Molecular Markers and the Pathogenesis of Adrenocortical Cancer Patsy S. H. Soon, Kerrie L. McDonald, Bruce G. Robinson and Stan B. Sidhu
The Oncologist 2008, 13:548-561. doi: 10.1634/theoncologist.2007-0243
The online version of this article, along with updated information and services, is located on the World Wide Web at: http://theoncologist.alphamedpress.org/content/13/5/548
Oncologist ® Endocrinology
Molecular Markers and the Pathogenesis of Adrenocortical Cancer
PATSY S. H. SOON,a,b KERRIE L. MCDONALD,ª BRUCE G. ROBINSON,a,c STAN B. SIDHUa,d
ªCancer Genetics, Kolling Institute of Medical Research, University of Sydney, ‘Department of Surgery, Bankstown Hospital and University of New South Wales, “Department of Endocrinology, and ªDepartment of Endocrine and Oncology Surgery, Royal North Shore Hospital, St. Leonards, Australia and University of Sydney
Key Words. Adrenocortical carcinoma . Adrenocortical adenoma . Adrenocortical tumors · Molecular marker
Disclosure: No potential conflicts of interest were reported by the authors.
ABSTRACT
Adrenal tumors are common, with an estimated inci- dence of 7.3% in autopsy cases, while adrenocortical carcinomas (ACCs) are rare, with an estimated preva- lence of 4-12 per million population. Because the prog- noses for adrenocortical adenomas (ACAs) and ACCs are vastly different, it is important to be able to accu- rately differentiate the two tumor types. Advancement in the understanding of the pathophysiology of ACCs is essential for the development of more sensitive means of diagnosis and treatment, resulting in better clinical outcome. Adrenocortical tumors (ACTs) occur as a component of several hereditary tumor syndromes, which include the Li-Fraumeni syndrome, Beckwith-
Wiedemann syndrome, multiple endocrine neoplasia 1, Carney complex, and congenital adrenal hyperpla- sia. The genes involved in these syndromes have also been shown to play a role in the pathogenesis of spo- radic ACTs. The adrenocorticotropic hormone- cAMP-protein kinase A and Wnt pathways are also implicated in adrenocortical tumorigenesis. The aim of this review is to summarize the current knowledge on the molecular mechanisms involved in adrenocor- tical tumorigenesis, including results of comparative genomic hybridization, loss of heterozygosity, and microarray gene-expression profiling studies. The On- cologist 2008;13:548-561
INTRODUCTION
Adrenal tumors are common, with an estimated incidence of 7.3% in autopsy cases [1]. A recent computed tomogra- phy (CT) study reported an overall 4.4% prevalence rate of adrenal lesions [2]. Adrenocortical carcinomas (ACCs), however, are rare, with an estimated prevalence of 4-12 per million population [3]. Because the prognoses for adreno- cortical adenomas (ACAs) and ACCs are vastly different, it is important to be able to accurately differentiate the two
tumor types. At present, the modified Weiss score is the most widely accepted pathological system for classifying adrenocortical tumors (ACTs) as benign or malignant [4]. This histopathological scoring system involves nine crite- ria-high mitotic rate, atypical mitoses, high nuclear grade, low percentage of clear cells, necrosis, diffuse architecture of the tumor, capsular invasion, sinusoidal invasion, and venous invasion. The presence of each criterion is given a score of 1, and a total score ≤2 is typically associated with
Correspondence: Stan Sidhu, MBBS, PhD, FRACS, Department of Endocrine and Oncology Surgery, Royal North Shore Hospital, St. Leonards, NSW 2065, Australia. Telephone: 61-2-94371731; Fax: 61-2-99268523; e-mail: stansidhu@nebsc.com.au Received De- cember 12, 2007; accepted for publication March 24, 2008. @AlphaMed Press 1083-7159/2008/$30.00/0 doi: 10.1634/theoncologist.2007- 0243
an ACA while a score ≥3 is indicative of an ACC [4-6]. In a study of 24 malignant and 25 benign ACTs, a Weiss score ≥3 had a sensitivity of 100% and a specificity of 96% in predicting malignancy. The authors noted, however, that the interobserver agreement for certain criteria, such as ar- chitecture, sinusoid invasion, nuclear grade, and venous in- vasion, was not reliable [4]. Furthermore, even when applied correctly, the Weiss system is far from infallible, and individual ACTs may go on to behave in a malignant manner despite initially receiving a Weiss score ≤2 [7-9]. Another shortcoming of this classification system is its re- striction to providing descriptive information only on the tumor’s morphology; consequently, it does not offer any molecular targets for therapy development.
Surgery is the mainstay of treatment for ACCs. Open adrenalectomy is recommended for excision of malignant primary adrenal lesions [10-12] because there have been some case reports that laparoscopic adrenalectomy for ACCs actually increases the risk for peritoneal dissemina- tion and metastasis [13-15]. Despite radical surgery with curative intent, for patients with localized ACCs, the ma- jority will develop metastases within 6-24 months of resec- tion [16].
About one fifth of the patients with ACC present with advanced disease that is not cured by surgery [17, 18]. The treatment of choice for these patients is mitotane (o,p’- dichlorodiphenyldichloroethane). Up to one third of pa- tients have at least a partial response to mitotane [19]. The largest series of mitotane use after surgery compared with surgery alone, in 177 patients with ACCs from eight centers in Italy and 47 centers in Germany, was published recently. All patients had radical resection with a follow-up of up to 10 years. Forty-seven of the 177 patients had mitotane after surgery, while the remainder of the patients had surgery alone. Mitotane treatment was associated with significantly longer recurrence-free survival and overall survival times compared with the control [16].
