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Association of Preoperative Plasma Levels of Vascular Endothelial Growth Factor and Soluble Vascular Cell Adhesion Molecule-1 With Lymph Node Status and Biochemical Progression After Radical Prostatectomy

From the Baylor Prostate Center and the Division of Male Reproductive Medicine and Surgery of the Scott Department of Urology; Department of Pathology and Molecular and Cellular Biology, Baylor College of Medicine; The Methodist Hospital, Houston; and Department of Urology, University of Texas Southwestern Medical School, Dallas, TX.

Address reprint requests to Kevin Mark Slawin, MD, Scott Department of Urology, Baylor College of Medicine, 6560 Fannin St, Ste 2100, Houston, TX 77030; e-mail: kslawin{at}www.urol.bcm.tmc.edu

	ABSTRACT

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION Authors' Disclosures of... REFERENCES

 
PURPOSE: Angiogenesis is a critical process for cancer progression. We tested whether elevated circulating levels of the angiogenesis-related markers vascular endothelial growth factor (VEGF) and/or soluble vascular cell adhesion molecule-1 (sVCAM-1) are associated with prostate cancer diagnosis, stage, progression, and metastasis.

PATIENTS AND METHODS: Plasma levels of VEGF and sVCAM-1 were measured on frozen, archival plasma obtained preoperatively from 215 consecutive patients who underwent radical prostatectomy for clinically localized disease, nine men with untreated prostate cancer metastatic to bones, and 40 healthy men without cancer.

RESULTS: Plasma levels of both VEGF and sVCAM-1 were highest in patients with bone metastases (P < .001). VEGF levels were higher in patients with clinically localized disease than in healthy controls (P < .001). VEGF levels were elevated in patients with biopsy and final Gleason sum 7 (P = .036 and P = .020, respectively) and extraprostatic extension (P = .047). Higher preoperative VEGF was independently associated with metastases to lymph nodes (P < .001). Both VEGF and sVCAM-1 were independently associated with biochemical progression after adjustment for the effects of standard preoperative features (P = .014 and P = .039, respectively). VEGF remained independently associated with biochemical progression after adjustment for standard postoperative features (P = .019).

CONCLUSION: Plasma levels of VEGF increased incrementally from healthy controls to patients with clinically localized disease to patients with lymph node and skeletal metastases. Higher preoperative VEGF was independently associated with metastases to lymph nodes and biochemical progression after surgery in both pre- and postoperative models. Plasma sVCAM-1 was elevated in men with bone metastases and was associated with biochemical progression in a preoperative model.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION Authors' Disclosures of... REFERENCES

 
Angiogenesis, the process of new blood vessel growth, is necessary for tumor growth and metastasis.¹ In prostate cancer, the conversion to an angiogenic phenotype has been associated with tumorigenesis^2,3 and late stages of tumor progression.^4,5 Angiogenesis has been studied primarily by directly examining new vessel growth within tumor specimens or experimental models. Commonly, microvessel density is determined by staining the neovasculature within a tumor with endothelial cell-specific antibodies and then counting the number of new blood vessels within vascular hotspots. Microvessel density has been shown to be higher and vascular networks more disorganized in malignant tissue than in normal prostate tissue.^6,7 Moreover, microvessel density has been associated with clinical and pathologic features of biologically aggressive prostate cancer, disease progression, and metastasis in some studies^8–12 but not in others.^13,14 Compounding this lack of clarity is the variability introduced through the use of different antibodies, widely varying interpretation, stratification criteria, and preanalytical protocols for tissue fixation and processing.

The conversion of these assays from tests that require tumor tissue specimens to assays that can be performed on blood would significantly increase their clinical value. Objective, standardized, and quantitative serum or plasma markers associated with tumor angiogenesis that also provide clinically useful data regarding tumor stage or risk of progression would enhance the ability of clinicians to use markers of this important biologic process to help guide therapy.

