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CLINICAL CASE SEMINAR |
Laboratoire d’Explorations Fonctionnelles Endocriniennes (C.G., V.G., Y.L.B.), Hôpital Trousseau, Assistance Publique-Hôpitaux de Paris, 75012 Paris, France; Services d’endocrinologie (M.-H.B.), d’anatomopathologie (N.B.), et de chirurgie viscérale (J.-L.P.) des Hôpitaux de Lyon, 69321 Lyon, France; and Department of Molecular Medicine (S.S., D.J.M., B.G.R.), Kolling Institute of Medical Research and University of Sydney, St. Leonards, 2065 NSW Australia
Address all correspondence and requests for reprints to: Dr. Christine Gicquel, Laboratoire d’Explorations Fonctionnelles Endocriniennes, Hôpital Trousseau, 26 Avenue Arnold Netter, 75012 Paris, France. E-mail: christine.gicquel{at}trs.ap-hop-paris.fr.
Abstract
The mechanisms of adrenocortical tumorigenesis are still not fully understood. Data from clonal analysis, comparative genomic hybridization, and allelotyping suggest that it involves a multistep process during which several genetic defects are progressively acquired, leading to the malignant transformation. The events involved in the first steps of this process are not well known, and most of the abnormalities described in adrenocortical tumors to date are associated with the malignant phenotype. We report a case that suggests that adrenocortical tumorigenesis may be a multistep process. A 43-yr-old patient underwent surgery for an incidentally discovered adrenal mass. Pathological analysis showed that this tumor consisted of two parts: a central part with features of malignancy surrounded by another part with a strictly benign appearance. These data were confirmed by molecular analysis and comparative genomic hybridization that were consistent with either a malignant or benign presentation. The apparently malignant part of the tumor exhibited molecular abnormalities [17p13 loss of heterozygosity (LOH), 11p15 uniparental disomy and overexpression of the IGF-II gene] as well as chromosomal gains and losses (comparative genomic hybridization) that have been previously described in malignant tumors. No abnormalities were found in the surrounding benign tissues. Although this observation is not definitive proof that adrenocortical tumorigenesis occurs via a multistep process, it suggests that there is a progressive change from the benign to the malignant state in some adrenocortical tumors.
WHETHER ADRENOCORTICAL CARCINOMAS develop from benign adenomas or occur as a separate disease has not been fully established. Data from clonal analysis (1, 2), comparative genomic hybridization (CGH; Refs.3, 4, 5, 6), and allelotyping with microsatellites (7) have suggested that adrenal tumorigenesis is a multistep process, with sequential progression from normal to adenomatous cells and eventually to malignant cells. Moreover, various molecular markers are strongly associated with the malignant phenotype. Abnormalities in the imprinted 11p15 region resulting in strong overexpression of the IGF-II gene (8, 9, 10), 17p13 LOH (11, 12), 2p16 LOH (7), and 11q13 LOH (7, 13, 14, 15) all are highly specific for malignant tumors, and some of them have been shown to have a prognostic value for the recurrence of localized tumors (16).
We report here an observation of a localized adrenal tumor composed of two different parts exhibiting different pathological features (benign and malignant). Pathological differences were confirmed by the analysis of molecular markers of malignancy (expression of the IGF-II gene and allelic status at the 11p15 and 17p13 loci) and by CGH patterns. This case supports the hypothesis that, at least in some tumors, there is a multistep adrenal tumorigenesis.
Case report
A 47-yr-old Caucasian male was referred for an incidentally discovered adrenal mass. This adrenal mass was diagnosed by computerized tomography (CT) scans of the abdomen performed for renal colic.
At diagnosis, the patient did not exhibit any endocrine symptoms. Hormonal evaluation revealed normal serum cortisol levels (345 nmol/liter; normal range, 165–785 nmol/liter) and a normal urinary excretion of free cortisol (233 nmol/d; normal range, 40–240 nmol/d). The serum ACTH level was decreased (1 pmol/liter; normal range, 2–11.2 pmol/liter). The dexamethasone suppression test (oral administration of 1 mg of dexamethasone at 2400 h the previous day) showed no suppression of serum cortisol level (lowest value, 358 nmol/liter). This functional pattern was suggestive of a preclinical adrenal Cushing’s syndrome. Serum dehydroepiandrosterone sulfate and 17-hydroxyprogesterone levels were normal, as was the urinary excretion of catecholamines and metanephrines.
An abdominal CT scan demonstrated a right adrenal mass measuring 5.5 x 4 cm. No secondary localizations were detected by chest x-ray or abdominal and thoracic CT scan.
The patient underwent laparoscopic surgery to remove the entire right adrenal gland.
The adrenal gland weighed 28 g and contained a well circumscribed and encapsulated, 4-cm, solitary tumor. Sectioning revealed that it consisted of two different parts, a bright yellow peripheral zone and a brown gelatinous central zone that measured 2.2 cm in diameter (Fig. 1A
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). The yellow component surrounding this central zone was composed of clear spongiocytic cells with very mild nuclear atypia, no mitosis, and no necrosis (Fig. 1C). Thus, the central zone exhibited five of the criteria proposed by Weiss (17), whereas the outer zone had none. No foci of vascular (venous or lymphatic) invasion and no capsular penetration was demonstrated in any part of the tumor. Immunohistochemical analysis was performed on formalin-fixed, paraffin-embedded material using monoclonal antibodies. Neither of the cellular components expressed cytokeratin (AE1, AE3), epithelial membrane antigen, chromogranin A, synaptophysin, or carcinoembryonic antigen. More than 10% of the central compact acidophilic cells and less than 1% of the spongiocytic cells were labeled with Ki 67 (Mib 1; DAKO Corp., Glostrup, Denmark).