Radiotherapy to the tumor bed has also been used as adjuvant treatment after radical resection of ACC. Re- ports with small numbers of patients have described re- sponse rates of up to 42%. Fassnacht et al. [20] compared a group of 14 patients who received radiotherapy to the tumor bed with a matched control group of 14 patients. The local recurrence rate was significantly lower in the radiotherapy group, at 14%, compared with 79% in the control group. The disease-free and overall survival times, however, were not significantly different between the two groups [20].
ACCs have a 10%-40% response rate to various che- motherapeutic agents. Chemotherapeutic drugs that have been reported to have some effect against ACCs include
etoposide, doxorubicin, and cisplatin. ACCs tend to express the multidrug resistance gene MDR-1, which results in the production of P-glycoprotein, which is involved in the re- moval of the drug from cancer cells. Because of this multi- drug resistance gene, single-agent chemotherapy is not favored for ACCs [21].
Advancement in the understanding of the pathophysiol ogy of ACCs is essential for the development of more sen- sitive means of diagnosis and treatment, resulting in better clinical outcome. The aim of this review is to summarize the current knowledge of the molecular mechanisms in- volved in adrenocortical tumorigenesis.
MOLECULAR ASPECTS OF ACCS
Hereditary Tumor Syndromes
ACTs can arise from several hereditary tumor syndromes. The causative genes in these syndromes have also been found to be involved in the tumorigenesis of some sporadic ACTs. Table 1 summarizes these hereditary tumor syn- dromes and the genes/chromosomal loci involved.
Li-Fraumeni Syndrome
Li-Fraumeni Syndrome (LFS; OMIM 151623) is an auto- somal dominant familial disease characterized by the early onset of tumors and multiple tumors in affected individuals. Families with this syndrome also have multiple affected family members. The most common tumor types that occur in LFS are soft tissue sarcomas, osteosarcomas, breast can- cer, brain tumors, leukemia, and ACCs [22, 23]. ACCs have been reported to occur in 3%-4% of patients with LFS, of- ten under the age of 20 [23]. Seventy percent of LFS cases are a result of a germline mutation in the TP53 gene [24]. A second variant is caused by a heterozygous germline muta- tion in the hCHK2 gene [25], while a four centi-Morgan re- gion on 1q23 has been implicated in a third variant [26].
Beckwith-Wiedemann Syndrome
Beckwith-Wiedemann syndrome (BWS; OMIM 130650) is a congenital overgrowth syndrome characterized by ex- omphalos, macroglossia, and gigantism in the neonate as well as the development of childhood tumors. These tu- mors, which include ACC, nephroblastoma, hepatoblas- toma, and rhabdomyosarcoma, occur in 5% of patients [27]. About 15% of cases with BWS are familial, with the re- mainder being sporadic. BWS is linked to the 11p15 chro- mosomal locus. This region is subject to parental imprinting, a genetic phenomenon in which specific genes are expressed solely either from the maternal or paternal al- lele. Genes located at 11p15 and implicated in the patho- genesis of BWS are the insulin-like growth factor 2 (IGF2),
| Table 1. Summary of hereditary tumor syndromes associated with ACTs | |||
|---|---|---|---|
| Hereditary tumor syndrome | Gene (chromosomal locus) | Manifestation of tumor syndrome | Prevalence of ACTs |
| Li-Fraumeni syndrome | TP53 (17p13), hCHK2(22q12.1), 1q23 | Soft tissue sarcoma, osteosarcoma, breast cancer, brain tumor, leukemia, ACC | ACC, 3%-4% |
| Beckwith-Wiedemann syndrome | IGF2, H19, CDKN1C, KCNQ1 (11p15) | Exomphalos, macroglossia, gigantism, ACC, nephroblastoma, hepatoblastoma, rhabdomyosarcoma | ACC, 5% |
| Carney complex | PRKAR1A (17q23-q24) 2p16 | Cardiac, endocrine, cutaneous, and neural myxomatous tumors, and pigmented lesions of the skin and mucosa | PPNAD, 90%-100% |
| Multiple endocrine neoplasia 1 | MEN1 (11q13) | Parathyroid, pancreatic islet cell, anterior pituitary and ACTs | ACT, 55%; ACC, rare |
| Congenital adrenal hyperplasia | CYP21B (6p21.3)-most common, CYP11B, CYP17A, HSD3B2 | Adrenal hyperplasia, virilization, salt-wasting | Adrenal tumors, 82%; hyperplasia, 100% |
| Abbreviations: ACA, adrenocortical adenoma; ACC, adrenocortical carcinoma; ACT, adrenocortical tumor; PPNAD, primary pigmented nodular adrenocortical disease. | |||
H19, and cyclin-dependent kinase inhibitor 1C (CDKN1C also known as p57kip2) genes. IGF2 is maternally imprinted, while H19 and p57kip2 are both paternally imprinted. Pater- nal uniparental isodisomy (duplication of the paternal allele and loss of the maternal allele) of 11p15 [28], germline mu- tations of p57kip2, or methylation of H19 and potassium channel, voltage-gated, KQT-like subfamily, member 1 (KCNQ1) have been implicated in the pathogenesis of BWS [27]. p57kip2 encodes a cyclin-dependent kinase (CDK) in- hibitor that belongs to the CIP/KIP family of cell-cycle reg- ulators. Overexpression of this gene arrests cells in the G1 phase of the cell cycle. H19 is transcribed to RNA but not translated to protein. It is thought to be involved in the reg- ulation of IGF2 [27].