Candidate circulating soluble markers that are associated with angiogenesis and cancer progression to metastasis include vascular endothelial growth factor (VEGF) and soluble vascular cell adhesion molecule-1 (sVCAM-1). VEGF, one of six members of the VEGF family of related proteins, is a homodimeric, heparin-binding glycoprotein that modulates angiogenesis though interaction with several specific receptors. VEGF promotes endothelial cell mitogenesis and survival, mediates chemotaxis of vascular endothelial cells, increases vascular permeability, inhibits maturation of antigen- presenting dendritic cells, and increases vasodilatation.^1,15–19 While normal prostatic epithelium and stroma and prostatic adenocarcinoma constitutively express VEGF,²⁰ malignant prostatic tissue produces significantly higher levels of VEGF than does benign prostatic tissue.²¹ Studies have shown a direct relationship between microvessel density and the level of VEGF expression during tumor progression.^22–24 Blood levels of VEGF have been reported to be significantly increased in patients with metastatic prostate cancer.²⁵ In addition, higher pretreatment plasma levels of VEGF have been shown to be independently associated with decreased survival in patients with hormone-refractory prostate cancer.²⁶ However, the prognostic significance of circulating levels of VEGF in patients diagnosed with clinically localized prostate cancer has not yet been reported.

VCAM-1 (CD 106) is a 90-kd transmembrane glycoprotein that is transiently expressed on the surface of vascular endothelial cells in response to vascular endothelial growth factors and cytokines.^27–29 During inflammation, endothelial expression of VCAM-1 regulates signal transduction and adhesion of leukocytes to the endothelium, via interaction with the beta-1 integrin very late antigen-4 on leukocytes.³⁰ Tumor necrosis factor-alpha, a cytokine known to be implicated in prostate stroma-epithelium interaction, has been shown to lead to a two-fold increase in expression of VCAM-1 in LNCaP prostate tumor cells.³¹ In addition, VCAM-1 has been shown to function as an adhesion molecule facilitating primary prostate cancer metastasis.³² Serum levels of soluble VCAM-1 have been shown to correlate closely with microvessel density in breast tumor specimens and to be strongly associated with disease stage, progression, and response to hormone therapy, suggesting a potential role for circulating soluble VCAM-1 as a surrogate clinical marker of local tumor angiogenesis and disease progression.³³ The clinical significance of circulating levels of sVCAM-1, like that of VEGF, in patients diagnosed with clinically localized prostate cancer has not yet been investigated.

We tested the hypothesis that plasma levels of VEGF and/or sVCAM-1 may serve as clinically useful biomarkers of tumor angiogenesis that would be associated with prostate cancer detection, stage, and prognosis. We evaluated the association of preoperative plasma levels of VEGF and sVCAM-1 with prostate cancer presence, invasion, progression, and metastasis in a large consecutive cohort of patients with clinically localized prostate cancer, who underwent radical prostatectomy and who had long-term follow-up, in men with metastatic skeletal disease, and in men without prostate cancer.

	PATIENTS AND METHODS

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Patient Population
All studies were undertaken with the approval and institutional oversight of the institutional review board for the Protection of Human Subjects at Baylor College of Medicine (Houston, TX). We evaluated plasma levels of VEGF and sVCAM-1 in nine men with bone scan-proven, metastatic prostate cancer and in 40 healthy patients without cancer. The patients with metastatic bone disease were not treated with either hormonal or radiation therapy before plasma collection. The healthy noncancer group was composed of two sets of consecutive patients who participated in the Baylor Prostate Center's weekly prostate cancer screening program, who had no previous history of any cancer or chronic disease, a normal digital rectal examination, and a prostate-specific antigen (PSA) level of less than 2.0 ng/mL, a PSA range that has an estimated probability of prostate cancer detection of less than 1% in the first 4 years after screening.³⁴

We also measured preoperative plasma levels of VEGF and sVCAM-1 levels in 215 consecutive patients who underwent radical prostatectomy for clinically localized prostatic adenocarcinoma. All patients underwent an extended lymph node dissection that included level I, II, and III lymph nodes as routine.³⁵ All 301 patients admitted to The Methodist Hospital (Houston, TX) with the intent to treat their clinically localized prostate cancer (cT1c-3a, NX, M0) with radical prostatectomy by surgeons of the Scott Department of Urology (Baylor College of Medicine, Houston, TX) during the period of November 1994 through December 1995 were potential candidates for this analysis. After consent was gained, preoperative plasma specimens were obtained from 252 of these men. Sixteen men initially treated with hormonal therapy, five who were treated with definitive radiotherapy, and one who was treated with cryotherapy before surgery were excluded from the analysis. No disease follow-up information was available for 15 men, and they were also excluded. This left 215 men for analysis. The mean patient age in this study was 61.8 ± 7.3 years (median, 62.6 years; range, 40 to 80 years). The operative surgeon assigned the clinical stage according to the 1992 tumor-node-metastasis system. Serum PSA was measured by the HybritechTandem-R assay (Hybritech Inc, San Diego, CA).

