CrossMark
The Genetics of Adrenocortical Tumors
Stéphanie Espiard, MDa,b,c, Jérôme Bertherat, MD, PhDa,b,c,d,*
KEYWORDS
. Adrenal cortex . Cancer . Cushing syndrome . PKA . TP53 . IGF2 . Wnt/B-catenin
· ARMC5
KEY POINTS
· Cushing’s syndrome due to primary bilateral adrenal hyperplasias is mainly due to germ- line alterations leading to cAMP/PKA pathway activation or ARMC5 inactivation.
· Cortisol-secreting adenomas are caused by somatic mutations of genes controlling the cAMP/protein kinase A (PKA) pathway, mainly the PKA catalytic subunit alpha.
· The most frequent somatic genetics alterations in adrenocortical carcinomas are muta- tions or deletions of TP53 and CTNNB1 or ZNRF3, altering the p53 and the Wnt/B-catenin pathways, respectively.
INTRODUCTION
Tumors of the adrenal cortex can be diagnosed by the investigation of clinical signs of steroid excess or incidentally on medical imaging. Most tumors are unilateral and can be classified as benign adrenocortical adenoma (ACA) or adrenocortical cancer (ACC). Benign adenomas can be nonsecretory or be responsible for various degrees of cortisol or aldosterone excess. ACAs are frequent in the general population, because it is the most frequent tumor found in adrenal incidentalomas that are present in 1% to 7 % of the general population. By contrast ACC is a rare tumor with an estimated
The authors’ work is supported in part by the ENSAT-CANCER Health-F2-2010-259735 (FP7 program), the Institut national de la santé et de la recherche médicale (S. Espiard is receiving a Poste Accueil), the E-rare # O1GM1407G from the European Community (Genomics of cAMP signaling alterations in adrenal Cushing), and the Conny-Maeva Charitable Foundation.
a Cochin Institut, INSERM U1016, 24 rue du Faubourg Saint Jacques, Paris 75014, France;
b Cochin Institut, CNRS UMR8104, 24 rue du Faubourg Saint-Jacques, Paris 75014, France;
” Paris Descartes University, 12 rue de l’Ecole de Médecine, Paris 75006, France; d Endocrinology Department, Center for Rare Adrenal Diseases, Hôpital Cochin, Assistance Publique Hôpitaux de Paris, 27 Rue du Fg-St-Jacques, Paris F-75014, France
* Corresponding author. Endocrinology Department, Hôpital Cochin, 27 Rue du Fg-St-Jacques, Paris F-75014, France.
E-mail address: jerome.bertherat@cch.aphp.fr
Endocrinol Metab Clin N Am 44 (2015) 311-334
http://dx.doi.org/10.1016/j.ecl.2015.02.004
prevalence between 4 and 12 cases per million. Bilateral tumors are less common and can be classified in 2 groups according to the size of the multiples nodules that develop in both adrenals: primary bilateral macronodular adrenal hyperplasia (PBMAH) and micronodular adrenocortical adrenal hyperplasia.
The current knowledge regarding the development of adrenal cortical tumors (ACTs) suggests that molecular alterations stimulating the proliferation of adrenocortical cells are drivers of this disorder. In this perspective, a genetic or/and epigenetic alteration would occur initially, followed by subsequent secondary events leading to tumor development. The molecular alterations initially described in ACT were identified by the study of rare familial tumor syndrome in which ACA or ACC were observed.1 These candidate gene approaches are based on the hypothesis that a germline alteration of a gene causing a hereditary familial tumor syndrome also occurs as a somatic event limited to a sporadic tumor. This approach was indeed very successful in identifying TP53 mutation or insulin-like growth factor 2 (IGF2) overexpression in ACC, and pro- tein kinase A (PKA) regulatory subunit 1-alpha (PRKAR1A) mutations in ACA.2-5 How- ever, using this approach, only genes that are already identified can be investigated. More recently, the development of pan-genomic approaches to study the genome and epigenome allowed for the identification of many new alterations in cancer.6 The appli- cation of these genomic tools to the study of ACT resulted in major advances in our understanding of the molecular genetics of these tumors. In particular, the use of next-generation sequencing in the past 2 years led to the identification of several new gene alterations in various types of ACT. This review describes the current knowl- edge on the genetics of ACT with a special emphasis on these recent advances (Table 1). Because alterations vary depending on the type of ACT, the review first describes bilateral tumors causing Cushing syndrome (CS), followed by cortisol- secreting adenomas and adrenal cancer.
