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Original Article |
Departments of Internal Medicine (G.P.B., A.M., A.S.), Oncology (A.G.B., M.M., P.V., A.G.N.), and Surgery (P.I., P.M.), University of Pisa, 56100 Pisa, Italy
Address all correspondence and requests for reprints to: Bernini Giampaolo, Department of Internal Medicine, University of Pisa, Via Roma 67, 56100 Pisa, Italy. E-mail: g.bernini{at}med.unipi.it.
Abstract
The angiogenic phenotype of 13 normal adrenal glands (N), 13 aldosterone-producing adenomas (APA), 12 cortisol-producing adenomas (CPA), 13 nonfunctioning adrenal cortical adenomas (NFA), and 13 adrenal cortical carcinomas (CA) was investigated. Intratumoral vascular density was explored by CD34, a marker of endothelial cells, and the angiogenic status was investigated by vascular endothelial growth factor (VEGF) expression, an important angiogenic factor expressed by tumoral cells. Vascular density, quantified as the number of vessels per square millimeter, was significantly lower (P < 0.0001) in CA (110.3 ± 27.8) than in N (336.6 ± 14.5), APA (322.8 ± 19.1), CPA (288.5 ± 14.3), and NFA (274.2 ± 19.8). VEGF expression, calculated as the percentage of positive cells, was significantly greater (P < 0.0001) in CA (85.3 ± 2.1) than in APA (56.5 ± 7.5), CPA (38.5 ± 7.0), N (33.1 ± 6.1), and NFA (0.76 ± 0.6). In APA, a negative relation between CD34 and plasma renin activity (P < 0.0002) and a positive association between CD34 and aldosterone levels (P < 0.05) was found.
In conclusion, the angiogenic phenotype of CA is characterized by VEGF overexpression but low vascularization, a finding suggesting a dissociation between angiogenic potential and neoangiogenic capabilities of these tumors. The lack of VEGF expression in NFA and the close association between angiogenesis and functional status in APA also suggest a possible influence of the angiogenic phenotype on hormonal secretion of these endocrine tumors.
SEVERAL EXPERIMENTAL AND clinical studies suggest that cancer growth and its metastatic spread are closely related to angiogenesis (1, 2, 3). Accordingly, a significant relationship between tumor aggressiveness and angiogenesis has been reported (4, 5, 6, 7) in various forms of human cancer. This association derives from studies that have evaluated the tumor angiogenic phenotype, including vascular density (VD) and the expression of angiogenic factors. To quantify vascularization, markers of endothelial cells, as such as Factor VIII, CD31, and recently CD34 (8, 9, 10, 11), have been used. To study the angiogenic status of tumors, the expression level of vascular endothelial growth factor (VEGF) (12, 13, 14, 15), an important angiogenic compound (12, 16), has been assessed. However, the true impact of angiogenesis on tumor aggressiveness is only partially known. Indeed, the above reported relationship does not seem to be a rule for all malignant tumors (17), in accordance with the fact that the formation of new vessels from existing vasculature is only one aspect of the metastatic power and growth of the cancer (8).
Another problem is the influence of angiogenesis on functional activity of endocrine tumors. It is likely that endocrine organs require adequate vasculature to facilitate access of hormone products to the circulation. Thus, a different angiogenic pattern might also condition the secretory status of some endocrine tumors.
Tumors arising from human adrenal cortex represent a group of lesions heterogeneous both in biological behavior (benign or malignant) and in functional status (hypersecretory or hormonally inactive). To date, these tumors have not received adequate investigation as far as angiogenesis is concerned, so that the angiogenic phenotype of these lesions is still unknown. The few data reported in the literature do not confirm the association between vascularity and malignancy because adrenocortical carcinomas seem to show no increase in VD (18), despite their well known invasive and metastatic capabilities (19, 20). In addition, these data (18) indicate that in cortical adenomas VD is likewise not enhanced and there is no difference in vascularization between nonfunctioning and functioning (aldosterone and cortisol) adenomas.
Here, we evaluated by immunohistochemistry the angiogenic phenotype of human normal and pathological adrenal cortex, including functioning and nonfunctioning adenomas and carcinomas. CD34 was used to explore VD, and VEGF was used to investigate the angiogenic status of these neoplasms.
