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Available online at www.sciencedirect.com

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Diagnostic and molecular aspects of adrenal cortical tumors Anne Marie McNicol, BSc, MBChB, MD, MRCP(UK), FRCPGlas, FRCPathn Molecular and Cellular Pathology, The University of Queensland, UQ Centre for Clinical Research, Royal Brisbane & Women's Hospital, Herston, Queensland 4029, Australia

AR T IC LE INFO

AB STR A C T Adrenal cortical diseases are relatively rare but tumors are the most common in diagnostic

Keywords:

practice. This is reflected by the content of this review. By studying familial syndromes in

Adrenal cortex

which they occur more frequently and the pathways involved in steroidogenesis and

Adenoma

cortical growth, the molecular genetics of these tumors is being unraveled. Genome-wide

Carcinoma

approaches have also been helpful. The emerging data may complement standard

Molecular

histological investigation in diagnosis and prognosis. & 2013 Published by Elsevier Inc.

Introduction The normal adult human adrenal gland weighs 4 g at surgical excision or in cases of sudden death. At hospital autopsy, the average is 6 g, reflecting the hypertrophy induced by adrenocorticotrophin (ACTH) in the stress of terminal illness. The adrenal gland comprises the outer cortex and inner medulla and is divided along the long axis into head, body, and tail with the alae as medial and lateral extensions. The medulla comprises about 10% of the total and is present in the head and body and focally in the alae.1 Where present, it lies centrally, although the border between it and the cortex is irregular. A cuff of cortex invaginates around the central vein. The cortex plays a pivotal homeostatic role, producing glucocorticoids, mineralocorticoids, and sex steroids and is made up of three zones with different histological features (Fig. 1). The zona glomerulosa (ZG) comprises small angular cells dispersed focally below the capsule. It produces aldosterone. The zona fasciculata (ZF) comprises large lipid-laden cells arranged in columns from the ZG or capsule to the inner zona reticularis (ZR). It is thought to be the main source of glucocorticoids (cortisol in the human gland). The cells of the ZR are eosinophilic and arranged in cords around vascular sinusoids. This zone is also able to produce cortisol and is the source of adrenal androgens.2 The cortex arises from the adrenogonadal primordium that develops from the n

Corresponding author. E-mail address: [email protected] (A.M. McNicol).

0740-2570/$ - see front matter & 2013 Published by Elsevier Inc. http://dx.doi.org/10.1053/j.semdp.2013.07.001

urogenital ridge.3 The Wilm's tumor gene (WT-1) and the WNT4 gene play early roles in development. Other important regulators include the transcription factor steroidogenic factor-1 (SF-1) and the nuclear receptor, dosagesensitive sex reversal, adrenal hypoplasia critical region gene 1 (DAX-1).4 Cholesterol is the precursor for all steroids and the enzymes involved in the process include members of the cytochrome P-450 family and 3-β-hydroxysteroid dehydrogenase (Fig. 2). Initially, cholesterol is transported to the inner mitochondrial membrane by steroid acute response (StAR) protein.5 The production of aldosterone is mainly under the control of the renin–angiotensin system, although potassium, ACTH, catecholamines, and prostaglandins also play a role; recent evidence suggests that endothelial cell-derived factors and adipokines are involved.6 Aldosterone is specifically produced by the ZG is because the enzyme aldosterone synthase is expressed only in that zone. The secretion of cortisol is controlled mainly by ACTH acting through the ACTH membrane receptor, a G protein-linked receptor, and cyclic AMP (cAMP). Cyclic AMP binds to the regulatory subunits of protein kinase A (PKA), influencing gene transcription.7 Other factors that may be involved include insulin-like growth factors (IGF-1 and IGF-2),8 adrenomedullin,9 and transforming growth factor β. Activin A may have an inhibitory role10 and catecholamines may also be involved 11,12

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Fig. 1 – Normal adult human adrenal cortex: ZG—zona glomerulosa; ZF—zona fasciculata; ZR—zona reticularis. The ZG is dispersed focally in the subcapsular area. The ZF comprises columns of lipid-laden cells. The ZR comprises eosinophilic cells arranged around vascular sinusoids.