Measurement of Plasma Levels of VEGF and sVCAM-1
Plasma samples were collected after a preoperative overnight fast on the morning of the day of surgery, at least 4 weeks after transrectal guided needle biopsy of the prostate. Blood was collected into VacutainerCPT^TM 8 mL tubes containing 0.1 mL of molar sodium citrate (Becton Dickinson Vacutainer Systems, Franklin Lakes, NJ) and centrifuged at room temperature for 20 minutes at 1500g. The top layer corresponding to plasma was decanted using sterile transfer pipettes. The plasma was immediately frozen and stored at –80 C in polypropylene cryopreservation vials (Nalgene, Nalge Nunc, Rochester, NY). It has been previously found that VEGF levels are higher when measured in serum than when measured in plasma.^36–38 Since VEGF is present in platelet granules and is released on platelet activation, the higher levels of VEGF in serum were likely as a result, at least in part, of release from damaged platelets, making the quantification of nonplatelet derived VEGF less accurate. Therefore, for VEGF, before assessment, an additional centrifugation step of the plasma was performed at 10,000g for 10 minutes at room temperature for complete platelet removal.³⁹ For quantitative measurements of VEGF and sVCAM-1 levels, we used quantitative immunoassays (R&D systems, Minneapolis, MN). Every sample was run in duplicate, and the mean was used. Differences between the two measurements for both plasma VEGF and sVCAM-1 were minimal (intra-assay precision coefficients of variation, 8.49% ± 11.10% and 4.86% ± 6.31%, respectively).

Pathologic Examination
All prostatectomy specimens were examined pathologically at our institution by a single pathologist (T.M.W.), who was blinded to clinical outcome. The radical prostatectomy specimens were processed by whole-mount technique, and pathologic parameters were evaluated in a manner previously described.⁴⁰ Total tumor volume was computed by computerized planimetry from the whole-mount sections for 184 of the 215 prostatectomy patients.⁴¹

Postoperative Follow-Up
Patients generally were scheduled to have a digital rectal examination and serum PSA evaluation postoperatively every 3 months for the first year, semiannually from the second through the fifth year, and annually thereafter. Biochemical progression was defined as a sustained elevation, on two or more occasions, of PSA > 0.2 ng/mL and was assigned to the date of the first value > 0.2 ng/mL in radical prostatectomy patients. Radical prostatectomy was aborted in two of the 11 patients who were found to have nodal metastases on frozen section analysis during the operation; these men are not excluded from the analysis. The two patients with metastases to regional lymph nodes whose prostates were not removed were categorized among those with progression from the day after surgery. Six patients (3%) received adjuvant radiation therapy before biochemical progression because of positive surgical margins. Three of them subsequently experienced PSA relapse and were considered to have disease progression from the date of the first value > 0.2 ng/mL. The other three patients were censored on the date of the last follow-up examination.

Statistical Analysis
The Mann Whitney U-test and the Kruskal Wallis test were used to test for differences in plasma VEGF and sVCAM-1 levels between clinical and pathologic features. Spearman's rank correlation coefficient was used to compare ordinal and continuous variables. Logistic regression was used for multivariate analyses of binary outcome variables. Multivariate survival analysis was performed with Cox proportional hazard regression model. Preoperative PSA level had a skewed distribution and therefore was modeled with a log transformation. Biopsy and radical prostatectomy Gleason sum were evaluated as grade 2 to 6 versus grade 7 to 10. Statistical significance in this study was set as P < .05. All reported P values are two-sided. All analyses were performed with SPSS statistical package version 11 for Windows (SPSS Inc, Chicago, IL).

	RESULTS

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Association of Plasma VEGF and sVCAM-1 With Prostate Cancer Presence and Metastasis
We assessed plasma VEGF and sVCAM-1 in nine patients with bone scan-proven, metastatic prostate cancer, 215 patients diagnosed with clinically localized prostate cancer, and 40 healthy controls (Table 1). Plasma levels of VEGF were higher in patients with skeletal metastases than those in patients with clinically localized disease (P < .001), which in turn were higher than those in healthy controls (P < .001). While plasma levels of VCAM-1 were highest in patients with skeletal metastases (P < .001), levels were higher in healthy controls than those in patients with clinically localized prostate cancer (P < .001).