Patients and Methods
Patients and pathology
The study, approved by the local Ethical Committee, involved 64 patients with nonfunctioning cortical adenomas (NFA; n = 13), aldosterone-producing adenomas (APA; n = 13), cortisol-producing adenomas (CPA; n = 12), and adrenal cortical carcinomas (CA; n = 13). NFA had been incidentally discovered, and the endocrinological investigation revealed normal catecholamines, glucocorticoids, androgens, and mineralcorticoids in all cases. Patients with APA showed the typical signs of hyperaldosteronism with hypertension (systolic blood pressure, 174 ± 7.0 mm Hg, mean ± SE; diastolic blood pressure, 107 ± 3.5 mm Hg), hypokalemia (2.9 ± 0.2 mmol/liter), low plasma renin activity (PRA) levels (0.050 ± 0.016 ng/liter), and high plasma aldosterone (1298.23 ± 171.98 pmol/liter). Patients with Cushing’s syndrome showed stigmata of the disease and a hormonal picture characterized by increase in plasma cortisol (894.2 ± 40.5 nmol/liter) and decrease in ACTH levels (1.87 ± 0.09 pmol/liter). In addition, all patients were unresponsive to low (1 mg) and high (8 mg) dexamethasone suppression tests. Patients with CA had been investigated for abdominal pain in 10 cases and for hirsutism in 3 cases; the latter showed high androgen levels with normal cortisol values, but 2 patients were unresponsive to dexamethasone (1 mg) administration. As controls, 13 normal adrenal glands (N) were studied after removal from patients with renal carcinomas. Clinical details of our population are shown in Table 1
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A total of 64 formalin-fixed, paraffin-embedded blocks of adrenal tissue were studied. To avoid possible discrepancies in the results, specimens were handled and processed in the same way before formalin fixation. Five-micrometer sections were stained with hematoxylin-eosin for histological evaluation. Additional 5-µm sections were used for immunohistochemistry.
Immunohistochemistry
Antibodies. The sections were incubated with the following primary antibodies: 1) mouse antihuman CD34 (QB-END10, DAKO Corp., Milan, Italy; dilution 1:100), and 2) rabbit antihuman VEGF (Oncogene Research Products, Cambridge, MA; dilution 1:100). For both, incubation time was 12 h at 4 C.
Method. Five-micrometer sections were deparaffinized in xylene and rehydrated in alcohols. Endogenous peroxide activity was blocked by incubating the slides in 1% hydrogen peroxide in methanol for 10 min. To unmask the antigens, the slides were microwave-treated in 10 mM citrate buffer (pH 6) for a total of 10 min. After nonspecific staining was blocked with normal serum, the sections were incubated with primary antibodies. CD34 and VEGF sections were then incubated for 30 min with biotin-labeled secondary antibody (dilution 1:500) and avidin-biotin-complex (Vector Laboratories, Inc., Burlingame, CA), respectively. 3-3' Diaminobenzidine tetrahydrochloride was used as chromogen. Finally, the sections were counterstained with hematoxylin, dehydrated, and mounted.
Controls. Vascular endothelial cells and normal keratinocytes were used as positive controls for CD34 and VEGF, respectively. Negative controls were obtained by omitting primary antibodies.
Evaluation of parameters
VD. Angiogenesis quantification (microvessel staining and counting) was evaluated as follows:
1) In pathological tissues, the three most vascular areas of the tumor were identified at low magnification as the hot spots. In each hot spot, vessels were counted on one x250 field (ocular lens, x10; objective lens, x25; field area, 0.74 mm2), and the mean of counts of these areas was recorded. Because our CA showed considerable heterogeneity, hot spots were carefully chosen with solid structure independent of the distance from the necrotic areas. By this procedure, we obtained a fairly representative picture of the entire tumor.
A single microvessel was defined as discrete clusters or single cells stained for CD34 clearly separated from adjacent vessels, tumor cells, and other connective tissue elements; the presence of a lumen was not required for scoring as a microvessel.
2) In normal glands, microvessel count was performed at x250 in three areas of the zona glomerulosa, zona fasciculata, and zona reticularis, respectively. The average was then calculated.
VEGF expression. This parameter was evaluated in a semiquantitative manner. The positivity index was obtained by counting almost 500 cells of lesions and of normal cortex and calculating the percentage of cells with VEGF cytoplasmic immunoreactivity.
The evaluation of both markers was performed in contiguous (5 µm) sections. The field of interest in the section used to count VD (CD34) was carefully highlighted. The contiguous section, immunostained with anti-VEGF, was then overlapped to identify the same field. When it was obtained, the evaluation of CD34 and VEGF was performed.
All parameters were determined independently by two pathologists (P.V. and A.G.B.), and discordant cases were solved by simultaneous review by both pathologists.