Carney Complex
Carney complex (CNC; OMIM 160980) is a dominantly inherited syndrome characterized by cardiac, endocrine, cutaneous, and neural myxomatous tumors, as well as pigmented lesions of the skin and mucosa [29-31]. Primary pigmented nodular adrenocortical disease (PPNAD), a main feature of CNC, is a rare cause of ad- renocorticotropic hormone (ACTH)-independent Cush- ing’s syndrome, usually in children and young adults [32]. There are two types of CNC-type 1 is caused by mutation of the protein kinase, cAMP-dependent, regu- latory, type 1, alpha (PRKAR1A) gene, located on 17q23- q24 [33], while type 2 has been attributed to the 2p16 chromosomal locus [34].
Multiple Endocrine Neoplasia 1
Multiple endocrine neoplasia 1 (MEN1; OMIM 131100) is an autosomal dominant syndrome characterized by the oc- currence of parathyroid, pancreatic islet cell, and anterior pituitary tumors [35]. In 55% of individuals diagnosed with MEN1, ACTs (typically ACAs) have also been reported [36-39]. ACCs have only rarely been reported with MEN1 [37-39]. The MEN1 gene, located on 11q13, encodes the menin protein. Although the function of menin is currently unknown, mutations have resulted in the loss of its function, suggesting that menin has tumor suppressor activities [40, 41]. Because menin is located in the nucleus, it is thought to play a role in the cell cycle, regulation of transcription, or DNA replication [35].
Congenital Adrenal Hyperplasia
Congenital adrenal hyperplasia (CAH) is an autosomal re- cessive disorder resulting from an enzyme deficiency in the cortisol synthesis pathway. Typically, the enzyme 21-hy- droxylase, which is encoded by cytochrome P450, family 21, subfamily B (CYP21B; OMIM 201910), is deficient. A lack of this enzyme leads to compensatory stimulation of the adrenal cortex by corticotrophin-releasing hormone and ACTH with consequent adrenal hyperplasia and overpro- duction of cortisol precursors, engendering higher levels of androgens. Deficiencies of 11 ß-hydoxylase, 17a-hydroxyl- ase, and 3ß-hydroxysteroid dehydrogenase are less com- monly the cause of CAH. Clinically, CAH is divided into a classic (severe) salt wasting or simple virilizing form and a nonclassic (mild) form [42-44]. Assessment for adrenal le-
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sions with abdominal CT in CAH patients and heterozy- gous carriers of CAH found that all CAH patients had adrenal hyperplasia as well as twice the rate of adrenal tu- mors, at 82%, compared with CAH carriers, at 45%. One in 11 patients with CAH and one in 20 carriers of CAH had adrenal tumors >5 cm [45]. Rarely, ACCs have been de- scribed in CAH [46, 47].
GENETICS OF SPORADIC ACTS
TP53 Gene
The TP53 gene is a tumor suppressor gene and is the most frequently mutated gene in human cancers [48]. The p53 protein plays a role as a transcription factor in regulation of the cell cycle, causing cell cycle arrest or cell death in re- sponse to DNA damaging agents such as radiation and vi- ruses [49]. Germline mutations of TP53 have usually been observed to occur in the highly conserved region of exons 5-8 [50].
TP53 mutation is thought to be a late event in the evo- lution of malignant transformation in sporadic ACTs. Mu- tations in exons 5-8 of TP53 have been found in 20%-27% of sporadic ACCs and 0%-6% of sporadic ACAs [51, 52]. Sequencing of exons 2-11 of TP53 in one study found mu- tations in 25% of sporadic ACCs, all of which occurred in exons 5-8. Patients with TP53 mutation showed a trend to- wards a shorter survival duration (p = . 098) [53]. Frequent somatic mutations in exon 4 of TP53, however, have been reported in 60% of ACAs and 50% of pheochromocytomas in Taiwanese patients [54], but these findings were not con- firmed in a study on white patients [55], suggesting that per- haps different ethnic backgrounds may result in different TP53 mutations.
The majority of TP53 mutations occur in its DNA-bind- ing domain. Less commonly, mutations in the oligomeriza- tion domain can occur [56]. Numerous missense mutations have been described for TP53 [57]. In ACCs, the R337H mutation in the oligomerization domain of the p53 protein results in the substitution of histidine for arginine at codon 337. This mutation is commonly found as a germline mu- tation in children with ACCs in southern Brazil [58]. It has been estimated that one in 10 carriers of this mutation de- velops ACC. In these cases of ACC, there is loss of het- erozygosity (LOH) of the normal wild-type TP53 allele with retention of the R337H mutated allele [59]. Other mis- sense mutations as well as nonsense mutations and dele- tions of TP53 have also been described [51-53, 60, 61].
The IGF2, p57kip2 (CDKN1C), and H19 Genes
Rearrangements, LOH (loss of one of two alleles of a gene), and abnormal imprinting of the 11p15.5 locus, resulting in
low p57kip2 and H19 and elevated IGF2 mRNA expression levels, have been reported in sporadic ACCs [62-64].
The IGF system is comprised of two peptide ligands (IGF1 and IGF2), two IGF receptors (IGF1R and IGF2R/ mannose-6-phosphate receptor), and six high-affinity bind- ing proteins (IGF binding proteins 1-6) [65]. In the adrenal gland, both IGF1 and IGF2 have growth-promoting as well as differentiating functions. They induce steroidogenesis in adrenocortical cells both in vitro and in vivo. IGF2 at high levels (50 times that of insulin) can also exert an insulin ef- fect, leading to hypoglycemia.
