Affiliations of authors: G. J. Byrne, A. Ghellal, J. Iddon, A. D. Blann, V. Venizelos, N. J. Bundred, Department of Academic Surgery, University Hospital of South Manchester, U.K.; S. Kumar, Department of Pathological Science, University of Manchester; A. Howell, Cancer Research Campaign Department of Medical Oncology, Christie Hospital National Health Service Trust, Manchester, U.K.
Correspondence to: Nigel J. Bundred, M.D., Reader in Surgical Oncology, University Hospital of South Manchester, Nell Lane, Manchester, M20 8LR, U.K. (e-mail: bundredn{at}fs1.with.man.ac.uk).
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ABSTRACT |
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INTRODUCTION |
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Surrogate markers of angiogenesis would facilitate the assessment of the response to angiogenesis inhibitors (1). Candidates for such biomarkers include glycoproteins that are produced and secreted by activated endothelial cells. These glycoproteins include vascular cell adhesion molecule-1 (VCAM-1) (6), endothelial selectin (E-selectin) (7), and von Willebrand factor (VWF) (8).
VCAM-1 is a 90-kd transmembrane glycoprotein that is expressed transiently on vascular endothelial cells in response to vascular endothelial growth factor (VEGF) and other cytokines (6). Endothelial expression of VCAM-1 plays a major role in adhesion of leukocytes to the endothelium in inflammation. In addition, endothelial cells expressing VCAM-1 bind melanoma cell lines, suggesting that VCAM-1 may function as an adhesion molecule to facilitate metastasis (6). Although VCAM-1 is expressed predominantly on activated endothelial cells, it is also found on dendritic cells and proximal renal tubule cells (6).
E-selectin (CD62E) (previously known as endothelial leukocyte adhesion molecule [ELAM-1]) is a transmembrane glycoprotein that, like VCAM-1, is expressed on endothelial cells in response to VEGF (9). E-selectin mediates adhesion of neutrophils, monocytes, and memory T cells to the endothelium and has, like VCAM-1, been implicated in metastasis (10).
VWF is produced by endothelial cells and platelets; however, it is not an adhesion molecule. Physiologic levels of VWF increase with platelet adhesion at the site of injury. VWF then forms a complex with factor VIII that facilitates normal blood clotting.
Soluble forms of several adhesion molecules (e.g., leukocyte-selectin [CD62L) and platelet-selectin [CD62P]) and growth factor receptors (e.g., c-erbB2) are known to be shed from the cell surface (11); VCAM-1 and E-selectin both have soluble forms. Soluble forms of VCAM-1 have been detected both in vitro (12) and in vivo (11,1315). Serum levels of soluble VCAM-1 are raised in patients with various malignancies, including breast and gastric cancers (16,17). The soluble form of E-selectin is present at higher levels in the serum of patients with certain cancers than in cancer-free control subjects (16). In addition, the serum VWF concentration has been shown to be increased in patients with advanced breast cancer as compared with normal control subjects (15). Koch et al. (18) have demonstrated that soluble forms of VCAM-1 and E-selectin released by leukocyte adhesion to endothelial cells are chemotactic attractants for endothelial cells.
Although immunohistochemical studies demonstrate that VCAM-1 and E-selectin are found on the membranes of malignant breast endothelial cells, they are not found on breast epithelial cells (19). Soluble VCAM-1 and E-selectin have been implicated in the mediation of angiogenesis (18). We, therefore, hypothesized that levels of VCAM-1 and E-selectin in the serum may provide surrogate markers of angiogenesis occurring in breast cancer. By contrast, we anticipated that VWF, which is released by all endothelial cells, would be a pan-endothelial marker that would not accurately report angiogenesis. To determine the potential of these serum glycoproteins to serve as biomarkers for angiogenesis, we examined whether serum levels of VCAM-1, E-selectin, or VWF are associated with standard prognostic factors in women with early and advanced breast cancers. We also tested whether microvessel density in tumors from women with early breast cancer correlates with serum levels of any of these endothelial cell-derived glycoproteins.