Clinical and pathological features of adrenal cortical disease Diseases of the adrenal gland can present with signs and symptoms of excess or reduced hormone secretion or with evidence of an adrenal mass or metastases from a carcinoma. Three classical syndromes are associated with hypersecretion of steroids. These include primary hyperaldosteronism (including Conn's syndrome), hypercortisolism (Cushing's syndrome), and hypersecretion of sex steroids (adrenogenital syndrome, virilization, or feminization). All may be associated with tumors or hyperplasia. Primary hyperaldosteronism: This accounts for 5–13% of cases of hypertension, mostly sporadic.13 About one-third are associated with an adrenal adenoma and a further 60% with bilateral idiopathic hyperplasia (idiopathic hyperaldosteronism, IHA). Unilateral hyperplasia accounts for 2% of cases.14 Cholesterol SCC

17α α

Pregnenolone

17α

17hydroxypregnenolone

Dehydroepiandrosterone

17-hydroxyprogesterone

Androstenedione

3βHSD

Progesterone 21

11-deoxycorcosterone

11-deoxycorsol

11β

Corcosterone

Corsol

Aldo

Aldosterone

Fig. 2 – Adrenal cortical steroidogenic pathway: SCC— cholesterol side chain cleavage enzyme; 3βHSD—3βhydroxysteroid dehydrogenase; 17α—17α-hydroxylase; 21— 21-hydroxylase; 11β—11β- hydroxylase; aldo—aldosterone synthase.

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Carcinomas are rare. A subset of patients have combined aldosterone- and cortisol-producing tumors.15 Where a tumor is present, the ZG may be normal or hyperplastic. It is unclear whether this hyperplasia is related to the pathogenesis of the disease or is the result of treatment. Cushing's syndrome: The clinical features of this syndrome are well recognized and include centripetal obesity, moon face, hypertension, striae, osteoporosis, and psychiatric symptoms. Two-thirds of cases are the result of hypersecretion of ACTH by the anterior pituitary gland, with 80–90% of these patients having a corticotroph adenoma. Most have bilateral diffuse cortical hyperplasia and the glands usually weigh 6–12 g, with broadening of the ZF and ZR and a relative prominence of ZR. Microscopic nodules are not uncommon, especially in the outer ZF. Of the patients, 10–20% have bilateral nodular hyperplasia, with nodules visible to the naked eye (Fig. 3) and the weights are higher and may be markedly different between the two glands. The intervening cortex is also hyperplastic. The relationship between the two is unclear, but they may be a continuum, with nodules developing in longer-standing disease. Ectopic ACTH syndrome underlies about 15% of cases, with ACTH and related peptides secreted by extra-pituitary tumors. Around half are associated with small cell lung carcinoma or bronchial carcinoid. Other tumors causing the syndrome include thymic carcinoids, neuroendocrine tumors of pancreas, medullary carcinoma of thyroid, and pheochromocytoma. The adrenal glands usually show bilateral symmetrical hyperplasia, weighing an average of 15 g each. Nodules are rare. Compact cells extend out close to the capsule and pleomorphism and mitotic figures are not infrequent. In adults, 15–20% have an adrenal tumor, equally split between benign and malignant. Females are more commonly affected. In children, more than half the cases are associated with tumor, the majority carcinoma. Because of the increased negative feedback to the pituitary, ACTH secretion is suppressed and the adjacent and opposite ZF and ZR are atrophic. Because of this, the ZG may appear more prominent. ACTH-independent macronodular adrenal hyperplasia (AIMAH) is a rare variant of the syndrome.16,17 The adrenal glands are often distorted and markedly enlarged. ACTH levels are suppressed. In many of these cases, the adrenal expresses inappropriate receptors and responds to ligands such as gastric inhibitory polypeptide (GIP), where the increase in cortisol secretion is related to food intake.18 Other receptors identified include those for vasopressin, luteinizing hormone,19 and serotonin.20 In the rare inherited Carney complex, the syndrome is associated with primary pigmented nodular adrenocortical disease (PPNAD).21,22 Both adrenal glands consist of multiple dark brown to black nodules, usually 1–3 mm in diameter. The combined weights range from 4 g to 21 g. The intervening cortex appears suppressed. Many of these cases are linked to the 17q22–q24 region. Heterozygous inactivating mutations of the protein kinase A regulatory subunit 1A gene (PRKAR1A) were reported initially in 45–65% of index cases of Carney complex, and may be present in about 80% of the families presenting with Cushing syndrome.23 The protein product is important in the cAMP pathway.