In sporadic ACCs, IGF1 has not been shown to be over- expressed [66]. IGF2, however, has frequently been re- ported to be overexpressed in ACCs compared with ACAs or normal adrenal cortices [67-69]. Higher IGF2 expres- sion levels are associated with a more malignant phenotype [66], and overexpression of IGF2 is associated with a higher risk for ACC recurrence [63]. Furthermore, LOH of the 11p15 locus has been demonstrated more frequently in ACCs than in ACAs-in 67% of ACCs versus 13% of ACAs. It is suggested that this LOH event leads to overex- pression of IGF2 because the maternal allele is lost while the paternal allele is duplicated, leading to a double dose of the expressed allele [62].
Studies with IGF2-transgenic mice have shown that the weights of the adrenal glands of these animals are signifi- cantly higher than those of controls, a result of hyperplasia of the zona fasciculata. Despite adrenal hyperplasia, over an 18-month period, these transgenic mice did not develop tu- mors in their adrenal glands, indicating that overexpression of IGF2 alone is insufficient to cause ACT formation and that other factors are required for tumorigenesis [70].
The p57kip2 gene is located within the 11p15 region and is paternally imprinted. It encodes a CDK inhibitor, which binds to cyclin-CDK complexes and inactivates their cata- lytic domain. It therefore functions as a negative regulator of cell cycle progression. By northern blot, p57kip2 mRNA was easily detected in normal adrenals and in all tumors with normal expression of IGF2, but was absent or low in tumors with overexpression of IGF2 and in the NCI-H295R adrenocortical cell line [71].
Somatic mutations of the p57kip2 gene are rare and do not account for the lower levels of p57kip2 mRNA and pro- tein expression in ACCs. Instead, other mechanisms, such as LOH and abnormalities of imprinting or methylation, could be responsible for the lower mRNA and protein levels with this gene [61].
MEN1 Gene
Because LOH of 11q13 occurs in about 20% of sporadic ACTs, and adrenal tumors occur in up to 40% of patients
from MEN1 kindreds, MEN1 was considered to be a prime candidate gene in the pathogenesis of these lesions. LOH of 11q13 is frequently found in ACCs but not in ACAs. Be- cause the MEN1 mRNA expression by quantitative poly- merase chain reaction and northern blot has been found to be similar in ACCs, ACAs, and normal adrenal cortices, and because no mutations within the MEN1 coding region were found in 33 sporadic ACTs and cell lines, it is unlikely that the MEN1 gene plays a prominent part in the pathogen- esis of sporadic ACTs [72-74].
PRKAR1A Gene
PRKAR1A is the main mediator of cAMP signaling [75]. One study found LOH of 17q22-24, the locus for PRKAR1A, in 23% of ACAs and 53% of ACCs. Direct se- quencing of the PRKAR1A gene revealed inactivating mu- tations in 10% of ACAs, with corresponding lower mRNA and protein levels in these tumors. These tumors were also smaller in size and exhibited paradoxical cortisol responses to dexamethasone, all features that are found in PPNAD. No mutations were found in ACCs. This is consistent with PPNAD, in which there has not been a case of ACC re- ported to date [76].
THE GUANINE NUCLEOTIDE-BINDING PROTEIN, ALPHA-STIMULATING ACTIVITY POLYPEPTIDE GENE
Activating somatic mutations of the guanine nucleotide- binding protein, alpha-stimulating activity polypeptide (GNAS) gene, located on 20q13.2, are responsible for the McCune Albright syndrome (MAS; OMIM 174800), a pe- diatric genetic disease. GNAS encodes the alpha subunit of the stimulatory G protein (Ga). In abnormal Gsa, there is a substitution of arginine 201 with histidine or cysteine, with subsequent lower GTPase activity, resulting in constitutive adenylate cylase activation and consequent cAMP signal- ing [77-79]. MAS is a sporadic disease that predominantly affects the skeleton, skin, and endocrine system. Classic manifestations include a triad of polyostotic fibrous dyspla- sia, large irregular café-au-lait spots, and endocrine dys- function, including precocious puberty, hyperthyroidism, gigantism, and Cushing’s syndrome. Because the genes in- volved in MAS and CNC both act on the cAMP pathway, the endocrine features of the two syndromes are very similar.
GNAS has been reported to rarely be mutated in sporadic ACAs. Mutation of codon 201 of GNAS has been reported in ACAs and tumors of patients with ACTH-independent macronodular adrenocortical hyperplasia (AIMAH) [80, 81]. There have been no reports, however, of GNAS muta- tions in sporadic ACCs.
SIGNALING PATHWAYS IN ACTS
Many different pathways are involved in cancer develop- ment. In ACTs, the ACTH-cAMP-protein kinase A (PKA) and Wnt pathways are thought to be important.
The ACTH-CAMP-PKA Pathway
The binding of ACTH to its receptor, a member of the G protein-coupled receptor family, results in the dissociation of the heterotrimeric Gs, causing the separation of the & subunit from the ß and y subunits and stimulation of ade- nylate cyclase, which in turn leads to the production of cAMP from ATP. cAMP then binds to the regulatory sub- units of PKA, releasing the catalytic subunits, which results in phosphorylation of proteins in the cytoplasm and nu- cleus, and subsequent signal transduction [82] (Fig. 1).
The ACTH-adenylate cyclase signaling pathway has been implicated in the pathogenesis of ACTs for a number of reasons. First, activating mutations of components of the adenylate cyclase pathway have been found in other human endocrine disorders, including toxic thyroid adenomas and acromegaly [83]. Second, there is a correlation between cir- culating ACTH levels and the size of the adrenal cortex, as seen in patients with CAH or Cushing’s disease [84]. Fi- nally, patients with CNC and MAS exhibit mutations in the PRKAR1A and GNAS genes, respectively. Both of these gene products are components of the cAMP pathway [33, 85]. No activating mutations of the melanocortin 2 receptor (MC2R; ACTH receptor) gene were found in 25 ACAs, 13 ACCs, and eight hyperplasias [81, 86, 87]. LOH of MC2R, however, was found in one of 16 ACAs and two of four ACCs [88], suggesting that loss of the ACTH receptor and response may play a role in adrenocortical tumorigenesis. Overall, ACTH is thought to have differentiating and growth inhibitory functions on adrenocortical cells [82, 89].