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PATIENTS AND METHODS |
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Patients With Early Breast Cancer
Women with newly diagnosed breast cancer (n = 93) provided venous blood immediately before surgery (axillary lymph node clearance combined with either mastectomy or wide local excision). The mean age of the patients at diagnosis was 57.8 years (range = 3295 years). Of the women with early breast cancer, 22 were premenopausal and 71 were postmenopausal. Blood was taken from a control group of 29 women with benign breast disorders (cyclical mastalgia [n = 17] or fibrocystic change [n = 12]) who had attended the same clinic. The commercial enzyme-linked immunosorbent assays (ELISAs) used to measure VCAM-1 and E-selectin had already reported a normal range of levels in healthy control women (VCAM-1: median value = 553 ng/mL [range = 395714 ng/mL]; E-selectin: median value = 46.3 ng/mL [range = 29.163.4 ng/mL]). We assayed serum levels in the 29 women with benign breast disorders and found that levels were in the normal range in these women as well.
An experienced breast pathologist assessed hematoxylineosin-stained specimens for tumor type, size, and grade (20) without knowledge of serum levels of VCAM-1, E-selectin, or VWF. The total number of lymph nodes in the axillary clearance specimens and the total number of involved lymph nodes were recorded for each patient. The development and site of a recurrent carcinoma were recorded at the date of presentation. Estrogen receptor was assessed in all tumors with the use of a standard immunohistochemical staining technique utilizing a primary mouse monoclonal antibody to human estrogen receptor (M7042; DAKO, Copenhagen, Denmark) as described previously (21).
Patients With Advanced Breast Cancer
Sequential serum samples were taken from 55 women with estrogen receptor-positive cancer who were commencing or switching hormonal therapy for progressing or newly diagnosed advanced breast cancer. All of the women had evidence of metastatic disease in the skeleton, as diagnosed by isotope bone scan and plain radiographs. In addition, 28 of the women had disease in one or more other sites (lung [n = 12], liver [n = 10], soft tissue [n = 8], adrenal [n = 1], or brain [n = 1]). Blood was taken immediately before the patient started or changed hormonal therapy and 3 months later. The women had a mean age of 56.4 years (range = 3781 years). At the time of breast cancer diagnosis, 13 of the women were premenopausal, and 42 were postmenopausal. Response to treatment was assessed by standard radiologic criteria at 3 and 6 months after initiation of or change in hormonal therapy or at disease progression (22). Each patient was assessed by a medical oncologist (A. Howell) without knowledge of serum concentrations of VCAM-1, E-selectin, and VWF.
VCAM-1, E-Selectin, and VWF Assays
Blood from all patients was centrifuged for 20 minutes at 3500g at 4 °C immediately after phlebotomy. The separated serum was then stored at -20 °C in 1-mL aliquots. Before analysis, samples were thawed slowly and mixed gently.
VCAM-1 and E-selectin were assayed with the use of commercial ELISA kits (R&D Systems Ltd., Oxford, U.K.) (11,12). Intra-assay precision for VCAM-1 and E-selectin assays was 6.7% and 5.1%, respectively. Interassay precision for VCAM-1 and E-selectin assays was 7.0% and 6.2%, respectively.
Serum concentrations of VWF were determined by ELISA, with the use of a technique described previously (23). The rabbit antihuman VWF antibody for this assay was obtained from DAKO (High Wycombe, U.K.).
