Contrast-enhanced ultrasonography for the detection and characterization of prostate cancer: Correlation with microvessel density and Gleason score

Transcription

1 Clinical Radiology 66 (2011) 732e737 Contents lists available at ScienceDirect Clinical Radiology journal homepage: Original Paper Contrast-enhanced ultrasonography for the detection and characterization of prostate cancer: Correlation with microvessel density and Gleason score J. Jiang, Y. Chen *, Y. Zhu, X. Yao, J. Qi Xinhua Hospital Affiliated to Shanghai Jiaotong University School of Medicine, 1665 Kongjiang Road, Shanghai , China article information Article history: Received 19 December 2010 Received in revised form 9 February 2011 Accepted 21 February 2011 AIM: To determine whether there is a correlation between the peak intensity of the lesion at contrast-enhanced ultrasonography and the microvessel density (MVD) and Gleason score in biopsy specimens of prostate cancer. MATERIALS AND METHODS: Contrast-enhanced ultrasonography using cadence-contrast pulse sequence (CPS) technology was performed in 147 patients with suspected prostate cancer before biopsy. An auto-tracking contrast quantification (ACQ) software was used to analyse the peak intensity (PI) of the lesion. The Gleason score and MVD immunoreactivity were determined in the prostate biopsy specimens. Ultrasound findings were correlated with biopsy findings. RESULTS: Prostate cancer was detected in 73 of 147 patients. The PI values of prostate cancer patients were significantly higher than those of non-malignant patients [9.81 (4.23) versus 5.69 (3.19) db; p < 0.01]. The mean (SD) PIs of prostate cancer lesions with a Gleason score of 6e9 were 7.08 (3.80), 8.65 (4.08), 9.76 (3.75), and 9.85 (4.13) db, respectively. The PI value increased significantly with a higher Gleason score (p < 0.01). The mean (SD) MVDs observed in prostate cancer lesions with a Gleason score of 6e9 were (10.54), (10.31), (9.29), and (11.82), respectively. There was a positive correlation between PI and MVD in prostate cancer, with a correlation coefficient of No correlation was found between PI value and age, prostate specific antigen (PSA) or prostate specific antigen density (PSAD) level (p > 0.05). CONCLUSION: The PI obtained by CPS harmonic ultrasonography appears to be of value as an indicator of MVD and increases with a higher Gleason score. CPS harmonic ultrasonography could be promising as a useful imaging technique in the detection and characterization of prostate cancer. Ó 2011 The Royal College of Radiologists. Published by Elsevier Ltd. All rights reserved. Introduction The annual incidence of prostate cancer has increased dramatically over the past decade such that prostate cancer * Guarantor and correspondent: Y. Chen, Xinhua Hospital Affiliated to Shanghai Jiaotong University School of Medicine, 1665 Kongjiang Road, Shanghai , China. Tel.: þ ; fax: þ address: joychen_1266@163.com (Y. Chen). is now the most commonly diagnosed visceral malignancy in men. 1 At present, a combination of prostate specific antigen (PSA) tests, digital rectal examination (DRE), and transrectal ultrasound imaging (TRUS) is usually applied for the detection of prostate cancer. 2 However, the existing diagnostic techniques lead to clinical over-staging in 8e45% of organ-confined prostate cancer and clinical understaging in 24e55% of locally advanced prostate cancer. 3e6 Radical surgery may be denied or overused due to staging /$ e see front matter Ó 2011 The Royal College of Radiologists. Published by Elsevier Ltd. All rights reserved. doi: /j.crad

