CLINICAL AND MOLECULAR ASPECTS OF ADRENOCORTICAL TUMOURIGENESIS
STAN SIDHU,*#1 CHRISTINE GICQUEL,# CHRISTOPHER P. BAMBACH,* PETER CAMPBELL, **** CHRISTOPHER MAGAREY, ** BRUCE G. ROBINSON### AND LEIGH W. DELBRIDGE*1
*University of Sydney Endocrine Surgical Unit and +Department of Endocrinology, Royal North Shore Hospital, Cancer Genetics, Kolling Institute of Medical Research, Royal North Shore Hospital, $Department of Medicine and “Department of Surgery, University of Sydney, ** Endocrine Surgical Unit, St George Hospital, ** Endocrine Surgical Unit, Liverpool Hospital, Sydney, Australia, Laboratoire d’Explorations Fonctionnelles Endocriniennes, Hôpital Trousseau, Paris, France
Adrenal masses are a common problem affecting 3-7% of the population. The majority turn out to be benign adrenocortical adeno- mas, which may be functional or non-functional. Much more rarely, these masses represent a primary adrenal carcinoma. It is becoming increasingly recognized that of the benign functioning adenomas or hyperplasias, the majority will hypersecrete aldoster- one and this will be more frequently detected when hypertensive populations are screened for this disease. In contrast, the incidence of primary adrenocortical carcinoma has remained steady and for this disease, surgery represents the mainstay of treatment. The advent of laparoscopic adrenal surgery has lowered the threshold size for recommending surgery for asymptomatic adrenal masses and as such, an increased proportion of adrenocortical cancers are being resected and detected at an earlier stage. Recent progress has been made in our understanding of the key genetic changes which underpin the biology of this disease. Progression from adrenal adenoma to carcinoma involves a monoclonal proliferation of cells which, among other defects, have undergone chromo- somal duplication at the 11p15.5 locus leading to overexpression of the IGF2 gene and abrogation of expression of the CDKN1C and H19 genes. TP53 is involved in progression to carcinoma in a subset of patients and the frequency of ACTH receptor deletion needs to be more fully explored. Other key oncogenes and tumour suppressor genes remain to be identified although the chromo- somal loci in which they lie can be identified at 17p, 1p, 2p16 and 11q13 for tumour suppressor genes and chromosomes 4, 5 and 12 for oncogenes.
Key words: adrenal cortex, adrenal cortical adenoma, adrenal cortical carcinoma, adrenal gland neoplasm, adrenal glands. Abbreviations: ACC, adrenocortical cancer; ACT, adrenocortical tumours; ACTH-R, ACTH receptor; BWS, Beckwith- Weidemann Syndrome; CC, Carney’s complex; CDK, cyclin dependent kinases; CGH, comparative genomic hybridization; CT, computed tomography; DHEAS, dehydroepiandrostenedione sulphate levels; GTP, guanine triphosphate; IGFBP, IGF- binding proteins; LFS, Li-Fraumeni Syndrome; LOH, loss of heterozygosity; MEN1, multiple endocrine neoplasia type 1; MRI, magnetic resonance imaging; PA, primary hyperaldosteronism; PAC, plasma aldosterone concentration; PRA, plasma renin activity.
EPIDEMIOLOGY OF ADRENOCORTICAL TUMOURS
Adrenal masses are a common problem affecting 3-7% of the population. Adrenocortical tumours (ACT) represent more than 80% of all adrenal masses. They may be benign or malignant, functional or non-functional. Depending on the series examined, adrenal masses are documented to exist in 3-7% of the popula- tion.1 The differential diagnosis of the adrenal mass detected during abdominal imaging for another reason, and in the absence of a previously diagnosed primary tumour, is shown in Table 1.2 The most common non-functional pathology is the adrenocortical
adenoma, followed by an adrenocortical cancer, phaeochromo- cytoma and a range of other benign pathologies. These may include gastrointesinal stromal tumours, schwannomas, fimbrial cysts and myelolipomas.
Hormonally active tumours are rarer. Primary hyperaldos- teronism (PA) is the most common and in 80% of instances is caused by an aldosterone producing adenoma. The remaining cases are attributable to bilateral (idiopathic PA) or more rarely unilateral hyperplasia (primary adrenal hyperplasia).3 PA was first described in 1955.4 Prevalence rates for PA have varied from 0.05% to 2% of the hypertensive population; however, in recent years prevalence estimates for this disease, which affects men and women equally in the 20-60 years age group, have been made at between 8% and 15% of the hypertensive population.5,6
In contrast to the several-fold increase in the number of diag- nosed cases of PA,3 the incidence of hypercortisolism has remained static at approximately one case per 100 000-500 000 people. Seventy per cent of cases are caused by a corticotropin secreting pituitary tumour (Cushing’s disease), 10% by an ectopic tumour secreting corticotropin and 20% by adrenal lesions. Most commonly this is an adrenal adenoma; however, adrenal hypercortisolism also arises from adrenocortical carci- noma, and bilateral macro or micronodular hyperplasia.7
S. Sidhu PhD, FRACS; C. Gicquel MD, PhD; C. P. Bambach MD, FRACS; P. Campbell FRACS; C. Magarey MS, FRACS; B. G. Robinson MD, FRACP; L. W. Delbridge MD, FRACS.
Correspondence: Dr S. Sidhu, Department of Surgery, Royal North Shore Hospital, St Leonards, New South Wales 2065, Australia.
