Molecular adrenocortical tumourigenesis
M. Reincke, F. Beuschlein, M. Slawik and K. Borm
Division of Endocrinology, Medical Department II, University of Freiburg, Germany
Abstract
Adrenocortical neoplasms are the most frequent abnormality of the adrenal cortex. Most of these lesions are clinically silent and are detected incidentally by ultrasound or com- puted tomography. The prevalence of these so-called ‘incidentalomas’ in the general population is around 1%, increasing with age and reaching 6% among those in the age range 60-70 years. In contrast, primary adrenocortical carcinoma, a highly malignant tumour, is rare, having an incidence of one case per million per year. Recent progress has been achieved in the understanding of adrenocortical tumourigenesis by mapping and identification of genes responsible for hereditary tumours that involve the adrenal gland. Investigation of the clonal composition of adrenal tumours demonstrates that adrenal car- cinomas are monoclonal, whereas adrenal adenoma may be polyclonal in approximately 25-40% of cases. Oncogenes and tumour-suppressor genes involved in adrenal carci- nomas include mutations in the p53 tumour-suppressor gene and rearrangements of the chromosomal locus 11p15.5 associated with IGF II hyperexpression. Constitutive acti- vation of the ACTH receptor-G protein-cAMP signal cascade does not play a role in adrenal tumour formation. Conversely, deletions of the ACTH receptor gene have recently been found in undifferentiated adenomas and in aggressive adrenocortical carcinomas, and, more recently, confirmed in a larger series of tumours. The available literature indi- cates that the signalling pathways of adrenocortical tumours are different from those of other endocrine neoplasms, such as pituitary and thyroid adenomas.
Keywords Adrenocortical adenoma, adrenocortical carcinoma, oncogene, tumour- suppressor gene, tumourigenesis, steroidogenesis. Eur J Clin Invest 2000; 30 (Suppl. 3): 63-68
Introduction
Primary adrenocortical tumours can be divided into benign adenomas and malignant carcinomas. Clinically silent adrenocortical adenomas are extremely frequent, as shown by autopsy studies and cross-sectional studies by abdominal CT [1]. The prevalence of these so-called ‘inci- dentalomas’ gradually increases with age, ranging from 3.0 to 7.0% in adults over 50-years old [2]. By definition, incidentalomas are discovered incidentally by abdominal CT or ultrasound scans performed for various unrelated reasons. The majority of these tumours are nonfunctional adrenal adenomas, which rarely cause clinical symptoms during follow-up [3]. In contrast, adrenocortical carci- noma is a highly malignant rare tumour with an approximate incidence of one new case per million per
year [4]. Some 70% of the patients suffer from an ad- vanced tumour stage with local invasion or distant meta- stasis at the time of diagnosis and can rarely be cured [5].
Hereditary tumour syndromes associated with adrenal pathology
Several hereditary tumour syndromes are associated with the formation of benign or malignant adrenocortical tumours. The discovery of the genetic events involved in these syndromes increases the understanding of adrenal tumourigenesis (Table 1).
Li-Fraumeni syndrome
The Li-Fraumeni syndrome was first described in 1969 [7]. It is a rare family tumour syndrome with a high incidence of breast cancer, leukaemias, soft tissue sarcomas, gliomas
Correspondence to: Martin Reincke, Abteilung Innere Medizin II, Klinikum der Albert-Ludwigs-Universität Freiburg,
Hugstetter Str. 55, 79111 Freiburg, Germany. Tel .: +49 761 270 3634, fax: +49 761 270 3413
| Clinical characteristics | Molecular defect | |
|---|---|---|
| Li-Fraumeni syndrome | Familial susceptibility to a variety of cancers including breast and adrenal cancers, gliomas, sarcomas | Germ-line mutations in the p53 tumour-suppressor gene (17p) |
| BWS | Neonatal macrosomia, macroglossia, omphalocele | Allelic loss of 11p15 (tumour-suppressor genes H19 and P57KIP2) |
| MEN 1 | Hyperparathyroidism, neuro-endocrine gut tumours, pituitary adenomas | Germ-line mutations in the menin gene (11q13) |
| Carney's complex | PPNAD, atrial myxomas, swannomas, lentigines, blue naevi of the skin/mucosa | Mutations in the protein kinase A type I-alpha regulatory subunit (2p16, Carney locus) |
| Congenital adrenal hyperplasia | Female/male pseudohermaphroditismus, cortisol deficiency, mineralocorticoid deficiency or excess | Inborn errors of cortisol biosynthesis enzymes resulting in chronic ACTH hypersecretion |
| Familial adenomatous polyposis | Multiple colonic adenomas and carcinomas, extra-adrenal tumours | Germ-line APC gene mutations (5q21) |
Modified after [2]. PPNAD = primary pigmented nodular adrenocortical disease; MEN 1 = multiple endocrine neoplasia type 1 (modified after [6]).