GENETICS OF BILATERAL ADRENOCORTICAL TUMORS
Bilateral tumors of the adrenal cortex are primary adrenal diseases causing CS char- acterized by the development of multiples nodules in both adrenals. In the micronod- ular forms (micronodular adrenocortical hyperplasia [MiAH]), the diameter of most adrenal nodules is less than 1 cm. MiAH is usually diagnosed in children and young adults. Primary pigmented nodular adrenocortical disease (PPNAD) represents the most frequent form of MiAH and is characterized by the pigmented aspect of the nod- ules. In the macronodular form (PBMAH), the nodules are greater than 1 cm and the total adrenal weight can reach more than 10 times the normal adrenal weight. Apart from very rare pediatric cases in patients with McCune-Albright, PBMAH is usually diagnosed in adults between 50 and 70 years of age. Illegitimate membrane receptors expression as well as intra-adrenal synthesis of adrenocorticotropic hormone (ACTH) have been identified in the pathophysiology of this disease.7-9 The bilateral nature of these tumors might lead to speculation that germline genetic or epigenetic factors play an important role in their development. Indeed many cases of PPNAD are part of a fa- milial syndrome, and some families with PBMAH have been reported as discussed later.
Micronodular Adrenocortical Hyperplasia and Primary Pigmented Nodular Adrenocortical Disease
Protein kinase A regulatory subunit 1-alpha
PPNAD is most often part of a multiple tumors syndrome termed Carney complex (CNC: Online Mendelian Inheritance in Man [OMIM] No. 160980), but it can be
isolated in 12% of cases. CNC is transmitted in an autosomal dominant mode. A PPNAD is detected in 60% of patients with CNC.10,11 The nonendocrine manifesta- tions of CNC are mainly cardiac myxomas, pigmented skin lesions (lentiginosis and nevi), and melanocytic schwannomas. The tumors of endocrine glands observed in CNC include somatotroph-pituitary adenomas, testicular Sertoli cell calcified tumors, benign thyroid nodules, differentiated thyroid cancer, and PPNAD.12,13 Genetic link- age analysis identified 2 loci in chromosome 2p16 and 17p22-24. Loss of heterozy- gosity (LOH) and copy number gains have been described in chromosome 2, but no causative gene at this locus has been found to date. 14 At the 17p locus, inactivating mutations of the PRKAR1A gene have been identified10,15,16 that may explain more than two-thirds of patients with CNC and probably more in typical familial forms of the disease. 10,17 Germline mutations of PRKAR1A lead to the inactivation of the pro- tein and overactivation of the cAMP/PKA pathway (Fig. 1). The mutations are mainly unique because usually each family has its own mutation and that mutation’s hot spots are very limited. The mutations are spread along the whole coding sequence and the adjacent intronic sequences. Eighty percent of the mutations lead to a pre- mature stop codon by nonsense or frame shift, and the mutant mRNA is degraded by the mechanism of nonsense-mediated mRNA decay (NMD).17 The other mutations escape NMD but lead to the production of defective proteins (longer, shorter, or with modified sequence). 17,18 The longest proteins could be degraded by the protea- some leading to haploinsufficiency.19 Additional somatic events in PRKAR1A, such as mutations or LOH in the nodules of PPNAD, suggest that it is a tumor suppressor gene. 17,18 Genotype/phenotype correlations have been established: isolated PPNAD is associated with the hot-spot c.709-7_709-2del,20 whereas mutations escaping NMD or exonic mutations lead to the occurrence of more manifestations of the CNC.10 Mice models confirmed that inactivation of PRKAR1A leads to tumorigen- esis. 13 Particularly, mice with specific inactivation of this gene in the adrenal cortex (AdKO mice) develop adrenal cortex hyperactivity and bilateral hyperplasia as observed in PPNAD.21 However, the role of additive effects of other pathways on the occurrence of tumors is suggested. Several studies have reported an activation of the Wnt/B-catenin signaling, mitogen-activated protein kinase (MAPK) signaling, or the mammalian target of rapamycin (mTOR) pathways and dysregulation of cyclins and the E2F family.22 In particular, PPNAD macronodules can harbor somatic sec- ondary mutations of CTNNB123,24 and overexpression of the Wnt/B-catenin signaling pathway targets is observed in PPNAD,25 suggesting a role of this pathway.