Email: sidhu@med.usyd.edu.au
Adrenocortical cancer (ACC) is a rare disease. Incidence figures from the New South Wales Cancer Council are not avail- able.8 Data from Swedish and American Registries estimate the incidence at between 0.5 and 2 cases per 1 million population per year.9,10 There is a bimodal age distribution with a peak incidence in children less than five years of age and in adults in the fourth to fifth decades of life.11 There is a definite female predominance and the aetiology of the disease is largely unknown with some suggestion of a causal link between ACC and cigarette smoking in men and the use of the contraceptive pill in women.12
CLINICAL FEATURES OF ADRENOCORTICAL TUMOURS
Adrenocortical tumours may be sporadic or occur as part of a hereditary tumour syndrome. The clinical and molecular charac- teristics of hereditary tumour syndromes associated with ACT formation are shown in Table 2.
Familial syndromes
Li-Fraumeni Syndrome (LFS, OMIM 151623)
First described in 1969, this syndrome is characterized by suscep- tibility to breast carcinoma, soft tissue sarcoma, brain tumours, oesteosarcoma, leukaemia and ACC.13 Possible component tumours are melanoma, gonadal germ cell tumours and carcinoma of the lung, pancreas and prostrate. These diverse tumours affect individuals at a young age and multiple primary tumours are seen in affected families. The underlying genetic abnormality is a germline mutation of TP53 located at 17p13 which is transmitted
| Histology | n (%) | Median size (cm) |
|---|---|---|
| Cortical adenoma | 166 (53) | 3.5 |
| Cortical carcinoma | 39 (12) | 7.5 |
| Phaeochromocytoma | 32 (10) | 5.0 |
| Myelolipoma | 26 (8) | 5.0 |
| Cystic lesions | 18 (6) | 4.2 |
| Tumours of neuronal lineage | 11 (3.5) | 6.0 |
| Metastases | 8 (2.5) | 6.5 |
| Others | 16 (5) | 4.7 |
in an autosomal dominant fashion.14 Recently, reports of germline TP53 mutations especially at codons 152, 158 and 337 have been described in children with apparently sporadic ACC without a classical history of LFS or Li-Fraumeni-like syndrome.15,16
Beckwith-Weidemann Syndrome (BWS, OMIM 130650)
This overgrowth disorder is characterized by macrosomia, macroglossia, organomegaly and developmental abnormalities (in particular abdominal wall defects with exomphalos). It pre- disposes to the development of embryonal tumours such as Wilms tumour, ACC, neuroblastoma and hepatoblastoma.17 Its incidence is estimated at 1 per 13 700 livebirths. BWS is related to genetic or epigenetic changes in the imprinted 11p15 region resulting in an increased level of IGF2.17
The 11p15 region is organized into two different clusters: a telomeric domain (BWSIC1) including the IGF2 and H19 genes and a centromeric domain (BWSIC2) including the CDKN1C (p57kip2), KCNQ1 and KCNQ1OT1 genes. The molecular abnor- malities involving the 11p15 region are genetic (11p15 paternal uniparental disomy or mutation in the CDKN1C gene) or more often epigenetic (isolated hypermethylation of the H19 gene or demethylation of the KCNQ1OT1 (LIT1), an antisense transcript within intron 10 of the KCNQ1 gene which is normally expressed from the paternal allele).
Carney’s Complex (CC, OMIM 160980 and 605255)
First described in 1985,18 this rare familial syndrome is character- ized by myxomatous masses (cardiac and cutaneous myxomas), spotty pigmented lesions of the skin and endocrine disorders including primary pigmented nodular adrenocortical hyperplasia producing hypercortisolism, pituitary adenomas producing growth hormone and various testicular tumours. CC is inherited as an autosomal dominant condition and the genes responsible have been mapped to 2p1619 and 17q22-24.20 Mutation analysis of the PRKAR1A gene (encoding a protein kinase A regulatory subunit 1-a) lying within the 17q 22-24 region, has shown germ- line mutations within a subset of patients with both sporadic and familial forms of this disease. It is believed that the PRKAR1A gene functions as a tumour suppressor.
Multiple Endocrine Neoplasia Type 1 (MEN1, OMIM 13100) MEN1 is transmitted in an autosomal dominant fashion with a high penetrance in an equal sex distribution. The principal clini- cal features are tumours of the parathyroid glands, anterior pitui- tary and endocrine pancreas with thymic carcinoids, thyroid adenomas, adrenocortical adenomas and adrenocortical cancers occurring with less frequency.21 MEN1 was mapped to 11q13 by linkage analysis and the gene responsible for the condition was
| Familial syndrome | Clinical features | Adrenal tumour | Chromosomal location | Gene |
|---|---|---|---|---|
| Li-Fraumeni | Breast carcinoma, soft tissue sarcoma, brain tumours, osteosarcoma, leukaemia | Adrenocortical carcinoma | 17p13 | TP53 |
| Beckwith-Weideman | Overgrowth syndromes, Wilms tumours, neuroblastoma, hepatoblastoma | Adrenocortical carcinoma | 11p15.5 | Unknown |
| Carney's complex | Cardiac and cutaneous myxomas, testicular tumours, pituitary tumours | Primary pigmented nodular hyperplasia | 17q22-24, 2p16 | PRKAR1A |
| Multiple endocrine neoplasia type 1 | Parathyroid, pituitary and pancreatic neuroendocrine tumours | Adrenocortical adenomas and carcinomas | 11q13 | MEN1 |
cloned in 1997.22 It encodes a 610 amino nuclear protein whose function is largely unknown.