Microvessel Count: Assessment of Angiogenesis
Microvessels in tumors from women with early breast cancer were visualized by immunostaining sections with antibodies to CD31, an endothelial cell antigen. Sections (5 µm) were cut from formalin-fixed, paraffin-embedded specimens of primary breast cancers. The specimens were dewaxed in xylene followed by four changes of ethanol, after which they were washed in tap water prior to staining. Endogenous peroxidase activity was blocked by treating the sections with 3% hydrogen peroxide in deionized water for 10 minutes. Tissue sections were put in 0.1 M citrate buffer (pH 6.0) and placed on a rotating table in a microwave oven. Heat pretreatment was carried out with two 15-minute cycles each at medium-high output (600 W). The sections were allowed to cool at room temperature and washed in Tris-buffered saline (TBS) prior to immunostaining. Nonspecific binding was blocked with 1% normal goat serum in TBS for 10 minutes. Serial sections were incubated with a 1 : 20 dilution of primary antibody (JC/70A monoclonal antibody to CD31; DAKO, Copenhagen) (2426). The slides were washed with TBS for 5 minutes, incubated with a 1 : 100 dilution of biotinylated secondary goat anti-mouse antibody (KO492; DAKO, Goldstrup, Denmark) in 1% normal goat serum in TBS for 30 minutes, and washed in TBS for 23 minutes. The streptavidinbiotin complex (1 : 100 in TBS; DAKO, Copenhagen) was applied for 30 minutes. The slides were then washed with TBS, treated with 0.08% diaminobenzidine (Sigma Chemical Co., St. Louis, MO) and hydrogen peroxide (0.3%) in deionized water, counterstained with hematoxylin, dehydrated, and mounted with DPX (a mixture of disterene, plasticizer, and xylene; BDH, Leicester, U.K.). Sections for which the primary or secondary antibody had been omitted were used as negative controls.
Microvessel density was quantified by light microscopy of labeled slides without knowledge of patient details. The most vascular areas in a tumor (i.e., the hot spots) were located at low magnification, and the vessels in these regions were counted with the use of a Chalkley point eyepiece graticule at 400x magnification (21). Any brown-staining endothelial cells or group of cells in contact with a spot in a graticule was counted as an individual vessel. The mean of four Chalkley counts for each tumor was calculated and used in statistical analysis. Microvessel density was assessed without knowledge of serum levels of VCAM-1, E-selectin, or VWF. Microvessel scores with the use of Chalkley counting ranged from 1 to 4 vessel counts, with 4 being the highest score and 1 being the lowest. We have previously validated this technique (21).
Statistical Methods
Statistical analysis was performed with the use of Pearson correlations for all paired continuous variables and Student's t tests for categorical analysis. Serum measurements of all three glycoproteins demonstrated skewed distributions; therefore, logarithms of the geometric means of variables were used in the analysis. Data for tumor grade and lymph node status for women with early breast cancer were assessed with the use of a one-way analysis of variance. For women with advanced breast cancer, logarithms of the geometric mean difference were analyzed with the use of a paired Student's t test. VCAM-1, E-selectin, and VWF serum levels were compared with vessel counts with the use of chi-square tests. All P values are two-sided.
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RESULTS |
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To investigate whether serum concentrations of activated endothelial cell molecules are associated with cancer, we compared serum levels of VCAM-1, E-selectin, and VWF in women with early breast cancer with those in control women with benign breast disease. In women with early breast cancer, serum concentrations of VCAM-1 (mean = 769.5 ng/mL; 95% confidence interval [CI] = 651887 ng/mL), E-selectin (mean = 54.3 ng/mL; 95% CI = 48.160.6 ng/mL), and VWF (mean = 141.6 ng/mL; 95% CI = 126.8156.5 ng/mL) were statistically significantly higher than those in control women (VCAM-1: mean = 483 ng/mL [95% CI = 448518 ng/mL]; E-selectin: mean = 43.5 ng/mL [95% CI = 40.346.8 ng/mL]; VWF: mean = 112.2 ng/mL [95% CI = 93.8128.6 ng/mL]) (P = .001, P = .003, and P = .031, respectively). In addition, serum levels of soluble VCAM-1 and E-selectin, but not of VWF, were significantly higher in women with histologic axillary lymph node metastases (40 [43%] of the 93 women with early breast cancer) than in lymph node-negative women or control women (P = .003 and P = .047, respectively; Fig. 1). VCAM-1 levels also were associated with the extent of tumor differentiation. Serum from women with well-differentiated tumors (grade 1 [n = 12]) had lower VCAM-1 levels (mean = 592 ng/mL; 95% CI = 520664 ng/mL) than serum from women with higher tumor grades (grade 2 [n = 34] or grade 3 [n = 47]) (mean = 793 ng/mL; 95% CI = 675911 ng/mL) (P = .003, two-sided Student's t test following logarithmic transformation). By contrast, serum E-selectin and VWF levels did not relate to tumor grade.