Phosphodiesterases
The phosphodiesterases (PDEs) hydrolyze cAMP and/or cGMP, decreasing cAMP levels after stimulation of the cAMP/PKA pathway (see Fig. 1). Pan-genomic single nucleotide polymorphism (SNP) analysis on DNA chips led to the identification of the 2q31-2q35 locus. Study of candidate genes in this region identified germline- inactivating mutations of PDE11A in patients with MiAH/PPNAD.26 Additionally, PDE11A rare variants with reduced enzymatic activity may play a role in the genetic predisposition to PPNAD; the frequency of these rare PDE11A variants is higher among patients with CNC with PRKAR1A mutations that develop PPNAD.27 An asso- ciation of these rare variants with PBMAH has also been observed.28,29
A germline-inactivating mutation of PDE8B on chromosome 5 has been described in a young girl who had a CS at 2 years of age.3º Somatic mutations of PDE8B have been described in PPNAD, PBMAH, and nonsecreting adenomas.31 PDE8B knockout mice present with increased corticosterone production. 32
| Table 1 Main genes associated with adrenocortical tumors | ||||
|---|---|---|---|---|
| Gene | Locus | Action | Alteration | Tumors and Associated Conditions |
| TP53 | 17q13 | Regulation of genes involved in cell cycle, apoptosis, senescence, DNA repair | Germ mutation Som mutation Som LOH | Li-Fraumeni syndrome (soft tissue sarcoma, breast cancers, brain tumors, ACC) Pediatric sporadic ACC Sporadic ACC |
| ZNRF3 | 22q12.1 | Inhibition of Wnt receptors, inhibition of Wnt/B- catenin pathway | Som mutation Som LOH | Sporadic ACC |
| CTNNB1 | 3q21 | Cytoplasmic/nuclear ß-catenin regulates genes expression involved in proliferation and differentiation | Som mutation | Sporadic ACC Nonfunctional ACA |
| IGFII | 11p15 | Growth factor involved in development and growth | Germ LOH/Epi-genetic alteration (overexpression) Som LOH/Epi-genetic alteration (overexpression) | Beckwith-Wiedemann syndrome (organomegaly, hemihypertrophy, Wilms tumor, ACC) Sporadic ACC |
| Menin | 11q13 | Regulation of genes involved in cell proliferation | Germ mutation Som mutation/LOH | Multiple endocrine neoplasia type 1 (hyperparathyroidism, pituitary adenomas, ACA or adrenal hyperplasia, ACC) Sporadic ACC |
| GNAS1 | 20q13 | Stimulation of adenylate cyclase, activation of the cAMP/PKA pathway | Postzygotic mutation Som mutation | McCune-Albright syndrome (fibrous dysplasia, café-au-lait spots, hyperfunction of endocrine glands, CPA) PBMAH, ACA |
| MC2R | 18p11 | ACTH receptor, activation of PKA pathway | Germ mutation | PBMAH |
|---|---|---|---|---|
| APC | 5q12-22 | Prevent -catenin accumulation, inhibition of Wnt/B-catenin pathway | Germ mutation Som mutation | Familial adenomatous polyposis (colon adenomas and carcinomas, pigmented retinal lesions, desmoids tumors, other malignant tumors, PBMAH, CPA, and ACC) Sporadic ACC |
| PRKAR1A | 17q22-24 | Inhibition of the catalytic subunit of PKA, inhibition of the cAMP/PKA pathway | Germ mutation | Carney complex (miAH, pituitary adenoma, testicular tumors, cardiac myxomas, lentiginosis) Isolated MiAH Sporadic CPA |
| Som mutation/LOH | ||||
| PDE11A | 2q31-35 | Hydrolysis of cAMP, inhibition of the cAMP/PKA pathway | Germ mutation | MiAH |
| PDE8B | 5q13 | Hydrolysis of cAMP, inhibition of the cAMP/PKA pathway | Germ mutation Som mutation | MiAH MiAH, PBMAH, nonfunctional ACA |
| ARMC5 | 16p11 | Regulation of apoptosis | Germ mutation Germ deletion | PBMAH |
| FH | 1q42 | Krebs cycle, amino acid metabolism | Germ mutation | PBMAH |
| PRKACA | 19p13 | Catalytic subunit of PKA, activation of PKA pathway targets | Som mutation Germ amplification | CPA MiAH, PBMAH |
Italic font style: rare feature.