2 J. Jiang et al. / Clinical Radiology 66 (2011) 732e errors. Therefore, improvement of detection, grading, and staging methods for prostate cancer is essential to provide a clear insight into the possibilities and risks of the available treatment options. Microvessel density (MVD) is increased in prostate cancer tissue due to the proliferation of neovessels and the increased MVD is associated with the prognosis and progression of prostate cancer, in terms of metastases, 7 stage of disease, 8 and disease-specific survival. 9 Therefore, visualization of the induced MVD may improve the detection and grading of cancer. The Gleason grading system is based on a microscopic characterization of the architectural criteria of prostate cancer. It is the most commonly used system for grading prostate cancer and is an important factor in the formulation of the therapeutic schedule and in the prognosis of prostate cancer. 10,11 Considering the value of the Gleason score and MVD, a non-invasive imaging technique that could reflect both the Gleason score and MVD would hold great promise in tumour detection and characterization. As prostate cancer is a multifocal and histologically heterogeneous disease, biopsies have limited use in the determination of all cancer sites and grades. 12,13 An imaging method that could indicate an increase in MVD and provide a predicable Gleason score could have value in choosing targets for prostate biopsies. This will lead to a change in biopsy strategies, bringing about a higher detection rate of prostate cancer and a more accurate biopsy Gleason grading, and hence, a more appropriate therapeutic strategy. Contrast-enhanced ultrasound imaging (CEUS) was developed to provide detailed and real-time perfusion imaging. It improved the visualization of tumour vascularity in many organs, including the prostate. 14,15 Nowadays, many CEUS techniques are available, among which the cadence contrast pulse sequencing (CPS) is worth mentioning. It can recognize and process the unique nonlinear fundamental and higher-order harmonic signals that are generated by the contrast agent, thus allowing for a significant increase in the specificity of contrast agent-totissue even at low mechanical index power levels. Furthermore, grey-scale CPS imaging is less affected by motion noise and blooming artefact, 16,17 so it is more sensitive in depicting neovascularity associated with prostate cancer. In addition, autotracking contrast quantification (ACQ) software further complements the CPS detection technology and facilitates the quantification evaluation of microvascularity. 16 This software allows a free choice of the region of interest (ROI) and a real-time observation of the enhancement pattern of contrast agent in the ROI. Moreover, it reconstructs the timeeintensity curve (TIC), from which the parameters of contrast agent perfusion in the ROI can be calculated easily thereby affording a more convenient and accurate method to evaluate the vascular perfusion of tumor. 17,18 Previous studies were inconsistent in terms of the value of CPS imaging in diagnosing prostate cancer. 18,19 Preliminary data suggested that the haemodynamic indices obtained from CPS imaging were different between low- and high-grade prostate cancer; however, it made no mention of the relationship between CPS parameters and the level of MVD in prostate cancer. 20 The current study was designed to clarify the potential of using CEUS with CPS technology in evaluating MVD and Gleason score in prostate cancer. The peak intensity (PI) of the tumours were evaluated using CPS harmonic imaging and the results were compared with pathological MVD and Gleason score to provide a non-invasive index for clinical tumour detection and tumour grading of prostate cancer. Materials and methods Patients Between March 2008 and September 2009, 147 consecutive patients with abnormal digital rectal examination findings or elevated serum PSA levels (4 ng/ml) without previous biopsy were studied prospectively by an experienced radiologist. All patients underwent a contrast-enhanced transrectal ultrasonography (TRUS) and, subsequently, a systematic eight core transrectal ultrasound-guided prostate biopsy as well as up to four cores of targeted biopsies from sonographic abnormal findings. The study was approved by our local Ethics Committee, and written informed consent was obtained from all patients. Imaging protocol Each patient was evaluated with ultrasonography at baseline and again during intravenous infusion of sulphur hexafluoride microbubbles (SonoVue, Bracco, Milan, Italy). The ultraound equipment used was a Sequoia 512 (Siemens Medical Solutions, Mountain View, CA, USA) with a transrectal probe (EV8C4-S). Normal grey-scale imaging was performed with a probe frequency of 7 MHz and a dynamic range of 80 db. For colour Doppler ultrasonography, the probe frequency was 6 MHz, and the colour Doppler gain was adjusted to maximize signal but eliminate colour noise from the tissue of the prostate. The colour Doppler window was set to include the entire gland. During the contrastenhanced ultrasound examinations, a fast bolus injection of 2.4 ml SonoVue was administrated intravenously followed by a 5 ml normal saline flush. The scanner was set in CPS mode with a probe frequency of 8 MHz. The acoustic power was set at a mechanical index of 0.11 and the dynamic range was fixed at 81 db. The CPS gain setting was set at automatic optimizing. The contrast imaging plane was chosen as the transverse plane of the sonographic abnormality, or the most hypervascular plane on colour Doppler imaging for patients with no suspicious baseline ultrasonography results. The entire examination was saved in DICOM format and transmitted to a workstation for further analysis. Image interpretation and analysis All contrast-enhanced ultrasound data were analysed on the workstation using Axius ACQ software (TOMTEC, Unterschleissheim, Munich, Germany) by a sonographer who was blinded to all clinical and pathological information. Considering that the enhancement degree of transition zone (TZ) would be affected by the acoustic

3 734 J. Jiang et al. / Clinical Radiology 66 (2011) 732e737 attenuation due to the probe frequency of 8 MHz used in CEUS examination, only the peripheral zone (PZ) was evaluated in this study for exact correlation of CEUS images with MVD results. Another cause was that the enhancement characterization of PZ lesions was entirely different from that of TZ lesions, which would be biased by the hypervascularity of the normal inner gland and coexisting benign prostate hyperplasia (BPH). Therefore, the ROI was placed and traced only on the PZ biopsy sites on contrast ultrasonographic images. For octant systematic biopsy cases, ROIs were drawn around the biopsy site and the diameters were set to approximately 5 mm. For targeted biopsy cases, the ROIs were drawn encompassing the sonographic abnormalities as closely as possible. The timeeintensity curve was reconstructed for each ROI and then the peak intensity (PI), which showed the value of the maximum intensity in the ROI that occurred after time zero (in db), was obtained. Pathological analysis All biopsy specimens were marked as to site of origin and analysed separately, and the pathological findings were categorized as malignant with an assigned Gleason score for each biopsy core, prostatic intraepithelial neoplasia (PIN), BPH, and inflammation. Prostate cancers with a Gleason score of 7 or above were categorized as high-grade tumours while the rest were categorized as low-grade tumours. When there was a mixed lesion with part of the lesion having a higher Gleason score and part having a lower Gleason score in the biopsy specimen, the specimen was assigned for a majority Gleason score. To assess MVD, immunohistochemical staining for anti-cd31 antibody (Dako, Glostrup, Denmark) using formalin-fixed, paraffinembedded biopsy specimens was performed. Microvessels labelled with the anti-cd31 antibody were quantified by the counting procedure of Weidner et al. 21 Briefly, the five most vascular areas (i.e., hot spots) with the highest numbers of microvessel profiles were chosen subjectively from each tumour section under a low-power lens (100), and then their microvessel numbers were counted under a high-power lens (400). The mean value of the microvessel numbers was the MVD value of the tumour. Statistical analysis All analyses were performed with SPSS 12.0 (SPSS Inc, Chicago, IL, USA). All data were described as means (SD). P-values less than 0.05 were considered to indicate a significant difference. Differences between benign and malignant patients were analysed using Student s t-test or the ManneWhitney test. PI and MVD values between nonmalignant