Vascular Density Is a Predictor ofCancer-Specific Survival in Prostatic Carcinoma

Ingela Franck Lissbrant,1 Par Stattin,2 Jan-Erik Damber,2 and Anders Bergh1*1Department of Pathology, Umeå University, Umeå, Sweden

2Department of Urology and Andrology, Umeå University, Umeå, Sweden

BACKGROUND. Microvessel density has been shown to give prognostic information in avariety of solid tumors, but its role in prostatic carcinoma needs further elucidation.METHODS. Intratumoral density of von Willebrand factor-positive microvessels was as-sessed in 98 cases of prostatic carcinoma, diagnosed at transurethral resection of the prostate(TURP) between 1975–1983, using two methods: 1) volume density of microvessels and 2)vascular count in the 2–3 most vascularized fields.RESULTS. Volume density and vascular counts were highly correlated. In Kaplan-Meyeranalysis, mean cancer-specific survival time for patients with a vascular count <135 wassignificantly longer than for patients with a vascular count >135 (P = 0.0064). The same resultsapplied to patients with WHO grade II tumors (P = 0.01). Excluding metastasis in a multi-variate analysis, both tumor stage and vascular count had an independent predictive value forcancer-specific survival in patients with WHO grade II tumors.CONCLUSIONS. Microvessel density may predict cancer-specific survival in prostatic car-cinoma. Prostate 33:38–45, 1997. © 1997 Wiley-Liss, Inc.

KEY WORDS: prostate; angiogenesis; prognosis

INTRODUCTION

Prostatic carcinoma is the most common canceramong men in the Western world. In 1994 it was pre-dicted that 38,000 men in the U.S. and more than35,000 men in the European Community would diefrom prostatic cancer [1]. Patients with prostatic car-cinoma have a diverse outcome and since treatment isassociated with substantial morbidity, the need for ac-curate prognostic markers cannot be overestimated[2,3]. Stage, grade, and serum prostate-specific antigen(PSA) are the strongest predictive tools available atpresent [4,5].

Tumor-associated angiogenesis, measured as vas-cular density, has been shown to give prognostic in-formation in a variety of solid tumors such as breastcancer, malignant melanoma, small-cell lung cancer,testicular cancer, ovarian carcinoma, and prostatic car-cinoma [6–11]. In order to grow and metastasize, solidtumors depend on angiogenesis [12]. Newly formedvessels provide tumor cells with oxygen and nutrientsas well as paracrine mediators produced by endothe-lial cells [13]. Initially, tumor cells lack the ability to

induce angiogenesis, and tumor growth does not startuntil tumor cells acquire the ability to induce neovas-cularization [14].

Tumor cells, intratumoral macrophages, mast cells,and endothelial cells have been found to produce sev-eral angiogenic factors, such as vascular endothelialgrowth factor (VEGF), basic fibroblast growth factor(bFGF), and angiogenin, as well as angiogenesis in-hibitors such as angiostatin and trombospondin [13].The balance between these angiogenesis regulatorsdetermines if neovascularization will occur or not [15].Only a small number of tumor cells acquire the abilityto induce angiogenesis [15]. Areas containing angio-genic tumor cells are recognized by intense neovascu-larization and high vascular density. Angiogenesis alsoplays a part in several steps of the metastatic cascade[16–18], and microvessel density and the number of tu-mor cells entering the circulation are correlated [19].

*Correspondence to: Dr. Anders Bergh, Department of Pathology,Umeå University, 901 87 Umeå, Sweden.Received 22 May 1996; Accepted 17 September 1996

The Prostate 33:38–45 (1997)

© 1997 Wiley-Liss, Inc.

Previous studies on angiogenesis in prostatic carci-noma have focused on the marked increase in vascu-larization in prostatic intraepithelial neoplasia andprostatic carcinoma compared to normal prostatic tis-sue [20–23]. Neovascularization has also been found tocorrelate with tumor volume in latent prostatic carci-noma [24]. Both angiogenesis and microvascular inva-sion have been shown to be independent predictors ofpathologic stage [25,26]. Moreover, an association hasbeen found between the risk of metastasis and tumorvascularization in prostatic carcinoma [11,27].