Abbreviations: ACA, adrenocortical adenomas; ACC, adrenocortical carcinoma; CPA, cortisol-producing adenoma; Germ, germline; LOH, loss of heterozygosity; MiAH, micronodular adrenal hyperplasia; PBMAH, primary bilateral macronodular adrenal hyperplasia; Som, somatic.
A Normal adrenal cell
B Adrenocortical hyperplasia
C Adrenocortical adenoma
ACTH
ACTH
ACTH
4)
5)
6)
1)
AAA
Y
a
AC
Y
a
AC
Y ₿
a
Y
a
AC
₿
₿
2)
₿
PDE
P
E
PDE
ATP CAMP
AMP
ATP
CAMP
AMP
ATP CAMP
AMP
PKA
PKA
PKA
R
R
1)
R
R
2)
R
R
R
R
C
C
C
R
R
R
R
C
3)
C
C
R
R
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
3)
CREB
CREB
C
CREB
pCREB
pCREB
pCREB
cre
cre
cre
Catalytic subunits of the protein kinase A
Recently, a comparative genomic hybridization (CGH) study on 35 patients with cortisol-secreting bilateral adrenal hyperplasia demonstrated that 5 patients harbored germline duplications of a region in chromosome 19p that included catalytic alpha subunit of PKA (PRKACA).33 Three patients were young boys with severe disease and micronodular or macronodular bilateral adrenal hyperplasia. The 2 other patients were a mother and her son with mild insidious Cushing syndrome diagnosed after the third decade and caused by bilateral macronodular hyperplasia. Thus, germline dupli- cation of PRKACA seems to also be a cause of bilateral adrenal hyperplasia, associ- ated preferentially with early severe Cushing syndrome (see Fig. 1B). Increased expression of the catalytic subunit alpha mRNA and protein, higher basal, and stimu- lated PKA activity were confirmed in tumor samples from these patients. 33 A germline triplication of chromosome 1p31.1, including PRKACB, has been recently described in a 19-year-old woman presenting with CNC complex without signs of PPNAD at the time of the publication. Young mice carrying a transgene PRKACB exhibited pituitary abnormalities but no adrenal disease. 34
Primary Bilateral Macronodular Adrenal Hyperplasia
PBMAH is characterized by bilateral adrenal hyperplasia associated with multiple nod- ules greater than 1 cm. Hypercortisolism is often associated, occurring generally late and insidiously. However, cortisol secretion can vary widely, as many forms diag- nosed after the investigation of adrenal incidentaloma present with subclinical Cush- ing syndrome and a moderate level of cortisol excess. This disease was previously referred to as ACTH-independent macronodular adrenal hyperplasia or massive macro-nodular adrenal dysplasia; but with the recent discovery that local, intra- adrenal secretion of ACTH is responsible for paracrine cortisol hypersecretion,9 the term PBMAH is now preferred.7 The bilateral nature of the disease and the description of familial forms have suggested a genetic origin. 35-39 PBMAH has served as a model to describe and establish the concept of illegitimate receptors in adrenal Cushing syn- drome. Aberrant expression of G protein-coupled receptors (vasopressin V1, serotonin [HT4], catecholamines, angiotensin, gastric inhibitory polypeptide, luteiniz- ing hormone, human chorionic gonadotropin, adrenergic receptors) is observed in most patients with PBMAH (see Fig. 1B).8,40,41 These receptors usually stimulate the cAMP/PKA signaling pathway, as does ACTH. However, no mutations of these re- ceptors have been found; the explanation of this abnormal expression has not yet been elucidated. Variants in PDE11A are found in more than one-quarter of patients with PBMAH.28,29 These variants lead to an activation of the cAMP/PKA pathway in vitro, suggesting that PDE11A variants with reduced enzymatic activity might play a role in the genetic predisposition to PBMAH.29 Rare mutations of the MC2R gene, encoding for the ACTH-receptor, have been reported in isolated cases of bilateral hy- perplasia (see Fig. 1B).42 In addition, bilateral hyperplasia has been described in other genetic diseases, including the McCune-Albright syndrome (GNAS1) (see Fig. 1B), 43,44 the multiple endocrine neoplasia type 1 (MEN1),45 the familial adenoma- tous polyposis (mutations of APC gene), 46,47 and the hereditary leiomyomatosis and renal cell cancer (fumarate hydratase [FH]).48,49 However, McCune-Albright is observed exclusively in rare cases of very young children; somatic GNAS1 mutations are very rare in adults with PBMAH.43 Mutations of MEN1, APC, or FH do not explain most of the cases of PBMAH in adult patients.