and adrenocortical carcinomas. Affected patients generally develop the first tumour before the age of 30 years, and second and third neoplasias are often observed, especially in patients previously treated with chemotherapy or ir- radiation [8]. The molecular basis of this disease has been elucidated by identification of germ-line point mutations in the p53 tumour-suppressor gene [9]. The second p53 allele is inactivated in tumour tissue by deletion of the short arm of chromosome 17 (17p), eliminating all wild- type p53 activity. Recently, p53 germ-line mutations have been found in children with adrenocortical carcinomas without a classical family history of the Li-Fraumeni syn- drome [10,11]. Similarly, germ-line mutations have been found in apparent sporadic adrenal cancer patients from Southern Brazil (Ana-Claudia Latronico, personal com- munication). Because of the consequences of germ-line p53 mutations for these individuals and their relatives, genetic testing has been recommended for risk assessment in childhood adrenocortical carcinoma.
Beckwith-Wiedemann syndrome
Beckwith-Wiedemann syndrome (BWS) is a rare con- dition (1/13700 live birth) characterized by macroglossia, gigantism, earlobe pits or creases, abdominal wall defects, and an increased risk for the development of Wilms tumours of the kidney, rhabdomyosarcoma, hepatoblas- toma, and adrenal carcinoma [12,13]. Although most BWS cases are sporadic, there are reports of families in which the disease segregates as an autosomal dominant trait with incomplete penetrance. BWS maps to chromo- some 11p15.5. Uniparental paternal isodisomie for this locus, which includes the IGF II gene, has been identified in affected individuals [14]. The complete loss of one IGF
II allele and a duplication of the remaining allele in association with IGF II overexpression has been demon- strated in tumours of the BWS and in sporadic adrenocortical tumours [14,15].
Multiple endocrine neoplasia type 1
The genetic defect responsible for multiple endocrine neoplasia type 1 (MEN 1) has been mapped to chromo- some 11q13 [16]. Tumourigenesis results from unmasking of a recessive mutation in the recently identified menin tumour-suppressor gene with the development of para- thyroid adenomas, pituitary adenomas and tumours of the endocrine pancreas [17]. Reports indicate that the adrenal gland is involved in a considerable proportion of patients (36-41%) with MEN 1. This lesion is often characterized as bilateral hyperplasia or adenomas, and occasionally even carcinomas [18]. Recently, in 12 patients with adrenocortical tumours out of a series of 33 patients with MEN 1, loss of constitutional heterozygosity for chromo- some 11q13 was demonstrated only in one patient with adrenocortical carcinoma, but not in 11 patients with benign adrenal lesions [18]. This suggests that adreno- cortical tumourigenesis is probably not a primary lesion in the MEN 1 syndrome. This is also supported by the finding that MEN 1 gene mutations are not found in patients with sporadic adrenocortical tumours.
Carney complex
The Carney complex, which is an autosomal dominant disorder, is characterized by the association of primary pigmented nodular adrenocortical disease, myxomas, par-
ticularly of the heart, and psammomatous melanotic swannomas involving the peripheral nervous system, spotty pigmentation and blue naevi of the skin or mucosa, and diverse endocrine neoplasms [19]. Testicular Sertoli cell tumours, GH-producing adenomas, thyroid follicular carcinomas, ovarian cysts, and adrenocortical tumours were associated with this familiar syndrome, whose chromosomal locus was recently mapped on 2p16 [19]. The gene has been cloned and shown to encode the protein kinase A type I-alpha regulatory sub- unit [20].
Familial adenomatous polyposis
Familial adenomatous polyposis (FAP) is an autosomal dominant inherited disorder characterized by the develop- ment of more than 100 adenomatous polyps in the colon and rectum, which can undergo malignant change. The incidence of FAP is between one per 17 000 and one per 5000. The condition has been causally linked to mutation of the adenomatous polyposis coli (APC) gene located at 5q21. Virtually all mutations in the APC gene are truncating mutations, resulting in loss of function of the APC protein. Spontaneous germ-line mutation of this gene occurs in many instances and accounts for the high incidence of FAP. Somatic mutations of the APC gene are also often observed in a variety of other human carcinomas. Patients with FAP sometimes develop various extracolonic manifestations, including adreno- cortical neoplasms [21,22]. Adrenocortical adenomas and carcinomas have been reported in FAP patients and, in a recent retrospective study among 162 FAP patients, the prevalence of adrenal incidentalomas was 7-4%, which is higher than the expected 0-6-3-4% reported in the literature [23].
