Significance of microvessel density in prostate cancer core biopsy

Background/Aim. In prostate tumors, angiogenesis, measured as microvessel density, is associated with tumor stage and Gleason score. The aim of this study was determine neovascularization of prostatic adenocarcinomas in core biopsies and corresponding prostatectomies. Methods. The study population included 61 patients who underwent radical prostatectomy (RP) for localized prostate carcinoma patients and did not receive chemohormonal, or radiation therapy before surgery. Tumor blocks were immunostained using the endothelial-specific antibody CD31 and subsequently evaluated at  400 magnification in both biopsies and corresponding prostatectomies. Results. When comparing microvessel density in core biopsies and corresponding prostatectomies, no statistically significant difference was found (p > 0.1). A statistically significant positive correlation was found when determining correlation between microvessel density (as linear and categorical variable, i.e. with the cut-off value of 48) that was associated with the Gleason score (p < 0.05) and tumor stage (p < 0.0001). There was no correlation between microvessel density and preoperative values of serum prostate-specific antigen (PSA) (p > 0.1). Conclusion. Microvessel density can be reliably applied to needle prostate biopsy specimens. Quantification of the microvascular density in biopsies is an accurate pre-operative predictor of tumor stage, discriminating between organ-confined and organ-extending neoplasms.


Introduction
The incidence of prostate cancer (PC) is on an exceptional increase in the whole world, thanks to early detection programs that include digital rectal examination, determining of serum prostate-specific antigen (sPSA), transrectal ultrasonography and needle biopsy of the prostate 1, 2 .This is a heterogeneous disease with unpredictable clinical flow from a relatively indolent disease to an aggressive form with rapid metastatic spreading of the disease and fatal outcome.Unfor-Correspondence to: Aleksandra Salapura Dugonjić, Cara Lazara 6, 78 000 Banja Luka, Republika Srpska.Phone: +387 51 463 273; Email: salapura_aleksandra@yahoo.com tunately, there are still no parameters that can be safely used to foresee whether it is a locally non-invasive prostate cancer (pT2) or invasive and metastasis expanded neoplasm (pT3).According to data from different researches published so far, PC in 24-60% patients was clinically under-staged before surgery, whereas in 8-45% of cases it was over-staged [3][4][5][6][7][8][9] .Potential biomarkers are still being researched as well as different methods of diagnostics, which could improve detection, preoperative grading and staging systems for prostate cancer in order to get a clearer picture about possibilities and risk of the application of adequate therapy procedures for each individual patient.Angiogenesis (neovascularization) is the process of creation of new functional capillary microvessels from the already existing vascular network.Vascularization of the primary tumor results in an expanded growth and the tumor then gets metastatic potential, whereas development of microvessels is necessary for growth of distant metastatic tumor hotspot 10 .
Tumor microvessels are not the same as the microvessels of normal tissue, they are heterogeneous in terms of organization, function and structure 11 .Angiogenesis is present in all the tumors, but with characteristic and significant differences between different types of tumors (the biggest intensity of angiogenesis was found with glioblastoma, followed by renal cell carcinoma, colorectal cancer, breast cancer, lung cancer, PC), but inside every individual type of tumor in different patients -individual tumors are well vascularized, while the others are poorly vascularlized 12 .The prognostic value of microvessel density (MVD), as a measure of tumor angiogenesis, is still unclear in the PC, particularly on samples of needle prostate biopsies [13][14][15] .Thus, the aim of this study was to determine prognostic significance of MVD, as a stage predictor in prostatic carcinomas in core biopsies (CB) and corresponding prostatectomies.

Methods
This retrospective study included 61 previously untreated patients with prostatic adenocarcinoma admitted with elevated serum PSA levels at the Clinic of Urology, Clinical Center of Vojvodina, in the period 2005-2006.All the patients underwent systematic sextant transrectal ultrasonography-guided core biopsies performed with an 18-gauge automated spring-loaded biopsy gun.The diagnosis of prostatic carcinoma in needle biopsies was followed by radical retropubic prostatectomy (RP) with bilateral pelvic lymphadenectomy.All tumors were primary diagnosed without previous therapy and none of the patients had clinical evidence of metastasis prior to surgery.Tumor grading on needle biopsies and prostatectomy specimens were undertaken according to Gleason.The final pathological stage on the whole mount prostatectomy specimens was determined according to the tumor-nodus-metastasis (TNM) system.