Recently, by a pan-genomic approach using SNP DNA chips to compare tumor and germline DNA in order to identify the region of chromosomal alterations in PBMAH, a copy neutral LOH has been demonstrated at 16p in surgically resected adrenal
nodules from patients with PBMAH.5º By whole-genome sequencing, inactivating mu- tations of armadillo repeat containing 5 (ARMC5) located at 16p have been identified. Assié and colleagues50 demonstrated mutations of ARMC5 in 55% of the 33 patients who underwent surgery for clinical Cushing syndrome. The mechanism of ARMC5 inactivation in patients with BMAH follows the Knudson 2-hit model of a tumor sup- pressor gene: at the germline DNA, one allele presents a mutation (or a large deletion); at the somatic level, the other allele is inactivated by another mutation or by a LOH at 16p.50 Patients in the cohort from the National Institutes of Health presented a germ- line ARMC5 mutation in 21% of the 33 index cases.51 Ten large PBMAH families have been screened by 3 different teams and 8 harbored an ARMC5 mutation.52-54 To date, 12 stops codons, 17 missenses, 21 frame-shifts, 2 deletions of one or more nucleotide acids, and 2 mutations in splicing sites have been described at the germline or the so- matic level.50-54 Besides, further studies are needed to determinate the implication in PBMAH of common variants described in the general population.51 All these muta- tions seemed unique, but the variants p.R898W50,51 and p.R267X50,54 have been found in 3 and 5 unrelated index cases (Fig. 2). The first preliminary genotype- phenotype correlation analysis suggests that ARMC5 is associated with larger hyper- plasia, more nodules, and more severe hypercortisolism.50,51 The function of ARMC5 and its signaling pathway are unknown. It contains 7 armadillo domains and a BTB domain. Such domains have been demonstrated in other proteins that are known to participate in protein-protein interactions. - catenin, the most-known armadillo pro- tein, is also involved in adrenal tumors. The first functional studies have shown a role of ARMC5 in apoptosis. Wild-type protein stimulates apoptosis in vitro, whereas this effect is lost with missense mutants. 50
somatic
protein
ARMC5
germline
LOH at 16p
microdeletion
p.K31X p.L40X
op.158NfsX45
p.A72LfsX36 p.R76X
p.A83RfsX51
pG57EfsX80
p.P93RfsX40
p.158NfsX45 ° **
p.A97GfsX4
p.P103RfsX22
pQ86X
p.A106Rfs*31 p.A106GfsX13
pA104GfsX7
p.S107GfsX9
p.A110RfsX9
p.A110PfsX27
143-
p.C139R
c.476-1G
c.453-475+5del28
p.A206DfsX22
Armadillo repeats
p.A296CfsX33
pR267X
p.R315W
p.R315Q, p.R315W
p.L331P
p.L318V
p.R364X
p.R362W p.R362L
p.L365P
p.W386X
p.E430X
p.L394P
p.E433X
p. T444PfsX17
444-
p.A492PfsX52
# p.T503PfsX34
pL548P
p.V584AfsX18
p.C579SfsX50
p.R593W **
ºp.R619X
p.R619X °
p.R654X
p.C657W p.C657R
p.F700del
BTB (POZ) domain
p.A702_S706del p.T715LfsX2
p.Y736S
748-
p.S779X
816-
p.H808P
p.R898W ***
935
Fig. 2. Germline and somatic mutations of ARMC5 in PBMAH. Location of mutations described to date50-54 on the ARMC5 protein according to the variant ENSP00000268314 (http://www. ensembl.org/). The location of domains is according to UniProtKB (Uniprot Q96C12-1, http:// www.uniprot.org/). Above: germline mutations. Below: somatic mutations and the mutations found on somatic DNA for which the germline or somatic level was not determinate (italic font). The open circle indicates mutations described in germline and somatic DNA. The asterisk indicates mutations described for several patients; each star represents one PBMAH index case. The number sign indicates that the mutation p.T503PfsX34 has been described in a meningioma in a patient presenting with a germline mutation of ARMC5.53
Finally, new candidate genes have been suggested by whole-exome sequencing (WES). DOT1L was mutated in 2 of 7 patients with PBMAH. This gene codes for a his- tone H3 lysine methyl-transferase that regulates gene transcription and cellular prolif- eration. A variant in HDAC9, a histone deacetylase was found in another tumor of this cohort.55 The gene endothelin receptor type A was identified as a putative gene of PBMAH by another WES study.56 Functional studies will be important to confirm the involvement of these newly described gene alterations in the pathophysiology of PBMAH.