Clonal analysis of adrenocortical tumours
Determination of the clonal composition of neoplastic tissues has been instrumental in establishing the cellular origin of many human tumours. The pathogenesis of can- cer is a multistep process during which an initiation event is followed by tumour promotion. The initiation event is widely regarded as a somatic mutation that occurs in oncogenes or tumour-suppressor genes. Adrenocortical steroid secretion is a complex process that is regulated by several hormones and growth factors, and which also con- trols adaptive processes such as hypertrophy and hyperplasia of the adrenal cortex. A polyclonal tumour would suggest that it developed from a group of cells under the common stimulus of growth factors of extra- or intra-adrenal origin. Conversely, a monoclonal tumour would suggest that it developed from a single genetically aberrant cell.
Three recent publications investigated the clonal com- position of adrenocortical tumours using X chromosome
inactivation analysis [24-26]. Gicquel et al. [24] found a monoclonal pattern in all four carcinomas studied. Of 14 benign adenomas, six tumours were monoclonal, whereas four adenomas clearly showed a polyclonal pattern. The study of Beuschlein et al. [25] revealed monoclonality in all carcinomas (n = 3) and in seven of eight adenomas. One cortisol-producing adenoma in this study was poly- clonal. These data demonstrate that adrenocortical carcinomas are generally monoclonal as a result of on- cogenic mutations of single cells with transformation and expansion into a malignant clone. Most of the adenomas also arise from oncogenic mutations, whereas a minority of benign adenomas is genetically hetero- geneous. These adenomas may have an oligo- or multicellular origin under the putative action of extra- adrenal or local growth factors. However, monoclonal and polyclonal adenomas might represent different stages of a common multistep process [26,27]. Based on this notion, the growth of polyclonal tumour tissue arises either from the proliferation of cells with a constitutive intrinsic growth potential or by stimulation of mitogens, followed by a secondary mutational event conferring a growth advantage of a particular clone leading to mono- clonality.
Cytogenetic aspects of adrenocortical tumourigenesis
The genetic aberrations involved in sporadic primary adrenocortical tumours are not completely understood. Previous studies have focused on selected chromosome regions. Yano et al. [28] studied loss of heterozygosity in one primary and eight recurrent adrenocortical carci- nomas and eight sporadic benign lesions. The carcinomas showed loss of heterozygosity on 17p, 11p and 13q. No genetic changes were found in any of the benign lesions. Karyotype analysis of adrenocortical tumours have revealed various results (for a review, see [7]).
More recently, using comparative genomic hybrid- ization (CGH), a high frequency of genetic aberrations was detected in adrenocortical carcinomas [29]. Losses most often involved the chromosomal regions 2, 11q and 17p, whereas gains took place at chromosomes 4 and 5. These results differ from our own experience using CGH in seven adenomas and 14 carcinomas [30]. Chromosomal gains were much more prevalent than losses and affected chromosomes 16p, 20q (each 11/14 tumours), 5, 7, 9q, 12q, 14q (each 7/14 tumours). Losses were mainly found at 9p. Total gains and losses were less frequent in large adenomas (tumour diameter > 4.0 cm) and absent in small adenomas (<4 cm). These studies show that adrenal carcinoma tumourigenesis involves amplifications of chromosomes not commonly affected in other human tumours, indicating the presence of new and possibly adrenal-specific oncogenes. This may give new insight into the mechanisms of adrenocortical tumourigenesis.