Sporadic adrenocortical tumours
Primary aldosteronism
Patients with PA are usually asymptomatic and when they do manifest symptoms these are usually non-specific. The excep- tions to this rule are patients of Asian descent in whom PA presents with significant paresis and neuromuscular symptoms.23 When symptomatic, patients complain of ailments relating to hypertension (headaches), hypokalaema (polyuria, muscle cramps) or lethargy and malaise.3 Patients with an aldosteronoma tend to have more severe symptoms, hypertension and hypo- kalaema compared to patients with adrenal hyperplasia, while patients with the rare glucocorticoid remediable PA present with symptoms at a younger age. This autosomal dominant condition is caused by the formation of a chimeric 11B hydroxylase/ aldosterone synthase gene fusing the five prime regulatory region of the 11- hydroxylase to the coding region of aldosterone synthase.24
Outpatient screening for PA was first suggested by Hiramatsu et al.25 and since then has been the accepted standard for initiat- ing investigation of patients with suspected PA. Our approach to referred hypertensive patients, with or without hypokalaema, is to have a mid-morning ambulant paired plasma aldosterone concen- tration (PAC) (ng/ml) and plasma renin activity (PRA) (ng/ml per h), followed by 24-h urine collection for electrolytes, creati- nine and aldosterone. A PAC/PRA greater than or equal to 40 with or without a 24-h urinary aldosterone (mcg/24 h) above a defined nomogram range relating urinary aldosterone to concur- rent 24 h excretion of sodium in normal volunteers, suggests the diagnosis. This is then confirmed by a sodium load test and famil- ial hyperaldosteronism is excluded by measurement of plasma aldosterone during 4-days of dexamethasone administration. Abdominal computed tomography (CT) scan and adrenal venous sampling is then undertaken and if this demonstrates lateralized aldosterone secretion, the patient is referred for laparoscopic adrenalectomy. Patients who have non-lateralized secretion are commenced on spironolactone for presumed idiopathic adrenal hyperplasia.
Hypercortisolism
Patients with hypercortisolism have a classic constellation of symptoms and signs including weight gain, centrifugal obesity, moon-like facies, acne, hypertension and diabetes. Serum cortisol levels and 24 h urinary-free cortisol excretion establish the diag- nosis and subtype discrimination occurs via plasma corticotropin levels, high dose dexamethasone-suppression testing and abdom- inal imaging.26 An adrenal cause for hypercortisolism is sug- gested by a low corticotropin level (<5 ng/L) and a lesion on radiological imaging. An elevated ACTH level (>5 ng/L) sug- gests either a pituitary tumour (Cushing’s Disease) or an ectopic lesion (ACTH-secreting tumour) which are evaluated with the high dose 8 mg overnight dexamethasone suppression test. Failure of ACTH levels to suppress suggest an ectopic ACTH- secreting tumour while suppression of corticotropin levels indicates Cushing’s disease. If the results are equivocal, bilateral inferior petrosal sinus sampling with measurement of the central to peripheral gradient may be required for corticotropin- dependent forms of the disease.27
Another recognized phenomenon is the patient with the inci- dental adrenal mass, without clinical overt evidence of hypercor- tisolism, but with subtle derangements in cortisol secretion that may be associated with obesity, hypertension or diabetes. The term ‘subclinical’ Cushing’s syndrome has been coined to account for the estimated 2-15% of patients with adrenal inciden- talomas whom demonstrate loss of the diurnal rhythm for cortisol secretion and resistance to dexamethasone suppression in the presence of a normal serum cortisol and 24 h urinary free cortisol secretion.28,29
Adrenocortical cancer
Adrenocortical cancer may present in a number of different fash- ions. Depending on the series reported, up to 60% are functional and secrete cortisol, oestrogen and androgens and manifest as Cushing’s syndrome, a virilizing or feminizing syndrome, but most commonly as a Cushing-virilizing syndrome.30,31 Functional tumours are more common in younger patients than older patients and presentation with PA is rare. Aggressive, advanced disease is more commonly associated with the Cushing-virilizing form compared with the pure virilizing syndrome alone. One-third present with a painful mass without hypersecretion which may be associated with malaise, weight loss, anorexia and lethargy. Approximately 10% of patients present with an incidentaloma. Unusual primary presentations include a varicocele or fever, sec- ondary to tumour necrosis or haemorrhage into the tumour or the effects of intracaval extension such as lower limb swelling or cardiac symptoms from more proximal extension into the right atrium. Presentation with a synchronous second malignancy occurs in up to 13% of patients and most commonly these are either a breast cancer, thyroid cancer or melanoma.11,30 Bilateral tumours larger than 5 cm are more likely caused by secondary disease.
The diagnosis is established using a combination of biochemi- cal screening and radiological imaging. Workup should include a full biochemical profile, serum cortisol, dehydroepiandrostenedi- one sulphate levels (DHEAS), sex hormones and their precursors as well as a 24-h urine collection for cortisol and ketosteroids. DHEAS is a good marker for malignancy because enzyme func- tion is defective in ACC with the subsequent tendency for steroid precursors to be secreted.32
Computed tomography images for ACC typical show an inho- mogeneous, irregular mass which may or may not be invading into surrounding structures. On enhancement, ACC have much higher attenuation values (>18 Hounsfield units) than adenomas.33
The use of magnetic resonance imaging (MRI) in ACC has proven useful. Characteristically, malignant lesions enhance on T2-weighted images when inphase/opposed-phase chemical shift imaging is applied. Malignant lesions fail to show loss of signal intensity on the opposed-phase sequence compared to benign lesions, which demonstrate significant signal intensity loss.34,35 Gadolinium washout studies have also been evaluated in ACC. MRI obtained after intravenous gadolinium reveal that ACC retain enhancement and have slower gadolinium washout com- pared with adenomas. CT and MRI are not only valuable for assessing the adrenal gland, but also provide useful information for operative planning. Tumour thrombus into the IVC is not an uncommon occurrence and precise information regarding the presence and extent of IVC infiltration is valuable for planning operative strategy.