CORTISOL-SECRETING ADENOMAS Activation of the Protein Kinase A Pathway
Alpha subunit of protein G
Beyond McCune-Albright in patients with pediatric PBMAH and rare adults, the gene coding for the alpha subunit of the protein Gs (GNAS1) can harbor somatic mutations in rare cortisol secreting tumors (see Fig. 1C).55,57-60 Whole-genome expression profiling (transcriptome) demonstrates activation of different oncogenic pathways such as Wnt/B-catenin pathways for PRKAR1A-mutated tumors and MAPK pathways for GNAS-mutated tumors.61
Catalytic alpha subunit of protein kinase A
Felix Beuschlein and colleagues33 followed quickly by 3 other teams recently identified somatic-activating mutations of the catalytic alpha subunit of PKA (PRKACA) in a sub- set of cortisol-producing adenomas (CPA) using WES (see Fig. 1C).55,58,62 The c.617A>G/p.L206R hot spot mutation was found in heterozygote state in more than 40% of the CPA considering these 4 cohorts.63 A unique mutation, the c.595_596insCAC/L199_Cys200insTrp, was found in one patient with a CPA.33 Muta- tions of PRKACA were mainly found in patients with clinically overt Cushing syndrome. Indeed, patients harboring a mutation are significantly younger at the time of the diag- nosis and present with Cushing’s syndrome and smaller tumors. 33,55,58,62 The hot spot p.L206R is located in a highly conserved active-site cleft of the catalytic subunits to which the regulatory subunits binds. In vitro experiments demonstrated that the 2 mu- tants are constitutively hyperactive and are resistant to inhibition by the regulatory subunits R1alpha58,62 and R2beta.33 The resistance to regulatory subunits R1alpha in- hibition is explained by a loss of interaction between the regulatory subunits and the L206RCa mutant. In adrenal tissue, this mutation leads to activation of the cAMP/ PKA pathway and high expression of steroidogenic enzymes.33,58 The constitutive activation of PKA can be suppressed in vitro by PRKACA inhibitors,55,62 opening a possible therapeutic opportunity.
Protein kinase A regulatory subunit 1-alpha
Somatic mutations of PRKAR1A have been described in CPA both by a candidate gene approach and more recently by WES.2,55 These mutations are inactivating mu- tations similar to the one observed in the germline DNA from patients with CNC. CPA with PRKAR1A mutations tend to be smaller and exhibit a paradoxic increase in urinary cortisol levels after dexamethasone suppression as often observed in pa- tients with CNC.2
Activation of the Wnt/B-Catenin Pathway
The canonical Wnt/B-catenin pathway regulates many cellular processes, including proliferation, differentiation, and apoptosis. In the absence of a Wnt ligand, ß-catenin is located in the cytoplasm, phosphorylated by a destruction complex of scaffolding
proteins, including Axin and APC and then targeted by E3 ubiquitin ligase for proteo- somal degradation. Binding of Wnt ligands to the receptor complex serpentine/low- density lipoprotein receptor leads to recruitment of the disheveled protein and disrup- tion of the destruction complex. Active B-catenin accumulates in the cytoplasm and translocates into the nucleus where it acts as a transcriptional coregulator.64 Tissier and colleagues65 first demonstrated the role of activation of the Wnt/B-catenin pathway in the pathogenesis of ACT. They showed by immunohistochemistry that B-catenin was accumulated in the cytoplasm and the nucleus of ACT in 27% of the benign and 31% of the malignant tumors. This particular pattern reflects the activation of this pathway.