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Recurrence did show an association with serum VCAM-1 levels (Table 1). Of the 93 women with early breast cancer, 16 have subsequently developed recurrent breast carcinoma (locoregional [n = 4], bone alone [n = 10], or mixed bone and visceral [n = 2]) after a median follow-up from the date of surgery of 38 months (range = 2272 months). The median time to recurrence was 21 months (range = 853 months). Preoperative serum VCAM-1 levels were higher in those women who developed early recurrence than in those who have remained cancer free to the end of the study (P = .01) (Table 1
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Levels of angiogenesis in women with advanced breast cancer cannot be assessed by microvessel density because the technique does not allow sequential assessment of angiogenesis in human metastases. Often biopsies cannot be performed on metastases and, if the lesion is excised, it cannot be assessed again. In animal models, angiogenesis is inhibited by hormonal therapy (27,28); therefore, we studied women with advanced breast cancer who were undergoing hormonal therapy to assess any changes in the levels of VCAM-1, E-selectin, and VWF. Of the 55 women with advanced breast cancer, 31 had a partial response (n = 10) or stable disease for more than 6 months (n = 21) after treatment with tamoxifen (n = 15), megestrol acetate (n = 10), goserelin (n = 3), or anastrozole (n = 3). Among women who showed a partial response to therapy, their serum VCAM-1 levels fell by an average value of 276 ng/mL (P = .043), whereas serum E-selectin and VWF levels did not change (Table 2). Among women whose disease was stable, there were no changes in serum levels of any of the three endothelial cell molecules.
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It has been shown previously that there is no difference in survival between patients who exhibit a partial response to hormonal therapy and those whose disease remains stable for 6 months (29). Thus, the two groups were also combined for analysis. Statistical comparison between this combined group with women whose disease progressed indicated that only VCAM-1 demonstrated a difference between responding and nonresponding tumors at 3 months (P<.001). The positive predictive value of no increase in serum VCAM-1 levels, indicating a response to therapy, was 90% (26 of 29) (Table 3). The sensitivity and specificity of a decrease in serum VCAM-1 levels were 98% and 80%, respectively (Table 3
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DISCUSSION |
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Our results demonstrate that serum levels of VCAM-1 are more closely associated than E-selectin and VWF serum levels with standard prognostic factors in early breast cancer. In addition, serum VCAM-1 levels correlate closely with tumor microvessel density, which is currently the standard method for measuring angiogenesis (4,5,8). Moreover, changes in levels of VCAM-1 in the serum of women undergoing hormonal therapy for metastatic breast cancer also paralleled responses of the epithelial cells to the endocrine therapy, suggesting that a "switch off" of angiogenesis is an early step in inhibition of tumor growth.
A study (17) showed serum VCAM-1 to be of prognostic value in patients with gastric cancer. However, given the large acute-phase reaction seen in gastric neoplasia, it is difficult to separate the acute-phase response from angiogenesis in this context. In the clearer context of breast cancer, we found that high serum levels of VCAM-1 identified tumors at risk of early relapse as well as lymph node status and microvessel density.
Angiogenesis proceeds at the same time as tumor growth and metastasis (13). The continuous formation of new capillaries, which is induced by tumor epithelial angiogenic factors (1,2), can be quantified by measuring microvessels (2426). While endothelial cell activation in vitro can be recognized by a sprouting phenotype (1,2,11), an angiogenic response in the cornea (18), or endothelial chemotaxis (18), recognition of endothelial cell activation in vivo is problematic (13). Microvessel counts have been shown to be an independent prognostic marker in breast cancer (8), but assessment of microvessel density relies on immunohistochemical staining of the primary tumor. Microvessel density thus provides a snapshot of angiogenesis that cannot be repeated once the primary tumor has been removed. The measurement of serum VCAM-1 levels, therefore, is potentially a simple surrogate method of determining levels of angiogenesis at any given time in women with early breast cancer. Serum VCAM-1 levels in women with advanced breast cancer potentially represent the state of continuing angiogenesis in metastases. The rise in VCAM-1 levels by 3 months correlated with hormone-nonresponsive disease, whereas a fall was seen in hormone-responsive disease, suggesting that a reduction in endothelial secretion of adhesion antigens occurs early in hormone-responsive tumors.