The Wnt Pathway
The Wnt family is comprised of a group of highly con- served growth factors with similar amino acid sequences, which play roles in developmental and homeostatic pro- cesses. The central event in the canonical Wnt signaling pathway is the accumulation of -catenin in the cytoplasm with subsequent translocation into the nucleus. Wnt binds to its receptor complex, which is composed of members of the frizzled family and low-density lipoprotein receptor- related protein. This results in the inhibition of the axin- adenomatous polyposis coli (APC)-glycogen synthase kinase 3B (GSK-3) complex, leading to a block in ß-catenin phosphorylation by GSK-3 and accumulation of ß-catenin in the cytoplasm. ß-catenin then translocates into the nu- cleus where it interacts with the T cell-specific transcrip- tion factor/lymphoid enhancer-binding factor 1 family of
Olicologist
ACTH
DAS
G protein coupled receptor
Extracellular
Adenylate cyclase
GY
Cytoplasm
GB
Gsa
GDP
GTP
P
Gsa
GY
GB
ATP
CAMP
Gene transcription
4 cAMPS
C
R
C
R
CREB
C
R
C
R
DNA
Active PKA
Inactive PKA
Nucleus
Nuclear membrane
Abbreviations: ACTH, adrenocorticotropic hormone; ATP, adenosine triphosphate; C, catalytic subunit of PKA; cAMP, cy- clic adenosine monophosphate; CREB, cAMP response element; Gsa, G-protein « subunit; GB, G-protein ß subunit; Gy, G- protein y subunit; GDP, guanosine diphosphate; GTP, guanosine triphosphate; P, phosphate; PKA, protein kinase A; R, regulatory subunit of PKA.
Adapted from http://www.biocarta.com/pathfiles/h_gsPathway.asp, with permission.
transcription factors to regulate transcription of Wnt target genes (Fig. 2A). In the absence of Wnt stimulation of its receptor, GSK-3 phosphorylates ß-catenin, resulting in its ubiquitylation and degradation by proteosomes [90] (Fig. 2B).
The Wnt pathway has been implicated in the pathogen- esis of several cancers [91-93], in particular, in patients with familial adenomatous polyposis and in the develop- ment of colorectal carcinomas [94]. The Wnt pathway was analyzed in 26 ACAs and 13 ACCs. Abnormal cytoplasmic and/or nuclear accumulation of B-catenin was found in 10 of 26 (38%) ACAs and 11 of 13 (77%) ACCs, with a focal pattern in ACAs and a diffuse pattern in ACCs. Abnormal immunohistochemisty (IHC) for ß-catenin was also more common in nonfunctioning (83%) than in functioning (25%) ACAs. The ß-catenin gene was screened for muta- tions, with similar frequencies of mutations found in both ACAs and ACCs-seven of 26 (27%) ACAs and four of 13 (31%) ACCs-and only in tumors displaying abnormal ß-catenin immunostaining. Because there is a higher rate of abnormal IHC for B-catenin in ACCs than in ACAs but similar rates of mutation of the B-catenin gene in the two groups, other components of the Wnt signaling pathway, such as APC or axin, may contribute to the pathogenesis of ACCs. Because B-catenin mutation was found in both
ACAs and ACCs, it may be an early step in a common mul- tistep pathogenesis of both ACAs and ACCs [95].
CLONAL ANALYSIS OF ACTS
Knudson [96] hypothesized that the pathogenesis of cancer involves a multistep process with an initiating event fol- lowed by events that result in tumor progression. The initi- ating event is thought to be a mutation in a single cell that confers upon it a growth advantage, resulting in monoclo- nal proliferation of that cell and cancer formation [96].
Clonal analysis of tumors determines the X inactivation patterns in females heterozygous for X-linked polymor- phisms. It is based on the rationale that only a single X chro- mosome is active in each somatic cell of a female. As a result, either the maternal or paternal X chromosome is ran- domly inactivated and this is transmitted in a highly stable fashion to the progeny cell [97]. Because X chromosome inactivation is random, it is expected that there are equal proportions of cells with the maternal and paternal X chro- mosomes. The presence of only either the maternal or the paternal X chromosome in all the cells indicates that the tu- mor is monoclonal.
Three clonal composition studies have shown that 60%- 100% of ACCs are monoclonal while 77.4%-100% of ad- renal hyperplasias and 12.5%-43% of ACAs are
A
PRESENCE OF WNT
Wnt
ZOUp
Frizzled receptor
L
Extracellular
R
P
Cytoplasm
Inhibition
Disheveled
B-catenin
APC
Axin
B-catenin
B-catenin
GSK-3
Transcription of target genes
Nuclear membrane
ß-catenin
Nucleus
TCF/LEF
DNA
B ABSENCE OF WNT
BAB
Frizzled receptor
L
Extracellular
R
P
Cytoplasm
Disheveled
Degradation of ß-catenin
P
B-catenin
Axin
APC
GSK-3
P
B-catenin
Repression of target genes
Nucleus
Nuclear membrane
TCF/LEF
DNA
Abbreviations: APC, adenomatous polyposis coli; GSK-3, glycogen synthase kinase 3ß; LRP, lipoprotein receptor-re- lated protein; P, phosphate; TCF/LEF, T cell-specific tran- scription factor/lymphoid enhancer-binding factor.