From these 61 patients, a total of 366 core biopsies were available, out of which 254 contained carcinomatous tissue (median 2; range 1-6 per case).These 254 biopsies were analyzed and only those containing at least two microscopic fields of neoplastic glands at x400 magnification were selected for determination of MVD.Insufficient tumor tissue was found in 55 core biopsies, and these biopsies were excluded.Finally, analyses were performed on 199 core biopsies.All RP specimens from 61 patients were evaluated in a standard fashion.Surgical margin (SM) sections from the apex and base were taken as shaved margins.Extraprostatic extension (EPE) was diagnosed if tumor was seen in the periprostatic soft tissue or was seen penetrating through a fibromuscular capsule and coming out on the other side.The seminal vesicles (SVs) were evaluated at the junction where they enter the prostate gland.All pelvic lymph nodes were evaluated for the presence of metastatic disease.All the cases were assigned with a Gleason sum (GS).After review of each case, the blocks with the highest GS and greatest density of tumor and those containing at least two microscopic fields of neoplastic glands at 400 magnification were selected for immunohistochemical staining.The study excluded the patients who had received prostaterelated therapy before RP, including androgen deprivation therapy, chemotherapy, radiation therapy, or other therapy.It also excluded the patients in whom there was no cancer remaining in the needle biopsy tissue to perform MVD analysis, as well as matched totally embedded RP specimens.
Serum PSA concentration was determined before RP and analyzed as a continuous and categorical variable with the cutoff value 10 ng/mL and 20 ng/mL.

Immunohistochemistry
Tumors from 61 patients -199 core biopsies with sufficient tumor areas, as well as sections from 61 selected tissue blocks of corresponding RP were analyzed by immunohistochemistry. Routine formalin-fixed, paraffin-embedded 3-4µm-thick sections from each patient were attached to silanized slides, sequentially deparaffinized and rehydrated.Access to tissue antigen sites for antibody attachment was enhanced by microwaving slides which were treated by citrate buffer for 20 minutes.Detection of microvessels was performed using a monoclonal antibody against the CD31 antigen (clone JC/70A; Dako, Glostrup, Denmark).Dilution of the primary antibody was 1 : 40 in Tris buffered saline (TBS) / 1% BSA / 1% human serum and were incubated for 30 minutes.The EnVision technique and development with the chromogen 3,3´diaminobenzidine tetrachloride (DAB) was used for visualization.Sections were lightly counterstained with hematoxilyn.Intense cytoplasmic immunoreactivity was observed in endothelial cells of small, medium-sized and large blood vessels in all study specimens.Normal prostate biopsy tissue served as a negative control after deletion of the primary antibody step and substitution of buffer during each run to suppress microvessel staining.

Determination of microvessel density
MVD was determined by light microscopy analysis for the areas of the tumor containing the most capillaries and small venules (microvessels, neovascular "hot spots") using the counting method introduced by Weidner and modified by Rogatsch 13,[16][17][18] .Prostate cancers are multifocal and heterogeneous in their MVD.The tumor area in CB and RP specimens containing the maximum number of descrete (brown) microvessels staining for CD31 was identified by scanning at low power (40 Salapura Dugonjić A, et al.Vojnosanit Pregl 2015; 72 (4): 317-327.and 100) 13,16,17 .These areas were most frequent at the margins of carcinoma.After identification of the three areas of highest neovascularization, individual microvessels were counted at 400 magnification, where one field is equivalent to 0.19 mm² representative 400 high power fields (Olympus BH-2 microscope, Olympus Optical Co. Ltd., Japan) 18 .Both isolated immunoreactive brown-staining endothelial cells and endothelial cell clusters, separate from adjacent microvessels clearly, tumor cells and connective-tissue elements, were considered countable vessels.Vessel lumens do not need to be considered as a microvessel and red blood cells were not used to define a vessel lumen.Exclusion of occasional immunoreactive macrophages and plasma cells was based on their morphological appearance 13,16,17 .The highest readings in CB and corresponding prostatectomy were expressed as the highest number of microvessels identified within any single 400 field.An average of multiple fields was not used.Assessment of MVD was done without knowledge of any clinicopathological data.MVD within normal prostate tissue and hyperplastic nodules served as internal control.