The objective of this study was to further investi-gate the relationship between microvessel density inprostatic carcinoma and cancer-specific survival, andto compare different techniques to estimate microves-sel density.

MATERIALS AND METHODS

Formalin-fixed and paraffin-embedded speci-mens from 98 tumors were randomly selected from a

series of prostate tumors consecutively diagnosed attransurethral resections (TUR) of the prostate at theCentral Hospital in Vasterås between 1975–1983.Mean age at diagnosis was 74 years (range, 52–95years). The patients had not received any cancertherapy prior to TUR. Staging was performed at timeof surgery, and local tumor stage was determined bydigital rectal examination according to the 1978 UnionInternationale Contre le Cancer (UICC) classifica-tion [28]. There were 9 T0 focal tumors, 25 T0 diffuse,19 T1, 31 T2, 5 T3, 5 T4 and 4 Tx tumors. Radionuc-lide bone scan was performed for M-staging; 11asymptotic patients had a positive bone scan indica-tive of metastatic disease, 57 patients had a negativebone scan, and in 30 cases no bone scan was per-formed. No lymph node staging procedures wereperformed. The tumor specimens were reexaminedand graded by one pathologist according to the WorldHealth Organization (WHO) classification [29]. Therewere 14 well-differentiated tumors (WHO grade I),53 moderately differentiated tumors (WHO gradeII), and 31 poorly differentiated tumors (WHO grade

Fig. 1. Sections of WHO grade II prostate tumors immunostained for von Willebrand factor, 200× magnification. A: Few immunostainedmicrovessels are observed in this tumor. The patient died 148 months after diagnosis. B: Numerous immunostained microvessels areobserved in the prostate tumor of this patient, who died 18 months after diagnosis.

Tumor Angiogenesis in Prostatic Carcinoma 39

III). After TUR, the patients were followed withsurveillance for a minimum of 11 years or until death.Castration was performed if the patient had symp-tomatic progression. Eight (8%) patients were stillalive in May 1994. Mean overall survival was 64months, 51 (56%) patients died from prostatic can-cer, and 39 (44%) patients died from other causes.Cause of death was determined by examination of pa-tient records.

Immunohistochemistry

Representative areas of prostatic cancer were iden-tified on hematoxylin/eosin-stained slides. Two tothree sections from each tumor were examined. Ad-jacent 4-mm-thick sections were deparaffinated andrehydrated according to standard procedures, washedwith phosphate-buffered saline (PBS) solution, andheated in a microwave oven at 800 W for a total of20 min in 0.01 M citrate buffer, pH 6, as describedearlier [30]. Endogenous peroxidase was blocked with3% hydrogen peroxide in methanol for 10 min. Thesections were treated with Pronase for 20 min, washedwith PBS, pretreated with 2% goat serum for 20 min,and incubated overnight with a rabbit antiserumagainst human von Willebrand factor (Dakopatts,Glostrup, Denmark) diluted to 1/200. The biotin strep-tavidin method was used (Vectastain Elite ABC kit;Vector Laboratories, Inc., Burlingame, CA) accordingto the manufacturer’s instructions. Briefly, the sectionswere incubated in chronological order with a biotinyl-ated multilink secondary antibody for 30 min, washedin PBS, incubated with alkaline phosphatase for 45min, and washed again in PBS. The sections were thendeveloped for 7 min using aminoethylcarbazole(AEC), rinsed briefly in water, lightly counterstainedwith Meyer’s hematoxylin solution, and mounted.

Microvessel Quantification

The criteria previously described for defining a mi-crovessel were used [6]. Any individual or cluster ofvon Willebrand factor-stained endothelial cell(s),separate from adjacent microvessels, tumor cells, orother connective tissue elements, was counted. Mi-crovessels both with and without lumen were in-cluded.

Two methods were used to estimate microvesseldensity:

1. Vascular count, i.e., the total number of microves-sels in the 2–3 most vascularized fields, or so-calledhot spots, at 200× (0.93 mm2) and 400× (0.23 mm2).

Hot spots were found by scanning the sections atlow power (100× magnification). Results of vascu-lar counts are presented as the highest (high) andmean count of microvessels.