Adapted from the Wnt homepage, http://www.stanford.edu/ ~rnusse/wntwindow.html#diagrams, with permission.
polyclonal [98-100]. Interestingly, one study examined both adrenal glands of a patient with AIMAH-a diffuse hyperplastic area and a <1-cm nodule from the right adre- nal gland and a 3.5-cm nodule from the left adrenal gland- and found polyclonality in the two right lesions and monoclonality in the left lesion, suggestive of a transition from hyperplasia to autonomous adenoma-like growth in larger nodules [99]. All three studies concur that ACCs are more often monoclonal, adrenal hyperplasia is more often polyclonal, and ACAs can be either monoclonal or poly- clonal. Polyclonality favors the idea that the tumor devel- oped from a group of cells under the common stimulus of a growth factor, while monoclonality suggests that it devel- oped from a single genetically aberrant cell. The presence of monoclonal and polyclonal ACAs could be a result of ei-
ther different pathological mechanisms or different stages of a common multistep process. It has been suggested that progression to a monoclonal tumor could occur as a result of a first event that initiates the growth of a polyclonal or partially monoclonal tumor with the maintenance of a nor- mal steroid secretory pattern, while a second event would confer a growth advantage in a selected clone of cells, with a concomitant loss of differentiated functions and an aber- rant steroid secretory pattern [98].
COMPARATIVE GENOMIC HYBRIDIZATION ANALYSIS
Comparative genomic hybridization (CGH) is a powerful tool for detecting genetic aberrations in tumors, and DNA copy losses identified on CGH have been shown to corre- late with LOH studies on the subchromosomal level. CGH analyses of ACAs and ACCs have identified clear differ- ences between the two groups [101-104]. There were more copy number changes in ACCs than in ACAs, with a mean number of 7.6-14 changes in ACCs versus 1.1-2 changes in ACAs [102, 103]. Smaller ACAs were less likely to have genetic abnormalities, and chromosomal changes occurred with increasing frequency with increasing tumor size. In ACCs, the number of genetic changes also increased with increasing tumor size [101-103]. The CGH data support the theory of an adenoma-to-carcinoma progression because there are more chromosomal changes in ACCs than in ACAs and the number of these changes increases with in- creasing tumor size.
LOH ANALYSIS
LOH analysis has been used to study chromosomal loci that have been linked to familial syndromes associated with ACTs. Numerous LOH studies have shown that LOH of 17p13 [63, 105, 106] (LFS), 11p15 [62, 63, 105] (BWS), 11q13 [73, 74, 107] (MEN1), 17q22-24 [76] (CNC), and 2p16 [107] (also CNC) tends to occur more frequently in sporadic ACCs than in ACAs (Table 2). Some of these stud- ies have also shown the absence of mutations in the genes involved with hereditary tumor syndromes, suggesting that there are other tumor suppressor genes within these loci that are involved in the pathogenesis of sporadic ACCs. How- ever, putative tumor suppressor genes that could play a role in tumorigenesis have yet to be elucidated.
CURRENT MOLECULAR MARKERS IN CLINICAL PRACTICE
Several studies have assessed the use of IHC molecular markers in discriminating ACCs from ACAs. The markers studied have included IGF2, Ki-67/MIB1, p53, murine double minute 2, p21, p27, cyclin D1, Bcl-2, topoisomerase
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| Table 2. Summary and evidence of genes involved in sporadic ACTs | |
|---|---|
| Gene (chromosomal locus) | Evidence of involvement in sporadic ACTs |
| TP53 (17p13) | Mutation of TP53 found in 20%-27% of ACCs and 0%-6% of ACAs [52, 53]; 17p13 LOH occurs in up to 87.5% of ACCs and up to 30% of ACAs [63, 105, 106] |
| IGF2 (11p15) | Overexpression of IGF2 mRNA in ACCs compared with ACAs [63, 67-69, 134]; 11p15 LOH occurs in up to 83% of ACCs and 34% of ACAs [62, 63] |
| PRKAR1A (17q23-q24) | LOH of 17q22-24 occurs in 53% of ACCs and 23% of ACAs; mutation of PRKARIA occurs in 10% of ACAs and not in ACCs [76] |
| MEN1 (11q13) | LOH of 11q13 occurs in 100% of ACCs and 25% of ACAs [73, 74, 107]; MEN1 mutation occurs in 7% of ACCs and ACAs [73, 74] |
| GNAS (20q13.2) | Mutation of GNAS occurs in ACAs and tumors of patients with AIMAH [80, 81] |
| Abbreviations: ACA, adrenocortical adenoma; ACC, adrenocortical carcinoma; ACT, adrenocortical tumor; AIMAH, adrenocorticotropic hormone-independent macronodular adrenocortical hyperplasia; LOH, loss of heterozygosity. | |
IIa, human epidermal growth factor receptor 2/neu, E- cadherin, and the retinoblastoma gene product [108-112]. Many of these molecular markers, however, lack specificity to achieve discrimination between ACCs and ACAs.
The utility of IGF2 and MIB1 (a mouse monoclonal an- tibody that recognizes a formalin-fixation resistant epitope on the cell proliferation-associated antigen Ki-67) IHC in discriminating between ACCs and ACAs was assessed in one study. Tumors were classified as ACAs or ACCs based on Weiss [5], Hough [113], and van Slooten [114] scores. For IGF2 IHC, 21 of 22 ACAs were negative and 13 of 17 ACCs were positive, giving a specificity of 95.5% and a sensitivity of 76.5%. For MIB1, 21 of 22 ACAs were neg- ative while 14 of 16 ACCs were positive, yielding a speci- ficity of 95.5% and a sensitivity of 87.5%. Combining IGF2 and MIB1 IHC yielded a sensitivity of 100% and a speci- ficity of 95.5% in differentiating ACCs from ACAs [110].