NP59 (I131-6B-iodomethylnorcholesterol) scanning has been advocated by some centres to differentiate benign from malignant
adrenal masses. The rationale for this application is that benign adrenal masses should take up isotope and enhance, whereas malignant or nonadenomatous masses will not. When used in conjunction with CT scanning, concordance between unilateral uptake, contralateral suppression and the presence of a mass indi- cates a benign functional adenoma.36
ACC staging
The most commonly used staging system for ACC was proposed by Macfarlane37 and modified by Sullivan.38 Essentially, stage 1 (<5 cm) and stage 2 (>5 cm) disease is confined to the adrenal gland and these patients have the best chance of cure. Stage 3 disease (local invasion with or without involved lymph nodes) and stage 4 disease (metastases or invasion of adjacent organs) have a characteristically poor prognosis. More recently, Icard et al.39 have suggested modifications of these staging systems whereby stage 1 and stage 2 reflect local disease, stage 3 loco- regional disease and stage 4 patients have distant metastases. This classification seems more logical because in patients with stage 3 tumours, complete resection is theoretically possible. Never- theless, 70% of patients present with either stage 3 or stage 4 dis- ease, while patients with the potentially curable stage 1 and stage 2 disease account for 30% of clinical presentations.
Incidentaloma
The evaluation of the patient with the incidental adrenal mass begins with the exclusion of a possible metastasis on historical grounds. Less than 5% of patients with secondary adrenal disease will have an occult primary. The most common primary tumours are lung, breast and colon cancer, followed by renal cell cancer, malignant melanoma, uterine or prostatic cancer. If the CT images are consistent with a myelolipoma, adrenal cyst or adrenal haemorrhage, then no further investigation is required. If there is a suspicion of malignancy then use of MRI and NP-59 as described previously enables discrimination between benign and malignant adrenal masses. Further evaluation aims to exclude hormonal function.
Biochemical screening for phaeochromocytoma should include plasma catecholamine levels and at least two sets of 24-h urine catecholamine levels to account for the episodic nature of secretion by these tumours.40 More recently, plasma levels of metanephrines and normetanephrines have been shown to have a higher sensitivity than other investigations for establishing the diagnosis of sporadic and familial phaeo- chromocytomas.41 Hypercortisolism and subclinical Cushing’s syndrome is excluded by the presence of a plasma cortisol which suppresses to less than 83 nmol/L following a single 3 mg overnight dexamethasone suppression test. A positive test requires further investigation including high dose dexametha- sone suppression, corticotropin releasing hormone test and analysis of diurnal rhythm. Patients with hypertension, with or without hypokalemia, should undergo an upright aldosterone/ plasma renin activity ratio screen to exclude PA. Less than 1% of patients with negative screening investigations will sub- sequently have a functioning adrenal tumour.42
TREATMENT AND OUTCOME Primary hyperaldosteronism
The treatment of patients with PA depends on the underlying pathology. Those patients with familial PA or bilateral adrenal
hyperplasia are best treated medically with spironolactone.3,5 Surgery is recommended for patients with unilateral disease be it an adenoma or nodular hyperplasia. The diagnostic combina- tion of CT scanning and adrenal venous sampling protects the surgeon from inadvertently removing an adrenal incidentaloma occurring in the presence of contralateral functioning adrenal hyperplasia. Traditionally, surgery for the adrenal glands was undertaken via a large incision using either the anterior, poste- rior or thoracoabdominal approach.43,44 Within the last decade, laparoscopic adrenalectomy has replaced open surgery in most units for small, benign adrenal tumours. The move to laparo- scopic adrenalectomy has occurred due to the significant advan- tages offered to patients in terms of postoperative wound pain, postoperative respiratory and wound complications, shorter hospital stay and earlier return to normal function.45-47 Patients with a small unilateral lesion and PA are therefore excellent candidates for the laparoscopic approach. Following surgery, patients can expect a complete cure in 40-60% of instances, a significant improvement in hypertension with fewer medica- tions in 30-40% of cases and in 10-20% of cases hypertension persists but is well controlled by medication. The main determi- nants of a complete cure following surgery are the presence of an adrenal adenoma, a favourable preoperative response to spironolactone, age less than 50 years, female sex, duration of hypertension less than five years and the absence of a family history of hypertension.48,49
Hypercortisolism
Bilateral total adrenalectomy is offered to patients who have Cushing’s disease and have failed transphenoidal microsurgery, with or without radiotherapy, patients with ectopic ACTH pro- duction, and patients with bilateral macro or micronodular hyper- plasia.7 Patients with severe psychosis or severe myopathy, who need rapid control of their hypercortisolism may benefit from bilateral adrenalectomy in preference to central treatments for Cushing’s disease. Unilateral adrenalectomy is appropriate for a solitary hyperfunctioning adenoma. Patients with hypercortiso- lism benefit from the laparoscopic approach because in the open era, wound infections, slow wound healing, wound dehiscence and haematoma were all more prevalent in this group. This seems intuitive, but has also been demonstrated in a Cushingoid porcine model of laparoscopic versus open posterior adrenalectomy where laparoscopic adrenalectomy was shown to be less catabolic and have fewer wound complications than open surgery.50 Never- theless, the efficacy of laparoscopic adrenalectomy in this group needs to be comparable with the results achieved during the open era.7 Data are emerging to this effect. Recent reports suggest that biochemical and clinical cures are being achieved using bilateral laparoscopic adrenalectomy for Cushing’s disease, ectopic ACTH syndrome and bilateral adrenal hyperplasia. At surgery, care must be taken to remove the adrenal gland in its envelope of fat to ensure no adrenal rests are left in situ. These may function at a later date as often bilateral adrenalectomy is end organ surgery and does not deal with the original adrenal stimulus.51 The other clinical dilemma is the approach to the patient with the adrenal mass and subclinical Cushing’s syndrome. One recent recommendation was to offer surgery to those less than 50 years of age, those with a suppressed plasma ACTH or those with obes- ity, diabetes or hypertension. Older patients, asymptomatic patients and patients with normal plasma ACTH level are treated non-operatively.52
Incidentaloma
One controversial area in adrenal surgery is the approach to patients with no history of other malignancy and an incidental adrenal mass which does not display any radiological stigmata of malignant disease. Various authors have made recommendations based on the estimated risk of malignancy. Copeland53 estimated that at 6 cm, 60 operations would have to be performed to remove one cancer. Ross and Aron,54 argue that in the absence of CT characteristics of malignancy, less than one in 10 000 lesions less than 6 cm in size represent an adrenocortical cancer. Their article established the practice of observing patients with incidental masses 3-6 cm in size and offering surgery to those with masses over 6 cm. However, since that initial report several groups have published a 10% incidence of small adrenocortical cancer (<5 cm) in surgical series of resected incidental adrenal masses.42,55 It is self-evident that ACC does not occur de novo at 6 cm and at some time has evolved from a smaller lesion. We would recommend adrenalectomy at 3 cm if patient age and health are acceptable. In a young patient with a potentially curable lesion, there is no technique allowing safe, effective, life- long surveillance. Serial CT if no change in size is detected, is usually ceased at 12-18 months.54 Currently these lesions are best treated with laparoscopic adrenalectomy.