2. Volume density (Vv%): volume vessels/volumereference tissue, i.e., the percentage of prostatictumor tissue occupied by vessel walls and luminawas assessed by a point-counting method, usinga 121-point eyepiece graticule at 400× magnifica-tion in 15–20 randomly chosen fields in each sec-tion. The number of graticule-crossing points(hits) overlaying von Willebrand factor-stainedcell(s) or vessel lumen and hits over reference space(tumor epithelium and tumor stroma) werecounted [31].

Both vascular counts and Vv% were examined with-

Fig. 2. Scatter matrix for vascular counts and volume density,showing high correlations for all microvessel density measure-ments. Individual Spearman rank correlation coefficients rangedbetween 0.83–0.99, P < 0.0001.

TABLE 1. Mean and Median Values for Vascular Countsand Volume Density

Variable n Mean Median

Volume density (%) 98 3.4 3.2200×, high 98 144 122200×, mean 98 136 115400×, high 98 54 49400×, mean 98 49 44

40 Lissbrant et al.

out knowledge of patient outcome and by one ob-server (I.F.L.).

Statistics

The Spearman rank correlation coefficient was usedto analyze correlations between different techniquesto estimate microvessel density. The Wilcoxon ranksum test was used to compare median vascular countsbetween WHO grade tumors, for patients with and

without metastasis as well as for tumor stage. Survivalanalysis was assessed using Kaplan-Meyer analysis.Comparison between groups were made using the logrank test. Cox multiple regression analysis was per-formed to evaluate the independent predictive values.P < 0.05 was considered statistically significant. Statis-tical analysis was performed using the Statistical Pack-age for the Social Sciences, SPSS 6.1 (SPSS Inc., Chi-cago, IL).

Fig. 3. Kaplan-Meyer survival plot of vascularcount at 200× high, cutoff value of 135, and can-cer-specific survival for all patients. Patients with<135 microvessels per field survived significantlylonger than patients with >135 microvessels perfield.

TABLE II. Kaplan-Meyer Survival Analysis ofMicrovessel Density (Vascular Counts and Volume

Density) and Cancer-Specific Survival, Showing MeanCancer-Specific Survival, Confidence Intervals, and

P-Values for All Patients*

Variable,vessels/field n

Meancancer-specific

survival,months

(all patients)Confidence

interval P-value

200×, high<135 55 117.0 94.7–139.4>135 43 65.2 52.5–78.0 0.0064200×, mean<125 55 117.2 94.9–139.6>125 43 65.1 52.4–77.7 0.0057400×, high<50 55 115.5 93.1–137.9>50 43 68.5 54.4–82.6 0.0164400×, mean<45 52 118.1 95.1–141.1>45 46 67.9 54.2–81.6 0.0078Volume density (%)<3% 53 120.2 93.8–146.5>3% 45 76.4 61.0–91.8 0.0327

*Both vascular counts and volume density significantly pre-dicted cancer-specific survival.

TABLE III. Kaplan-Meyer Survival Analysis ofMicrovessel Density (Vascular Counts and Volume

Density) and Cancer-Specific Survival, Showing MeanCancer-Specific Survival, Confidence Intervals, and

P-Values for Patients With WHO Grade II Tumors*

Variable,vessels/field n

Meancancer-specific

survival,months(WHO

grade II)Confidence

interval P-value

200×, high<135 28 131.1 103.6–158.6>135 25 62.7 53.3–88.8 0.0148200×, mean<125 29 131.4 104.0–158.8>125 24 69.9 52.1–87.8 0.0106400×, high<55 30 130.2 103.1–157.4>55 23 69.9 52.0–87.8 0.0088400×, mean<50 29 130.4 103.0–157.9>50 24 72.2 52.8–91.6 0.0142Volume density (%)<3% 26 134.9 101.8–168.0>3% 27 82.0 62.5–101.4 0.0591

*Vascular counts significantly predicted cancer-specific sur-vival.

Tumor Angiogenesis in Prostatic Carcinoma 41

RESULTS

Microvessel Quantification

Microvessels both in benign prostate tissue and inprostate cancer were stained by von Willebrand factor.Microvessels in benign tissue served as internal con-trols. Prostate tumors were notably heterogeneous intheir microvessel density (Fig. 1).

