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The Molecular Pathogenesis of Hereditary and Sporadic Adrenocortical and Adrenomedullary Tumors

Pediatric and Reproductive Endocrinology Branch, National Institute of Child Health and Human Development, National Institutes of Health (C.A.K., K.P., G.P.C.), Bethesda, Maryland 20892; and Department of Medicine III, University of Leipzig (C.A.K.), 04103 Leipzig, Germany

Address all correspondence and requests for reprints to: Christian A. Koch, M.D., FACP, Department of Medicine III, University of Leipzig, Phil.-Rosenthalstrasse 27, 04103 Leipzig, Germany. E-mail: kochc{at}exchange.nih.gov.

Abstract

Modern imaging modalities lead to frequent detection of adrenal masses, most of them incidental findings. Although the majority of adrenocortical and adrenomedullary tumors are benign, there are no reliable clinical and laboratory markers to distinguish most of them from malignant neoplasms. The molecular mechanisms underlying the pathogenesis of these tumors have recently begun to be unraveled. A fruitful avenue for the elucidation of tumorigenesis has been the study of adrenal tumors that are manifestations of hereditary or postzygotic genetic syndromes, because one knows the "first hit", i.e. the primary gene defect. In contrast, in sporadic adrenal tumors the first hit, possibly a somatic mutation of a tumor-related gene, is unknown, and therefore the sequence of genetic alterations is difficult to establish. In this article we review in addition to our own work the literature on molecular aspects of adrenocortical and adrenomedullary tumorigenesis.

ADRENAL TUMORS HAVE been identified in up to 9% of all autopsy cases, with most of them appearing in hypertensive and diabetic subjects (1, 2, 3). Most of these masses (found in 7% of patients older than age 50 yr) are detected incidentally, during assessment of patients by modern imaging modalities. Although the majority of adrenal tumors are nonfunctional, as demonstrated by biochemical investigations, some are hormone secreting and responsible for endocrine pathology (4, 5). The clinician often faces the difficult task of assessing a patient with an adrenal incidentaloma for the presence of subclinical disease or for the possibility and potential of malignancy. If initial imaging characteristics suggestive of malignancy, such as intratumor necrosis, irregular margins and local metastasis, are not present, regular imaging follow-up should be performed to help detect malignant masses in a timely fashion. Generally, adrenal tumors with a malignant potential grow faster than those with a benign phenotype. However, it should be noted that even adrenal tumors smaller than 2 cm at detection might already be metastatic (6).

The molecular pathogenesis steps of hereditary/genetic adrenal tumors that occur after the "first hit" (the primary gene defect) are not well understood, whereas those of adrenal tumors that occur sporadically are even more obscure. In addition, histological or molecular tumor markers that can reliably distinguish between benign and malignant adrenal masses are generally lacking (6, 7, 8, 9, 10, 11, 12). In 1984, Weiss (7) suggested the introduction of a malignancy score determined by histopathological features of adrenocortical tumors. He acknowledged three limitations of his multifactorial analysis. First, only 25 patients with adrenal adenomas and 18 with carcinomas were studied. Second, the follow-up period of benign tumors was between 5 and 11 yr, whereas it was up to 23 yr for malignant tumors. Third, tumor tissue could be heterogeneous within the same lesion or classified as borderline, and therefore the score, even if established by experienced pathologists, could not be completely reliable.

Some limitations also characterize the diagnostic and prognostic values of genetic and molecular markers studied by Gicquel et al. (9). For the 96 patients diagnosed with localized adrenal disease, the follow-up ranged between 5 months and 11.5 yr. A longer follow-up period might have demonstrated a higher number of malignant adrenal tumors, initially classified as benign lesions. Gicquel et al. (9) found loss of heterozygosity (LOH) at 17p13 in 23 of 76 (30%) tumors restricted to the adrenal gland and in 11 of 13 (85%) adrenal cancers. Similarly, LOH at 11p15 was observed in 32 of 94 (34%) tumors restricted to the adrenal gland, whereas it was present in 15 of 18 (83%) of adrenal carcinomas. Furthermore, overexpression of IGF2 was demonstrated in 26 of 94 (28%) tumors restricted to the adrenal gland, whereas it was shown in 15 of 18 (83%) of adrenal cancers. These findings suggest that none of the aforementioned genetic and molecular alterations would reliably predict malignancy.

Elucidating the molecular pathogenesis of adrenal tumors may help improve the diagnosis and treatment of patients with such masses. Here, we review genetic/molecular aspects of adrenal tumorigenesis in masses arising from the adrenal cortex and medulla. These structures have embryologically different progenitor cells: the adrenal cortex anlagen appear as a thickening of the coelomic epithelium in the fourth week of gestation, whereas neural-crest derived chromaffin cells migrate into the adrenal primordium by the eighth week of gestation and remain as discrete islands until the first postnatal week, when they progressively form a rudimentary adrenal medulla (13).

Adrenocortical tumors

Genetic tumor syndromes.

Adrenocortical nodules and tumors have been detected in several familial or sporadic genetic syndromes and in many cases appear to be manifestations of one and the same molecular defect. Studying such patients clinically and genetically may help unravel the molecular pathogenesis of the much more common sporadic adrenal tumors. Below we provide a brief description of genetic syndromes that have been associated with adrenal tumors.

The Li-Fraumeni syndrome.

This autosomal dominant familial cancer syndrome was first described in 1969. It is associated with breast cancer, brain tumors, soft tissue sarcomas, leukemia, and adrenocortical carcinoma (14). The frequency of adrenal cancer in patients with the classic Li-Fraumeni syndrome is about 1% (15). Patients with this syndrome generally develop tumors before age 30 yr, and most of them have germline mutations in the tumor suppressor gene p53 located at chromosomal locus 17p13 (16, 17, 18). Children with p53 germline mutations may have an adrenocortical tumor as the sole manifestation of Li-Fraumeni syndrome, as discovered by analyzing 55 patients, including 18 children, with apparently sporadic adrenocortical tumors (19). Seventeen of these children had virilization, and 8 of them also had hypercortisolism (19). Tumors of patients with p53 germline mutations frequently show LOH by deletion of the wild-type p53 allele at chromosome 17p13 (19, 20). Reduced tumor suppressor activity of p53 may facilitate tumorigenesis and tumor progression by abnormal cell cycle regulation, increased growth, and decreased apoptosis. In the control of cell growth and death, p53 interacts with Mdm2, a cellular protooncogene, in an autoregulatory loop. Mdm2 protein can block the transcriptional activity of p53, help export this protein into the cytoplasm, or promote its degradation. Important for the various cellular responses to p53 are several target genes including growth arrest genes, such as p21 and 14-3-3, as well as DNA repair and apoptosis genes (reviewed in Ref. 21).

The Beckwith-Wiedemann syndrome.