Angiogenesis takes place continuously at the advancing edge of tumors or metastatic deposits (1,2). It may occur not just by endothelial cell proliferation but also by co-opting of host blood vessels, endothelial cell migration, and capillary cell morphogenesis (13). Serum VCAM-1 levels in patients with advanced breast cancer represent an accurate measure of continuing endothelial activation and angiogenesis, as indicated by the rise in VCAM-1 levels within 3 months in women whose breast cancer was progressing and by the fall in these levels in women whose breast cancer showed a partial response to endocrine therapy. These observations suggest that, in hormone-responsive tumors, a reduction in the surface area of activated endothelium leads to a reduction in shed VCAM-1 into the serum. Once blood vessels have become established in tumors, the endothelium expresses different antigens (19,21,24). The factors controlling expression of all of these endothelial antigensin particular, soluble VCAM-1are not clear.
In tumors, endothelial VCAM-1 plays a major role in the adhesion of leukocytes to the endothelium, suggesting that cellular adhesion and angiogenesis are linked (18,32). The adhesion molecule vß3 integrin is a marker of angiogenic vascular tissue in wound granulation tissue (33), and both soluble E-selectin and soluble VCAM-1 are angiogenic in a corneal model (18). The mechanism underlying the shedding of VCAM-1 and E-selectin is not yet known, but shedding is believed to occur after adhesion of inflammatory or tumor cells to activated vascular endothelium (32). VCAM-1 expression by endothelial cells, although tissue and organ dependent (34), is induced by VEGF, tumor necrosis factor-
, interleukin 1ß, and interferon gammaall of which have been implicated in the angiogenic response (19,35,36). For example, Jallal et al. (37) have described the release of a 90-kd protein by tumor cells that increases the expression of VCAM-1 in tumor vessels. However, the same protein leads to tumor regression and infiltration by natural killer cells and macrophages when given locally or systemically in nude mice models (37).
Increased expression of VCAM-1 on activated endothelium may facilitate transmigration of monocytes and T lymphocytes across the endothelium because these cell types express VLA4, the ligand for VCAM-1 (38). The binding of monocytes to endothelial cells and the concomitant release of cytokines by both endothelial cells and monocytes have been reported to lead to the release of soluble VCAM-1 from the endothelial cells (18); the soluble VCAM-1, in turn, binds to adjacent endothelial cells (via its ligand, VLA4), potentiating the angiogenic effects of cytokines released by the tumor itself on endothelial cells and enhancing tumor angiogenesis (18). Increases in endothelial VCAM-1 expression may, therefore, be one mechanism to increase the immune response to tumors, but this mechanism is ultimately harmful to the host by increasing angiogenesis and metastasis (37). Thus, the tumor may effectively hijack the normal tissue process of angiogenesis to provide itself with a blood supply.
Endocrine agents (e.g., tamoxifen and medroxyprogesterone acetate) are known to induce tumor regression by acting through steroid receptors in tumor epithelial cells (27,28). In addition, medroxyprogesterone acetate inhibits tumor growth and neovascularization in the rabbit cornea (39). Tamoxifen has also been shown to inhibit angiogenesis and endothelial cell growth in human tumors (27) and in animal models (28), although whether the inhibition occurs directly or indirectly, via inhibition of epithelial cell secretion of cytokines, is unclear. Whatever the mechanisms, our data suggest that inhibition of endothelial cell activation and a fall in serum VCAM-1 levels are key early events in the response of tumors to hormonal therapy. The fall in serum VCAM-1 levels occurred early and was not associated with a flare or rise in responding tumors, which is often seen with clinical epithelial cell markers, such as MUC1 and CA15-3 (40).