| Gene | Prevalence of mutations | Author | Year |
|---|---|---|---|
| Signal transduction | |||
| Constitutive activating | 0/25 tumours | Latronico et al. [32] | 1995 |
| ACTH receptor mutations | 0/16 tumours | Light et al. [33] | 1995 |
| ACTH receptor deletions | 1/16 adenomas | ||
| 2/4 carcinomas | Reincke et al. [34] | 1997 | |
| Constitutive activating angiotensin II | 0/55 adenomas | ||
| type 1 receptor mutations | 0/1 carcinomas | Sachse et al. [35] | 1997 |
| G-Protein mutations (Gas) | 0/11 tumours | Lyons et al. [36] | 1990 |
| 0/18 tumours | Reincke et al. [37] | 1993 | |
| G-Protein mutations (Gai2) | 3/11 tumours | Lyons et al. [36] | 1990 |
| 0/18 tumours | Reincke et al. [37] | 1993 | |
| 0/18 tumours | Gicquel et al. [38] | 1995 | |
| Calcium-dependent | Normal in 17/17 tum. | Latronico et al. [39] | 1994 |
| proteinkinase C activity | |||
| Ras mutations | 0/17 tumours | Moul et al. [40] | 1993 |
| 0/33 tumours | Ohgaki et al. [41] | 1993 | |
| 3/24 carcinoma (N-ras) | Yashiro et al. [42] | 1994 | |
| 4/32 adenomas (N-ras) | |||
| Growth factors | |||
| IGF II overexpression/LOH 11p15 | 27/29 carcinomas | ||
| 3/35 adenomas | Gicquel et al. [15,43] | 1997 | |
| IGF II overexpression | 5/6 carcinomas | ||
| 0/15 adenomas | Ilvesmäki et al. [44] | 1993 | |
| Tumour-suppressor genes | |||
| p53 exon 4 | 11/15 adenomas | Lin et al. [45] | 1994 |
| 0/19 adenomas | Reincke et al. [46] | 1996 | |
| p53 exon 5-8 | 3/15 carcinoma | Ohkagi et al. [41] | 1993 |
| 0/18 adenomas | |||
| 5/13 carcinomas | Reincke et al. [47] | 1994 | |
| 0/5 adenomas | |||
| APC gene | 0/4 carcinomas | Wakatsuki et al. [21] | 1998 |
| 0/6 adenomas | |||
| p57KIP2 and H19 | Low expression in | Liu et al. [48] | 1997 |
| 3/10 adenomas | |||
| 6/6 carcinomas | |||
| MEN 1 gene | Point mutations | ||
| 0/25 adenomas | Heppner et al. [49] | 1999 | |
| 0/7 carcinomas | |||
| 0/2 adenomas | Kjellman et al. [50] | 1999 | |
| 0/11 carcinomas | |||
| 1/14 adenomas | Schulte et al. [51] | 1999 | |
| 1/14 carcinomas | Schulte et al. [52] | 2000 |
Modified after [6].
Oncogenes and tumour-suppressor genes ACTH receptor mutations and adrenal tumours
Cyclic AMP is a key second messenger involved in hor- mone hypersecretion and/or increased cell proliferation in a variety of endocrine tissues. Constitutive activation of key regulatory proteins of cAMP, such as G-protein coupled receptors and GTP binding proteins, have been implicated in the pathogenesis of diseases such as acromegaly and toxic thyroid adenomas.
Adrenocortical tumourigenesis differs from pituitary and thyroid tumourigenesis, because activation of the
cAMP/protein kinase A pathway seems to be of little importance in the development of adrenocortical neo- plasms. ACTH is the main hormone regulating steroid hormone secretion but it fails to cause adrenocortical hypertrophy in the absence of innervation by the splanchnic nerve. ACTH in physiological concentrations does not stimulate cell proliferation of adrenocortical cells, and even pharmacological doses of ACTH induce only moderate cell growth [31]. In keeping with these findings, activating mutations of neither the ACTH recep- tor nor the a-chain of the Gs have been identified in benign or malignant adrenocortical tumours (Table 2). In contrast, activating mutations of the Gi2 - one of the
adenylyl cyclase inhibitory G-proteins - were found in very few adrenocortical tumours, but not in a variety of other endocrine and nonendocrine tumours (Table 2).
These data suggest that, in the adrenal cortex, the ACTH/Gs/protein kinase A signaling pathway is pref- erentially important for steroid hormone secretion and, hence, for maintenance of a highly differentiated cellular phenotype, but is of relatively low importance for cellular proliferation. This is supported by the recent finding of deletions of the ACTH-R gene in undifferentiated adrenocortical tumours. Of 16 patients with benign lesions, mutational loss of the ACTH-R gene by deletion was present in one oncocytic nonfunctional adenoma, but not in 15 hyperfunctioning adenomas. Of four informative patients with adrenocortical carcinomas, loss of hetero- zygosity of the ACTH receptor gene was present in two cases. Both patients had advanced tumour stages and exhibited a more rapid course than carcinoma patients without LOH. Northern blot experiments showed reduced expression of ACTH-R mRNA in the tumours with LOH of the ACTH-R gene, suggesting functional significance of this finding at the transcriptional level [36]. These data were recently confirmed in a larger series of tumours using a polymorphic marker within intron 1 of the ACTH receptor gene. This demonstrates that the ACTH receptor may act as a tumour-suppressor gene. Allelic loss of the ACTH-R gene in adrenocortical tumours can result in loss of differentiation, a characteris- tic feature of human tumourigenesis that is associated with clonal expansion of a malignant cell clone.
IGF II, p53 and other oncogenes
Using the ‘candidate gene approach’, several studies have investigated the prevalence of putative adrenal oncogenes and tumour-suppressor genes (Table 2). Most of these studies showed a low prevalence of mutations. However, IGF II overexpression and mutations in the p53 tumour- suppressor gene have often be demonstrated in adreno- cortical carcinomas.
Acknowledgements
Supported by a grant of the Mildred-Scheel-Stiftung, Bonn, to M.R.
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