Follow-up
After surgery, all NFA, APA, and CPA were cured, whereas among CA, 12 patients had died of local and/or diffuse metastases and 1 patient was alive at the time this manuscript was written (Table 2).
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All data are expressed as mean ± SEM. Values obtained for each variable to compare groups were analyzed by using the unpaired t test (for age comparison) and Wilcoxon’s test. Spearman correlation coefficients (r) were used as parameters of association. Statistical difference was accepted at P value less than 0.05.
Results
Clinical details of all groups (Table 1
)
Age proved to be significantly higher in patients taken as controls (who were patients with renal carcinomas but normal adrenals) than in those with functioning and nonfunctioning adenomas. There was no statistical difference in age between CA, APA, CPA, or NFA. The smallest tumor size was observed in APA, whereas the greatest size was found in CA.
Clinical details of patients with CA (Table 2)
Tumor size of patients with CA ranged between 60 and 250 mm. Four tumors showed no necrosis or low necrosis, whereas nine had moderate or wide necrotic areas. The majority of tumors belonged to stage II-III, and survival time ranged between 3 and 100 months (mean, 24.5 months).
VD
Mean (±SE) VD, quantified as the number of vessels per square millimeter, was significantly lower (P < 0.0001) in CA (110.3 ± 27.8) than in N (336.6 ± 14.5), APA (322.8 ± 19.1), CPA (288.5 ± 14.3), and NFA (274.2 ± 19.8). VD did not differ in N and APA, whereas it was lower in NFA (P < 0.01) and CPA (P < 0.02) than in N (Fig. 1, top). Analytical data indicated that 12 of 13 CA, 6 of 13 NFA, and 4 of 12 CPA had CD34 values below the lower limit (270 number of vessels/mm2) of the normal, set at the 10th percentile of distribution calculated in our control group (Fig. 1, bottom).
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shows a clearly different immunostaining between CA and normal specimens. The number of microvessels was lower in the former than the latter.
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The expression level of VEGF, calculated as percentage of positive cells, was significantly higher (P < 0.0001) in CA (85.3 ± 2.1%) than in N (33.1 ± 6.1%), CPA (38.5 ± 7.0%), APA (56.5 ± 7.5%), and NFA (0.76 ± 0.6%). VEGF staining in APA, but not in CPA, was greater than in N (P < 0.02). APA and CPA expressed more VEGF (P < 0.0001) than NFA. NFA showed the lowest VEGF expression, and therefore the expression level proved lower (P < 0.0001) than in the other groups (Fig. 3, top). Analytical data indicated that all CA had VEGF expression values equal to (n = 2) or above (n = 11) the upper limit (70%) for normal, set at the 90th percentile of our controls, whereas all NFA had expression values equal to (n = 2) or below (n = 11) the lower limit (5%) for normal VEGF (10th percentile of our controls). The majority of APA (9 of 13) and CPA (10 of 12) had VEGF levels in the normal range (Fig. 3, bottom).
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shows that adrenocortical cells of CA exhibited intense cytoplasmatic VEGF staining, whereas this was not the case for adrenocortical cells of NFA.
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In APA, a negative relation between VD and PRA (r = 0.852; P < 0.0002) and a positive association between VD and aldosterone levels (r = 0.521; P < 0.05) was found. In APA and in NFA, VD and VEGF did not correlate with age, body mass index, blood pressure, mass size, potassium, or hormones. In CPA, no association was found between hormones and angiogenic markers. In CA, neither immunohistochemical marker correlated with age, mass size, tumor necrosis, disease stage, and patient survival. Finally, in no group were correlations between VD and VEGF found.
Discussion
Few studies in the literature have evaluated angiogenesis in human adrenal pathological cortex. Here, we included a relatively large number of specimens, in particular carcinomas that represent an infrequent form of human cancer.
Our results show a clearly different angiogenic pattern between carcinomas and adenomas or normal cortex. Differences were detected both by a marker of VD (CD34) and by an index of angiogenic status (VEGF).
In our experimental conditions, adrenal carcinomas appeared to have high angiogenic potential because they showed increased VEGF expression in comparison to that observed in normal tissue and in adenomas. Our data are fully in agreement with a recent study in which the mean cytosolic concentration of VEGF in 13 adrenal CA was higher than in adenomas and transitional tumors (21). The present data also indirectly confirm a report (22) demonstrating that patients with adrenal carcinomas have serum VEGF levels significantly higher than patients with adenomas and normal subjects. Our study was not planned to evaluate whether VEGF overexpression was primary or secondary to the presence of wide necrotic areas found in our tumors. The former hypothesis, however, is likely because VEGF in tumor cells seems to be constitutively expressed at high levels regardless of ambient O2 tension (12), whereas in normal tissue VEGF is up-regulated and its mRNA is stabilized under conditions of hypoxia (23).