In a multivariate logistic regression analysis (Table 2) that adjusted for the effects of preoperative VEGF, preoperative sVCAM-1, and clinical stage, only preoperative PSA and biopsy Gleason sum were associated with organ confined disease (P = .008 and P < .001, respectively). However, in a model that adjusted for the effects of preoperative sVCAM-1, preoperative PSA, and biopsy Gleason sum, only preoperative plasma VEGF and clinical stage were associated with metastases to regional lymph nodes (P < .001 and P = .019, respectively).

View larger version (22K): [in this window] [in a new window]   Fig 1. Kaplan-Meier estimates of progression-free probability for the 215 patients with clinically localized prostate cancer treated with radical prostatectomy stratified into groups above or below the median vascular endothelial growth factor (VEGF) level of 9.9 pg/mL.

View larger version (22K): [in this window] [in a new window]   Fig 2. Kaplan-Meier estimates of progression-free probability for the 215 patients with clinically localized prostate cancer treated with radical prostatectomy stratified into groups above or below the median soluble vascular cell adhesion molecule-1 (sVCAM-1) level of 493.8 ng/mL.

	DISCUSSION

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION Authors' Disclosures of... REFERENCES

In our study, preoperative plasma VEGF was significantly associated with biochemical progression in this large cohort of consecutive patients with long-term follow-up after radical prostatectomy. This association remained significant after adjustment for the effects of standard preoperative and postoperative features. George et al²⁶ recently reported that plasma levels of VEGF are independently associated with overall survival in 197 patients with hormone-refractory prostate cancer. In addition, we found that preoperative plasma levels of VEGF were elevated in patients with established features of biologically aggressive prostate cancer such as biopsy or final Gleason sum 7, extraprostatic extension, and metastases to regional lymph nodes. Tissue expression of VEGF has been shown to be directly associated with tumor Gleason sum in studies using immunohistochemical staining⁴² and rapid colorimetric in situ hybridization.⁴³ Gleason grade is one of the strongest predictors of biologic behavior of prostate cancer, such as metastatic potential.¹⁴ Higher VEGF expression has also been associated with more advanced pathologic stages.⁴⁴ However, of all the adverse pathologic features, the presence of histopathologically evident prostate cancer metastases to pelvic lymph nodes is the strongest predictor of future disease progression to distant metastases, regardless of apparent success in eradicating local disease.^45–48 Thus, while the pT category of the primary tumor is a substantial stratification variable in determining who may or may not benefit from radical surgery, the nodal tumor burden is the most significant pathologic prognostic factor in patients with prostate cancer.

We found that preoperative plasma VEGF was an independent predictor of metastases to regional lymph nodes. Conventional imaging tools used for lymph node staging (eg, computed tomography, magnetic resonance imaging, and positron emission tomography) lack sensitivity for detecting small but clinically significant lymph node metastases.^49–52 It has been reported that approximately 10% to 25% of patients undergoing radical prostatectomy for clinically organ-confined disease will be found to harbor lymph node metastases.^53–55 Preoperative predictive models that consider established markers such as serum PSA, clinical stage, and Gleason grade can provide an estimate of the risk of nodal metastasis but still are imperfect for determining prognosis in individual patients.^56,57 Preoperative plasma VEGF may improve early identification of patients who harbor lymph node metastases, thereby providing surgeons with the opportunity for intensive and meticulous lymphadenectomy (which has been suggested to be curative in some men with low volume metastases),³⁵ for selecting patients best suited for clinical trials of early systemic intervention, for sparing men who have undergone prostatectomy from the morbidity associated with ineffective local adjuvant or salvage radiation therapy, and/or in predicting patient outcome. Although the topic of the extent of lymph node sampling remains controversial, there is recent evidence that an extended pelvic lymphadenectomy is associated with a high rate of lymph node metastases outside of the fields of standard lymphadenectomy.^35,58 In our study, a standard pelvic lymph node dissection was performed in each patient with a mean of 9.9 ± 5.1 lymph nodes per patient removed at the time of radical prostatectomy.

We confirmed that plasma levels of VEGF are greatly elevated in patients with regional and distant metastases compared with those in patients with nonmetastatic prostate cancer or those in healthy subjects.^25,59 There is overwhelming evidence for a causal involvement of the VEGF in prostate cancer invasion and metastasis, and inhibition of the VEGF expression has been shown to alter these processes. VEGF is a multifunctional cytokine that promotes endothelial cell proliferation and migration in various models.^16–18,60 Moreover, it induces the urokinase plasminogen activator and its receptor, leading to degradation of the extracellular matrix facilitating endothelial cell and tumor cell migration and invasion. Furthermore, VEGF is a potent vascular permeability factor causing leaky vessels. Several studies have shown that VEGF may play a role in prostate cancer's organ tropism in metastatic spread to bone.^61,62 Using an athymic mouse model injected with DU145 human prostate cancer cells, Borgstrom et al⁶³ showed a complete inhibition of neovascularization and tumor growth after the initial prevascular angiogenesis-independent growth phase.