Microvessel density was estimated by two differentmethods, i.e., vascular counts (microvessels per area)and volume density (volume vessels per unit tissuevolume, Table I). High and average vascular counts at200× and 400× magnification and vascular density(Vv%) were all significantly associated with each other(Fig. 2). These results indicate that counting the totalnumber of microvessels in only one field gives thesame information as counting three fields or volumedensity in 15 randomly chosen fields.

Survival Analysis

Exploring data from vascular counts at 200× high inKaplan-Meyer analysis, starting at the median value(122) as a cutoff point, significant differences in can-cer-specific survival were found in the range 120–170,with the most distinct difference in survival time at acutoff value of 135 (n = 51, P = 0.0064, Fig. 3). Intra-tumoral microvessel density, using either vascularcounts or Vv%, was correlated with cancer-specificsurvival in univariate analysis (Table II). Microvesseldensity was a predictor for overall survival as well (P= 0.0016).

When analyzing WHO grade II tumors, all mi-crovessel density measurements except volume den-sity remained significant predictors of cancer-specificsurvival (Table III). Patients with WHO grade II tu-mors with <135 microvessels survived an average of131 months, compared to a mean survival time of 63

months for patients with >135 microvessels per field(P = 0.0148, n = 53, Fig. 4).

Significant differences in median vascular countswere found between grade I tumors and, grade IIand grade III tumors, respectively (P = 0.018 and P =0.004), in the Wilcoxon rank sum test. However, nodifferences in median vascular counts were foundbetween grade II and grade III tumors (P = 0.164,Fig. 5) and for patients with and without metastasis(P = 0.94). This was also true when comparing vas-cular counts for different stages categorized intothree different groups: T0 focal and diffuse, T1 andT2, and T3 and T4 (P = 0.33, P = 0.28, and P = 0.58,respectively).

When evaluating the independent predictive valuesfor metastasis, tumor stage, microvessel density,WHO grade, and age in a Cox multiple regressionanalysis, only metastasis had an independent predic-tive value for cancer-specific survival (Table IV). The

Fig. 5. Box plot for WHO grade and vascular count at 200×high. Each box shows the median, quartiles, and extreme valueswithin each category. Significant differences in median vascularcount were found between WHO grade I tumors, and WHOgrade II and III tumors, respectively (P = 0.018 and P = 0.004).

Fig. 4. Kaplan-Meyer survival plot of vascularcount at 200× high, cutoff value of 135, and can-cer-specific survival for patients with WHO gradeII tumors. Patients with <135 microvessels perfield survived significantly longer than patientswith >135 microvessels per field.

42 Lissbrant et al.

results were the same for patients with WHO grade IItumors. When metastasis was excluded in the analysisof this group, both tumor stage and vascular counthad independent predictive values (Table V).

DISCUSSION

The present investigation is based on a group ofhomogeneously treated prostatic cancer patients. Af-ter diagnosis, they were all followed with surveillance,and no intervention was made until symptoms ap-peared. Moreover, the follow-up time was long. Alto-gether, this means that these patients showed approxi-mately the natural history of the disease. This studyshows, for the first time, a statistically significant cor-relation between microvessel density and cancer-specific survival in univariate analysis. As expected,metastasis had the strongest predictive value in a mul-tivariate analysis.

Patients with WHO grade I tumors generally havea good prognosis, whereas patients with WHO gradeIII tumors have a poor outcome. Since WHO grade IItumors, which account for the majority of prostaticcancers [32,33], differ in their malignant characteristicsand progression, their prognosis is unpredictable.There is consequently an urgent need to identify prog-nostic markers for this large group of patients. In thisstudy, microvessel density had an independent pre-dictive value for cancer-specific survival in patientswith WHO grade II tumors. Further studies areneeded to elucidate whether microvessel density pre-dicts survival in the large group of medium-grade tu-mors using other histological grading systems, such asGleason score.

The methodology of evaluating tumor angiogenesishas been a matter of extensive discussion in angiogen-esis research [34–37]. To evaluate microvessel density,it is probably important to identify all intratumoralmicrovessels. Immunostaining techniques for identi-fying von Willebrand factor, CD31, CD34, and Ulex

europaeus agglutinin I are the most common at present,but none of them stain all microvessels [34]. It remainsto be shown whether other, possibly more sensitivemethods than von Willebrand factor, could improvethe prognostic value of vascular density measure-ments.