In any case, VEGF represents a pivotal molecule in angiogenesis, and its overexpression endows the tumor with the potential to synthesize new vessels and therefore to induce tumor growth and metastasis formation (24, 25). However, despite high VEGF, our carcinomas appeared to have a reduced vascularization, lower than adenomas and normal cortex. Similar results were found in another series of adrenocortical carcinomas (18), and low vascularization was also observed in a recent report (26) in pituitary adenomas in comparison to normal tissue. The low vascularization that we found has several explanations. First, VEGF expressed in adrenal carcinomas could be functionally inactive or antagonized by other angiogenic inhibitors. Second, the role played by VEGF in the adrenal cortex may be secondary, given the recent identification of EG-VEGF (27), a molecule expressed only in adrenal tissue with a different structure from VEGF and almost identical functional activity. This protein could be low in adrenal CA and may provide an explanation for our data.
Despite the low vascularization, the survival time of our patients with carcinomas was very short. This finding apparently disagrees with the demonstration that neoangiogenesis is associated with malignancy and that VD is an independent prognostic indicator of the risk of future metastases and death in several human cancers (4, 5, 6, 7, 28, 29). However, even in the presence of low VD, malignant tumors may preserve their aggressiveness. Indeed, neoangiogenesis is only one aspect of cancer metastatic power (8), and tumoral vessels, even if scanty, may have efficient rheology permitting local diffusion and metastases. Nor can it be ruled out that the diffusion of our tumors was not hematogenous but occurred through lymphatic pathways undetected by the immunohistochemical study we used (only VEGF-C, a particular isoform of VEGF, seems to be a specific factor for lymphangiogenesis; Refs. 30 and 31).
Our study also involved a group of functioning adenomas. CPA showed an angiogenic phenotype comparable to normal specimens, without correlations with cortisol and ACTH levels. In APA, tumor vascularization was quantitatively similar to normal but was negatively related to PRA and positively associated with aldosterone. In addition, the angiogenic potential of APA proved to be elevated because VEGF expression levels were significantly higher than in normal cortex. Thus, an association between angiogenesis and functional status seems to be present in APA. Accordingly, we found that nonfunctioning adenomas have a VD lower than normal cortex and express very low levels of VEGF. Although we studied only 13 specimens, this finding is unlikely to be due to chance or technical problems because it was observed in virtually all cases, at least as far as VEGF expression is concerned. Thus, a relation between low angiogenic power and lack of hormonal activity seems to exist in nonfunctioning adenomas.
In conclusion, our results show that the angiogenic phenotype of human adrenocortical carcinomas is characterized by VEGF overexpression but low vascularization, a finding suggesting a dissociation between angiogenic status and neoangiogenic capabilities of these tumors. In addition, we observed a lack of VEGF expression in nonfunctioning cortical adenomas and a close association between angiogenesis and functional status in aldosterone-producing adenomas, pointing to a possible influence of the angiogenic phenotype on hormonal secretion of these endocrine tumors.
Acknowledgments
We are grateful to Dr. Gianfranco Argenio for his critical revision of this manuscript.
Footnotes
Abbreviations: APA, Aldosterone-producing adenomas; CA, adrenal cortical carcinomas; CPA, cortisol-producing adenomas; N, normal adrenal glands; NFA, nonfunctioning adrenal cortical adenomas; PRA, plasma renin activity; VD, vascular density; VEGF, vascular endothelial growth factor.
Received November 12, 2001.
Accepted July 22, 2002.
References
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  R. Libe, A. Fratticci, and J. Bertherat Adrenocortical cancer: pathophysiology and clinical management Endocr. Relat. Cancer, March 1, 2007; 14(1): 13 - 28. [Abstract] [Full Text] [PDF]
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|
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 |
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 P. Huber, C. Mallet, E. Faure, C. Rampon, M.-H. Prandini, O. Feraud, S. Bouillot, and I. Vilgrain ACTH depletion represses vascular endothelial-cadherin transcription in mouse adrenal endothelium in vivo J. Mol. Endocrinol., February 1, 2005; 34(1): 127 - 137. [Abstract] [Full Text] [PDF]
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