Statistics
The correlation of MVD (in a categorical and continuous fashion) in biopsies and corresponding prostatectomies was calculated using the MANOVA.Microvascular counts in organ confined (pT2) versus organ-extending tumors (pT3) were compared with MANOVA and χ2 test.To determine the relationship between tumor grade, final pathological stage and MVD in biopsies and prostatectomies, the median value, i.e.48 of microvessel counts of 61 tumors was set as the cut-off point.The χ2 test, MANOVA, discriminative analysis, Pearson coefficient (χ) multiple correlation coefficient (R) were applied to identify the associations between MVD counts of 48 and less of 48 and more than 48 and final pathologic results, using a significance level of 0.05, and level of 0.001 for a very high statistical significance.

Clinical findings
The mean age was 66 years (SD ± 5.28; range from 52 to 78) at the time of surgery.The pretreatment serum PSA ranged from 2.8 to 73.3 ng/mL (mean 14.73 ± 12.75 ng/mL) (Table 1).PSA (continuous variable) was examined for the association with pT (MANOVA: p = 0.817, that is χ 2 test: p = 0.602) and GS (MANOVA: p = 0.901, that is χ2 test: p = 0.949) both as a continuous variable and in a categorical fashion (< 10 ng/mL vs 10-20 ng/mL vs > 20 ng/mL).No statistically significant association was seen (Tables 2 and 3).
Final pathological staging in RP of 61 tumors fulfilling the selection criteria recorded 37 (61%) as pT2, and 24 (39%) as pT3 (organ-extended) (Table 1).The 24 pT3 cases demonstrated EPE in all the cases of which seminal vesicles invasion in 19 (31%).These scores correlated significantly with tumor stage when analyzed by the χ 2 test (p < 0.0001 for biopsies and prostatectomies, respectively) as shown in Table 2. Regional lymph node metastases were present in 6 (10%) cases, all of them were pT3 and GS ≥ 7 (GS 7-2 cases, GS 8-3 patients and GS 9-1 case).А statistically significant correlation was found between metatases in regional lymph nodes on one side and pT (p = 0.001; χ 2 test), and GS (p = 0.062 χ 2 test) (Tables 2 and 3) on the other side.The median Gleason score for all tumors was 6 (range 4-8) in core biopsies and 7 (range 4-9) in prostatectomies.There was a significant discordance between biopsy and matched prostatectomy grades.Needle core biopsy underestimated tumor grade in 39% of cases (15 cases: GS6→GS7; 4 cases: GS7→GS8; 2 patients: GS6→GS8; 2 cases: GS5→GS6; and 1 patient in GS5→GS7, GS4→GS6, GS 7→GS 9) and overestimated in 1% (1 case: GS 8→GS 7).pT2 tumors scored 6 (range 4-8) in biopsies and 7 (range 4-9) in RP; the median score in carcinomas staged as pT3 was 7 (range 4-8) in core biopsies and in RP (range 4-9).These scores correlated significantly with the tumor stage when analyzed by the χ 2 test (p < 0.0001 for biopsies and prostatectomies, respectively) as shown in Tables 2 and 3.The median number of biopsies per case involved by cancer was 3 (range 1-5).Immunostaining for CD31 exhibited intense and homogeneous staining of the endothelial cells of blood microvessels on all the 61 examined cases, as evidenced by positive staining of non-tumor-associated vessels (Figures 1 and 2).It did not react with lymphatic endothelium or fibroblasts.Immunoreactivity was also observed in a small number of macrophages and plasma cells.MVD ranged from 22 to 89 (mean 49.84 ± 13.36) in core biopsies and 46.85 ± 14.47 in prostatectomies).When comparing these values, no statistical significance was found (MANOVA: p = 0.239).MVD (continuous variable) was examined for the association with pT both as a continuous variable and in a categorical fashion (pT2 vs pT3).A very high statistical significance was evident when the microvessel density in prostatectomies and the final patho-logical stage were compared (MANOVA: p = 0.001) (Table 4, Figure 3).This difference in MVD between pT2 and pT3 tumors was confirmed by univariate analysis when the mean microvessel count of all tumors, ie, 48, was set as a cut-off value (χ 2 : p = 0.017) (Figure 3, Table 4).A statistically significant correlation was found between metatases in regional lymph nodes (pN0 vs pN1) and MVD (MANOVA; p = 0.071 for continous and χ 2 : p = 0.038 for categorical variable) (Ta-ble 4).MVD was examined for association with GS both as a continuous variable and in a categorical fashion (≤ 48 vs > 48).Although a statistically significant discrimination between low-and high-grade tumors was found (MANOVA: p = 0.044 for continuous and χ 2 test: p = 0.039 for categorical variable), it did not reach the level of final pathological stage.An increase in MVD was not associated with pretreatment PSA as a continous variable (MANOVA: p = 0.393) or as a categorical variable (χ 2 test : p = 0.279) (Table 4).All those variables previously analyzed (clinical and pathological characteristics) were statistically significantly associated with final pathological stage (pT).Additionally, univariate analyses (Pearson test, multiple correlation coefficient) and multivariate analyses (MANOVA; discriminative analysis) were done in order to determine the most significant factors in prediction organ-confined (pT2) versus organextending prostate cancer (pT3).The Gleason score (p < 0.001) and metastases in regional lymph nodes (p = 0.001) showed a strong statistical significance.MVD also showed statistical significance (p = 0.017), but not so strong as previously mentioned (Table 5).Preoperative serum PSA alone was not significant in predicting final pathological stage (p = 0.322) (Table 6).

