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Original Studies |
INSERM U-244, Département de Biologie Moléculaire et Structurale/Biochimie des Régulations Cellulaires Endocrines, Commissariat à l’Energie Atomique (F.d.F., E.M.C., J.J.F.), F-38054 Grenoble, France; Service de Biochimie A, Centre Hospitalier Régional Universitaire de Grenoble (F.d.F., M.E.A., E.M.C.), 38700 La Tronche, France; Laboratoire d’Explorations Fonctionnelles Endocriniennes, Hôpital Trousseau (C.G.), 75012 Paris, France; and Clinique des Maladies Endocriniennes et Métaboliques, Hôpital Cochin (X.B.), 75014 Paris, France
Address all correspondence and requests for reprints to: Dr. J. J. Feige, INSERM U-244, Département de Biologie Moléculaire et Structurale/Biochimie des Régulations Cellulaires Endocrines, Commissariat à l’Energie Atomique de Grenoble, 17 rue des Martyrs, F-38054 Grenoble Cedex 9, France.
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Abstract |
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Introduction |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Despite some recent progress in the understanding of the pathogenesis of adrenocortical tumors, reliable markers to predict their natural evolution are still missing. Moreover, in many cases the benign or malignant nature of a localized tumor cannot be established with certainty, reflecting the lack of absolute clinical, biological, or histological criteria. Interestingly, the recent characterization of specific genetic rearrangements [uniparental disomy at the 11p15 locus, loss of heterozygosity at the 17p13 locus, and major overexpression of the insulin-like growth factor II (IGF-II) gene], which are highly frequent in carcinomas (>80%), but are observed at a low frequency in adenomas (<12.5%), offers new potential prognostic criteria that remain to be evaluated in a prospective study with long-term follow-up of patients (2, 3, 4, 5).
Angiogenesis, the biological process by which new blood capillaries are formed from preexisting microvessels and venules, is an essential step in the progression of a variety of solid tumors (6). It has been proposed from the results of experimental animal tumor models and from the observation of human breast and cervical carcinomas that induction of angiogenesis (often termed the angiogenic switch) is a discrete component of tumor evolution activated during the early premalignant stages of tumor development (7). Angiogenesis appears to be a limiting step for subsequent tumor growth beyond the size of a few cubic millimeters (7) and for metastasis formation (8). Under physiological conditions, the vasculature is quiescent in the adult, except for some hormonally controlled processes, such as ovulation, menstruation, implantation, and pregnancy. The biochemical characterization of a number of peptides and proteins that stimulate or inhibit angiogenesis has led to the concept of angiogenic balance (9, 10). The angiogenic status of a given cell type under any physiological or pathological situation results from the balance between the biological activities of both angiogenic and angiostatic factors. This concept is supported by the observation that tumor angiogenesis may be induced by either increased expression of angiogenic factors (10, 11) or decreased expression of angiostatic factors (10, 12, 13, 14).
The angiogenic status of adrenocortical tumors has not been studied in detail to date. The normal adrenal gland is a highly vascularized tissue irrigated centripetally by a network of fenestrated capillaries (called sinusoids) originating from capsular arterioles (15, 16). Early, but still valid, histological observations of the adrenal cortex have established that each endocrine cell from the glomerulosa, the fasciculata, or the reticularis zone is in direct contact with a capillary sinusoid (17). A recent comparison of the vascular patterns of adrenocortical tumors with that of the normal adrenal cortex showed that the sinusoidal network in adenomas was indistinguishable from that in the normal gland, whereas carcinomas showed a disorganized vasculature, with large vessels interspersed with irregular networks of microcapillaries (18).
In the present study we measured the abundance of three angiogenesis markers [namely vascular endothelial cell growth factor A (VEGF-A), platelet-derived endothelial cell growth factor (PD-ECGF), and thrombospondin-1 (TSP1)], in a series of 43 benign and malignant adrenocortical tumors. These markers were selected on the basis of their reported implication in cancer angiogenesis. VEGF-A is a potent and selective growth factor for micro- and macrovascular endothelial cells (19) and an inducer of vascular permeability (20). The dramatic alterations of yolk sac and embryonic vascularization observed in mouse embryos heterozygous for a disrupted VEGF-A gene (21, 22) have definitely established VEGF-A as a pivotal molecule in angiogenesis. Overexpression of VEGF has been reported in a number of tumor types and appears of significant prognostic value in breast, stomach, and colon cancers (23, 24). Although it was initially purified from platelet extracts as an inducer of thymidine incorporation into endothelial cells (25), PD-ECGF does not stimulate the proliferation of endothelial cells (26). However, it is angiogenic and promotes tumor growth in vivo (25). Its sequencing revealed complete identity with the enzyme thymidine phosphorylase that converts thymidine to thymine (27), an enzyme overexpressed in various neoplastic tissues (28, 29, 30, 31, 32). TSP1 is a multimodular secreted protein that associates with the extracellular matrix and possesses a variety of biological functions, including a potent antiangiogenic activity (33). TSP1 expression has been shown to be down-regulated in a number of experimental tumor models, and this regulation has been correlated with the decreased expression of tumor suppressor genes (34, 35). On the other hand, TSP1 has been reported to be overexpressed in a number of malignant tissues and to be present in higher than normal levels in the plasma of cancer patients (36, 37). This discrepancy may be related to different contributions of the stroma to the tumor mass from one type of tumor to the other; a detailed immunohistochemical study of the localization of TSP1 in human breast neoplasms has shown that TSP1 is overexpressed in the stroma, but is absent from the tumor epithelial cells (38).