This syndrome occurs sporadically or in an autosomal dominant pattern with variable expressivity. Affected patients have macroglossia, abdominal wall defects, gigantism, and an increased risk of developing Wilms’ tumors of the kidney, hepatoblastoma, rhabdomyosarcoma, and adrenal carcinoma (22). Beckwith-Wiedemann patients with adrenal cancer presented primarily with virilization caused by tumor production of testosterone, dehydroepiandrosterone and its sulfate, or other androgenic steroids (23, 24). Recently, however, a 20-yr-old woman with Beckwith-Wiedemann syndrome presented with hypertension and increased catecholamine secretion due to bilateral pheochromocytomas (25). Family studies revealed a gene locus at 11p15.5, which includes the IGF2 and the p57/KIP2 genes, the latter encoding the p57 tumor suppressor protein (26). p57 is a negative regulator of cell proliferation and inhibits G₁ cyclin/cyclin-dependent kinase (CDK) complexes (27). Individuals with Beckwith-Wiedemann syndrome often have uniparental paternal isodisomy for the IGF2 locus. Overexpression of IGF2 is seen in several types of neoplasias, including Wilms’ tumor, hepatoblastoma, colon cancer, renal cell carcinoma, and adrenocortical tumors (28, 29, 30).

Usually autosomal genes are expressed from both parental alleles. However, some genes, including IGF2, H19, and p57, demonstrate functional imprinting, a phenomenon that indicates that only one parental allele (either maternal or paternal) is expressed while the other allele is silenced. Upon activation of the silenced gene copy, biallelic expression takes place, which might influence cell growth. Whereas normally the paternal IGF2 allele is transcribed, H19 and p57 genes are expressed by the maternal allele. Duplication of the paternal 11p15 allele containing the IGF2 gene locus and/or loss of the maternal allele are frequently found in adrenal cancer (28, 29).

Although H19 accounts for approximately 3% of embryonic mRNA, this is not translated into protein. H19 may function as a tumor suppressor gene by inhibiting IGF2 gene expression (31). Indeed, deletion of the maternal H19 gene promoter facilitates expression of the normally restrained maternal IGF2 gene and results in a growth advantage (32). In adrenal cancer this is associated not only with increased transcripts of IGF2 and decreased mRNA expression of H19 and p57, but also with elevated IGF-II protein compared with normal adrenocortical tissue (29, 33). Local auto- or paracrine effects through the IGF system could be exerted in adrenal cancer, granted that IGF-I and IGF-II receptors are present in adrenocortical tumors (reviewed in Refs. 29 and 30). However, most likely IGF-II is a tumor progression rather than an initiating factor, as indicated from its frequent overexpression in malignant adrenocortical tumors (27 of 29 tumors) compared with benign adenomas (3 of 35 tumors) (9, 33).

Carney complex.

This hereditary syndrome, described in 1980, is characterized by spotty skin pigmentations, atrial and peripheral myxomas, psammomatous melanotic schwannomas, and endocrine tumors, including primary pigmented nodular adrenocortical disease and pituitary tumors secreting GH (34). Among 338 patients with Carney complex, primary pigmented nodular adrenocortical disease occurred in 88 (26%); this often remains undetected if not properly screened for, as subclinical, atypical, or periodic Cushing’s syndrome are frequent occurrences in this condition (35, 36). Twenty-four-hour urinary free cortisol excretion may be normal, but the diurnal rhythm of cortisol secretion as well as 1 mg dexamethasone suppression testing may be abnormal, the latter frequently causing paradoxic stimulation of cortisol secretion. The adrenal nodules in Carney complex are usually small (approximate average diameter, 6 mm) and have a brown or black color in a background of internodular adrenal cortex atrophy (37). The inheritance pattern of Carney complex is autosomal dominant. At least two chromosomal loci have been identified: 2p16 and 17q22–24 (38, 39, 40, 41, 42). Despite microdissection analyses of tumors from affected patients, no deletions were found at 2p16 and 17q22, implying that the responsible gene might be an oncogene rather than a tumor suppressor gene (39, 42). By contrast, LOH at 17q was reported in tumors from other groups of patients with Carney complex (38, 40, 41). Using the candidate gene approach and positional cloning, PRKAR1A, a gene encoding protein kinase A regulatory subunit 1, was found mutated in a subset of patients with Carney complex (38, 40). Analysis of protein kinase A activity in tumors associated with Carney complex revealed decreased basal activity but increased cAMP-stimulated activity compared with tumors not associated with Carney complex.

In contrast to these findings of aberrant cAMP-dependent protein kinase A activity, another second messenger-dependent protein kinase, calcium-dependent protein kinase C, has been reported to have the same activity in sporadic benign and malignant adrenocortical tumors and normal adrenal tissue (43).

Multiple endocrine neoplasia type 1 (MEN1).

MEN1 is an autosomal dominant tumor syndrome. Approximately 35% of patients with MEN1 have adrenal nodules (44, 45). This prevalence is approximately 4 times higher than that (<9%) of adrenal incidentalomas in individuals without MEN1. These nodules may arise in the cortex or the medulla of the adrenals of these patients. The majority of these adrenocortical tumors are nonfunctional, yet some secrete cortisol causing Cushing’s syndrome, and a few secrete aldosterone causing primary hyperaldosteronism (44, 46). A recent study of 66 patients with MEN1 germline mutations reported 18 patients (27%) with adrenal tumors. Eleven patients had nonfunctional adrenocortical tumors, whereas cortisol-secreting adrenal tumors were found in 6 patients, 3 of whom had benign tumors and 3 of whom had adrenal cancer. Also, an MEN1 patient has been reported with a catecholamine-secreting pheochromocytoma (47). The gene responsible for MEN1 is located at 11q13 and encodes the novel protein menin. LOH studies of tumors from patients with MEN1 indicate that the menin gene may be a tumor suppressor gene. Menin is widely expressed in adult human tissues, especially in proliferating ones (48).

LOH analyses of adrenocortical nodules from MEN1 patients have only been reported in two studies (45, 46). Beckers et al. (46) found LOH at 11q13 in an aldosterone-producing adrenal adenoma, whereas Skogseid et al. (45) did not find LOH at 11q13 in adrenocortical nodules from 12 patients with MEN1. One important aspect in such investigations is the underdiagnosis of LOH by contamination of tumor with normal tissue, especially before sophisticated microdissection procedures were developed (49).

Interestingly, unilateral pheochromocytomas have been reported in seven patients with MEN1. All seven tumors showed LOH of the wild-type allele around the menin locus, implicating inactivation of MEN1 in adrenomedullary tumorigenesis (reviewed in Ref. 44). In these studies the follow-up of the respective patient is important, especially when considering that in genetic syndromes, such as MEN1, tumors that are part of the respective syndrome usually also occur in the contralateral organ.

Familial hyperaldosteronism (FH).

Primary hyperaldosteronism was first described by Conn who suggested a very high prevalence of this disorder (50). However, in a study of normotensive and hypertensive patients with adrenal nodules, Kaplan and Cook (51) demonstrated that secretion of aldosterone in 43 hypertensive patients was similar to that in 39 normotensive ones, suggesting that primary hyperaldosteronism was rarely the cause of essential hypertension. One important aspect in this context is the identification of an adrenal nodule as an aldosteronoma, especially when a patient has hypertension and hyperaldosteronism, as indicated by an aldosterone/plasma renin activity ratio of more than 20. In general, aldosterone-producing adrenal lesions (hyperplasia, nodule, and carcinoma) account for less than 1% of patients with hypertension (52). The distinction between hyperplasia and nodule/adenoma is important, because patients with primary FH may have a detectable adrenal adenoma in one gland but none (yet) in the "hyperplastic" contralateral adrenal, making them candidates for aldosterone antagonist treatment rather than for surgical adrenalectomy.