Discussion
If prostate cancer is diagnosed on time, whether primary or recurrent, it may be curatively treated.The process of the very diagnostics, screening and staging of the disease is controversial, due to limitation in its disclosure 14 .As a result of widespread testing of patients for sPSA over the past decade, most patients with prostate cancer now present with the clinically localized disease, and their tumors are rarely graded with Gleason scores < 6 19 .In general, serum PSA levels correlate with a larger tumor volume, advanced pathologic stage and higher grade [20][21][22] .Although higher grade cancer produces less PSA per cell, when compared to lower grade tumors, overall, poorly differentiated tumors are associated with higher PSA levels as these tumors tend to be larger and of a more advanced stage 23 .There are exceptions with very high grade prostate cancers which are so poorly differentiated that associated serum PSA levels are disproportionally low 20 .In our study, a statistically significant correlation was not found between serum PSA levels (as continuous and categorical variables) and stage of disease (pT2 vs pT3), and also between sPSA and tumor grade (Gleason score) (<7 vs ≥ 7).Preoperative serum PSA in our investigation, as in some other studies, showed that it could not give useful pathologic correlations on individual basis, for each patient 5,9,22,[24][25][26] .A significant overlap of sPSA values between different tumor stages (pT2 vs pT3) did not enable clear distinction between these values (regarding organconfined prostate carcinoma versus organ-extending carcinoma) in relation to locally limited PC, namely locally invasive PC.
Clinical doctors classify patients with newly diagnosed PC by stage and grade.This classification is important because of the extraordinary variability in the potential for disease progression.Tumor grade, stage, and the presence of competing medical hazards are the most powerful predictors of survival 19 .There is a significant discordance between biopsy and matched prostatectomy grades.Needle core biopsy underestimates tumor grade in 33-45% of cases and overestimates in 4-32% [27][28][29] .Grading errors are common in biopsies with small amounts of tumor and low-grade tumor 30 .In our study, Gleason score values before and after the surgery differed in 40.98% of the patients.Needle core biopsy underestimated tumor grade in 39.34% of the cases, with the greatest discordance in distinguishing GS 6 from GS 7. Needle core biopsy overestimated tumor grade in one patient 81.63% (GS 8 versus GS 7 on matched prostatectomy).Judging by univariate and multivariate studies, tumor grade is one of strongest and most useful prognostic parameters which forecast tumor stage 20,27,31 .This possibility of forecasting can be applied to almost every determination of pathologic tumor stage, including EPE (extraprostatic extension), SVI (seminal vesicle invasion), regional node metastasis, and bone metastasis.In research, patients are usually grouped according to the Gleason score as low risk (GS < 7), medium-risk (GS 7) and high-risk groups (GS 8-10).In some studies, patients with the Gleason score 7 have the same disease outcome and behavior of PC as the ones with GS 8-10, so some researchers put them in the same group 18 .According to some researchers, GS ≥ 8 determined on needle core biopsy as a strong prognostic factor which indicates the possibility of the existence of regional lymph node metastasis 32 .Both sPSA and GS can provide significant prognostic data when their values are at either high (sPSA < 20 ng/mL, GS 8-10), or low (sPSA < 4 ng/mL, GS 2-4) level.However, the majority of patients are exactly in the middle, namely with GS 7 and intermediary level sPSA 22 .Our study shows a statistically high correlation between tumor stage and grade, namely 95.8% of patients with GS ≥ 7 had locally invasive PC (pT3).Also, all the patients with regional lymph nodes metastases (pN1) had poorly differentiated PC (GS ≥ 7).A need for correct preoperative tumor stage determination is essential, especially after studies on massive tumor series clinically diagnosed as organ-confined prostate cancer (T2), out of which approximately 24-60% after RP had pathologic confirmation of locally invasive and metastatically spread disease (pT3, pN1) (1.3-8).In 8-45% patients PC were preoperatively clinically over-staged 9 .In our research, after radical prostatectomy, 61% PC were in tumor stage pT2, and 39% were in a stage pT3, while as 10% patients had metastatically spread disease.