The data reported here indicate that the mean cytosolic concentration of VEGF-A in the population of malignant tumors is higher than that in benign or transitional tumors. Conversely, the mean cytosolic TSP1 concentration is lower in malignant and transitional tumors than in adenomas. Thymidine phosphorylase activity does not vary significantly among the three populations. The pertinence of these markers as prognostic factors for localized or metastatic tumor recurrence has been evaluated.
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Subjects and Methods |
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Forty-three adult patients (10 men and 33 women), 23–79 yr old,
with sporadic adrenocortical tumors surgically removed between 1990 and
1998 were included in this study. The median postsurgery follow-up of
these patients was 44 months (range, 5.2–104 months). The hormonal
status and the stage of the tumors as localized, regional, or
metastatic were evaluated as previously described (39).
Seventy-two percent of the patients (n = 31) were referred because
of endocrine symptoms, and 86% (n = 37) exhibited an abnormal
hormonal profile. Histological features, including high mitotic rate,
atypical mitoses, high nuclear grade, low percentage of clear cells,
necrosis, diffuse architecture of tumor, capsular invasion, sinusoidal
invasion, and venous invasion, were carefully analyzed according to the
method of Weiss (40, 41). Eighteen tumors without any of
these histological features were classified as benign, 5 of them
presenting with bilateral adenomas (macronodular hyperplasia). Twelve
localized tumors with 1–3 of these histological features were
classified as transitional. Thirteen tumors with more than 3 of these
features or with documentation of metastasis were classified as
malignant tumors (41). Clinical, pathological, and
hormonal data are summarized in Table 1
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Liquid nitrogen-frozen tumors were homogenized at 4 C in 10 mmol/L Tris-HCl buffer, pH 7.4, containing 1 mmol/L ethylenediamine tetraacetate, 1.5 mmol/L MgCl2, 0.5 mmol/L dithiothreitol, 1 mmol/L phenylmethylsulfonylfluoride, 10 µg/ml leupeptin, and 10% glycerol. The homogenate was centrifuged at 800 x g for 10 min to pellet the nuclei, and the supernatant was then centrifuged at 100,000 x g for 1 h. The protein content of the cytosols (100,000 x g supernatants) was determined using the bicinchoninic acid method (Pierce Chemical Co., Rockford, IL).
Assessment of thymidine phosphorylase activity
Thymidine phosphorylase activity in tumor cytosols was determined by spectrophotometric methods as described previously (42). Briefly, 50–100 µL of cytosolic fractions were incubated for 3 h at 37 C with 10 mmol/L thymidine in a final volume of 1 mL 50 mmol/L potassium phosphate buffer, pH 7.4. At the beginning and the end of the reaction, 0.5-mL aliquots of each sample were mixed with 1 mL 0.5 mol/L NaOH. The amount of thymine formed was determined spectrophotometrically at 300 nm. One unit of activity is defined as the formation of 1 µmol thymine/h. Parallel analysis of seven samples of human breast tumors by Western blotting using a monoclonal antibody to PD-ECGF (a gift from Dr. Roy Bicknell) showed a statistically valid correlation between the values of enzymatic activity and the intensities of immunoreactive signals (regression coefficient = 0.98).
Determination of TSP1 and VEGF-A protein expression
TSP1 concentrations in the tumor cytosols were determined by a sandwich enzyme-linked immunosorbent assay (ELISA) developed in the laboratory. The immobilized antibody was a commercial rabbit polyclonal antibody raised against human TSP1 (Calbiochem, La Jolla, CA). The mouse monoclonal antihuman TSP1 antibody used in the second step was from Roche (Meylan, France). The detection limit of the assay was 5 ng/ml. VEGF-A was quantitated using the Quantikine kit from R and D Systems, Inc. (Oxon, UK). This commercial ELISA does not cross-react with placenta growth factor. Moreover, it detects the different isoforms of VEGF-A, among which VEGF121 and VEGF165 are the most abundantly expressed.