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carcinoma may also present with distant metastasis, abdominal or loin pain, abdominal fullness, or more general features of malignancy such as weight loss.

Adrenal cortical adenoma

Fig. 3 – Adrenal gland from a patient with Cushing's disease (pituitary-dependent Cushing's syndrome) showing diffuse and nodular hyperplasia.

Virilization and feminization: Excess production of sex steroids causes virilization, feminization, or precocious puberty, depending on the age and sex of the patient and the nature of the steroids secreted. Congenital adrenal hyperplasia is a group of autosomal recessive diseases associated with inherited defects in steroidogenesis.24–29 Most present early in life. The lack of negative feedback to the pituitary by cortisol causes an increase in ACTH secretion and marked adrenal cortical hyperplasia. The clinical presentation depends on the enzyme involved and is related to the range of steroids produced. Adrenal cortical tumors can also produce sex steroids, usually androgens. In adults, this is more common in association with carcinoma.

General aspects of adrenal cortical tumors In the past, benign adrenal cortical tumors usually presented only if they secreted excess steroids and produced a clinical syndrome. However, adrenal cortical nodules can be found in up to 53% of adrenal glands at autopsy.30,31 They are often multiple and are usually regarded as hyperplastic. Single nodules are regarded as adrenal cortical adenomas (ACA) and are present in 5% of autopsies.30 Adrenal lesions are now often identified by imaging when the abdomen is screened for the investigation of other disease—“incidentalomas.”32 They are more common with increasing age, seen in 7% of those over 70 years.33 Most of these are nonfunctional adrenal cortical adenomas, but some may be associated with sub-clinical Cushing's syndrome or primary hyperaldosteronism.34 Both adenomas and hyperplastic nodules can show marked degenerative change, with fibrosis, myxoid change, and sometimes the presence of angiomatoid blood vessels and hemorrhage.35 Functional incidentalomas are usually removed, but there is still debate as to what to do with non-functional lesions.36 Because malignant tumors tend to be larger than benign tumors, larger lesions may be removed but the threshold for surgery may vary. Smaller lesions can be rescanned at intervals to assess whether they are growing. Other lesions identified in this way include pheochromocytoma and metastases. Adrenal cortical

Single nodules are usually regarded as adenomas, and clonal analysis shows that most are monoclonal, supporting this approach.37–39 They are more common in women. Double adenomas have been reported occasionally.40,41 Adenomas often weigh less than 50 g but may be large.42 They are intra-adrenal and may be unencapsulated, have a pseudocapsule, or a true capsule. Histologically they comprise predominantly lipid-laden cells resembling ZF, arranged in an alveolar pattern interspersed with short cords. Focal groups of compact cells may be seen. Mitotic activity is rare. In patients with Conn's syndrome, there may be cells resembling ZG and others showing mixed features of ZG and ZF—‘hybrid’ cells (Fig. 4). When spironolactone has been administered, small whorled intracytoplasmic inclusions (spironolactone bodies) may be present in the tumor cells and in the ZG and outer ZF. Compact cells often predominate in tumors associated with virilization. This may cause problems in assessing malignant potential as outlined below.

Adrenal cortical carcinoma This is a rare but aggressive tumor, with a prevalence of between 0.5 and 2 per million.43–45 It accounts for up to 2% of all malignancies.46–48 Surgery is still the mainstay of treatment. The outcome remains poor 49,50 but is better if the patient is treated at a specialized center.51 Over half show local invasion or metastases at the time of presentation and this is associated with poorer survival; one study reporting median survival of 15 months compared to 101 months for intra-adrenal tumors.52 Functioning tumors account for between 24% and 74% of cases.42,53,54 Cushing's syndrome is most common, sometimes accompanied by virilization. Conn's syndrome and feminization are rare.