and malignant lesions were compared using Student s t-test. PI and MVD values among lesions with different Gleason scores were compared by using analysis of variance (ANOVA). Pearson correlation coefficients were calculated to analyse the correlation between PI and MVD at pathology. Table 1 Clinical characteristics of patients. Non-malignant Prostate cancer p No. of patients Age, years (9.38) (45e83) (7.18) (52e86) <0.01 PSA, ng/ml (8.22) (0.3e41.1) (17.71) (3e100) <0.01 PSAD, ng/ml/ml 0.22 (0.13) (0.01e0.56) 0.49 (0.46) (0.07e2.38) <0.01 Data are mean (SD) (range). PSA, prostate specific antigen; PSAD, prostate specific antigen density. Results At pathological examination, the final diagnosis of the 147 patients was benign in 71 cases and premalignant or malignant in 76 cases. The former included 47 cases of BPH and 24 cases of BPH with inflammation infiltration. The latter included three cases of PIN and 73 cases of prostate cancers. After 1-year follow-up observation for the nonmalignant cases, two patients were excluded because of higher PSA velocity and two patients were lost to follow-up. Thus after excluding three patients with PIN there were 73 prostate cancer patients and 67 non-malignant patients enrolled in the present study. Table 1 demonstrates the clinical characteristics of the patients. In total, there were 678 biopsy cores from PZ analysed by ACQ software, including 216 cores with pathologically proven malignancy from 73 prostate cancer patients and 462 cores from 67 non-malignant patients. The mean PIs of prostate cancer and non-malignant patients were 9.81 (4.23) and 5.69 (3.19) db, respectively. The MVDs of prostate cancer and non-malignant patients were (10.31) and (9.89), respectively. The PI and MVD values of prostate cancer patients were significantly higher than those of non-malignant cases (p < 0.01) (Table 2). Table 3 shows that the PIs of prostate cancer lesions with a Gleason score of 6e9 were 7.08 (3.80), 8.65 (4.08), 9.76 (3.75) and 9.85 (4.13) db, respectively. The PI value increased significantly with a higher Gleason score (p < 0.01). In addition, the PI value of high-grade prostate cancer was significantly higher than that of low-grade lesions [9.25 (4.09) versus 7.08 (3.80); p < 0.01; Figs 1 and 2]. The MVDs observed in prostate cancer lesions with a Gleason score of 6e9 were (10.54), (10.31), (9.29), and (11.82), respectively (Table 3). The mean MVD of highgrade prostate cancer was significantly higher than that of low-grade prostate cancer [59.04 (10.32) versus (10.54); p < 0.01; Figs 1-2]. There was a positive correlation between PI and MVD in prostate cancer, with a correlation coefficient of 0.617(p < 0.01; Fig 3). No correlation was found between PI value and age, PSA or PSAD level (p > 0.05). Table 2 The peak intensity (PI) and microvessel density (MVD) between non-malignant and malignant biopsy cores. Non-malignant Prostate cancer p PI, db 5.69 (3.19) 9.81 (4.23) <0.01 MVD, n/high power field (9.89) (10.31) <0.01 Data are mean (SD). PI, peak intensity; MVD, microvessel density.

4 J. Jiang et al. / Clinical Radiology 66 (2011) 732e Table 3 The peak intensity (PI) and microvessel density (MVD) of prostate cancer lesions with Gleason scores of 6e9. Biopsy Gleason score 6(n¼66) 7 (n ¼ 92) 8 (n ¼ 33) 9 (n ¼ 25) PI, db 7.08 (3.80) 8.65 (4.08) 9.76 (3.75) 9.85 (4.13) MVD, n/high (10.54) (10.31) (9.29) (11.82) power field Data are mean (SD). PI, peak intensity; MVD, microvessel density. Discussion Evaluation of tumour vascularity is particularly important in tumour detection and characterization. Studies of MVD within the prostate show a clear association between increased MVD with the presence of carcinoma and with a higher tumour grade. 22e25 Thus, quantitative assessment of MVD can provide critical data to guide therapeutic decisions. Unfortunately, the microvessels that proliferate in prostate cancer are below the resolution of conventional Doppler imaging; only the larger feeding vessels are visualized by this type of imaging. 