In the past, FH was underdiagnosed, and many patients underwent unilateral adrenalectomy for adenoma with recurrence of hyperaldosteronism from the contralateral adrenal later in life. This observation implies that there may be a transition from adrenal hyperplasia to adenoma or tumor formation. Often, investigators use the terms diffuse hyperplasia, nodular hyperplasia, and nodule/adenoma with an arbitrary cut-off for an adrenal lesion to refer to nodule when the diameter of the latter exceeds 1 cm (reviewed in Ref. 53). Genetic analyses of such adrenal lesions may be of great help in defining their pathogenesis and nomenclature.

In a subset of families with primary FH, aldosterone production can be suppressed by dexamethasone administration through decreasing ACTH secretion. Almost all of these affected individuals have FH type 1, whereas patients with FH who do not respond to dexamethasone administration are determined to have FH type 2 (54). Importantly, both FH type 1 and FH type 2 are often transmitted in an autosomal dominant fashion and medically treated. The genetic basis for FH type 1 is the formation of a hybrid gene caused by the fusion of the corticotropin-regulated promoter of the 11ß-hydroxylase gene (CYP11B1) and the angiotensin II-regulated aldosterone synthase gene (CYP11B2) at 8q24 (55). In FH type 2, the responsible gene remains unidentified, but has been linked to 7p22 (56). The precise mechanisms of adenoma/tumor formation in patients with FH types 1 and 2 remain unknown.

Congenital adrenal hyperplasia.

These autosomal recessive disorders are caused by impaired cortisol secretion from the adrenal cortex and subsequent elevation of ACTH. More than 95% of affected patients have germline mutations in the gene coding for 21-hydroxylase at 6p21.3, and approximately 80% of these patients have uni- or bilateral nodular adrenal hyperplasia (57). Because up to 45% of heterozygous carriers have macronodular adrenal disease, investigators analyzed patients with apparently sporadic adrenocortical tumors for mutations in the 21-hydroxylase gene, but detected such a defect in only a small subset (58, 59).

Mutations in the transcription factor gene encoding steroidogenic acute regulatory protein have been reported to cause congenital lipoid adrenal hyperplasia, a rare disease characterized by markedly enlarged adrenals and deficient adrenal steroidogenesis often leading to salt wasting, acidosis, and death in infancy. In some cases it is associated with parotid tumor development (60, 61).

The McCune-Albright syndrome.

The McCune-Albright syndrome is a sporadic postzygotic genetic disease characterized by pigmented café-au-lait patches of the skin, polyostotic fibrous dysplasia of the bones, endocrinological abnormalities including precocious puberty, thyroid nodules and thyrotoxicosis, GH-secreting pituitary adenomas, and Cushing’s syndrome caused by primary adrenocortical hyperfunction (62, 63, 64). Of 158 patients with McCune-Albright syndrome, 5 had hypercortisolism caused by nodular adrenocortical disease (65). Other reports of patients with adrenal lesions in the McCune-Albright syndrome also described hypercortisolism but no other adrenal hormone oversecretion phenomena (62, 63, 64, 66). Somatic, activating mutations in the -chain of the stimulatory G protein GNAS1 (G_s), at 20q13.2 are responsible for this syndrome and result in stimulation of cAMP, a second messenger in the hormonal signal transmission system of many endocrine tissues. Activated mutant G proteins cause increased intracellular cAMP concentrations and eventually hormone overproduction, as implicated in GH-secreting pituitary adenomas and toxic/hormone-hypersecreting thyroid nodules (67). Mutations in the G protein genes G_s (GNAS1) and the cAMP inhibitory G_i2 (GNAI2) occur in only a very small subset of adrenocortical tumors (68, 69, 70, 71, 72).

Apart from mutant G proteins, ectopic G protein-coupled receptors may play a role in adrenocortical tumorigenesis. Aberrant expression of ectopic membrane hormone receptors, including those for gastric inhibitory polypeptide, ß-adrenergic agonists, vasopressin, LH, and IL-1, were identified in some (cortisol-producing) adrenal adenomas (reviewed in Ref. 73). How exactly these receptors and mutant G proteins itself cause adrenal tumor formation, however, remains unknown. Of note, malignant transformation of these adrenal masses has not been reported as yet.

Sporadic adrenocortical tumors.

Somatic genetic alterations that are identified in sporadic adrenal tumors are difficult to place in a context of tumor formation and progression. Alterations of gene regulation and expression are numerous in cancer, and this is also true for adrenocortical tumors. Cell replication errors may lead to numerical changes in chromosomes, chromosomal translocations, amplification and/or loss of genes, somatic sequence alterations in specific genes including DNA repair genes, and other genomic changes. One avenue of investigation represents the analysis of adrenocortical tumors for alterations in genes that are known to be associated with hereditary tumor syndromes as those discussed above. Thus, candidate genes include p53, MEN1, IGF2, and genes and receptors involved in signal transduction systems. Another important step in analyzing adrenal and other tumors is to determine whether they are mono- or polyclonal. Further analyses may include comparative genomic hybridization studies and allele typing using microsatellite markers to find hints for involvement of genes with tumor suppressor or oncogenic function.

The main hormones regulating adrenocortical hormone production are ACTH and angiotensin II. No mutations have been found in the angiotensin II type 1 receptor gene ATR1 at 3q21–25 in human adrenal tumors, but moderate ATR1 overexpression has been reported (74, 75, 76, 77). Upon binding of ACTH to its receptor, the ACTH (melanocortin type 2) receptor, activation of the adenyl cyclase/cAMP/cAMP-dependent protein kinase A pathway causes phosphorylation of proteins that mediate steroidogenesis (reviewed in Ref. 73). The ACTH receptor is a G protein-coupled receptor and is encoded by a gene mapping to chromosome 18p11 (78, 79). Mutation analysis of the coding region of the ACTH receptor gene in 41 adrenocortical tumors was negative, but deletions of this receptor were identified in a small subset of adrenocortical adenomas and carcinomas (80, 81, 82). The ACTH receptor gene was deleted in 1 nonfunctional adrenal adenoma, but was not deleted in 15 hyperfunctional (autonomously hormone-producing) adrenal adenomas (80). Whether such genetic alterations are involved in the tumorigenesis of adrenocortical tumors remains unknown.

Clonal analyses.

The cellular origin of adrenocortical tumors is still unknown, although clonal analyses of adrenal cortex lesions have helped better define the nature of adrenocortical tumors. Usually the development of cancer and tumors, in general, is regarded as a multistep process. One tumor-initiating mutation in a single cell may equip this cell with a selective growth advantage, enabling it to become a tumor. This tumor would then be called monoclonal, as it derived from a single genetically aberrant cell. By contrast, a polyclonal tumor would develop from a group of aberrant cells arising in parallel.

There are several technical issues that may confound the interpretation of clonality data, including tissue contamination and reporting bias (skewing the methylation pattern toward polyclonality), inconsistent DNA amplification and methylation (skewed clonality pattern in normal tissues), the relation between tumor and sample size, and the timing of sampling during tumor evolution (83). Only one of three reported studies on the clonal analysis of adrenocortical tumors mentioned the duration of the follow-up of patients with benign adrenal adenomas/hyperplasias (84), although this feature is important as differences in the rate of growth may distinguish benign from malignant adrenocortical tumors (84, 85, 86). Diaz-Cano et al. (84) noted that the distinction between adrenocortical nodular hyperplasia and adenoma was arbitrary, and classified the adrenocortical lesions they investigated according to WHO criteria (87). In one study polyclonality was evident in 14 of 18 cases of adrenocortical hyperplastic lesions (84), whereas in another it was evident in 5 of 5 cases (85). In adrenal adenomas (nonmetastatic at the time of investigation), polyclonality was present in 3 of 22 cases (84), 6 of 14 lesions (85), and 1 of 8 cases (86). By contrast, monoclonality was found in 4 of 4 adrenal carcinomas (85), and in another study it was found in 3 of 3 cases (86).