Fig. 4 – Adrenal adenoma causing Conn's syndrome (Original magnification  400). In the upper left of the figure, the cells resemble zona fasciculata. In the lower part, there are cells resembling zona glomerulosa and hybrid cells, having features of both.

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Most weigh more than 100 g although some would use 50 g as the threshold for suspicion of malignancy.55 They usually range in size from 3 cm to 40 cm. Some are encapsulated, but many are adherent to, or invade, fat or adjacent organs. On slicing, they often show lobulation with fibrous bands and areas of necrosis and hemorrhage (Fig. 5). Where metastases are present, these are commonly found in liver, lung, retroperitoneum, lymph nodes, and bone.54,56 Histologic examination shows a less ordered structure than in adenomas. Trabecular and diffuse patterns are common. Some may show focal or widespread myxoid change.57 Nuclear pleomorphism is seen (Fig. 6) and mitoses, including atypical forms, are often present. There may be confluent necrosis. Capsular and vascular invasion can often be identified along with infiltration of local tissues. Malignancy is defined both by local invasion and metastasis.

Histologic diagnosis of malignancy While the diagnosis of malignancy is easy in most cases, all intra-adrenal tumors must be assessed histologically for malignant potential. Tumors should be widely sampled and any suspicious areas processed. A number of multifactorial systems have been developed55,58–60 but the most commonly used is that by Weiss.59–61 Nine histological features are assessed (Table 1) and the presence of three or more indicates malignant potential. An alternative is the modified Weiss index (Table 1).62 This omits histological features that had poor inter-observer correlation in a validation study and incorporates the others into a weighted score. This system has performed well in diagnosing malignancy when compared to van Slooten and Weiss.63 In a few cases, the different systems may give different diagnoses, and occasionally a diagnosis of indeterminate or borderline tumor may have to be made.

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Fig. 6 – Adrenal cortical carcinoma showing significant nuclear pleomorphism. (Original magnification  400.)

A different approach has recently been proposed, starting from an analysis of the sensitivity and specificity of each of the individual Weiss features in diagnosing malignancy.64 Based on the observation that diffuse architecture was seen in 76% of carcinomas compared to 9% of adenomas, the group examined the reticulin network histologically. They found that disruption was 100% sensitive and 96% specific for identifying tumors with a Weiss score ≥3. The additional presence of one of the following features—mitotic rate 45 per 50 high-power fields (HPF), necrosis, and venous invasion—gave 100% sensitivity and specificity. Proliferative activity, as defined by the Ki-67 index, is usually higher in carcinomas than in adenomas, but the threshold has varied in many of the studies.61 We have found in our own practice that an index greater than 5% is seen only in carcinoma.65–67 However, a low index does not define benign behavior. Immunopositivity for p53 is seen in about 50% of carcinomas and rarely in adenomas 65,68 and carcinomas overexpress IGF-2.69,70 There are two main areas of difficulty in applying the Weiss criteria, pediatric tumors and oncocytic tumors. Adrenal tumors in children often show features associated with malignancy in adults such as pleomorphism, proliferation, and necrosis, and yet behave in a benign fashion.71 These will not be dealt with further. Oncocytic tumors resemble those at other sites and are composed of large eosinophilic cells with accumulation of mitochondria. Originally reported as non-functioning and benign,72 functional73 and malignant74,75 variants have now been described. The problem is that most have less than 25% clear cells, pleomorphic nuclei, and diffuse architecture, a combination giving a Weiss score of 3, and thus a malignant diagnosis. A modification of the Weiss approach has been suggested (Table 2).76 Three major criteria and four minor criteria have been proposed and tumors are defined as malignant, of uncertain malignant potential or benign, based on the presence or absence of these. This approach has been validated in a recent study that also suggested that oncocytic carcinomas have a better outcome than the usual type.77

Prognostic features of carcinoma Fig. 5 – Adrenal cortical carcinoma showing areas of necrosis and hemorrhage. The normal adrenal gland is seen to the right.