Another concern has been the microvessel-countingtechnique [38]. Therefore, we wanted to determine ifcounting hot spots promotes a bias, since this requiressubjective selection of the most vascularized area. Pos-sibly, it would be more accurate to use standard ste-reological techniques [31] to measure the mean intra-tumoral volume density of microvessels in randomlychosen fields in each section. However, the differentmeasurements were highly correlated, which suggeststhat the easiest and most rapid technique, i.e., count-ing the total number of microvessels in the most vas-cularized field, could be recommended. The reason asto why an estimate of mean vessel density (Vv%) iscorrelated to an estimate of the maximal vessel density(hot spot) is unknown. This correlation may, however,suggest that an estimate of mean microvessel densityobtained from several random needle biopsies couldhave a predictive value. Further studies are needed totest this hypothesis. The advantages and drawbacks ofmanual vs. computerized counting techniques makefor another area that needs to be investigated [38].

The reason why increased microvessel density iscorrelated to poor prognosis in patients with prostatictumors is unknown. Interestingly, no relationship wasfound between intratumoral microvessel density andtumor cell- and endothelial cell-proliferation in eitherbreast [39,40] or prostatic [41] carcinoma, suggestingthat increased vascular density makes tumors aggres-sive by other mechanisms than by stimulating tumor-cell proliferation. The previous observation, that in-creased vascularization increases metastatic capacityin prostate cancer [11,27], is thus the most likely ex-planation for the correlation between vascular densityand prognosis.

TABLE IV. Cox Multiple Regression Analysis for Independent Predictors of Cancer-Specific Survival: Metastasis,WHO Grade, Stage, Vascular Count, and Age*

Covariate/reference groupRelative risk(all patients)

P-value(all patients)

Relative risk(WHO grade II)

P-value(WHO grade II)

Metastasis/no metastasis 4.7 0.001 5.0 0.005WHO grade I/WHO grade II 0.3 0.30WHO grade III/WHO grade II 2.0 0.08T1, T2/T0 2.4 0.06 3.0 0.06T3, T4/T0 2.7 0.08 3.5 0.11Vessel count at 200× high, >135/<135 1.4 0.36 1.7 0.25Age >71/<71 years 1.0 0.92 1.1 0.89

*All patients and patients with WHO grade II tumors were analyzed. Metastasis was the only independent predictor.

Tumor Angiogenesis in Prostatic Carcinoma 43

CONCLUSIONS

The present study suggests that microvessel den-sity is a prognostic factor in prostate cancer.

ACKNOWLEDGMENTS

Mrs. Birgitta Ekblom, Ms. Pernilla Andersson, andMrs. Elisabeth Dahlberg have contributed to this pa-per by their skillful technical assistance. Dr. Lars Karl-berg, Department of Urology at the Central Hospitalof Vasterås, kindly provided the series of prostate can-cer specimens. This study was supported by grantsfrom the Swedish Cancer Society (project 1760), Uni-versity Hospital in Umeå, the Maud and Birger Gus-tavsson Foundation, and the Lion’s Research Founda-tion, University of Umeå.

REFERENCES

1. Boyle P, Maisonneuve P, Napalkov P: Geographical and tem-poral patterns of incidence and mortality from prostate cancer.Urology 1995;46:47–55.

2. Pedersen KV, Herder A: Radical retropubic prostatectomy forlocalised prostatic carcinoma: A clinical and pathological studyof 201 cases. Scand J Urol Nephrol 1993;27:219–224.

3. Moreno JG, Ahlering TE: Late local complications after defini-tive radiotherapy for prostatic adenocarcinoma. J Urol 1992;147:926–928.

4. Gleason DF, Mellinger GT: Prediction of prognosis for prostaticadenocarcinoma by combined histological grading and clinicalstaging. J Urol 1974;111:58–64.

5. Oesterling JE: Prostate specific antigen: A critical assessment ofthe most useful tumor marker for adenocarcinoma of the pros-tate. J Urol 1991;145:907–923.