Analysis at the chromosomal 11p15 locus and evaluation of IGF-II messenger RNA (mRNA) content
Allelic loss at the 11p15 locus in tumors was investigated by Southern blot analysis as previously described (3). Tumor IGF-II mRNA contents were evaluated by dot-blot analysis as previously described (3, 4) and compared with normal adrenal content. When the tumor IGF-II mRNA content was more than 10-fold larger than that in normal adrenals, the tumors were classified as overexpressing IGF-II.
Statistical analysis
Differences in angiogenic or angiostatic factor concentrations among the groups were analyzed by the Mann-Whitney U test.
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Results |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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An enzymatic assay was used to measure the thymidine phosphorylase
activity of the angiogenic factor PD-ECGF in tumor cytosols. Thymidine
phosphorylase activity of the adenomas (mean, 4.4 U/g; range, 1–15
U/g) was not significantly different from that of the carcinomas (mean,
5.1 U/g; range, 3–8 U/g) or that of the transitional tumors (mean, 4.4
U/g; range, 3–8 U/g; Table 2 and Fig. 1A).
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Concentrations of TSP1 were significantly higher in the cytosols
of adenomas (mean, 142 µg/g; range, 40–390 µg/g) than in
carcinomas (mean, 69 µg/g; range, 8–344 µg/g) or transitional
tumors (mean, 53 µg/g; range, 0–161 µg/g; Table 2
and Fig. 1B). The mean cytosolic TSP1 concentration determined from two normal
human adrenals was 52 ± 12 µg/g. Using a threshold value of 57
µg/g (corresponding to the 75th percentile of the carcinoma values),
89% of the adenoma samples presented TSP1 concentrations above
threshold, whereas only 25% of the carcinoma and 33% of the
transitional tumor samples did so.
Conversely, levels of VEGF-A were significantly lower in the cytosols
of adenomas (mean, 44.3 ng/g; range, 9–81 ng/g) and transitional
tumors (mean, 83.2 ng/g; range, 3–141 ng/g) than in those of
carcinomas (mean, 404 ng/g; range, 30–1370 ng/g; Table 2 and Fig. 1C).
The mean cytosolic VEGF-A concentration determined from two normal
human adrenals was 111 ± 23 ng/g. Using a threshold value of 107
ng/g (corresponding to the 25th percentile of the carcinoma values),
100% of the adenomas and 72% of the transitional tumors showed VEGF-A
concentrations under threshold, whereas 75% of the carcinomas had
VEGF-A concentrations above threshold. However, no significant
correlation was observed between tumor weight and VEGF-A
concentrations.
Correlations between genotypic and biochemical features of adrenocortical tumors
The population of tumors was split into two subgroups according to
either one of the following two genotypic features: overexpression of
the IGF-II gene (Fig. 2, A and B) or
allelic loss at the 11p15 locus (Fig. 2, C and D), and the
distribution of cytosolic VEGF-A and TSP1 concentrations in each of
these subpopulations was determined. It clearly appeared that tumors
with IGF-II overexpression had significantly higher VEGF-A
(P < 0.01) and lower TSP1 (P < 0.001)
concentrations than tumors without IGF-II overexpression. A similar,
although less significant (P < 0.05), cosegregation
was observed between 11p15 allelic loss and either high VEGF-A or low
TSP1 values.
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Of the 19 patients with transitional and malignant tumors who were
regularly followed-up, 6 showed tumor recurrence within a median time
period of 14.3 months (range, 3.2–32.1 months), whereas the other 12
did not recur over a median follow-up period of 58.9 months (range,
40.5–97 months). As shown in Fig. 3A, tumors from the patients (with a carcinoma or a transitional tumor)
that showed recurrent tumor growth contained higher VEGF-A
concentrations than those that did not, although the low number of
recurrence events makes this difference barely significant. In
contrast, TSP1 did not appear to be a useful marker to discriminate
between recurring and nonrecurring tumors (Fig. 3B).
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Discussion |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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PD-ECGF is a misnamed angiogenic factor that stimulates angiogenesis in vivo although it is not mitogenic for endothelial cells in vitro (26). PD-ECGF is thymidine phosphorylase, and its enzymatic activity was not significantly different among the three groups of adrenocortical tumors examined: adenomas, transitional tumors, and carcinomas. In contrast to other tumor types (30, 32), it did not appear to be a relevant marker of adrenocortical tumors and was not further studied.