Both the van Slooten and Weiss scores are reported to correlate with metastatic behavior and with cancer specific

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Table 1 – Histological criteria for the assessment of malignant potential in adrenal cortical tumors. Weiss system59,60 √ √ √ √ √ √ √ √ √

High nuclear grade (Fuhrman grade 3/4) Mitotic rate 45/50 HPF Atypical mitoses Clear cells comprising o25% of the tumor Diffuse architecture (4one-third of the tumor) Necrosis Venous invasion Sinusoidal invasion Capsular invasion

Modified Weiss system62

√  2 √ √  2 √

√

HPF—high-power fields (  40). The features to be assessed in each analysis are marked with a tick. Each is given a score of 1 if present and 0 if absent. In the modified system, nuclear grade, diffuse architecture, and venous and sinusoidal invasion are not assessed. The scores for mitotic rate and clear cell component are doubled. The total score is calculated and a score of 3 or more in either system indicates malignant potential.

survival in metastasizing tumors.63 A Weiss score of ≥6 was associated with poorer disease-free and overall survival.78 Patients with a Ki-67 index of 43%66 or 47%78 had a lower disease-free survival and the Ki-67 index had an inverse correlation with survival.79 A mitotic rate of 420 per 50 HPF has been associated with lower median survival.60 This has been used in the grading of carcinomas, tumors with ≤20 mitoses in 50 HPF defined as low grade and those with 420 as high grade.60,80 Large vessel extension, as defined by invasion of the wall or intraluminal extension into the inferior vena cava or renal vein, has been linked to a poorer prognosis.81 The most important factor influencing the prognosis is tumor stage. The International Union against Cancer (UICC) currently has a TNM classification and staging system82 (Tables 3 and 4). A recent study by the European Network for the Study of Adrenal Tumors (ENSAT) on 416 cases has suggested a modification that results in better indication of prognostic risk83 based on analysis of risk factors in Stage III patients. This led to redefining T3 to include tumor associated with venous tumor thrombus in the vena cava or renal vein and reassigning particular criteria to different stages (Tables 3 and 4). This system has been validated as superior to the UICC staging in predicting outcome in a second set of 573 cases from North America.84 The addition of tumor grade to ENSAT staging reportedly gives improved stratification of outcome.85 In a separate study, tumor stage and mitotic activity defined three prognostic groups in the carcinomas.64

These approaches require further testing, but may lead to modification of staging in future.

Immunohistochemistry Occasionally, histological features are ambiguous and a confident diagnosis of a cortical tumor is not possible on routine hematoxylin and eosin staining. Immunohistochemistry is useful in this situation. General neuroendocrine markers (e.g. synaptophysin) are not helpful in distinguishing them from pheochromocytoma, as they may be positive in adrenal cortical tumors.86 However, chromogranin is negative in cortical tumors and almost always positive in pheochromocytoma. Conversely, the majority of cortical tumors will be positive for inhibin-α and/or with Melan A (clone A103)87–89; only a very small minority of pheochromocytomas and paragangliomas also express these markers. Cortical tumors rarely stain strongly for cytokeratin, particularly in the absence of antigen retrieval. A recent publication has reported that tumors with widespread myxoid features express neurofilament,57 although the significance is unclear.

Molecular aspects of adrenal cortical tumors There are a number of comprehensive reviews on this topic.80,90–95 Adrenal cortical tumors occur more frequently

Table 2 – Assessment of malignant potential in oncocytic adrenal cortical tumors.76,77 Major criteria

Minor criteria

Mitoses 45/50 HPF Atypical mitoses Venous invasion

Large size (410 cm/4200 g) Necrosis Capsular invasion Sinusoidal invasion Interpretation Malignant Oncocytic adrenocortical carcinoma Borderline Oncocytic adrenocortical neoplasm of uncertain malignant potential Benign Oncocytoma

Any major criterion Any minor criterion Absence of major and minor criteria HPF—high-power fields (  40).