6. Weidner N, Folkman J, Pozza F, Bevilacqua P, Allred EN, MooreDH, Meli S, Gasparini G: Tumor angiogenesis: A new signifi-cant and independent indicator in early-stage breast carcinoma.JNC 1992;84:1875–1887.

7. Srivastava A, Laidler P, Davies RP, Horgan K, Hughes LE: Theprognostic significance of tumor vascularity in intermediate-thickness (0.76–4.0 mm thick) skin melanoma. A quantitativehistologic study. Am J Pathol 1988;133:419–423.

8. Fontanini G, Bigini D, Vignati S, Basolo F, Mussi A, Lucchi M,

Chine S, Angeletti CA, Harris AL, Bevilacqua G: Microvesselcount predicts metastatic disease and survival in non-small celllung cancer. J Pathol 1995;177:57–63.

9. Olivarez D, Ulbright T, DeRiese W, Foster R, Reister T, EinhornL, Sledge G: Neovascularization in clinical stage A testiculargerm cell tumor: Prediction of metastatic disease. Cancer Res1994;54:2800–2802.

10. Hollingsworth HC, Kohn EC, Steinberg SM, Rothenberg ML,Merino MJ: Tumor angiogenesis in advanced stage ovarian car-cinoma. Am J Pathol 1995;147:33–41.

11. Weidner N, Carroll PR, Flax J, Blumenfeld W, Folkman J: Tumorangiogenesis correlates with metastasis in invasive prostate car-cinoma. Am J Pathol 1993;143:401–409.

12. Folkman J: What is the evidence that tumors are angiogenesisdependent? JNC 1990;82:4–6.

13. Folkman J: Tumor angiogenesis. In Mendelsohn J, Howley PM,Israel MA, Liotta LA (eds): ‘‘The Molecular Basis of Cancer,’’Philadelphia: W.B. Saunders Company, 1995:206–224.

14. Folkman J, Watson K, Ingber D, Hanahan D: Induction of an-giogenesis during the transition from hyperplasia to neoplasia.Nature 1989;339:58–61.

15. Folkman J: Angiogenesis in cancer, vascular, rheumatoid andother disease. Nat Med 1995;1:27–31.

16. Fidler IJ, Ellis LM: The implications of angiogenesis for the bi-ology and therapy of cancer metastasis. Cell 1994;79:185–188.

17. Dvorak HF, Nagy JA, Dvorak JT, Dvorak AM: Identification andcharacterization of the blood vessels of solid tumors that areleaky to circulating macro molecules. Am J Pathol 1988;133:95–109.

18. Gross JL, Moscatelli D, Rifkin DB: Increased capillary endothe-lial cell protease activity in response to angiogenic stimuli invitro. Proc Natl Acad Sci USA 1983;80:2623–2627.

19. Liotta LA, Kleinerman J, Saidel GM: Quantitative relationshipsof intravascular tumor cells, tumor vessels, and pulmonary me-tastases following tumor implantation. Cancer Res 1974;34:997–1004.

20. Siegal JA, Yu E, Brawer MK: Topography of neovascularity inhuman prostate carcinoma. Cancer 1995;75:2545–2551.

21. Brawer MK, Bigler SA, Deering RE: Quantitative morphometricanalysis of the microcirculation in prostate carcinoma. J CellBiochem [Suppl] 1992;16:62–64.

22. Bigler SA, Deering RE, Brawer MK: Comparison of microscopicvascularity in benign and malignant prostate tissue. HumPathol 1993;24:220–226.

23. Fregene TA, Khanuja PS, Noto AC, Gehani SK, Van EgmontEM, Luz DA, Pienta KJ: Tumor-associated angiogenesis in pros-tate cancer. Anticancer Res 1993;13:2377–2381.

24. Furusato M, Wakui S, Sasaki H, Ito K, Ushigome S: Tumour

TABLE V. Cox Multiple Regression Analysis for Independent Predictors of Cancer-SpecificSurvival Excluding Metastasis*

Covariate/reference groupRelative risk(all patients)

P-value(all patients)

Relative risk(WHO grade II)

P-value(WHO grade II)

WHO grade I/WHO grade II 0.93 0.91WHO grade III/WHO grade II 1.6 0.14T1, T2/T0 3.3 0.002 3.9 0.008T3, T4/T0 3.1 0.03 5.8 0.02Vessel count at 200× high, >135/<135 1.6 0.11 2.7 0.02Age >71/<71 years 1.1 0.68 0.9 0.70

*All patients and patients with WHO grade II tumors were analyzed. For patients with WHO grade II tumors, both tumor stage andmicrovessel density were independent predictors of cancer-specific survival.