Since 1971, when Folkman determined that tumor growth and dissemination depended on angiogenesis and also that tumors, along with inflammatory cells and related vasculature, created a complex ecosystem which communicates through chemical signals, many studies have been made in order to support this theory 33 .MVD varies widely depending on tumor type; all tumors, including the ones with smallest MVD, depend on angiogenesis 34 .
Reference publications contain numerous conflicting studies which relate to the possibility of angiogenesis to predict pathological stadium for patients with clinically organconfined prostate carcinoma.Many studies proved the connection between MVD and GS, disease stage, as well as possibilities of metastatic spread of carcinoma in future, while as in other studies MVD in relation to stage pT has not shown superior predictive value (Table 6).Still, most researchers estimate that MVD increased values have a prognostic significance in estimation of biological behavior of PC.In a Weidner et al. 35 research, patients with metastasis had double higher values of MVD in relation to locally invasive PC, and higher values of MVD were related to higher Gleason score, but only in poorly differentiated PC.Offersen et al. 36 points to the fact that the maximum value, and not the median one MVD, is significantly associated with survival estimation for patients with CP.In a Bostwick-lead multiinstitutional study, logOMVD (optimized microvessel density) was statistically significantly correlated with GS and pre-operative sPSA values, as well as with pT3.However, disease outcome forecasted by OMVD did not relate to patients who had organ-confined disease (pT2) and GS 6-9 21 .In our research, higher MVD continual and categorical variables were correlated with poorly differentiated prostate carcinoma, namely GS ≥ 7, with higher tumor stage (pT3), namely they were higher in metastatic spread disease (pN1).Such results have been confirmed in studies of many au-Salapura Dugonjić A, et al.Vojnosanit Pregl 2015; 72 (4): 317-327.thors 4, 6, 18, 37-41 .Our study does not show a statistically significant correlation with pre-operative values of sPSA, which is in line with certain findings listed in the literature 5,9,25,42 .In a tissue microarray study (TMA), used on the largest number of samples so far (3261 RP), Erbersdobler et al. 43 prove using univariate analyses, a significant correlation between an increased MVD and advanced stadium of PC, pT3 (p < 0.001), as well as a higher GS, (GS ≥ 7) (p < 0.001), but also points out to the existence of significant differences between tumors, taking into account their localization, that is, that the transitional zone tumors (TZ) have a lower MVD, compared to the peripheral zone (PZ) tumors.However, MVD has not been proved to be an independent prognostic parameter in multivariant analyses, instead it is closely connected to the other factors contributing to tumor aggression.The authors point out that if the antiangiogenic therapy for prostate cancers has not been established yet and if it starts being applied, knowing the differences in MVD between individual tumors and tumor locations (TZ versus PZ tumors) would become significant 43 .Steiner et al. 15 has established a mild correlation between mRNA of individual endothelial factors (CD31, CD 34) in prostate cancer tissue compared to the histologically determined MVD, even though higher values of histologically determined MVD were statistically significantly related to higher GS and stadium, pT3 < 0.001).