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Table 3 – Comparison of criteria for UICC/TNM7145 and ENSAT83 staging for adrenal cortical carcinoma. Criteria

UICC/TNM7

ENSAT

T1 T2 T3

Tumor ≤5 cm, no invasion Tumor 45 cm, no invasion Tumor any size, locally invasive but not involving adjacent organs Tumor any size, invading adjacent organs or with distant metastases Negative regional nodes Positive regional nodes No distant metastases Distant metastases

Same as TNM7 Same as TNM7 Tumor infiltration into surrounding tissues

T4 N0 N1 M0 M1

in a number of familial syndromes and this has guided research on sporadic tumors. These include Beckwith–Wiedemann syndrome, linked to the 11p15 locus with paternal disomy and overexpression of IGF-296; Li–Fraumeni syndrome, associated with germline mutations in the TP53 tumor suppressor gene on 17q13.197; familial adenomatous polyposis98,99; and multiple endocrine neoplasia type 1 syndrome (MEN1) with mutations in the MEN1 gene on 11q13.100,101 Comparative genomic hybridization (CGH) and interphase cytogenetic studies have demonstrated widespread chromosomal changes in carcinomas and fewer in adenomas,102–104 with increasing numbers of changes in larger tumors.102 Poorer outcome in carcinoma has been linked to an increasing number of chromosomal changes.105 Further investigations have shown loss of heterozygosity (LOH) at 2p16 (92%), 11q13 (≥90%), and 17p13 (≥85%) in carcinomas.106 LOH at 18p11.2, the locus for the ACTH-receptor gene has been reported in two of four carcinomas, but not in adenomas.107 LOH at 17p13 and at 11p15 has been reported to correlate with tumor recurrence after complete surgical removal.103 Despite the LOH at 17p13, somatic mutations of TP53 are found in only 25–35% of sporadic adult carcinomas,68,108–110 suggesting another tumor suppressor gene (TSG) at this locus. Germline mutations of TP53 are more common in pediatric carcinomas than in adults,111,112 with a particular mutation (R337H) accounting for the very high incidence in Southern Brazil.113 About 25–40% of patients with MEN1 develop adrenal disease, usually non-functional adenomas or hyperplasia.106,114 Carcinomas are rare. In sporadic carcinomas, although LOH is common at 11q13, somatic mutations in MEN1 gene are rare,106,114 again suggesting another TSG.

Tumor invasion into adjacent organs or venous tumor thrombus in vena cava or renal vein Same as TNM7 Same as TNM7 Same as TNM7 Same as TNM7

The most common abnormality in carcinoma is overexpression of IGF-2, reported in about 90% of cases.115–117 The IGF-2 gene is located at 11p15 where there are complex interactions of a number of genes.118–120 It is maternally imprinted and expressed from the paternal gene. In adrenal carcinomas, there is usually loss of the maternal allele and duplication of the paternal allele. The Wnt/β-catenin pathway is involved in a range of pathologies including PPNAD, hyperplasias, adenoma, and carcinomas.121–125 Cytoplasmic and nuclear staining were described in 11 of 13 cases of carcinoma125 and activating mutations of β-catenin gene (CTNNB1) in 27% of adenomas and 31% of carcinomas.124,125 This pathway appears to interact with cAMP, and this is of interest because of the roles of these pathways in adrenal development and steroidogenesis. Activating mutations of the ACTH-receptor have not been identified126,127 and mutations in G proteins are rare.128,129 Gene profiling studies on adrenal cortical tumors are in agreement that there are differences between benign and malignant lesions80,130–136 although the molecular signatures described differ, except for the overexpression of IGF-2 in carcinoma. Interestingly, there are now ongoing clinical trials targeting the IGF-1 receptor through which IGF-2 signals.137 Nevertheless, they all demonstrate that genes involved in cell proliferation are dysregulated in carcinoma. In addition, all provide evidence that, based on hierarchical clustering, there are at least two groups of carcinomas with differences in survival. One study indicates that a 10-gene panel gives prognostic information additional to that associated with grade and this is now used in clinical practice.80 There have now been a number of studies of microRNA (miR) expression in a range of adrenal cortical diseases,138–144 but the numbers of cases are still small, so the significance of the findings requires further validation. However, differences

Table 4 – Comparison of UICC/TNM7145 and ENSAT83 staging for adrenal cortical carcinoma.