44 Lissbrant et al.

angiogenesis in latent prostatic carcinoma. Br J Cancer 1994;70:1244–1246.

25. Brawer MK, Deering RE, Browne M, Preston SD, Bigler SA:Predictors of pathologic stage in prostatic carcinoma. The role ofneovascularity. Cancer 1994;73:678–687.

26. Salomao DR, Graham SD, Bostwick DG: Microvascular invasionin prostate cancer correlates with pathologic stage. Arch PatholLab Med 1995;119:1050–1054.

27. Wakui S, Furusato M, Itoh T, Sasaki H, Akiyama A, Kinoshita I,Asano K, Tokuda T, Aizawa S, Ushigome S: Tumor angiogen-esis in prostatic carcinoma with and without bone marrow me-tastasis: A morphometric study. J Pathol 1992;168:257–262.

28. Union Internationale Contre le Cancer (UICC): ‘‘TNM Classifi-cation of Prostate Tumours,’’ 2nd ed. Berlin: Springer, 1978.

29. Mostofi FK, Sesterhenn IA, Sobin LH: ‘‘International Histologi-cal Classification of Prostate Tumours,’’ Geneva: WHO, 1980.

30. Taylor CR, Shi SR, Chaiwun B, Young L, Imam SA, Cote RJ:Strategies for improving the immunohistochemical staining ofvarious intranuclear prognostic markers in formalin-paraffinsections: Androgen receptor, estrogen receptor, progesterone re-ceptor, p53 protein, proliferating cell nuclear antigen, and Ki-67antigen revealed by antigen retrieval techniques. Hum Pathol1994;25:263–270.

31. Weibel ER: ‘‘Stereological Methods, Vol 1: Practical Methods forBiological Morphometry,’’ London: Academic Press, 1979.

32. Partin AW, Coffey DS: Benign and malignant prostatic neo-plasms: Human studies. Recent Prog Horm Res 1994;49:293–331.

33. Gronberg H, Bergh A, Damber J-E, Jonsson H, Lenner P, Ång-

strom T: Prostate cancer in northern Sweden. Incidence, sur-vival and mortality in relation to tumour grade. Acta Oncol1994;33:359–363.

34. Weidner N: Intratumor microvessel density as a prognostic fac-tor in cancer. Am J Pathol 1995;147:9–19.

35. Craft PS, Harris AL: Clinical prognostic significance of tumourangiogenesis. Ann Oncol 1994;5:305–311.

36. Weidner N: Current pathologic methods for measuring intratu-moral microvessel density within breast carcinoma and othersolid tumors. Breast Cancer Res Treat 1995;36:169–180.

37. Axelsson K, Ljung BE, Moore DH II, Thor AD, Chew KL, Edg-erton SM, Smith HS, Mayall BH: Tumor angiogenesis as a prog-nostic assay for invasive ductal breast carcinoma. JNCI 1995;87:997–1008.

38. Barbareschi M, Gasparini G, Morelli L, Forti S, Dalla Palma P:Novel methods for the determination of the angiogenic activityof human tumors. Breast Cancer Res Treat 1995;36:181–192.

39. Vartanian RK, Weidner N: Correlation of intratumoral endothe-lial cell proliferation with microvessel density (tumor angiogen-esis) and tumor cell proliferation in breast carcinoma. Am JPathol 1994;144:1188–1194.

40. Fox SB, Gatter KC, Bicknell R, Going JJ, Stanton P, Cooke TG,Harris AL: Relationship of endothelial cell proliferation to tu-mor vascularity in human breast cancer. Cancer Res 1993;53:4161–4163.

41. Vartanian RK, Weidner N: Endothelial cell proliferation in pros-tatic carcinoma and prostatic hyperplasia: Correlation withGleason’s score, microvessel density, and epithelial cell prolif-eration. Lab Invest 1995;73:844–850.

Tumor Angiogenesis in Prostatic Carcinoma 45