Contrary to the previously reported research results, certain authors have reached completely opposite conclusions in their studies 41,44 .Silberman et al. 41 determined a correlation between MVD and tumor progression after RP, but not with pathologic stage in patients who had GS 5-7.A correlation between MVD and disease stage, as well as metastasis, was not proved in the work of Matsushima et al. 45 , while a correlation between MVD and Gleason grade was statistically almost significant.Rubin et al. 25 also did not find a correlation between MVD and GS, tumor stage, positive surgical margins or seminal vesicle invasion, but also not with increased postoperative sPSA values as a sign of disease recurrence.By using multivariate analysis (using estimation p53, retinoblastoma, chromogranin A and MVD), Krupski et al. 46 has determined that MVD values showed no prognostic significance of importance in comparison to p53 and retinoblastoma in estimation of patient survival.Gettman et al. 5 found no correlation between OMVD and DNA ploidy, Gleason grade, pathologic stage, or with sPSA (preoperative serum PSA), neither the application of univariate and multivariate analysis proved OMVD as a predictor of clinical or biochemical disease recurrence.By using the image analysis system for determination MVD, van Niekerk et al. 47 determined no consistent increase of MVD in TZ tumors in terms of the surrounding unchanged tissue of prostate and benign tissue hyperplasia, unlike PZ prostate cancer, which had almost double increase of MVD value, explaining this with intrinsic biological differences between these two zonal types of tumors (such as heterogeneous of microvasculature of TZ tumor), which, at least partly, condition their different biological behavior.In this study, no correlation was found between MVD and Gleason score of TZ and PZ tumor.Unlike van Niekerk et al, Tretiakova et al. 48in their research using computer analysis of MVD conclude that MVD is not statistically significantly increased in PC compared to the normal surrounding tissue of the prostate, as well as neither in low grade PC (GS ≤ 3 + 4) compared to high grade PC (GS ≥ 4 + 3), as well as that MVD cannot be considered a useful prognostic parameter.Taverna et al. 49,50 , examining two-dimensional geometrical complexity of vasculature of PC, divided the patients in two groups, taking into account increase/decrease of fractal dimension of tumorous vascular surface and surrounding non-tumorous tissue, establishing that the patients with a lower tumorous vascular surface had a worse clinical outcome, that is, that the tumor progression was not dependant on angiogenesis.At the end, Taverna et al. 51 leave an open question as to whether angiogenesis is a "canonic hallmark" of PC and point out that there are no powerful methods of quantifying the reversal of neovascularity.
The majority of these studies, using different antibodies, methods of counting and selection, show some significant correlations between MVD and poorly differentiated PC and shorter patient survival, suggesting that MVD is a strong measurer of tumor angiogenic activity.The controversy of results is a consequence of practical problems which limit the usage of MVD measuring on surgical material, namely there is no consensus neither on vessels counting nor on "cut-off'" value which would differentiate/separate high-and low-grade neoplasms.Different methodologic problems occur while counting blood vessels, such are different observations by different pathologists, even the same pathologist, during the first count/selection of areas with most intensive neovascularisation ('hot-spot'), as well as heterogeneity of tumor, which remain unresolved and can therefore influence on results of immunohistochemistry analysis.The number of published results on MVD up to now is 14-300, and along with that 'cut-off', which varies between 23-160 35,39,43,52,53 .In our research the mean value of MVD on samples of needle core biopsies was 49.84 ± 13.36 (22-78), and on samples of matched RP 46.85 ± 14.47 (28-89), and after their comparison there was no statistically significant difference, while as 'cut-off' value was 48.If one neglects the variations in patient selection (for example, Tretiakova et al. 48divide 67% of patients with GS 7 PC to low grade PC group (≤ 3 + 4) and high grade PC group (≥ 4 + 3), one finds that those different values are mostly conditioned by different techniques of tumor blood vessels counting.These differences can be a consequence of endothelial antibodies choice, selection of vascular parameters, choice of tumor field in which measurement is done, vessel counting method, determination of 'cut-off' values which is used in correlation analysis along with other clinic and pathologic variables and survival, as well as wrong statistical methods which are used.Selection of 'cut-off' values was based on personally estimated median value of MVD, bellow which the prognosis was good, and above it bad, and therefore it had to be seen arbitrary until valid values were identified.Such differences can depend on the fact whether MVD is estimated on periphery, or in the center of tumor 39 .As it was shown that there was a strong correlation in MVD values gained by using different antibod- ies, where CD-31, which we used in our study, was shown to be more