Stage 1 Stage 2 Stage 3

UICC/TNM7

ENSAT

pT1 N0 M0 pT2 N0 M0 pT1–2 N1 M0

Same as TNM7 Same as TNM7 Tumor with any one of following: Involved lymph nodes Extra-adrenal tissue infiltration Venous tumor thrombus in renal vein or inferior vena cava Any tumor with distant metastases

pT3 N0 M0 Stage 4

pT3 N1 M0 pT4 N0 M0 pT1–4 N0–1 M1

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have been identified between normal adrenals and hyperplasias142,144; also between normal and benign and malignant tumors. Upregulation of Mir483-5p has been associated with malignancy in two studies.139,143

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Conclusion The diagnosis and prognosis of adrenal cortical carcinoma has been largely dependent on morphologic analysis alone. Treatment options have been limited in what is an aggressive tumor. Our understanding of the molecular changes underlying tumorigenesis and the signaling pathways involved will permit the development of targeted therapies. More refined morphologic analysis coupled with molecular information will also provide better prognostic information in these cases.

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re fe r en ces 22. 1. Dobbie JW, Symington T. The human adrenal gland with special reference to the vasculature. J Endocrinol. 1966;34:479–489. 2. Nguyen AD, Conley AJ. Adrenal androgens in humans and nonhuman primates: production, zonation and regulation. Endocr Dev. 2008;13:33–54. 3. Keegan CE, Hammer GD. Recent insights into organogenesis of the adrenal cortex. Trends Endocrinol Metab. 2002;13: 200–208. 4. Beuschlein F, Keegan CE, Bavers DL, et al. SF-1, DAX-1, and acd: molecular determinants of adrenocortical growth and steroidogenesis. Endocr Res. 2002;28:597–607. 5. Miller WL. Steroidogenic acute regulatory protein (StAR), a novel mitochondrial cholesterol transporter. Biochim Biophys Acta. 2007;1771:663–676. 6. Willenberg HS, Schinner S, Ansurudeen I. New mechanisms to control aldosterone synthesis. Horm Metab Res. 2008;40: 435–441. 7. Soon PS, McDonald KL, Robinson BG, et al. Molecular markers and the pathogenesis of adrenocortical cancer. Oncologist. 2008;13:548–561. 8. l'Allemand D, Penhoat A, Blum W, et al. Is there a local IGFsystem in human adrenocortical cells? Mol Cell Endocrinol. 1998;140:169–173. 9. Albertin G, Forneris M, Aragona F, et al. Expression of adrenomedullin and its receptors in the human adrenal cortex and aldosteronomas. Int J Mol Med. 2001;8:423–426. 10. Vanttinen T, Liu J, Kuulasmaa T, et al. Expression of activin/ inhibin signaling components in the human adrenal gland and the effects of activins and inhibins on adrenocortical steroidogenesis and apoptosis. J Endocrinol. 2003;178:479–489. 11. Haidan A, Bornstein SR, Glasow A, et al. Basal steroidogenic activity of adrenocortical cells is increased 10- fold by coculture with chromaffin cells. Endocrinology. 1998;139: 772–780. 12. Haidan A, Bornstein SR, Liu Z, et al. Expression of adrenocortical steroidogenic acute regulatory (StAR) protein is influenced by chromaffin cells. Mol Cell Endocrinol. 2000;165: 25–32. 13. Rossi GP. Prevalence and diagnosis of primary aldosteronism. Curr Hypertens Rep. 2010;12:342–348. 14. Young WF. Primary aldosteronism: renaissance of a syndrome. Clin Endocrinol (Oxf). 2007;66:607–618. 15. Willenberg HS, Spath M, Maser-Gluth C, et al. Sporadic solitary aldosterone- and cortisol-co-secreting adenomas:

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