Based on these limited data, it appears that adrenocortical carcinomas are monoclonal, whereas the majority of benign adrenocortical lesions are polyclonal. One might speculate that tumorigenesis develops on the grounds of polyclonal adrenocortical cell aggregates, some of which gain a selective growth advantage, giving rise to a monoclonal tumor. However, taking the aforementioned issues of clonality interpretation into account, it remains unclear whether a clonality assay helps differentiate benign and malignant adrenocortical lesions.

Gene amplifications and losses.

Methods to identify amplifications or losses of specific genes include comparative genomic hybridization (CGH) and allele typing using polymorphic microsatellite markers. As mentioned earlier, all studies concerning adrenocortical tumors must be carefully examined, granted that many investigators did not refer to a specific follow-up time, the natural history being important in classifying an adrenocortical tumor as benign or malignant. In the first study using CGH in 22 adrenocortical tumors, malignancy was defined by histological criteria, tumor size, and clinical outcome, with a follow-up time as long as 8.8 yr (range, 0.3–8.8 yr) (88). In this study no gains or losses were identified by CGH in adenomas less than 5 cm. Adenomas more than 5 cm size (follow-up time, 3 yr) had 1 chromosomal gain or loss or both, whereas adrenal carcinomas (the smallest being 7 cm; range, 7–20 cm) frequently had losses involving chromosomes 2, 11q, and 17 p (4 of 8 tumors) and gains at chromosomes 4 and 5 (4 of 8 tumors). The same investigators validated their CGH findings on chromosomal losses in a later study using microsatellite markers with a detailed analysis of region 2p16 (89). In a less defined study with regard to clinical data (i.e. follow-up in many cases was unknown, classification of benign and malignant lesions was not stated), another group (90) identified in 41 adrenocortical tumors (12 carcinomas, 23 adenomas, and 6 hyperplasias) gains of chromosome 17 or 17 q only in 2 of 6 hyperplastic lesions, predominantly gains of chromosomes 17 and 9q32 in adrenocortical adenomas, and losses of 1p21–31, 2q, 3p, 3q, 6q, 9p, and 11q14-qter as well as gains of 5q12, 9q32-qter, 12q, and 20q in adrenal carcinomas.

A very recent study reported an equal distribution of chromosomal gains and losses in benign and malignant adrenocortical tumors, although the genetic events in both groups were quite different (91). Limitations of this study concern 1) the classification of benign vs. malignant tumors, which was not based on the presence of metastases but on modified Weiss histological criteria (7); and 2) the variable follow-up period for the group of benign tumors, which was up to 41 months. These investigators analyzed 18 benign and 13 malignant adrenocortical tumors. Benign adrenal masses smaller than 5 cm frequently had a gain in chromosome 4, whereas malignant (>5 cm) adrenal tumors had gains in chromosomes 5, 12, and 19. Losses in the benign group were limited to chromosome 3q, whereas they occurred at chromosomes 1p, 11, 17p, and 22 in the malignant group. This is in contrast to earlier CGH studies showing gains at chromosome 4 mainly or exclusively in malignant tumors (88, 90, 92). Sidhu et al. (91) proposed activation of a protooncogene on chromosome 4 as an early event in adrenocortical tumorigenesis and the presence of 4 or more CGH alterations in a tumor suggestive of the malignant phenotype. Their CGH data in malignant adrenocortical tumors were similar to those in the study by Kjellman et al. (88). A fourth CGH study consisting of 14 adrenocortical carcinomas and 7 adenomas (no data on the follow-up period were provided) is at variance with these findings by reporting chromosomal gains to be more prevalent than losses (92). Tumors less than 4 cm did not have any gains or losses. Gains involved chromosomes 5, 7, 8, 9q, 11q, 12q, 14q, 16, 17q, 19, 20, and 22q, whereas losses were found at chromosomes 9p and X.

Two other groups studied CGH in childhood adrenocortical tumors (93, 94). Figueiredo et al. (93) found gain of 9q34 in 8 of 9 sporadic adrenocortical tumors (6 carcinomas and 3 adenomas; unclear classification, short follow-up) in addition to gains at many other chromosomes as well as losses at 2q, 3, 4, 9p, 11, 13q, 18, 20p, and Xq. The other group (94) confirmed high level amplification of chromosome 9q34 in 13 of 20 adrenocortical tumors. These findings of numerous chromosomal gains and losses in benign and malignant childhood adrenocortical tumors stand in contrast to CGH analyses of adrenocortical tumors in adults. Before concluding that the genetic background in childhood adrenocortical tumors is different from that in adults, one should consider the possible flaws of the individual studies (for instance, classification of benign vs. malignant tumors) as well as the methodological limitations of CGH.

To elucidate the pathogenesis of adrenocortical tumors, the precise definition of adenoma vs. carcinoma is of utmost importance, as is the identification of precursor lesions. Without this critical information, it is difficult to determine the sequence of events from tumor initiation to progression. The assumption that putative oncogenes at regions of chromosomal gain represent the first step in tumor development, and regions of chromosomal loss represent putative tumor suppressor genes allowing subsequent steps of tumor progression may sound plausible to many investigators, but remains speculative.

Specific gene alterations.

Gene defects in genetic tumor syndromes such as MEN1 may also be involved in the pathogenesis of sporadic tumors. Therefore, adrenocortical tumors have often been analyzed for MEN1, G_s, RAS, H19, IGF2, p57/KIP2, and p53 gene alterations (Table 1).

The roles of IGF2, H19, and p57/KIP2, all located at 11p15, in the pathogenesis of adrenocortical tumors was mentioned above under the heading Beckwith-Wiedemann syndrome. Overexpression of IGF2 is frequently found in malignant sporadic adrenocortical tumors and may serve as a molecular prognostic marker (9, 29, 33). Genetic alterations in p57/KIP2 (CDK inhibitor 1C) were sought in adrenocortical tumors, because adrenal cancers frequently showed allelic loss at 11p15. However, no somatic mutations were found in this gene in 75 sporadic adrenocortical tumors (101), but low p57/KIP2 expression was demonstrated in 3 of 10 adrenal adenomas and in 6 of 6 carcinomas (102). Reduced or absent function of this gene seems to lead to enhanced activity of G₁ CDK complexes with possibly subsequent promotion of cell proliferation (27). Consequently, investigators examined adrenocortical tumors for abnormalities in other inhibitors of CDKs. One such protein, P16, whose gene is located at 9p21, is an inhibitor of the CDK 2A gene. One of 7 benign and 3 of 7 malignant adrenocortical tumors showed loss of 1 p16 allele and absent p16 protein by immunohistochemistry (103). p21, a CDK inhibitor that can be induced by p53, was overexpressed in 70% (25 of 38) of adrenocortical cancer samples (11). Down-regulation of p21, however, did not affect the prognosis of patients with adrenal cancer in this study. This suggests a role for these CDK inhibitors in only a small subset of adrenocortical cancers.