Transcription factors have also been used as possible molecular markers that can differentiate ACCs from ACAs. A member of the nuclear receptor family of transcription factors, steroidogenic factor 1 (SF1) maps to 9q33.3. It has a key role in the development and function of the adrenal cortex [115]. A study on SF1 knockout mice demonstrated that these mice died on postnatal day 8 with severe adreno- cortical insufficiency resulting from an absence of the ad- renal glands [116]. SF1 heterozygous mice have also been found to develop adrenal insufficiency [117]. In CGH stud- ies of 11 pediatric ACTs, James et al. [118] found that 9q34 showed amplification in 10 of the 11 ACTs. This finding was confirmed by Figueiredo et al. [119], who went on to use fluorescence in situ hybridization to confirm that there was a higher SF1 copy number in these tumors. Another study from this group also showed a higher SF1 copy num- ber in eight of 10 pediatric ACTs. SF1 protein levels, how- ever, were noted to be higher in all ACTs than in the normal
adrenal cortex [120]. IHC with SF1 has not been shown to differentiate between ACCs and ACAs [121], but it is use- ful in distinguishing between primary ACC and metastasis from other sites [122].
GATA6 is from the GATA family of transcription fac- tors, which is characterized by binding to the DNA consen- sus sequence (A/T)GATA(A/G). GATA6 plays a role in cellular maturation and differentiation [123]. GATA6 pro- tein expression has been found to be significantly lower in ACCs than in ACAs on IHC. Accordingly, ACTs with Weiss scores of 4-9 had a significantly lower GATA6 level than ACTs with Weiss scores of 1-3 [121].
Vascular endothelial growth factor (VEGF) plays a piv- otal role in the regulation of both normal and tumor angio- genesis [124]. Angiogenesis is critical for tumor growth and metastasis [125]. VEGF has been found to be increased in the majority of cancers and is associated with a poorer outcome [126-129]. One study assessed VEGF expression in 18 ACAs (Weiss score, 0), 12 transitional tumors (Weiss score, 1-3), and 13 ACCs (Weiss score >3) by enzyme- linked immunosorbent assay, and found that VEGF levels were significantly lower in ACAs and transitional tumors than in ACCs; these VEGF levels were unrelated to tumor weight. The VEGF levels for transitional tumors or ACCs that recurred were also higher than for those that did not re- cur [130]. Serum VEGF levels have also been assessed in patients with ACCs versus patients with ACAs, and they were not found to be significantly different between the two groups [131]. VEGF, however, as a molecular marker for ACC, has not been integrated into clinical practice.
THE HUNT FOR NEW MOLECULAR MARKERS
While IGF2 and MIB1 are promising diagnostic markers, they do not predict the clinical behavior of ACCs. Measur- ing LOH at 17p13 has been suggested as a new molecular
| Study | Samples | Upregulated genes | Downregulated genes |
|---|---|---|---|
| Giordano et al. [67] | 11 ACCs, 4 ACAs, 1 macronodular hyperplasia, 3 normal adrenal cortices | IGF2; ubiquitin carrier protein E2-C (UBCH10); KIAA0101; secreted phosphoprotein 1 (SPP1); chromosome 20 open reading frame 1 (C20ORF1) | Alcohol dehydrogenase 1 (ADH1); ADH2; tropomodulin (TMOD); stromal cell-derived factor 1 (SDF1); KIAA1024 |
| de Fraipont et al. [68] | 24 ACCs (Weiss score ≥4); 33 ACAs (Weiss score <4) | IGF2; TGFß2; FGFR1; FGFR4; macrophage stimulating 1 receptor (MSTĪR); TGFBR1; KCNQ1 overlapping transcript 1 (KCNQ10T1); glyceraldehyde-3- phosphate dehydrogenase (GAPD) | Steroidogenic acute regulatory protein (StAR) Cytochrome CYP11A; hydroxy-delta-5- steroid dehydrogenase, 3 beta- and steroid delta-isomerase 1 (HSD3B1); CYP11B1; CYP21A2; CYP17; protein phosphatase 1, catalytic subunit, alpha isoform (PP1A); S100 calcium binding protein B (S100B); glypican 3 (GPC3); inhibin «-chain (INHA); cAMP response element modulator (CREM); retinoblastoma 1 (RB1); nonmetastatic protein 23 (NM23H5); TGFB3 |
| Velázquez et al. [135] | 7 ACCs, 13 ACAs | Ubiquitin specific peptidase 4 (USP4); ubiquitin fusion degradation 1 like (UFDIL); inositol polyphosphate phosphatase-like 1 (INPPLI); aquaporin 3 (AQP3); H3 histone, family 3B (H3F3B) | Chemokine (CXC motif) ligand 10 (CXCL10); retinoic acid receptor responder 2 (RARRES2); aldehyde dehydrogenase 1 family, member A1 (ALDH1A1); cytochrome b reductase 1 (CYBRD1); glutathione S-transferase A4 (GSTA4) |
| Slater et al. [69] | 10 ACCs, 10 ACAs | Cathepsin H (CTSH); mucolipin 3 (MCOLN3); FGFR1; aldo-keto reductase family 1, member C1 (AKRIC1); fibronectin 1 (FN1) | MGC5306; cytoplasmic FMR1 interacting protein 2 (CYFIP2); Purkinje cell protein 4 (PCP4); glutaminyl- peptide cyclotransferase (QPCT); paralemmin (PALM) |
| West et al. [134] | 18 ACCs, 5 ACAs, 1 indeterminate ACT | Thyroid hormone receptor interactor (TRIP); delta-like 3 (DLL3); FLJ22814; dual oxidase 2 (DUOX2); FLJ10458 | Phenylalanine hydroxylase (PAH); major histocompatibility complex, class II, DR alpha (HLA-DRA); pleiomorphic adenoma gene-like 1 (PLAGLI); CYP11B1; HLA-DPA1 |
The first four studies compare expression profiles of adult ACCs versus ACAs, while the last study compares expression profiles of pediatric ACCs versus ACAs.