14 CEUS represents one practical approach to detect these microvessels in the prostate. Furthermore, the CPS imaging, one of the CEUS techniques, together with the ACQ software is a promising technique to quantify tumour vascularity by calculating the parameters of contrast agent perfusion such as PI. A series of clinical trials have demonstrated a definite correlation between PI and MVD in some malignant tumours including hepatocellular carcinoma (HCC), breast carcinoma, and gastric carcinoma. 26e29 However, the microvascularity evaluation of prostate cancer by PI on the CPS image has not been reported. In the present study, by studying the relationship between the MVD and PI values in the prostate, significant differences were found for both values between prostate cancer and non-malignant patients. The mean PIs of prostate cancer and non-malignant patients were 9.81 (4.23) and 5.69 (3.19) db, respectively. The MVDs of prostate cancer and non-malignant patients were (10.31) and (9.89), respectively. The PI and MVD values of prostate cancer patients were both significantly higher than those of non-malignant patients (p < 0.01). As for the values of MVD and PI in cancer lesions with different Gleason scores, MVD and PI increased with higher Gleason scores. In addition, there was a positive correlation between PI and MVD in prostate cancer, with a correlation coefficient of (p < 0.01). This correlation might be related to the formation of new microvessels associated with the growth of malignancies. As the carcinoma progresses, the number of microvessels increases and the vascular structural abnormalities, such as calibre variations, elongation and coiling, non-hierarchical vascular networks, disturbed normal dichotomous branching, and an incomplete vascular wall may be more likely to appear, which cause numerous functional impairments such as increased transcapillary permeability, increased interstitial pressure, and newly formed arteriovenous shunts. 30 On CEUS imaging, the development of neovascularity and structural abnormalities of angiogenesis in the prostate cancer is demonstrated as an increased PI. There have been other studies about angiogenetic evaluation of malignant tumours by PI obtained from CEUS. Wang et al. 26 reported that the PI in HCC was significantly higher than that of peripheral tissue, which indicated the high blood perfusion of HCC. Moreover, the PI of HCC was significantly correlated with MVD, and thus, they came to the conclusion that tumour vascularity could be evaluated by CEUS. As for CEUS imaging of prostate cancer, although no previous study has investigated the correlation between PI and MVD, Sedelaar et al. 31 had demonstrated selective enhancement of regions with increased MVD in prostate cancer. Their study data showed that the mean MVD count of the enhanced side was on average 1.93 times higher than the unenhanced side, and that CEUS has the potential to visualize lesions with increased MVD, which was helpful in the detection of prostate cancer. In the present study the CPS haemodynamic parameters were compared between lesions with different Gleason scores to investigate whether the parameters could reflect Figure 1 Contrast-enhanced TRUS showing a focal area of prostate cancer in a 76-year-old man with a Gleason score of 3þ3 and a PSA level of 5.87 ng/ml. (a) Contrast imaging presents a poorly enhanced region in the left PZ and the PI of the lesion was only 7.29 db. (b) Pathological examination demonstrated that there were a few microvessels (stained in brown) in the lesion and the MVD was 57 (anti-cd31 antibody, original magnification 400).