We found that patients with prostate cancer have higher plasma levels of VEGF than healthy controls. Duque et al²⁵ found a tendency for plasma VEGF to be higher in patients with clinically localized prostate cancer (n = 54) than in controls (n = 26). In contrast, one other study did not find any statistically significant difference in serum levels of VEGF between controls (n = 21), patients with benign prostatic hyperplasia (n = 9), and patients with clinically localized prostate cancer (n = 16).⁶⁴ However, this study suffered from a small sample size and, more importantly, relied on serum samples, which are known to be less reliable than plasma samples for measuring VEGF levels.^36–39 Indeed, VEGF measured in serum samples is released from platelets on activation after venipuncture.^37–39 To ensure complete platelet removal, we rapidly processed citrated plasma samples and performed an additional centrifugation. This has been demonstrated to be the only suitable protocol, better reflecting any disease-related overspill of VEGF into the circulation.^37,39 Interestingly, we found a significant correlation between plasma levels of VEGF and the total and transitional zone volume of the prostate, two established markers of benign prostatic hyperplasia. The association of VEGF with this and other pathologies together with the overlap in VEGF levels between men with cancer and controls make it questionable whether this marker can be used for early diagnosis of prostate cancer.

In our study, plasma sVCAM-1 levels were highest in patients with bone metastases. Previous studies have shown an association between sVCAM-1 and metastasis to bone^65,66 and to the brain.⁶⁷ However, we found that sVCAM-1 levels were lower in patients with clinically localized diseases than those in healthy subjects. Serum sVCAM-1 levels have previously been shown to be higher in patients with prostate cancer than in healthy controls.⁶⁸ These authors did not, however, differentiate between metastatic and nonmetastatic disease. Interestingly, these authors also found that serum levels of sVCAM-1 are elevated in patients with benign prostatic hyperplasia. In concordance with this finding, we found a direct correlation between serum sVCAM-1 levels and the transition zone volume of the prostate. Plasma sVCAM-1 levels were not associated with any clinical or pathologic features of patients undergoing radical prostatectomy for clinically localized disease. While prediction of final pathologic features is important, nomograms incorporating biomarkers that can predict disease progression in patients undergoing radical prostatectomy would provide a more useful adjunct for the management of patients with prostate cancer.

We found that preoperative plasma sVCAM-1 level was an independent predictor of prostate cancer progression in patients undergoing radical prostatectomy, when adjusted for standard preoperative but not postoperative features. A systematic survey of angiogenic markers in breast cancer patients indicated that serum sVCAM-1 is an accurate marker of tumor angiogenesis and prognosis in breast cancer.³³ These authors reported that serum sVCAM-1 was the marker most closely associated with standard prognostic factors in early breast cancer. In addition, serum sVCAM-1 levels correlated closely with tumor microvessel density. This study did not, however, investigate VEGF levels.³³

In conclusion, preoperative plasma VEGF was independently associated with metastases to lymph nodes and biochemical progression after radical prostatectomy. If validated, preoperative plasma VEGF may aid in the selection of patients undergoing radical prostatectomy who may benefit from more extensive lymph node dissection and treatment regimens combining modalities. Further research is necessary to determine whether plasma VEGF is a surrogate marker of the development of new blood vessels in prostate cancer, and thus can facilitate the assessment of the response to angiogenesis inhibitors. In contrast, plasma sVCAM-1 has restricted usefulness as a staging tool for guiding therapy and predicting outcome in patients with clinically localized prostate cancer, and therefore has limited clinical utility. Several limitations in this study should be considered. Some variables that were inconclusive as a result of limited statistical power may attain statistical significance if the sample size or the length of follow-up is increased.

	Authors' Disclosures of Potential Conflicts of Interest

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The authors indicated no potential conflicts of interest.

	NOTES

 
S.F.S. is supported by the Austrian Program for Advanced Research and Technology, and V.A.A. is supported by the American Cancer Society (grant No. IRG 93-034-06).

Drs Shariat and Anwuri contributed equally to this study.

Authors' disclosures of potential conflicts of interest are found at the end of this article.

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Submitted September 26, 2003; accepted December 29, 2003.

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