Adrenocortical cancer
Surgery remains the cornerstone of treatment for stages 1-3 adrenocortical cancer. The strongest predictor of good outcome is the ability to perform a curative resection. Mean survival times for unresectable disease are less than 12 months; however, 5 year actuarial survival for those who undergo complete resec- tion range from 32% to 48%.39,56 The recommended surgical approach is to remove en bloc all disease including contiguous structures, such as the liver, kidney, spleen and pancreas. This is usually best achieved via a subcostal incision.39 Increasingly, laparoscopic resection of large potentially malignant tumours up to 10 cm is being reported.46 However, given that the best chance of cure remains complete local resection, it would seem prudent to minimize the potential for spillage of tumour cells by resect- ing these large lesions through an open incision. If local recur- rence occurs following complete local resection, then the literature would support an aggressive approach to resecting recurrent disease, as this offers the patient the best chance of long-term cure.31
The mainstay of systemic therapy for ACC is the adrenolytic agent, mitotane. Originally trialed in dogs, this agent has been used for the treatment of metastatic disease as well as an adju- vant agent following complete resection. The benefits of mito- tane seem to be very stage specific. Those with stage 4 disease show a response in up to 50% of instances.57 Adjuvant mitotane following complete resection has not shown to provide any sur- vival benefit.39 Experience with the use of single and multiagent chemotherapy, including etoposide, doxorubicin and cisplatin, has demonstrated a low response rate and a short response dura- tion. Part of the refractoriness to chemotherapy may be explained by the fact that ACC expresses the multidrug resist- ance gene.58
Prognosis
The overall survival for patients with ACC is 38% at 5 years. When stratified for stage, it is 66% for stage 1 tumours, 58% for
stage 2 tumours, 24% for stage 3 disease, and 0% for stage 4 disease.39 Various groups have attempted to predict prognosis on clinical, histological and molecular grounds. Clinically, it is accepted that curative resection, recent diagnosis and local stage convey a more favourable prognosis.39 Stojadinovic et al.,59 per- formed multivariant analysis of a large number of histological and molecular markers in a large cohort of ACC and demon- strated, like Weiss et al.,60 that a mitotic rate of greater than 5 per 50 high power field is an adverse prognostic histological feature. They also analysed expression of several cell cycle proteins including Ki-67, p53, mdm-2, bcl-2, p21 and p27. However, they could not identify a profile with a significant predictive value for metastatic disease following complete local resection.
Gicquel et al.61 have published the only long-term, large cohort study examining the prognostic significance of three molecular markers, 17p13 loss of heterozygosity, 11p15.5 loss of hetero- zygosity and IGF2 gene overexpression in adrenocortical cancer. Evaluating the prognostic value of these markers in localized tumours, they showed that these three molecular markers (previ- ously reported as being strongly associated with the malignant phenotype) are strong predictors of disease-free survival in uni- variate analysis. 17p13 LOH is also a molecular marker of inde- pendent prognostic significance in a multivariate analysis taking into account biological and pathological data.
MOLECULAR ASPECTS OF ADRENOCORTICAL TUMOURIGENESIS
Cancer is the end result of the clonal expansion of a population of cells, which have acquired a number of non-lethal genetic altera- tions favouring uncontrolled cell proliferation or inhibition of cell death. In many cases, the accumulation of somatic mutation is the end product of tissues subjected to a number of environmental stresses such as chemical agents, viruses, or radiation exposure. Alternatively up to 10% of cancers arise from a germline muta- tion predisposing to a familial cancer syndrome.62
Numerous generic alterations affecting key cancer forming genes have been identified. These alterations fall into four categories:
(1) Subtle sequence changes: these changes affect the coding region of a gene and involve a nucleotide(s) substitution, deletion or insertion.
(2) Alterations in chromosome number: gain or loss of whole chromosome(s) are found in nearly all human cancers.
(3) Chromosomal translocation: fusion between different regions of two chromosomes may confer a tumourigenic advan- tage to the fusion product by activating a proto-oncogene.
(4) Gene amplification: multiple copies of a chromosomal region containing a growth-promoting gene are amplified.
The end result is that such changes contribute to genetic insta- bility and allow tumour formation and progression.62 The genetic alterations which permit neoplastic development affects two key classes of genes: oncogenes and tumour suppressor genes.