Comparison of TSP1 and VEGF-A concentrations in adenomas and carcinomas revealed statistically significant differences. Mean VEGF-A and TSP1 concentrations in carcinomas were, respectively, increased and decreased compared with those in adenomas, suggesting that overexpression of this angiogenic factor and decreased expression of this antiangiogenic protein are associated with the malignant phenotype. No major difference in the concentrations of these factors was observed between localized and more invasive carcinomas (either regional or metastatic at diagnosis), suggesting that these alterations in the angiogenic status of the tumors precede the invasive step.
More interestingly, the analysis of transitional tumors (i.e. tumors presenting histological alterations intermediate between those of benign and malignant tumors) revealed that the mean TSP1 concentration was significantly lower than that in adenomas (and similar to that in carcinomas), whereas VEGF-A concentrations were not statistically different from those in adenomas (and were lower than those in carcinomas). Although these variations were not observed in 100% of the tumors of each population, 78% (7 of 9) of the carcinomas with low TSP1 concentrations also had elevated VEGF concentrations, whereas this correlation was observed in only 14% (1 of 7) of the transitional tumors. This suggested that the decrease in TSP1 expression may be an earlier event than the increase in VEGF-A expression during tumor progression from adenomas to carcinomas. Several genetic studies addressing tumor clonality or search for chromosomal aberrations have supported the hypothesis that the formation of adrenocortical carcinomas is the result of a multistep tumorigenic process (43, 44). To our knowledge, the early decrease in tumor TSP1 concentration observed in this study is the first biochemical alteration to be characterized that would allow discrimination between premalignant and malignant stages of adrenocortical tumors. Decreased TSP1 expression has been observed in several tumor cell lines, such as human glioblastoma, breast, and lung carcinoma cell lines (12, 34, 45), as well as in p53-deficient fibroblasts from Li-Fraumeni patients (46) and in the cytosols of bladder cancers (35). In several of these examples, a correlation has been established between the loss of wild-type p53 expression and decreased TSP1 expression (47). p53 mutations have been identified in approximately 30% of sporadic adrenal carcinomas, but have not been detected in adenomas from Caucasian patients (48). A Taiwanese group identified mutations within exon 4 of the p53 gene (an unusual hotspot, as 90% of p53 mutations in human tumors are located within exons 5–8) in 60% of benign adrenocortical adenomas (49), but this was not confirmed in a larger series of 27 tumors from Europe and the U.S. (50). It would be informative to determine whether the decreased expression of TSP1 observed in 60% of the carcinomas and 50% of the transitional tumors in our study correlates with p53 mutations.
Abnormalities of the IGF-II locus at chromosome 11p15 resulting in overexpression of this maternally imprinted gene are frequently observed in transitional and localized malignant adrenocortical tumors but rarely in adenomas (4). It was therefore not surprising to observe that the tumors with these alterations also had higher VEGF-A and lower TSP1 contents than tumors with normal IGF-II levels. Among the 38 patients with localized or regional tumor who were included in this study, 6 showed tumor recurrence after primary tumor resection. Although these 6 tumors had significantly higher VEGF-A concentrations (P < 0.05) than the nonrecurrent tumors, the size of this cohort needs to be increased to definitely establish the prognostic value of VEGF-A concentrations with a higher statistical confidence. Such a correlation between high VEGF-A concentrations and tumor recurrence has been established in a number of other tumor types, including primary breast cancer (51) and gastric carcinoma (52).
The alterations in TSP1 and VEGF-A expression observed in adrenocortical tumors suggest that a change in the angiogenic phenotype accompanies tumor progression from adenomas to carcinomas. This is in agreement with the histological observations showing a normal vasculature in the adenomas and a more disorganized and irregular microvasculature in the carcinomas (18). This also supports the possibility of using inhibitors of angiogenesis such as TSP1 or angiostatic fragments of this large multimodular protein to block neovascularization and, therefore, tumor growth. Overexpression of TSP1 in a number of tumor cell lines, including breast and skin carcinomas and glioblastomas, has been shown to revert their transformed phenotype and their ability to form tumors in nude mice (14, 34, 53, 54). It will be worth investigating whether TSP1-based antiangiogenic therapy may represent a more efficient alternative or a complement to the classically used antimitotic treatments such as mitotane (55). In particular, our study allows definition of a subset of tumors showing low TSP1 and normal VEGF-A concentrations as a transitional group that may be particularly responsive to antiangiogenic therapy.
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Acknowledgments |
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Footnotes |
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Received March 4, 2000.
Revised July 11, 2000.
Accepted August 11, 2000.
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Localized tumors; 
, tumors presenting extensions at diagnosis
(either regional or metastatic). The statistical significance of
differences between groups was evaluated using the Mann-Whitney U test.