A very important growth factor besides IGF-II is epidermal growth factor (EGF). Its receptor (EGFR) has been studied in a small number of adrenal tumors. By immunohistochemistry, EGFR was overexpressed in benign and malignant adrenocortical tumors (104, 105, 106), whereas EGF itself was not detected (104). Instead, TGF was overexpressed in adrenal cancer (104). TGF is a natural ligand for EGFR.

The p53 gene has been studied in many tumors, including adrenocortical tumors, because of its frequent mutation in cancers and its known role in regulating the cell cycle (21). p53 has been classified as a tumor suppressor gene and is mutated in the germline of most patients with Li-Fraumeni syndrome. As this syndrome is associated with adrenocortical tumors, it is conceivable that p53 may also play a role in the pathogenesis of sporadic adrenal tumors. Although allelic loss at 17p13, the locus for the p53 gene, and somatic p53 mutations frequently occur in adrenal cancer, they are uncommon in benign adrenocortical tumors, suggesting that genetic alterations in p53 are rather involved in tumor progression than initiation (107). Only 1 study from Taiwan reported p53 mutations in adrenocortical adenomas, but the classification of benign vs. malignant adrenal tumors, including the follow-up period of the affected Taiwanese patients, has remained unclear (108). By contrast, p53 mutations (predominantly in exons 5–8) were reported in up to 70% of adrenal cancers (101, 107, 109, 110). However, it remains unknown whether biallelic inactivation of p53 occurred in these tumors, because investigators reported either allelic loss at 17p13 or somatic p53 mutations, but not whether both events (biallelic inactivation of p53) took place in the respective tumor. Nevertheless, LOH at 17p13 has been suggested as a molecular prognostic marker for sporadic adrenocortical tumors (8, 9).

The RAS gene family encodes G proteins, which are involved in signaling pathways through modification of intracellular cAMP concentrations (111). Overexpression of RAS may lead to constitutive signal transduction and/or cell proliferation (112). RAS mutations were first identified in 12.5% of adrenocortical tumors, with an equal prevalence in benign and malignant tumors (113). A subsequent study reported somatic Kras mutations and an overexpression of Kras in 33% of benign adrenocortical tumors, whereas somatic Hras mutations were not detected (114). However, previous studies did not show this prevalence rate of RAS mutations (109, 115).

The RET protooncogene at chromosome 10q11.2 encodes a tyrosine kinase receptor that is involved in the control of cell differentiation and proliferation (116). Germline mutations in RET are found in almost all patients with MEN type 2 (MEN2). Importantly, RET is only expressed in certain tissues, such as the neural crest-derived parafollicular C cells in the thyroid gland and extra- and intraadrenal chromaffin cells. Overrepresentation of mutant RET may initiate tumorigenesis of medullary thyroid carcinoma and pheochromocytoma (117, 118). Interestingly, up to 70% of papillary thyroid carcinomas, especially when radiation induced (e.g. victims of Chernobyl nuclear reactor accident), have chromosomal RET rearrangements (reviewed in Ref. 116). Although adrenocortical tumors are not part of MEN2, these tumors may have genetic alterations involving RET. The analysis of 21 sporadic adrenocortical tumors revealed 1 aldosterone-producing tumor with a point mutation in RET, and RET/PTC1 rearrangements in 2 tumors, 1 cortisol-producing and 1 aldosterone-producing (119).

Telomerase is the ribonucleoprotein enzyme that elongates telomeres, i.e. chromosomal DNA ends. In most normal somatic cells, telomerase is repressed, whereas this enzyme is reactivated in transformed cells. Recently, a considerable amount of data have been gathered on telomerase activity in human cancers, including endocrine tumors. In general, the presence or absence of telomerase activity in adrenocortical tumors seems to have no major role as a prognostic marker, although some studies suggest that malignant adrenal tumors have increased telomerase activity. Hirano et al. (120) examined 41 adrenocortical tumors for telomerase activity and found 2 tumors that were classified as malignant with increased telomerase activity compared with 39 tumors classified as benign, of which 4 showed increased telomerase activity. During follow-up, 2 of these 4 tumors became malignant, as indicated by the development of metastases. The follow-up period in telomerase-positive tumors was maximally 50 months, and in all tumors it was at least 6 months. Teng et al. (121) reported an increased telomerase activity in 5 of 23 adrenal tumors. All 3 malignant adrenocortical tumors were positive in the telomeric repeat amplification protocol (TRAP), whereas only 1 of 19 benign adrenal adenomas was TRAP positive. In that study the classification of malignant tumors and the follow-up period of all tumors were not stated. Kinoshita et al. (122) demonstrated increased telomerase activity by TRAP in 1 of 13 adrenocortical tumors with an unknown follow-up period. Mannelli et al. (123) detected telomerase activity in 7 adrenal carcinomas and in 9 of 11 adrenal adenomas. Malignancy was defined by macro- and microscopic histological criteria with a follow-up for at least 2 yr for patients with tumors classified as benign. Bamberger et al. (124) found very low (<10% of positive control) telomerase activity in 3 of 3 adrenal incidentalomas, 6 of 6 cortisol-secreting adenomas, 6 of 6 aldosterone-secreting adenomas, and 7 of 7 adrenal cancers. Telomerase activity in pheochromocytomas (125) is mentioned under Adrenomedullary tumors.

Adrenomedullary tumors

Genetic syndromes and familial tumors.

Tumors arising within the adrenal medulla comprise pheochromocytomas and ganglioneuromas. Approximately 90% of all pheochromocytomas are sporadic, and the remainder are familial. Pheochromocytomas occur in three hereditary syndromes: MEN2, von Hippel-Lindau (VHL) disease, and neurofibromatosis type 1 (NF1). Recently, germline mutations in the genes for succinate dehydrogenase subunits D and B (SDHD and SDHB), which encode mitochondrial enzymes involved in the respiratory chain, were identified in patients with apparently nonsyndromic pheochromocytomas (126). It is unclear why less than 2% of patients with NF1, less than 36% of patients with germline mutations in the VHL gene, and less than 50% of patients with germline mutations in the RET gene develop a pheochromocytoma (127, 128, 129). Furthermore, it remains unexplained why in hereditary pheochromocytoma syndromes, the prevalence rates of bilateral pheochromocytomas vary in the respective syndrome; less than 78% of patients with MEN2 have bilateral pheochromocytomas, whereas bilateral pheochromocytomas occur in less than 50% of patients with VHL disease and in less than 25% of patients with NF1 (127, 128, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145). In addition, it remains puzzling why unrelated as well as related patients with one of the aforementioned hereditary syndromes and identical germline mutations develop the respective pheochromocytoma at widely different ages, i.e. in the first months of life or after the eighth decade.

VHL syndrome.

VHL disease affects about 1 in 36,000 individuals and consists of a variety of neoplasms and masses, including renal cell carcinomas; cysts involving the kidney, pancreas, and epididymis; hemangioblastomas of the central nervous system; and pheochromocytomas (146, 147). The gene responsible for VHL disease is the VHL tumor suppressor gene, located on chromosome 3p25/26. Less than 36% of patients with a VHL germline mutation develop a pheochromocytoma. Most VHL mutations associated with pheochromocytoma also predispose the patient to renal cell carcinoma, a combination defined as VHL disease type 2. Up to one third of VHL-associated pheochromocytomas do not oversecrete catecholamines. Therefore, patients affected with such tumors may have normal blood pressure and no pheochromocytoma-related symptoms (127). The biochemical and histopathological profiles of VHL-associated pheochromocytomas may help distinguish them from other pheochromocytoma types. VHL pheochromocytomas predominantly secrete norepinephrine, compared with MEN2-related pheochromocytomas which mostly secrete epinephrine (148). Histologically, VHL pheochromocytomas have a prominent amphophilic and clear cell cytoplasm pattern, as opposed to MEN2-associated pheochromocytomas (149).