Oncogenes
These genes normally function by modulating cell growth and differentiation, but through genetic alteration have lost this abil- ity. Oncogenes encode for growth factors, growth factor rec- eptors, with and without tyrosine kinase activity, membrane bound signal transduction proteins, cytoplasmic serine threonine kinases and nuclear transcription factors. Mutation, chromosomal
amplification and chromosomal translocation serve to activate oncogenes in one of these key pathways, thereby leading to unregulated cell growth and transcription.63
Tumour suppressor genes
In contrast to oncogenes, tumour suppressor genes such as retino- blastoma, APC (adenomatous polyposis coli) and TP53 have an inhibitory effect on cell growth and survival and in the case of TP53, activation leads to cell death. According to Knudson’s 2-hit hypothesis64 both alleles of a tumour suppressor gene, and in Knudson’s original description this related to the retino- blastoma gene, need to be inactivated for tumourigenesis to pro- ceed. In those with the inherited form of the disease, the first hit is in the germline and is present in every cell of the body. The second hit is a mutation or deletion in the remaining wild type allele occurring at the somatic level and therefore predisposing to cancer formation.
ADRENOCORTICAL TUMOURIGENESIS
Much remains to be elucidated regarding the pathogenesis of ACT although progress is being made.65 Many studies have focused on genome wide screening techniques (e.g. comparative genomic hybridization (CGH) or loss of heterozygosity (LOH)) studies using multiple microsatellite markers, to identify key chromosomal regions implicated in adrenal tumour formation. Other studies have focused on interrogating key tumour onco- genes or tumour suppressor genes which have been shown to be the molecular defect predisposing to familial cancer syndromes expressing ACT as part of their phenotype (Table 2).
Clonal analysis
Examining tumour clonality is intrinsic to establishing tumour progression pathways. Polyclonality suggests that tumour cells are under the influence of local or systemic stimuli, whereas monoclonality suggest that tumour progression is the end result of intrinsic genetic mutation. By examining the pattern of X-chromosome inactivation in heterozygous female tissues, it has been established that adrenocortical cancer con- sists of a monoclonal population of cells, that nodular hyper- plastic adrenal tissue are composed of a polyclonal population of cells and that adrenal adenomas may be either monoclonal or polyclonal.66,67 The genetic heterogeneity evidenced in adrenal adenomas may be explained by different pathogenic molecular pathways or, alternatively, these tumours may be at different stages of a common multistep pathway. Concerning the latter, it seems plausible that extrinsic factors such as mitogens or growth factors may cause increased cell prolifera- tion, which renders cells more susceptible to oncogene or tumour suppressor gene mutation, and that once a subclone of cells acquires a genetic advantage over competing subclones, selective proliferation and tumour replacement with the advantaged clone occurs.65
Cytogenetic analysis of ACT
Loss of heterozygosity analysis is an established technique that has been successfully applied to tumour suppressor gene identifi- cation.22 Yano et al.68 were the first to screen a panel of benign and malignant ACT for LOH at three loci: 11p, 13q and 17p. Of
patients heterozygous for markers at these loci, four of six (66%), three of six (50%) and six of six (100%) ACC showed LOH at 11p, 13q and 17p, respectively. No LOH was observed in the benign tissues. Kjellman et al.,69 performed genome wide LOH using multiple microsatellite markers in 39 ACC and 21 adeno- mas. The vast majority of LOH was detected in ACC and involved chromosomes 2, 4, 11 and 18. Further fine mapping of regions at 2p16 and 11q13, identified a minimal region of loss within 1 cM of the Carney complex locus at 2p16 and a region at 11q13, which includes the MEN1 locus. Following exclusion of MEN1 mutations in their ACT, it was suggested that these two regions harbour putative tumour suppressor genes as LOH was almost exclusively seen in ACC.
In contrast to LOH, constitutional chromosomal alterations involving both gain and loss may be identified using CGH. CGH is a molecular cytogenetic technique that permits genome wide screen- ing of tumour DNA for gain or loss of DNA copy sequences.70 Regions of gain may harbour potential proto-oncogenes, while regions of copy sequence loss may contain tumour suppressor genes. This then allows further fine mapping of regions considered impor- tant in the development of specific tumour types.
Our group has recently screened a panel of 32 ACT (13 ACC, 18 benign tumours, one indeterminate tumour) and shown that DNA copy genes were more prevalent in ACC (100%) compared with the adrenocortical adenomas (61%).71 Interestingly, 86% of the changes seen in the adenomas were also evidenced in the can- cers, albeit in lesser numbers, supporting the concept of a multi- step progression pathway from normal adrenal gland to adrenal adenoma to ACC. Regions of DNA copy gain were seen on chro- mosomes 4, 5, 12 and 19. Regions of DNA copy loss were fre- quently seen on chromosomes 1p, 17p, 2q and 11q. Our overall experience is consistent with the experience of Kjellman et al.72 and to a lesser extent Zhao et al.73 suggesting that activation of a proto-oncogene(s) on chromosome 4 may be an early event in adrenal tumourigenesis with progression from adenoma to carci- noma involving activation of oncogenes on chromosomes 5 and 12 and inactivation of tumour suppressor genes on chromosomes 1p and 17p.
ONCOGENES
Abnormalities of the imprinted 11p15 chromosomal region and the insulin-like growth factor system in adrenocortical tumours
The insulin-like growth factor (IGF) system is involved in the development and maintenance of differentiated adrenocortical functions and its role has been largely documented in adreno- cortical tumours.