The Wnt/B-catenin pathway is activated in 25% to 50% of ACA according to B-catenin immunohistochemistry analysis.24,65,66 This activation is explained in 70% of the cases by CTNNB1 mutations (Fig. 3).66 CTNNB1 mutations are more frequent in nonsecreting ACA than cortisol-secreting ACA. 66
ADRENOCORTICAL CANCER
Cancer of the adrenal cortex is a rare tumor with an overall poor prognosis. 67 It can be responsible for steroid excess, typically cortisol and androgens; but mineralocorti- coids and steroid precursors can be also oversecreted. Despite an overall poor outcome there is a certain heterogeneity, and some tumors do not recur after removal or progress slowly when metastatic. Molecular genetics can now give some clue as to the cause of this clinical heterogeneity.
Genes and Pathways Involved in Adrenocortical Cancer
Tumor protein p53
Li-Fraumeni syndrome and germline mutation of TP53 Li-Fraumeni syndrome (LFS) (OMIM No. 151623) is an autosomal disorder characterized by predisposition and early onset of several cancers, including sarcoma, breast carcinoma, brain tumors, leuke- mia, and ACC.68,69 Germline mutations of the tumor protein p53 gene (TP53) located at 17p13.1 have been reported in 29% to 90% of cases.68-71 Pediatric ACCs are related to TP53 germline mutations in more than 70% of cases in Europe and North America.72 The incidence of pediatric ACC is remarkably high in southern Brazil, where more than 90% of patients carry the germline TP53 mutation p.R337H. In the state of Paraná in Brazil, a systematic screening of newborns found the hot spot in 0.27% of the 171,649 newborns screened. Around 50% of the carriers’ children were followed dur- ing 3.0 to 6.7 years; the penetrance of ACT was 2.39%.73 In sporadic ACC in adult pa- tients, germline mutations of TP53 are found in between 3.9% and 5.8% of cases. Several patients did not fulfill the diagnostic criteria for LFS.74,75 Twenty-five percent of TP53 mutations appear de novo.76 Beyond these mutations, it has been shown recently that TP53 polymorphisms in adult patients seem to influence overall survival.77
TP53 signaling pathway in sporadic adrenocortical cancer The protein p53, named as the guardian of the genome has a fundamental role in the cellular response to stress, oncogene activation, or DNA damage by regulating the cell cycle and apoptosis. It is the most altered gene in sporadic cancers.76 At the somatic DNA level, mutation of TP53 is a frequent event occurring in between 16% and 70% of ACC if the whole gene is sequenced.78,79 TP53 is considered a tumor suppressor gene, and both alleles are supposed to be inactivated in the tumor tissue. LOH at 17p13 is observed in 85% of ACCs.80 However, other mechanisms may lead to TP53 inactivation because LOH and mutation are not associated in every case.79 The presence of an abnormal nuclear staining of TP53 by immunohistochemistry correlates well with TP53 mutations and
A
Normal regulation of ßcatenin signaling
B Normal activation of ßcatenin signaling
C Activation in adrenocortical carcinomas
3)
ZNRF3
ZNRF3
LRP5/6
Wnt
Frizzled
Frizzled
LRP5/6
Frizzled
LRP5/6
Dvl
Dvl
Axin
P P
1)
Degradation
Axin
Axin
APC
CK1
GSK3
APC
APC
ßcatenin
2)
ßcatenin
CK1
ßcatenin
GSK3
CK1
GSK3
P
P
Degradation
ßcatenin
ßcatenin
ßcatenin
ßcatenin
ßcatenin
ßcatenin
ßcatenin
ßcatenin
LEF/TCF
ßcatenin
LEF/TCF
ßcatenin
ßcatenin
could be used as a diagnostic tool.79 Somatic mutations of TP53 are associated with aggressive tumors and poor outcomes. 79,81