Pheochromocytomas in VHL disease typically develop according to Knudson’s two-hit model, an inherited germline mutation of VHL and loss of function of the wild-type allele of the VHL gene. Bender et al. (150) studied 35 pheochromocytomas from patients with VHL germline mutations for biallelic inactivation of the VHL gene and found such inactivation in 32 tumors. The remainder were investigated for hypermethylation of CpG islands in the VHL gene promoter. However, this mechanism of VHL inactivation could not be found. A possible explanation for the inability of the investigators to detect biallelic VHL inactivation, including LOH in 3 of 35 pheochromocytomas, may be a technical problem of performing LOH analyses on nonmicrodissected tissue (49). Smaller studies of VHL-associated pheochromocytomas report LOH at 3p25/26 at lower frequencies. Khosla et al. (151) performed LOH studies on 41 pheochromocytomas, 1 of which was VHL disease associated, and revealed LOH at 3p25/26. Crossey et al. (152) identified LOH at the VHL gene locus in 1 of 2 VHL pheochromocytomas. Zeiger et al. (153) found LOH in 3 of 4 VHL-associated pheochromocytomas and in 4 of 9 sporadic pheochromocytomas. Prowse et al. (154) detected biallelic inactivation of VHL in 2 of 5 pheochromocytomas from patients with VHL disease.

Although these data suggest that pheochromocytoma in patients with VHL germline mutations is genetically part of VHL disease, the exact mechanisms of tumorigenesis remain unknown. The finding of biallelic inactivation of VHL in renal carcinomas and cysts from patients with VHL germline mutations, renal cysts being entirely benign structures, indicates that not all cells with biallelic inactivation of VHL become tumors, and that the time point of the second hit during cell development and other factors may determine the fate of the affected cell (155). In a series of 18 pheochromocytomas, 12 had loss of 1p. Among these tumors, 2 were VHL-associated pheochromocytomas, 1 of which had 1p loss (156). Lui et al. (157) identified 1p loss in 6 of 36 VHL-related pheochromocytomas and loss of chromosome 11 in 31 of 36 (86%) tumors. None of the tumors had somatic mutations in the MEN1 gene at 11q13 or the SDHD gene at 11q23. Reduced NF1 expression was found in 1 of 2 VHL-related pheochromocytomas (158).

MEN2.

MEN2 is an autosomal, dominantly inherited cancer syndrome caused by germline mutations in RET. It affects about 1 in 40,000 individuals and is characterized by medullary thyroid carcinoma, pheochromocytoma, and parathyroid hyperplasia/adenoma (159). RET is a protooncogene located at chromosome 10q11.2. It consists of 21 exons, 6 of which are hot spots for mutations. These are exons 10, 11, and 13–16. During their lifetime, approximately 50% of patients with RET germline mutations in these hot spot exons clinically develop a pheochromocytoma. Data from autopsy studies on the (true) prevalence of pheochromocytoma in individuals with RET germline mutations are unavailable.

Medullary thyroid carcinoma is a high grade malignancy and can develop in the first year of life in patients with RET germline mutations. This is the basis of performing prophylactic thyroidectomy with lymph node dissection in these patients (129). By contrast, it is unclear when patients with RET germline mutations develop pheochromocytoma. This becomes especially important when examining a patient with MEN2 and a known RET germline mutation for signs and symptoms suggestive of pheochromocytoma, positive biochemical tests, but negative high resolution imaging studies. Such a patient may have adrenal medullary hyperplasia, which has been regarded as a precursor lesion for pheochromocytoma (133). An important question in this setting is how hyperplasia, especially when described as nodular, and how pheochromocytoma, are defined. Some investigators use an arbitrary cut-off of 1 cm to distinguish nodular adrenal medullary hyperplasia (<1 cm) and pheochromocytoma (134). Since MEN2-related pheochromocytomas rarely metastasize (in <5% of patients), and approximately 50% of patients with MEN2-related pheochromocytoma are asymptomatic, annual surveillance starting at age 6 yr rather than prophylactic adrenalectomy is recommended (140, 159). Patients with pheochromocytoma-related symptoms often have palpitations and sweats besides paroxysmal or sustained hypertension, because MEN2-associated pheochromocytomas are mostly adrenergic, i.e. predominantly epinephrine secreting (148).

In patients with MEN2, germline mutations in RET are responsible for the development of pheochromocytoma, but the precise mechanisms of tumorigenesis in vivo are poorly understood. Loss of chromosome 1p has been identified in 9 of 9 and in 5 of 5 MEN2-associated pheochromocytomas (156, 157). Lui et al. (157) also found loss of 11p in 2 of 5 MEN2-related pheochromocytomas. Interestingly, the chromosomal area 1p may play a role in the development of neuroblastoma (160). A responsible gene for neuroblastoma, however, has not yet been identified (161). Reduced NF1 expression was found in 5 of 14 MEN2-related pheochromocytomas, suggesting a possible role of the NF1 gene and the ras pathway in tumor formation (158).

Recent studies shed some light on the pathogenesis of MEN2-associated pheochromocytomas (117, 162, 163). Transgenic mice heterozygous for the M918T RET mutation developed hyperplasia of parafollicular C cells and adrenomedullary hyperplasia without progression, respectively, to medullary thyroid carcinoma or pheochromocytoma. By contrast, mice homozygous for this RET mutation developed an earlier onset of C cell and adrenomedullary hyperplasia as well as progression to medullary thyroid carcinoma and pheochromocytoma (162). Somatic RET mutations in tumors from patients with RET germline mutations have rarely been reported (163, 164) (Table 2). In a study of nine MEN2-associated pheochromocytomas, five tumors had duplication of the mutant RET allele in trisomy 10, and two tumors showed loss of the wild-type RET allele (117). Chromaffin cells with such an allelic imbalance between mutant and wild-type RET may gain a growth advantage and develop into a pheochromocytoma (Fig. 1).

View larger version (105K): [in this window] [in a new window]   Figure 1. Model for tumorigenesis in MEN2-associated pheochromocytomas. In a patient with MEN2, each cell of the target organs (adrenal medulla) carries a RET germline mutation in the heterozygous state with one intact wild-type RET allele. Selected chromaffin cells undergo a second hit through duplication of the mutant RET allele in trisomy 10 or loss of the wild-type allele, giving these cells a growth advantage with subsequent tumor formation. M, Mutant RET allele; WT, wild-type RET allele; C10, chromosome 10.

NF1.