The 11p15 imprinted chromosomal region
We and others showed that strong overexpression of the IGF2 gene, which encodes a fetal growth factor, is a frequent feature of the malignant state, occurring in about 90% of malignant tumours but not in benign tumours.74-76 The IGF2 gene maps to the 11p15 region which is submitted to parental imprinting. The mechanism of IGF2 gene overexpression is, at least partly, related to pathological imprinting of the 11p15 region. Indeed, most tumours with overexpression of the IGF2 gene also exhibit paternal isodisomy (loss of maternal allele and duplication of the paternal active IGF2 allele) or, less frequently, loss of
imprinting (with maintenance of the maternal allele but a paternal-like IGF2 gene expression pattern)76 (Fig. 1). Genetic or epigenetic changes in the imprinted 11p15 region resulting in an increased level of IGF2 have also been implicated in Beckwith-Wiedemann syndrome. The 11p15 imprinted region includes other candidate genes for adrenocortical tumourigene- sis. The H19 gene, an untranslated RNA with a putative growth suppressor function, and the CDKN1C gene encoding a G1 cyclin dependent kinase inhibitor from the CIP/KIP family, are
highly expressed in normal adrenocortical tissues and are expressed from the maternal allele. H19 and CDKN1C expres- sion therefore is abrogated in most malignant tumours76-78 (Fig. 1). Although the exact role of H19 in adrenal tumourigen- esis remains unclear, the abrogation of CDKN1C gene expres- sion associated with an overexpression of G1 cyclins and G1 cyclin dependent kinases (CDK) leads to a breakdown of cell cycle regulation from overactivity of G1 cyclin-CDK com- plexes (Fig. 1).
maternal allele
A
paternal allele
CDKN1C
IGF2
H19
malignant tumour
Leu Tum
B
mat -
paternal allele
IGF2
H19
pat
CDKN1C
paternal allele
Paternal Isodisomy
CDKN1C
IGF2
H19
benign malignant
100-
cyclin E (AU)
100
CDK2 (AU)
4
IGF2
4
A
4
50
50
H19
0
A
CDKN1C
&
normal
benign malignant
normal
benign malignant
Abnormal mRNA expression of 11p15 genes
C
Overexpression of G1 cyclin and CDK
Abrogation of CDKN1C expression
benign
malignant
cyc E CDK2
Overactivity of cyclin-CDK complexes in malignant tumours
The IGF system
The IGF system is complex and comprises several elements. The biological activity of IGF1 and IGF2 is modulated by two structurally different IGF receptors and six IGF-binding pro- teins (IGFBP). IGF2 messenger ribonucleic acid are efficiently translated and malignant tumours contain large amounts of IGF2 protein, which is partly in the prohormone form.79 How- ever, IGF2 effects are restricted to tumours and systemic plasma levels of IGF2 are always in the normal range. The type 1 insulin-like growth factor receptor which mediates the prolif- erative effects of both IGF1 and IGF2 is also overexpressed in malignant tumours74 and we have showed that IGF2 is directly involved in proliferation of the human adrenocortical carci- noma NCI H295R cell line and acts through the IGF1 recep- tor.80 The type 2 insulin-like growth factor receptor (IGF2R), by ensuring the clearance and the degradation of IGF2, has an antiproliferative function. Loss of heterozygosity (LOH) at the mannose-6-phosphate/IGF2R locus is a frequent event in adrenocortical tumours and supports the hypothesis that it may function as a tumour suppressor gene in adrenocortical tumourigenesis.81
IGFBP also modulate the biological effects of IGF. Analysis of the IGFBP expression profile in H295R cells and adrenocortical tumours showed an enhanced IGFBP-2 content in H295R cells and malignant adrenocortical tumours overexpressing the IGF2 gene.79,80 Several studies have suggested that IGFBP-2 expres- sion may be associated with malignancy and a recent report sug- gests that IGFBP-2 enhances cell proliferation in adrenal tumours by an IGF-independent mechanism.82
Finally, all data concerning the IGF system and the 11p15 genes are consistent with a major role of dysregulation of the imprinted 11p15 region and the IGF system in transition from benign to malignant adrenocortical tumours. Whatever are the exact mechanisms for this dysregulation, these molecular markers could permit a more accurate diagnosis of malignancy and probably also a better assessment of prognosis of adreno- cortical tumours.
ACTH cyclic AMP/protein kinase A pathway
The ACTH receptor (ACTH-R) belongs to the superfamily of G-protein coupled seven transmembrane domain receptors. The trimeric G-protein is composed of three different polypeptide chains (a, B and 8) and is responsible for trans- membrane signal transduction once ligand activation of ACTH-R occurs. The a subunit, designated Gs, binds and hydrolyses guanine triphosphate (GTP) and when GTP is bound, the active Gs stimulates adenyl cyclase to produce many molecules of cyclic AMP from adenosine triphosphate. Few mutations have been found in this pathway in ACT. Of 25 adenomas and 13 carcinomas studied, no activating muta- tions of the ACTH-R have been identified.83,84 Similarly, mutations of the G proteins, Gs and Gi, in four separate studies have revealed only three mutations at codon 179 in 76 ACT.85-88 This evidence suggests that activation of ACTH-R and its downstream signalling pathway are implicated in cel- lular differentiation and steroid hormone secretion rather than ACT progression. Further support for this paradigm comes from a study by Reincke et al.89 where allelic loss of ACTH-R was examined in 16 benign and four malignant ACT. LOH for the ACTH-R was observed in two of four informative cancers,
but not in 15 hyperfunctioning adenomas, suggesting a role for the ACTH-R in cellular de-differentiation.