5 736 J. Jiang et al. / Clinical Radiology 66 (2011) 732e737 Figure 2 Contrast-enhanced TRUS showing a focal area of prostate cancer in an 85-year-old man with a Gleason score of 4þ5 and a PSA level of ng/ml. (a). Contrast imaging presents a marked enhanced region in the right PZ and the PI of the lesion was db. (b) Pathological examination demonstrated that there were rich microvessels (stained in brown) in the lesion and the MVD was 90 (anti-cd31 antibody, original magnification 400). the change in Gleason scores. The results showed that haemodynamic parameters were significantly different between the various Gleason score groups, with a trend towards higher Gleason scores with increasing PI (p < 0.01). In addition, The PI values of high and low-grade prostate cancer were 9.25 (4.09) and 7.08 (3.80), respectively. The PI of high-grade prostate cancer was significantly higher than that of low-grade lesion (p < 0.01). Mitterberger et al. 32 also discussed the relationship between the CEUS appearance and the Gleason score. They performed contrast-enhanced colour Doppler targeted biopsies plus 10 core systematic biopsies in 690 patients with a clinical suspicion of prostate cancer during the administration of the ultrasound contrast agent SonoVue. The Gleason score of all 180 cancers detected on contrast-enhanced colour Doppler targeted biopsy was found to be 6 or higher (mean 6.8), whereas the Gleason scores of all 166 cancers detected on systematic biopsy ranged from 4 to 6 (mean 5.4). Then they concluded that CEUS-targeted biopsy can detect cancers with higher Gleason scores, and that CEUS is helpful in the grading of prostate cancer. Although the correlation between prostate cancer Gleason score and haemodynamic parameters was not assessed in their study, the positive conclusion agrees with the present study. The main limitation of the present study was that the pathology evaluation was limited by the lack of correlation to prostatectomy Gleason score. Previous researchers demonstrated that the biopsy specimen usually undergraded the prostatectomy Gleason score and the most common undergrading was by 1 or 2 score points. 33 However, the discrepancies were more likely to occur in prostate cancer patients with biopsy Gleason scores of 2e4. K oksal et al. found the accuracy was 15, 97, and 100% for Gleason scores of 2e4, 5e7, and 8e10 on needle biopsy, respectively. 34 In the present study, no patient had a designated Gleason score lower than 5, and the study group consisted of 59 (81%) patients with Gleason scores of 6e7 and 14 (19%) with Gleason scores of 8e10. Considering the composition ratio of Gleason scores among the patients, the present results were less affected by the discrepancies between the prostatectomy and biopsy specimens. In conclusion, the PI obtained using CPS harmonic ultrasonography correlates with the Gleason score and MVD in the prostate cancer. CPS harmonic ultrasonography could be a promising imaging technique for the detection and characterization of prostate cancer. This technique may bring about a higher detection rate of prostate cancer and more accurate biopsy Gleason grading, thereby providing a more appropriate therapeutic strategy. Acknowledgements Financial support from the Science and Technology Commission of Shanghai Municipality Foundation ( , ) is gratefully acknowledged. References Figure 3 Significant correlation between PI and MVD of prostate cancer. 1. Jemal A, Siegel R, Xu J, et al. Cancer statistics, CA Cancer J Clin 2010;60:277e Wijkstra H, Wink MH, de la Rosette JJ. Contrast specific imaging in the detection and localization of prostate cancer. World J Urol 2004;22:346e50.

6 J. Jiang et al. / Clinical Radiology 66 (2011) 732e Reese AC, Sadetsky N, Carroll PR, et al. Inaccuracies in assignment of clinical stage for localized prostate cancer. Cancer 2011;117:283e9. 4. Yoon F, Rodrigues G, D Souza D, et al. Assessing the prognostic significance of transrectal ultrasound extracapsular extension in prostate cancer. Clin Oncol 2006;18:117e Eisenberg ML, Cowan JE, Davies BJ, et al. The importance of tumor palpability and transrectal ultrasonographic appearance in the contemporary clinical staging of prostate cancer. Urol Oncol 2009; doi: / j.urolonc Eisenberg ML, Cowan JE, Carroll PR, et al. The adjunctive use of power Doppler imaging in the preoperative assessment of prostate cancer. BJU Int 2010;105:1237e Bono AV, Celato N, Cova V, et al. Microvessel density in prostate carcinoma. Prostate Cancer Prostatic Dis 2002;5:123e7. 