Molecular analysis
Informed consent for the analysis of leukocyte and tumor DNA and for access to the information collected was obtained from the patient in accordance with national ethical rules.
17p13 Allelic status and tumor IGF-II mRNA content were determined as previously described (16). 11p15 Allelic status was performed by multiplex-PCR using highly polymorphic short-tandem repeat markers within tyrosine hydroxylase, D11S2362, and D11S922. Amplification products were separated on a 6% denaturing polyacrylamide gel on an ABI PRISM sequencer model 373 A Genetic Analyzer (PE Applied Biosystems, Foster City, CA). Results were analyzed with the Genescan PCR analysis software (version 1.2.2-1; PE Applied Biosystems). The methylation status of the H19 and the KCNQ1OT genes was analyzed as previously described (18). The restriction pattern of tumor DNA was compared with that of leukocyte DNA.
As shown in Fig. 2, the central part of the tumor displayed a complete LOH at the p53 gene locus (17p13.3; Fig. 2A), a uniparental disomy at the 11p15 locus (Fig. 2B), and an overexpression of the IGF-II gene (72-fold higher than in the benign peripheral tissue; Fig. 2C). These abnormalities were not found in the surrounding benign part of the tumor, where the restriction pattern at the 17p13.3 and 11p15.5 loci was the same as in leukocyte DNA and the IGF-II mRNA content was the same as in normal adrenal glands (Fig. 2, A–C).
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The technique was performed as previously described (6). For each tumor sample, 15–20 metaphase spreads were captured, and only metaphases containing high-quality fluorescence intensities were included in the final analysis. After exclusions, the observations from 9–10 metaphase spreads for each tumor sample were pooled to obtain the mean ratio. Ratios of at least 1.20 were considered as gains of genetic material, whereas ratios below 0.80 were considered as losses of genetic material.
The central part of the tumor exhibited numerous CGH changes, with gains at chromosomes 4, 5p, 10, 12p, 16p, 19, and 20 and losses at chromosomes 2 and 11 (Fig. 3). The surrounding benign tissue did not show any CGH change.
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During the last decade, our understanding of the mechanisms of adrenal tumorigenesis has advanced, and there is considerable evidence suggesting a stepwise progression from adenoma to carcinoma because a particular clone of cells gains a growth advantage over competing subclones as a result of advantageous genetic events (1, 2, 3, 4, 5, 6, 7). A large body of evidence suggests that accumulated genetic changes underlie the development of neoplasia. This multistep process is well illustrated by the model of colorectal cancer (19).
A variety of genetic abnormalities have been described in adrenocortical tumors, most of them being associated with the malignant phenotype (for reviews, see Refs.20, 21, 22). The events involved in the formation of adenoma are not well known. However, recent data suggest that a change in the angiogenic phenotype (23) or chromosomal instability (21) have already occurred in premalignant tumors.
We report the case of a patient with an incidentally discovered adrenal tumor. This tumor was apparently localized in the adrenal and exhibited two very different pathological areas: a malignant central part surrounded by benign tissue. These pathological differences are reflected by genetic abnormalities and data from CGH.
Indeed, 17p13 LOH, uniparental disomy at 11p15, and overexpression of the IGF-II gene are highly specific of malignant tumors and are associated with a risk of recurrence in strictly localized tumors (16).
The CGH abnormalities described in the central malignant part of the tumor are also in accordance with previous studies. The chromosome gains observed in the malignant part of the tumor were the same as those previously described (5, 6). Losses at chromosomes 2 and 11q have also been described in malignant tumors (3, 4, 6). CGH is theoretically unable to detect chromosomal rearrangements such as uniparental isodisomy (24) and losses at 11p15 locus have been exceptionally reported in CGH studies of adrenocortical tumors. In the malignant part of this tumor, the loss of chromosome 11 concerns both the long and the short arms and can reflect the methodological limitations of CGH (6).
Our data indicate a multistep adrenal tumorigenesis. However, we cannot definitively exclude the alternative hypothesis that these two tumors arose separately from separate clones. The finding of at least one of the DNA rearrangements in both the central (i.e. malignant) and the peripheral (i.e. benign) parts of the tumor would strongly support the multistep tumorigenesis hypothesis. However, the absence of the DNA rearrangement in the peripheral part does not rule out this hypothesis. Because the patient was a male, the assessment of clonality by the analysis of X inactivation patterns was not possible.
That is the first description of an adrenocortical tumor with two different pathological components. Although our findings do not provide definitive proof for a multistep process of adrenocortical tumorigenesis, they strongly suggest that such a process is involved in at least some adrenocortical tumors.
Footnotes
This work was supported by the Assistance Publique Hôpitaux de Paris, the University Paris VI, Institut National de la Santé et de la Recherche Médicale (U515), and Programme Hospitalier de Recherche Clinique Grant AOM 95201 for the Comete Network.
Abbreviations: CGH, Comparative genomic hybridization; CT, computerized tomography; LOH, loss of heterozygosity.
Received July 18, 2002.
Accepted November 20, 2002.
References
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  B. Allolio and M. Fassnacht Adrenocortical Carcinoma: Clinical Update J. Clin. Endocrinol. Metab., June 1, 2006; 91(6): 2027 - 2037. [Abstract] [Full Text] [PDF]
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A. Thorin-Savoure, F. Tissier-Rible, L. Guignat, A. Pellerin, X. Bertagna, J. Bertherat, and H. Lefebvre Collision/Composite Tumors of the Adrenal Gland: A Pitfall of Scintigraphy Imaging and Hormone Assays in the Detection of Adrenal Metastasis J. Clin. Endocrinol. Metab., August 1, 2005; 90(8): 4924 - 4929. [Abstract] [Full Text] [PDF]
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