Abbreviations: ACA, adrenocortical adenoma; ACC, adrenocortical carcinoma; ACT, adrenocortical tumor; CYP, cytochrome P450; FGFR, fibroblast growth factor receptor; IGF, insulin-like growth factor; TGF, transforming growth factor.
marker with the capacity to predict tumor recurrence [63]. A combination of diagnostic and prognostic markers as ad- juncts to the Weiss score could be the most feasible.
Microarray gene-expression analysis is a high-through- put technique that allows the simultaneous analysis of the expression of thousands of genes in a tissue. By comparing the gene-expression profiles of two different groups, such
as ACCs and ACAs, it is possible to identify genes that are significantly upregulated or downregulated in one group relative to the other. The assumption is that these genes are in some way involved in the pathogenesis of these tumors. Overexpressed genes specific to ACCs have the potential to become therapeutic targets. At the time of writing this re- view, five microarray gene-expression profiling studies
Olicologist
comparing ACCs with ACAs had been conducted, four spe- cifically examining adult tumors and one examining pedi- atric tumors. A summary of the top genes identified to be significantly differentially expressed between ACCs and ACAs are listed in Table 3.
Because of the low incidence of ACC, the majority of the microarray gene-expression profiling studies were per- formed using a small number of samples. This, in part, ex- plains the disparity in genes identified across all of the studies. In addition, the different microarray platforms, dif- ferent software and algorithms used to analyze the data, and different significance level cutoffs used also influence the gene lists. Contamination with normal adrenocortical, med- ullary, or stromal tissue could also account for differences in expression profiles. One gene, however, IGF2, was found to be overexpressed in all studies comparing ACCs and ACAs.
Several studies have noted the prognostic significance of molecular markers in ACCs [63, 68, 132]. In one study, an IGF2 gene-related cluster was identified that could se- lect the subgroup of patients with ACCs who were at a high risk for recurrence and who would therefore benefit from adjuvant therapy [68]. This IGF2 gene-related cluster con- tained eight genes. Ninety percent of tumors with low ex- pression of the IGF2 gene-related cluster were ACAs, while 75% of tumors with high expression of these genes were ACCs. In contrast a 14-gene steroidogenesis cluster could identify ACAs with high accuracy. Analyzing a sub- group of 40 tumors with follow-up data showed that either the IGF2 or steroidogenesis cluster of genes alone was not as effective as the Weiss score in terms of predicting ma- lignancy and postoperative recurrence [68]. Another study found that LOH of the 17p13 locus in a cohort of 96 local- ized ACTs was a strong predictor of a shorter disease-free survival time, with a relative risk of 21.5 by multivariate analysis [63]. Volante et al. [132] studied the protein ex- pression of matrix metalloproteinase type 2 (MMP2), also known as gelatinase A, by IHC in a cohort of 50 ACCs and 50 ACAs. They showed that MMP2 was detected in one of
50 (2%) ACAs and 37 of 50 (74%) ACCs (p <. 001). MMP2 protein expression in ACCs was focal in two thirds of cases and diffuse in the remainder. It was also noted that more diffuse expression of MMP2 in ACCs was associated with shorter overall and disease-free survival times [132]. Interestingly, Kjellman et al. [133] assessed MMP2 mRNA in 16 ACCs and 14 ACAs by an mRNA in situ hybridization technique and found that 13 of 16 (81%) ACCs and one of 14 (7%) ACAs expressed MMP2 mRNA. However, the MMP2 mRNA was actually found in surrounding stromal tissue and not in the neoplastic cell itself [133]. Serum lev- els of MMP2 have not been found to be useful in predicting either ACC or ACA [131].
CONCLUSION
Progress into the elucidation of the genes and pathways in- volved in the pathogenesis of ACC has been slow largely because of the rarity of this tumor. The TP53, IGF2, H19, p57kip2, and MEN1 genes are involved in adrenocortical carcinogenesis, as are the ACTH-cAMP-PKA and Wnt pathways. CGH and LOH studies, however, have impli- cated the involvement of many chromosomal regions in which oncogenes or tumor suppressor genes have yet to be identified. Further work is therefore needed to better under- stand the pathogenesis of ACCs.
ACKNOWLEDGMENTS
P. S. H. Soon is supported by the National Health and Med- ical Research Council of Australia, New South Wales Can- cer Institute and Royal Australasian College of Surgeons. The authors would like to thank Dr. Diana Benn for her crit- ical review of the manuscript.
AUTHOR CONTRIBUTIONS
Conception/design: Patsy S. H. Soon, Kerrie L. McDonald, Bruce G. Robin- son, Stan B. Sidhu
Collection/assembly of data: Patsy S. H. Soon
Data analysis and interpretation: Patsy S. H. Soon
Manuscript writing: Patsy S. H. Soon, Kerrie L. McDonald, Bruce G. Robin- son, Stan B. Sidhu
Final approval of manuscript: Patsy S. H. Soon, Kerrie L. McDonald, Bruce G. Robinson, Stan B. Sidhu
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