In patients undergoing hormonal therapy, changes in serum VCAM-1 levels provide a surrogate measure of angiogenesis, allowing rapid assessment of response to therapy rather than the static measurement obtained by microvessel density. Chemotherapy should theoretically reduce angiogenesis even more expeditiously than hormonal therapy, in which reduction in serum VCAM-1 levels occurs within 3 months. We are currently examining whether measurement of serum VCAM-1 levels in women undergoing primary medical chemotherapy for locally advanced breast carcinoma provides an earlier marker of response to therapy than does clinical examination or radiologic assessment.
Angiogenesis in women with advanced breast cancer is inhibited by endocrine therapy in hormone-responsive tumors (27,28). In this study, serum VCAM-1 levels but not E-selectin levels rose rapidly in women whose disease progressed on endocrine therapy but remained static in women whose disease remained stable or who had a partial response to therapy. The level of VWF, a pan-endothelial cell marker, although raised in breast cancer, did not correlate with VCAM-1 levels, microvessel density, or tumor response. Like VCAM-1, E-selectin is also expressed at higher levels in activated endothelium within breast tumors as compared with normal breast endothelium (19), but serum VCAM-1 (not E-selectin) more closely reflected vessel counts, prognostic factors, and clinical progression.
The finding that VCAM-1 levels were a good marker of angiogenesis but E-selectin levels were a poor marker was not entirely surprising because antiangiogenesis drugs have been shown to increase the expression of E-selectin but not VCAM-1 on breast tumor endothelial cells (41). The reason for this effect is unknown, but it may be due to increased endothelial turnover or increased intravascular shedding of VCAM-1 from activated endothelial cells relative to E-selectin. No increases in serum VCAM-1 levels were seen in patients with hormone-responsive tumors, but increases in serum VCAM-1 levels observed in five patients who were subsequently determined to have stable disease at 6 months (Table 3) may reflect a late response of these tumors to hormonal therapy, a well-recognized phenomenon (22). Levels of several epithelial tumor markers (e.g., CA15-3 and carcinoembryonic antigen [CEA]) may rise initially in responding tumors in the so-called "flare response" (40). Because the major source of VCAM-1 in tumors is endothelial cells, not epithelial cells (9), serum VCAM-1 may prove to be an early and sensitive marker of disease progression in hormone-nonresponsive tumors. By contrast, we have not seen any increases in VCAM-1 levels in the serum of patients with hormone-responsive tumors.
Ideally, we would have compared serum VCAM-1 levels with immunohistochemical expression of VCAM-1 to confirm that increases in serum VCAM-1 levels were derived directly from the endothelium. Unfortunately, there is, as yet, no reliable antibody for VCAM-1 staining in paraffin sections. Frozen-section immunohistochemistry studies show that 10%40% of breast cancers have endothelial cells that express VCAM-1, depending on the antibody and techniques used (19,34,42), which implies that serum VCAM-1 is rapidly turned over and secreted from activated endothelium. The currently used breast tumor markers (e.g., CA15-3 and CEA) are secreted by epithelial cells (40). The combination of an endothelial marker (i.e., serum VCAM-1) and epithelial markers may be better than multiple epithelial tumor marker measures in predicting breast cancer response to hormonal therapy because measurement of two different tumor cell compartments will likely improve early assessment of response.
Our data, from what is, to our knowledge, the first systematic survey of angiogenic markers in breast cancer patients, indicate that serum soluble VCAM-1 is an accurate marker of tumor angiogenesis in breast cancer. For a biomarker to be useful as a monitor of a pathological process, it must be easily measurable, must reflect accurately the pathological process that it is designed to measure, and must provide the clinician with an answer that can be easily interpreted. Serum VCAM-1 fulfills these criteria as a clinical measure of tumor angiogenesis in breast cancer.
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NOTES |
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G. J. Byrne was a recipient of a Tom Jones Memorial Fellowship from the University of Manchester. J. Iddon was a recipient of a Royal College of Surgeons of England Research Fellowship. Funding for the study was provided by the Manchester Surgical Research Trust.
We thank Alison Wynn Hann, Medical Statistician, University Hospital of South Manchester, who provided statistical assistance for the study. We are also grateful to our consultant pathologist colleagues for their diagnosis and pathological assessment of the tumors.
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Manuscript received July 19, 1999; revised May 25, 2000; accepted June 13, 2000.
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