8. Bostwick DG, Wheeler TM, Blute M, et al. Optimized microvessel density analysis improves prediction of cancer stage from prostate needle biopsies. Urology 1996;48:47e Lissbrant IF, Stattin P, Damber JE, et al. Vascular density is a predictor of cancer-specific survival in prostatic carcinoma. Prostate 1997;33: 38e Epstein JI, Allsbrook Jr WC, Amin MB, et al. The 2005 International Society of Urological Pathology (ISUP) Consensus Conference on Gleason Grading of Prostatic Carcinoma. Am J Surg Pathol 2005;29: 1228e Epstein JI. What s new in prostate cancer disease assessment in 2006? Curr Opin Urol 2006;16:146e Heidenreich A, Aus G, Bolla M, et al. EAU guidelines on prostate cancer. Eur Urol 2008;53:68e Fuchsjager M, Shukla-Dave A, Akin O, et al. Prostate cancer imaging. Acta Radiol 2008;49:107e Halpern EJ. Contrast-enhanced ultrasound imaging of prostate cancer. Rev Urol 2006;8 (Suppl. 1):S29e Wink M, Frauscher F, Cosgrove D, et al. Contrast-enhanced ultrasound and prostate cancer; a multicentre European research coordination project. Eur Urol 2008;54:982e Phillips P, Gardner E. Contrast-agent detection and quantification. Eur Radiol 2004;14 (Suppl. 8):P4e Siosteen AK, Elvin A. Intra-operative uses of contrast-enhanced ultrasound. Eur Radiol 2004;14 (Suppl. 8):P87e Seitz M, Gratzke C, Schlenker B, et al. Contrast-enhanced transrectal ultrasound (CE-TRUS) with cadence-contrast pulse sequence (CPS) technology for the identification of prostate cancer. Urol Oncol Aigner F, Pallwein L, Mitterberger M, et al. Contrast-enhanced ultrasonography using cadence-contrast pulse sequencing technology for targeted biopsy of the prostate. BJU Int 2009;103:458e Zhu Y, Chen Y, Jiang J, et al. Contrast-enhanced harmonic ultrasonography for the assessment of prostate cancer aggressiveness: a preliminary study. Korean J Radiol 2010;11:75e Weidner N, Semple JP, Welch WR, et al. Tumor angiogenesis and metastasisdcorrelation in invasive breast carcinoma. NEnglJMed1991;324:1e Wilson NM, Masoud AM, Barsoum HB, et al. Correlation of power Doppler with microvessel density in assessing prostate needle biopsy. Clin Radiol 2004;59:946e Erbersdobler A, Isbarn H, Dix K, et al. Prognostic value of microvessel density in prostate cancer: a tissue microarray study. World J Urol 2010;28:687e Strohmeyer D, Strauss F, Rossing C, et al. Expression of bfgf, VEGF and c-met and their correlation with microvessel density and progression in prostate carcinoma. Anticancer Res 2004;24:1797e Yang J, Wu HF, Qian LX, et al. Increased expressions of vascular endothelial growth factor (VEGF), VEGF-C and VEGF receptor-3 in prostate cancer tissue are associated with tumor progression. Asian J Androl 2006;8:169e Wang Z, Tang J, An L, et al. Contrast-enhanced ultrasonography for assessment of tumor vascularity in hepatocellular carcinoma. J Ultrasound Med 2007;26:757e Du J, Li FH, Fang H, et al. Correlation of real-time gray scale contrastenhanced ultrasonography with microvessel density and vascular endothelial growth factor expression for assessment of angiogenesis in breast lesions. J Ultrasound Med 2008;27:821e Shiyan L, Pintong H, Zongmin W, et al. The relationship between enhanced intensity and microvessel density of gastric carcinoma using double contrast-enhanced ultrasonography. Ultrasound Med Biol 2009;35:1086e Zhao H, Xu R, Ouyang Q, et al. Contrast-enhanced ultrasound is helpful in the differentiation of malignant and benign breast lesions. Eur J Radiol 2010;73:288e Folkman J. Angiogenesis in cancer, vascular, rheumatoid and other disease. Nat Med 1995;1:27e Sedelaar JP, van Leenders GJ, Hulsbergen-van de Kaa CA, et al. Microvessel density: correlation between contrast ultrasonography and histology of prostate cancer. Eur Urol 2001;40:285e Mitterberger M, Pinggera GM, Horninger W, et al. Comparison of contrast enhanced color Doppler targeted biopsy to conventional systematic biopsy: impact on Gleason score. J Urol 2007;178:464e Tomioka S, Nakatsu H, Suzuki N, et al. Comparison of Gleason grade and score between preoperative biopsy and prostatectomy specimens in prostate cancer. Int J Urol 2006;13:555e Koksal IT, Ozcan F, Kadioglu TC, et al. Discrepancy between Gleason scores of biopsy and radical prostatectomy specimens. Eur Urol 2000;37: 670e4.