sensitive, showing 18-33% higher results of microvessel counting, MVD, than some other antibodies which are usually used (for example, CD34 is detectable, except in endothelial cells, in mesenchymal and inflammatory cells and lymphatic vessels), the biggest discrepancies related to other of the listed reasons 18,45 .Studies that could not confirm the prognostic significance of MVD were the ones which mostly differed from the methodology described by Weidner et al. 16,17,35 .Each of the previously mentioned authors used numerous modifications of this method (e.g., determination of the so-called 'hot spots', namely areas with the highest number of blood vessels, how many fields are counted and where, whether focuses overlap).This study also used a modification of the Weidner 13 method of microvessel counting on needle biopsy and matched radical prostatectomy specimens on a high-power microscopic field, 400.This method selection in our research has a foundation in certain studies on different tumors with good correlations between results of blood vessels calculation at 400 magnification in relation to 200 magnification 16 .The approach chosen in this investigation has also beensupported by several studies performed on other human tumor types showing good correlations between vascular performed in 400 versus 200 magnifications 13,18 .MVD values in our study are higher in relation to previous studies 35,53 .A possible explanation of such results is, as Rogatsch et al. 18 stressed, that higher resolution 400 results in MVD value increase by 11-33% when it is compared to 200 magnification as shown breast carcinoma 16,17 .Application of image analysis system in histologically determined MVD, as was suggested by a few groups of researchers is more expensive, more demanding, unsuitable for routine application, and is not more accurate in comparison to calculation done by other researchers in person 5,6,21,25,[47][48][49] .Precisely, these differences in the manner of determination of MVD in many studies, disable their adequate interpretation and comparison, thus a consensus-agreed methodology for determination of MVD could be used to provide more proper comparison and interpretation of MVD values when compared to clinical and pathological parameters.
Having in mind the values of GS and MVD, noninvasive imaging technique that can reflect both GS and MVD to be able to provide timely diagnostics and determination of PC characteristics 14 .Histological heterogeneity and multifocality of PC limit use of needle biopsy in determination of all carcinomas grades and sites 9 .What would be valuable for choosing targets for prostate biopsies would be an imaging method, which could indicate increasing in MVD and it could also provide a foreseeable Gleason score.This should result in a change of biopsy strategy, the outcome of which would be a higher detection rate of prostate cancer and a more accurate Gleason grading, meaning, a more adequate therapeutic strategy 9 .In line with this, the most used conventional imaging methods today are ultrasonography with moleculary targeted constrast microbubbles (CEUS) and magnetic resonance imaging (MRI), amended with moleculary, metabolic and functional imaging techniques 14 .Some researchers correlate the results obtained by imaging methods with histologically determined MVD. Lee et al. 54 evaluated tumorous angiogenesis using the mouse xenograft model injected with human PC-3 prostate cancer cells, using contrast-enhanced sonography, establishing a statistically significant correlation of the US maximum intensity and CD31-positive microvesel count.Jiang et al. 9 established on samples of needle prostate biopsies, that the peak intensity of prostate cancer at CEUS was statistically significantly increased with a higher GS and histologically determined MVD.Osimani et al. 55 showed that in PC blood volume and permeability surface area product measurements obtained with perfusion computed tomography had the highest correlation with immunohistochemical markers of angiogenesis, MVD 55 .Unlike them, Franiel et al. 56 , using MRI perfusion and blood volume hotspots with histological MVD, determined no significant correlation, explaining this with heterogeneous vascularization of the normal and tumorous prostate tissues, as well as different thickness of MRI slices, that is, histological paraffin blocks, but also with technical limitations of MRI, suggesting that the computer-based 3D prostate model could be used in the future to provide a more accurate correlation of histological and MRI imaging findings.Even though the prognostic value of microvessel density in prostate cancer is contradictory and microvessel density is not recommended for routine application by the World Health Organization, it is still the subject of research, particularly in the samples of needle prostate biopsies, where its prognostic significance is still unclear 57 .

Conclusion
Although the number of patients in this study was small, the obtained results indicate that quantification of microvascular density in biopsies is an accurate pre-operative predictor of tumor stage, discriminating between organ-confined and organ-extending neoplasms.

Table 4 Microvessel density (MVD) and clinical and pathological results in prostate carcinoma
NB -needle biopsy; RP -radical prostatectomy.