncol Biol Phys 2007; 67: 84-90.

 6. Borre M, Nerstrom B, Overgaard J. Association between immunohistochemical expression of vascular endothelial growth factor (VEGF), VEGF-expressing neuroendocrine-differentiated tumor cells, and outcome in prostate cancer patients subjected to watchful waiting. Clin Cancer Res 2000; 6: 1882-1890.

 7. Zeng Y, Opeskin K, Baldwin ME, et al. Expression of vascular endothelial growth factor receptor-3 by lymphatic endothelial cells is associated with lymph node metastasis in prostate cancer. Clin Cancer Res 2004; 10: 5137-5144.

 8. de la Taille A, Katz AE, Bagiella E, et al. Microvessel density as predictor of PSA recurrence after radical prostatectomy. A comparison of CD34 and CD31. Am J Clin Pathol 2000; 113: 555-562.

 9. Weidner N, Carroll PR, Flax J, et al. Tumor angiogenesis correlates with metastasis in invasive prostate carcinoma. Am J Pathol 1993; 143: 401-409.

10. Luczynska E, Gasinska A, Wilk W. Expression of Ki-67 (MIB-1) and GLUT-1 proteins in non-advanced prostatic cancer. Pol J Pathol 2012; 63: 272-277.

11. Wu T TL, Wang JS, Jiann BP, et al. Expression of vascular endothelial growth factor in Taiwanese benign and malignant prostate tissue. J Chin Med Assoc 2007: 70: 380-384.

12. Ferrer FA, Miller LJ, Andrawis RI, et al. Vascular endothelial growth factor (VEGF) expression in human prostate cancer: in situ and in vitro expression of VEGF by prostate cancer cells. J Urol 1997: 157: 2329-2333.

13. Brawer MK, Deering RE, Brown M, et al. Predictors of pathologic stage in prostatic carcinoma. The role of neovascularity. Cancer 1994; 73: 678-687.

14. Barth PJ, Weingärtner K, Köhler HH, Bittinger A. Assessment of the vascularization in prostate carcinoma: A morphometric investigation. Hum Pathol 1996; 27: 1306-1310.

15. Borre M, Offersen BV, Nerstr?m B, Overgaard J. Microvessel density predicts survival in prostate cancer patients subjected to watchful waiting. Br J Cancer 1998; 78: 940-944.

16. Rubin MA, Buyyounouski M, Bagiella E, et al. Microvessel

density in prostate cancer: lack of correlation with tumour grade, pathological stage, and clinical outcome. Urology 1999; 53: 542-547.

17. Silberman MA, Partin AW, Veltri RW, Epstein JI. Tumor angiogenesis correlates with progression after radical prostatectomy but not with pathologic stage in Gleason sum 5 to 7 adenocarcinoma of the prostate. Cancer 1997; 79: 772-779.

18. Rogatsch H, Hittmair A, Reissigl A, et al. Microvessel density in core biopsies of prostatic adenocarcinoma: a stage predictor? J Pathol 1997; 182: 205-210.

19. Bono AV, Celato N, Cova V, et al. Microvessel density in prostate carcinoma. Prostate Cancer Prostatic Dis 2002; 5: 123-127.

20. Erbersdobler A, Isbarn H, Dix K, et al. Prognostic value of microvessel density in prostate cancer: a tissue microarray study. World J Urol 2010; 28: 687-692.

21. Arakawa A, Soh S, Chakraborty S, et al. Prognostic significance of angiogenesis In clinically localized prostate cancer (staining for Factor VIII-related antigen and CD34 antigen. Prostate Cancer and Prostatic Dis 1997; 1 32-38.

22. Bettencourt MC, Bauer JJ, Sesterhenn IA, et al. CD34 immunohistochemical assessment of angiogenesis as a prognostic marker for prostate cancer recurrence after radical prostatectomy. J Urol 1998; 160: 459-465.

23. Mucci LA, Powolny A, Giovannucci E, et al. Prospective study of prostate tumor angiogenesis and cancer-specific mortality in the health professionals follow-up study. J Clin Oncol 2009; 27: 5627-5633.

24. Taverna G, Grizzi F, Colombo P, Graziotti PP. Microvessel density estimate: friend or foe in the light of prostate vascular system complexity? World J Urol 2010; 28: 405-406.

25. Hlatky L, Hahnfeldt P, Folkman J. Clinical application of antiangiogenic therapy: microvessel density, what it does and doesn’t tell us. J Nat Can Inst 2002; 94: 883-893.

26. Taverna G, Colombo P, Grizzi F, et al. Fractal analysis of two-dimensional vascularity in primary prostate cancer and surrounding non-tumoral parenchyma. Pathol Res Pract 2009; 205: 438-444.

27. Joseph IB, Nelson JB, Denmeade SR, Isaacs JT. Androgens regulate vascular endothelial growth factor content in normal and malignant prostatic tissue. Clin Cancer Res 1997; 3: 2507-2511.

28. Wegiel B, Evans S, Hellsten R, et al. Molecular pathways in the progression of hormone-independent and metastatic prostate cancer. Curr Can Drug Targets 2010; 10: 392-401.



Address for correspondence



Anna Gasinska


Department of Applied Radiobiology

Centre of Oncology – Maria Sklodowska-Curie Memorial Institute

Garncarska 11

31-115 Cracow, Poland

tel. +48 12 423 10 73

fax +48 12 422 66 80

e-mail: z5gasins@cyf-kr.edu.pl
Copyright: © 2013 Polish Association of Pathologists and the Polish Branch of the International Academy of Pathology This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International (CC BY-NC-SA 4.0) License (http://creativecommons.org/licenses/by-nc-sa/4.0/), allowing third parties to copy and redistribute the material in any medium or format and to remix, transform, and build upon the material, provided the original work is properly cited and states its license.
FEATURED PRODUCTS
Quick links
© 2019 Termedia Sp. z o.o. All rights reserved.
Developed by Bentus.
PayU - płatności internetowe