NF1 affects about 1 in 4000 individuals, whereas pheochromocytoma occurs in less than 2% of patients with NF1 (128). NF1 is inherited in an autosomal dominant fashion. Clinically, pheochromocytomas in patients with NF1 appear at a later age than in patients with MEN2 or VHL disease. Of 125 patients with NF1, 27 (22%) had no symptoms attributable to pheochromocytoma. NF1-associated pheochromocytomas primarily secrete norepinephrine and rarely epinephrine (128). The NF1 gene is believed to be a tumor suppressor gene mapped to chromosome 17q11.2 (167). It is large (open reading frame, 8454 nucleotides) and cumbersome to analyze for mutations. Affected patients have typical pathognomonic clinical features of NF1, including neurofibromas and typical skin café-au-lait spots. NF1 encodes for neurofibromin, a protein that has similarities to RAS/GTPase-activating protein. RAS has a role in controlling cell growth and differentiation through several signaling pathways, including that of cAMP (111, 112).

Tumor formation in patients with NF1 has been proposed to occur according to Knudson’s two-hit model, which requires biallelic inactivation of a tumor suppressor gene. Mice heterozygous for one mutant NF1 allele develop pheochromocytoma in 50% of cases (168). In patients with NF1-associated pheochromocytoma, but unknown germline mutations in NF1, loss of the wild-type NF1 allele could be shown in three of seven tumors (169). In another study examining neurofibromin expression, which is normally present in the adrenal gland, six of six pheochromocytomas from patients with unknown NF1 germline mutations had no neurofibromin expression. LOH could be shown in one of these six tumors (170). Of note, loss of neurofibromin was seen in tumors from patients with and without NF1 (167). Therefore, the exact molecular pathogenetic pathways for formation of a pheochromocytoma in patients with NF1 remain unknown.

Sporadic tumors.

As in the adrenal cortex, sporadic tumors in the adrenal medulla represent the majority of tumors occurring in this location. Allele losses on chromosomes 1p, 3p, 3q, 17p, and 22q are common findings in both familial and sporadic pheochromocytomas (150, 151, 156, 157, 171, 172, 173, 174, 175, 176, 177, 178). It is not clear how these allele losses are involved in tumorigenesis or tumor progression of pheochromocytomas, especially in tumors in which the first hit remains unknown.

CGH analyses.

There are three studies using comparative genomic hybridization to analyze sporadic and familial pheochromocytomas for chromosomal losses or gains (157, 173, 175). Dannenberg et al. (173) reported CGH results in 29 sporadic pheochromocytomas. Patients with these tumors tested negative for germline mutations in RET and VHL and had no features of NF1. Four of 29 pheochromocytomas were extraadrenal. Nineteen of 29 tumors were classified as benign, and 10 were malignant, using the presence of metastases as malignancy criterion. The average follow-up period was 7.6 yr in this study. The mean size of benign tumors was 7 cm (largest tumor, 15 cm), and that of malignant pheochromocytomas was 11 cm (smallest tumor, 4 cm). Chromosomal losses were more frequent than gains. No amplifications were detected. Losses on 1p and 3 were more frequent in benign tumors than in malignant pheochromocytomas. Overall, losses occurred most frequently on 1p (86%), 3q (52%), 6q (34%), 3p (31%), 17p (31%), and 11q (28%). Gains were seen on 9q (38%) and 17q (31%).

Erdstrom et al. (175) found similar patterns of CGH alterations in 13 sporadic and 6 familial (MEN2A and NF1) pheochromocytomas. Among these tumors, 4 had been classified as malignant, but only 2 of the 4 sporadic pheochromocytomas were metastatic. Extensive local invasion and/or the presence of metastases were used as criteria for malignancy. The follow-up period of patients in this study was not available in all cases. CGH results revealed frequent losses on chromosomes 1p (83%), 3q (39%), 11p (17%), 3p (17%), 4q (17%), and 11q (13%). Gains were observed predominantly on 16p (9%), 17q (17%), 19q (26%), and 19p (26%). Losses on chromosome 11q were more common in malignant than in benign tumors.

Lui et al. (157) investigated 36 VHL-related and 5 MEN2-associated pheochromocytomas and found loss of 1p in all MEN2 pheochromocytomas, but only in 6 of 36 VHL pheochromocytomas, whereas loss of 11p occurred in 31 of 36 (86%) VHL tumors and in only 2 of 5 MEN2 pheochromocytomas. Chromosomal gains were rarely found.

Although CGH studies and other investigations using microsatellite analyses indicated that the most frequent chromosomal loss in pheochromocytomas occurred at 1p, an area that is also the most frequently affected in neuroblastomas/neuroblastic type tumors, no specific gene alterations at this chromosomal region, which contains multiple genes, were shown to be involved in the development of pheochromocytomas or neuroblastomas.

Specific gene alterations.

A subset (<10%) of sporadic pheochromocytomas have somatic mutations in RET, VHL, or SDHD (179, 180, 181, 182, 183), suggesting that these genes contribute to pheochromocytoma pathogenesis in a subset of tumors. van der Harst et al. (184) identified somatic RET mutations in 4 of 27 benign and in 1 of 29 malignant pheochromocytomas. The follow-up in this study is unknown, making the interpretation of these findings difficult. Of note, apparently sporadic pheochromocytomas may turn out to be part of a familial syndrome, as demonstrated by van der Harst et al. (185), who identified germline mutations in the VHL gene in 6 of 68 patients with apparently sporadic pheochromocytomas. In a recent study of 271 patients presenting with nonsyndromic pheochromocytoma, almost one quarter had germline mutations in VHL, RET, SDHD, or SDHB (126). Whether this justifies performing germline mutation testing for VHL, RET, SDHD, or SDHB in all patients presenting with pheochromocytoma needs to be determined.

As in adrenocortical tumors, genetic alterations in p53 have been investigated in adrenomedullary tumors. Yoshimoto et al. (186) found somatic p53 mutations in 6 of 33 pheochromocytomas. Interestingly, 3 of these 6 cases showed overexpression of p53 protein in tumor cell nuclei by immunohistochemical staining. Herfarth et al. (187) found LOH at 17p13, the p53 gene locus, in 4 of 22 pheochromocytomas, but no p53 mutations. Immunohistochemical analyses did not reveal any overexpression of p53 protein in these 22 pheochromocytomas.

De Krijger et al. (188) performed an immunohistochemical study of 29 malignant and 85 benign pheochromocytomas using antibodies directed against the p53, Bcl-2, and c-erbB2 proteins. Malignant tumors had a higher frequency of p53 and Bcl-2 protein expression than benign pheochromocytomas, suggesting antiapoptotic mechanisms in the pathogenesis of malignant pheochromocytomas. These data on p53 and its protein are in conflict with the available results of CGH studies and microsatellite analyses, which do not suggest LOH at 17p and/or somatic biallelic inactivation of p53 in many pheochromocytomas. In addition, another inhibitor of apoptosis, survivin, could not be shown to be expressed at greater levels in malignant pheochromocytomas than in benign tumors (12).

Reduced or absent NF1 gene expression was found in one of four sporadic adrenal pheochromocytomas, suggesting a minor role of neurofibromin in the pathogenesis of these chromaffin cell tumors (158).

Allelic loss at 3p, an area harboring many genes, including VHL and RASSF1A, was reported to occur in approximately 20% of sporadic pheochromocytomas (see above). No mutations in RASSF1A at 3p21, but hypermethylation of the RASSF1A promoter region, were identified in 5 of 23 (20%) sporadic pheochromocytomas and in 37 of 67 neuroblastomas (171). How this mechanism contributes to tumorigenesis or tumor progression in these tumors needs to be clarified.