RAS ONCOGENES
Ras proteins are membrane associated proteins involved in down- stream signalling once ligand stimulation of growth factor recep- tors occurs. The three ras proteins (H, N and K) possess intrinsic GTPase activity and are one of the most commonly mutated oncogenes in human cancers.90 Few ras oncogene mutations in ACT have been demonstrated in three series.91-93
TUMOUR SUPPRESSOR GENES Tp53
The TP53 gene is a tumour suppressor gene located at 17p13.1 and encodes for a 393 amino acid protein. More than half of human cancers have a TP53 mutation. The p53 protein acts as a transcription factor and is known to regulate the expression of many genes including p21, mdm1, gadd45, cyclin G, bax, IGF2, IGFBP2 and IGFBP3.94 Once activated by genotoxic stress (e.g. carcinogens, X-rays) or non-genotoxic stress (e.g. hypoxia) TP53 acts to control cell growth and regulate genomic integrity principally by induction of target genes which arrest the cell cycle in the G1-S phase and induce cellular apoptosis.95 The prevalence of TP53 muta- tions was first explored by Oghaki et al.91 and Reincke et al.96 and then later by Lin et al.97 TP53 was implicated as a strong candidate gene in adrenocortical tumourigenesis because it is commonly mutated in other cancers, 17p LOH has been reported in ACC and not adrenocortical adenomas68 and ACC is a manifestation of Li-Fraumeni syndrome in which patients carry a germline TP53 mutation. However, the studies of Oghaki et al.91 and Reincke et al.96 showed mutations in only three of 15 (20%) and three of 11 (27%) ACC, respectively, when exons 5-8 were sequenced. Lin et al.,97 reported muta- tions in 11 of 15 (73%) adrenocortical adenomas studied for TP53 mutation and nine of 11 (82%) of these mutations were seen in exon 4. Subsequently Reincke et al.,98 sequenced exon 4 in 19 adrenocortical adenomas without finding any mutation and suggested the discrepancy between results was due to either environmental factors or technical errors in sequencing methodology.
More recent interest in the role of TP53 in ACT progression was sparked by a report by Ribeiro et al.99 demonstrating a germline R337H mutation in exon 10 of TP53 in 35 of 36 (97%) children from southern Brazil who had apparently sporadic ACC. This finding was verified by Latronico et al.16 who also examined the tumours and germlines of 37 adults from Brazil with ACT and discovered that five of 37 (13.5%) of the tumours showed the R337H mutation in exon 10. This same mutation was present in the germline of one of three patients in whom blood was available for analysis. DNA analysis of 120 alleles of unrelated controls did not reveal this mutation, demonstrating that the R337H mutation is not widespread in the Brazilian pop- ulation. It is now acknowledged that the R337H mutation is a signature low penetrance allele causing an exclusive predisposi- tion to ACC.100 Most recently, a group from Italy has published a 70% mutation rate in 10 ACC examined for mutation in TP53.101
MEN1 gene
The MEN1 gene functions as a tumour suppressor and mutation in one of the nine coding exons predisposes to multiple endocrine neoplasia. LOH at 11q13 has been described in greater than 90% of informative ACC in three different series compared with an incidence of LOH at 11q13 in less than 30% of informative ade- nomas, implicating the MEN1 gene in ACC formation. However, the same groups demonstrating LOH at 11q13 in ACC have reported only one mutation in 32 ACC in whom the MEN1 gene was sequenced.69,102,103 Similarly, one mutation in 41 adrenal ade- nomas was found. These results suggest that as yet an unidenti- fied tumour suppressor gene at this locus is implicated in ACC formation.
P16
The p16 tumour suppressor gene lies within the chromosomal region 9p21 and mutation is frequently seen in malignant human cell lines. P16 codes for a cyclin kinase inhibitor which binds to cyclin dependent kinases 4 and 6 to stop progression of the cell cycle from the G1 to the S phase. LOH at 9p21 was seen in three of seven informative ACC and one of seven informative adeno- mas and subsequent immunohistochemistry for p16 showed absence of immunostaining in those tumours with LOH.104 These results implicated p16 as having a role in ACT progression; how- ever, when sequenced in 10 adenomas and 10 cancers, no muta- tions were identified.105
SUMMARY
By examining genes implicated in familial cancer syndromes and using genome wide surveillance methodology (e.g. CGH) key chromosomal regions of interest and certain key genes have been implicated in the pathogenesis ACT formation. Progress in this field has been slower than in other areas of cancer research (e.g. colon cancer, breast cancer) principally because of the rarity of the tumours involved. In order to ensure continued progress in the field, a coordinated approach to identification of patients with adrenal disease and collection of their tumours following surgical removal is required.
The current status of tumour progression models in ACT is sum- marized in Figure 2. Little is known about mechanisms of progres- sion from normal adrenal gland to adrenal adenoma. However, certain key genetic events in progression to ACC are known. Pater- nal isodisomy at 11p15.5 with overexpression of IGF2 and abroga- tion of expression of the CDKN1C and H19 genes is a key event in ACC formation. TP53 is involved in progression to carcinoma in a subset of patients and the frequency of ACTH-R deletion needs to be more fully explored. Other key oncogenes and tumour suppres- sor genes remain to be identified although the chromosomal loci in which they lie can be identified at 17p, 1p, 2p16 and 11q13 for tumour suppressor genes and chromosomes 4, 5 and 12 for onco- genes. Newer techniques such as gene expression profiling or chro- mosome specific gene profiling will permit more targeted gene interrogation based on cytogenetic data published to date. Such an advance will greatly accelerate progress in this field of research.
IGF2 Overexpression CDKN1C and H19 abrogation of expression TP53 mutation ACTH-R deletion
Specific genes
Polyclonal cell expansion
Monoclonal expansion
Normal Adrenal
Gain of 4
Adrenal Adenoma
Loss of 17p and 1p Gain of 5 and 12
ACC
Chromosomal loci
IGF system and 11p15 17p13
1p21, 2p16, 9p21, 11q13,12p12-14, 18p11
angiogenic factors, metalloproteinases.
ACKNOWLEDGEMENTS
This work would not have been possible without the support of the Royal Australasian College of Surgeons Research Founda- tion. At the time of writing, SS was in receipt of the Sir Roy McGaughey research scholarship.
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