The protooncogene c-erbB-2 may play a role in pheochromocytoma progression, as indicated by an immunohistochemical study of 34 pheochromocytomas (27 were sporadic) that revealed a higher intracytoplasmic granular c-erbB-2 staining in malignant tumors (189).

Deletion analysis of the p16 tumor suppressor gene has been negative in 26 pheochromocytomas (22 were sporadic), suggesting no major role of this gene in the pathogenesis of pheochromocytoma (190). Similarly, no somatic mutations could be identified in the c-mos protooncogene and the endothelin-B receptor (191).

Local growth factors, including IGF-II, may also play a role in the pathogenesis of pheochromocytoma, as IGF-II transcripts are expressed in these tumors (29, 192). However, the IGF system has been better explored in adrenocortical tumors, in which it seems to predominantly play a role in tumor progression (9). Using immunohistochemistry, EGFR was overexpressed in four of seven pheochromocytomas (105).

Somatic mutations in GNAS1, Kras, or Hras have not been detected in pheochromocytomas (72, 114).

In both familial and sporadic pheochromocytomas, accumulation of mutations and deletions in numerous genes may develop during tumor evolution and progression. How these genetic alterations affect biochemical pathways and stimulate tumorigenesis remains to be shown. Combining clinical and morphological observations with genetic investigations may help in this endeavor.

Ganglioneuroma.

Ganglioneuroma is a rare neuroectodermal tumor related to neuroblastoma. In less than 21% of cases, ganglioneuromas occur in the adrenal gland (193). The so-called composite tumors, which make up to 4% of intraadrenal tumors, consist of pheochromocytoma associated with neuroblastoma, ganglioneuroblastoma, or ganglioneuroma (194). There are apparently two groups of neuroblastomas: one with LOH at 1p36 combined with less favorable prognosis, and another without LOH at this locus and good prognosis. Using microdissection, we recently analyzed a 5-cm adrenal ganglioneuroma for LOH at 1p34–36 and 17p13 and did not detect it. This might indicate that this ganglioneuroma was pathogenetically related to and possibly preceded by a neuroblastoma without 1p36 LOH. This further suggests that this locus may not be involved in tumorigenesis of this tumor type, if a tumor suppressor gene is held to be causative. The lack of LOH at the p53 locus might indicate that p53 is not involved in ganglioneuroma formation or progression. However, we did not screen for p53 mutations. Other investigators found low p53 content in four ganglioneuromas and three myelolipomas, but did not analyze these tumors for p53 mutations (195). It remains unclear why p53 protein was reduced in these tumors and what role this phenomenon plays in their pathogenesis

Clonal analyses.

As in tumors of the adrenal cortex, clonality analyses of tumors occurring in the adrenal medulla might help elucidate their pathogenesis. Clonal proliferation of cells can lead to neoplastic growth, especially when associated with abnormally low apoptosis rates of these selected cells, which results in a dominant monoclonal cell population. When studying tumors for clonal patterns, certain limitations apply, as outlined above (see Adrenocortical tumors). In the 1970s, Baylin et al. (196) analyzed one MEN2A-associated pheochromocytoma for clonality using the X-linked glucose-6-phosphate dehydrogenase assay and proposed tumorigenesis through a combination of events that rendered multiple clones of cells susceptible to a final monoclonal growth by clone selection.

One problem that needs to be addressed in such studies is the definitions of tumor and hyperplasia (197). For adrenomedullary tumors, nodule size has been proposed as a differentiation criterion: chromaffin cell conglomerates/aggregates smaller than 1 cm are defined as hyperplasia, whereas such conglomerates are called pheochromocytoma when they are larger than 1 cm (134). Accepting these criteria, a recent study reported that in 30 informative adrenomedullary nodules from 11 patients with MEN2A, 27 nodules were monoclonal and 3 nodules were polyclonal by X-chromosome inactivation analysis (198). Of 29 informative patients with pheochromocytoma, 23 tumors showed a monoclonal pattern (6 malignant and 17 benign tumors, including 9 MEN2A-associated and 8 sporadic ones), and 6 pheochromocytomas were polyclonal (all sporadic, 1 benign and 5 locally invasive). The investigators of this study emphasized that their microdissection technique taking more than 100 cells would exclude nonrandom skewness or patch size variability. Based on their results, they concluded that polyclonal diffuse adrenomedullary hyperplasia would result in monoclonal nodular hyperplasia by early clonal expansions. According to their results, the distinction between hyperplasia and pheochromocytoma by the criterion nodule size would not be necessary.

Telomerase.

Telomerase activity has been correlated with the differentiation grade of a tumor, generally implying that malignant tumors have a higher telomerase activity than benign ones because of reactivation of telomerase in malignantly transformed cells. This topic was discussed above under the adrenocortical tumors heading. In the studies of telomerase activity in pheochromocytomas, the follow-up period of the respective patients was rarely reported. Hirano et al. (120) reported elevated telomerase activity in 1 of 6 benign pheochromocytomas. Kubota et al. (125) described increased telomerase activity in 3 of 3 malignant and none of 16 benign tumors in a follow-up of at least 3 yr. Teng et al. (121) found increased telomerase activity in an adrenal ganglioneuroma. Bamberger et al. (124) detected high telomerase activity in only 1 malignant pheochromocytoma compared with very low activity in 7 of 7 benign and 2 of 3 malignant pheochromocytomas. Kinoshita et al. (122) identified increased telomerase activity in 2 of 7 benign pheochromocytomas with an unknown follow-up period. Based on these studies, telomerase activity cannot be used as a reliable prognostic marker in patients with pheochromocytoma. Furthermore, the role telomerase plays in the process of adrenomedullary tumor evolution remains unknown.

Summary

Adrenocortical and adrenomedullary tumors arise from different progenitor cells, and most occur sporadically, associated with multiple somatic genetic alterations. It is difficult to identify the exact order in which these genetic changes/events take place in adrenal tumor evolution and progression. Also, it remains challenging to find reliable genetic and molecular diagnostic and prognostic markers. Such studies on adrenal tumors that occur in genetic syndromes, such as FH for adrenocortical tumors or MEN2 for adrenomedullary tumors, may facilitate the unraveling of the molecular pathogenesis of these masses. Duplication of the mutant RET allele in trisomy 10 or loss of the wild-type RET allele was recently proposed as the second hit in MEN2-associated pheochromocytomas. Somatic alterations, such as mutations of genes known to be responsible for some hereditary syndromes (i.e. MEN1 and VHL), are only rarely found in sporadic adrenal tumors. Further studies on the function and interaction of candidate genes and their encoded proteins are needed (199).

Acknowledgments

We thank Drs. Z. Zhuang, A. Vortmeyer, S. Huang, S. Pack, I. Lubensky, G. Eisenhofer, and S. Bornstein for their long-term collaboration and support. We also thank Dr. O. Rennert for his review of the manuscript and constructive comments.

Footnotes

Abbreviations: CDK, Cyclin-dependent kinase; CGH, comparative genomic hybridization; EGF, epidermal growth factor; EGFR, epidermal growth factor receptor; FH, familial hyperaldosteronism; LOH, loss of heterozygosity; MEN1, multiple endocrine neoplasia type 1; MEN2, multiple endocrine neoplasia type 2; NF1, neurofibromatosis type 1; SDHD and SDHB, succinate dehydrogenase subunits D and B; TRAP, telomeric repeat amplification protocol; VHL, von Hippel-Lindau.

Received July 12, 2002.

Accepted August 22, 2002.

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