Other actors of the p53 pathways have been recently shown to play a role in the pathogenesis of ACCs. Overexpression of PTTG1, which encodes securin, a negative regulator of p53, was identified as a marker of poor survival.82 Loss of retinoblastoma (Rb) protein has been found by immunohistochemistry in 27% of aggressive ACCs. This defect is related in most cases to mutations of the RB1 gene or its allelic loss.83 Recently, Assié and colleagues78 have studied by several genomic approaches a large European cohort of more than 120 ACCs. Mutations of RB1 were found in 7% of tumors. Eleven percent of the tumors of this cohort harbored mutations of CDKN2A, and 2% exhibited high-level amplification of CDK4. Thus, overall, 33% of the tumors had alterations of the p53 pathway (Fig. 4). Similar findings have been reported in a small series by WES.58
The Wnt/6-catenin signaling pathway
Familial adenomatous polyposis Patients with familial adenomatous polyposis (FAP) (OMIM No. 175100) or Gardner syndrome present multiple colonic polyps and an
Carcinomas (ACC)
Adenomas (ACA)
CHROMOSOMES
Poor prognosis
Good prognosis
High mitotic grade
Alteration
Loss/Gain
LOH
Expression
Cluster
C1A
C1B
IGF2
GENES
TP53
Mutations
CTNNB1
ZNRF3
EPIGENETICS
Cluster
Mi3
Mi2
Mi2
Mi1
miRNAs
miR-483
miR-195
DNA methylation
CIMP
High
Low
Non
PATHWAYS
P53-Rb
Alteration
Wnt- ßcatenin
Present
Moderate
Absent
increased risk of early colon carcinomas. Pigmented retinal lesions, desmoids tumors, osteomas, thyroid adenoma/carcinomas, and other different malignant tumors have also been described.84 Adrenocortical tumors, especially nonfunctional nodular hy- perplasia, CPA, and ACC affects 7% to 13% of patients.64,85 FAP is caused by germ- line inactivating mutation of APC, a tumor suppressor gene that inhibits Wnt/B-catenin signaling (see Fig. 3). According to the Knudson’s model, ACC in patients with FAP exhibits somatic APC mutations as a second hit.86
Activation of the Wnt/B-catenin pathway in sporadic adrenocortical cancer Activation of the Wnt/B-catenin signaling pathway is observed according to ß-catenin immuno- histochemistry in a third of ACC.46,65 Activation of B-catenin is mainly related to muta- tion of its gene CTNNB1.46,65 Sequencing of other actors of the pathway, such as AXIN1, AXIN2, and WTX, failed to show frequent somatic mutations in sporadic ACC.86-88 However, mutations of APC were recently found in 2% of cases from a large cohort of ACC.78 Consistently, transcriptome studies have shown an overexpression of Wnt/B-catenin target genes in ACC.89 Mutations of CTNNB1 or a histologic pattern of its activation are associated with poor outcomes. 46,81 In addition to the observation of a late occurrence of malignancy features in the hyperplastic adrenals from mice with constitutive activation of B-catenin, 90,91 this suggests that Wnt/B-catenin activation is a late driver of tumorigenesis. 64,89 In childhood ACTs, a histologic pattern of B-catenin activation is found in 70% of ACT; but CTNNB1 mutations are not a common event (6%), and it can be associated with TP53 mutations. A decreased expression of Wnt inhibitor genes is observed. Nevertheless, an association with poor prognosis is also found.92
Zinc and ring finger 3 (ZNRF3)
ZNRF3 is a cell-surface transmembrane E3 ubiquitin ligase that is a negative regulator of the Wnt/B-catenin pathways (see Fig. 3). ZNRF3 leads the Wnt-LRP6 receptors complex to degradation. ZNRF3 is regulated by the R-Spondin protein that regulates the association of ZNRF3 with the related leucine-rich repeat-containing G protein- coupled receptors LGR4. This association results in membrane clearance of ZNFR3 and activation of the Wnt/B-catenin pathway.93 Recently, ZNRF3 appeared as the most frequently altered gene (21%) in ACCs. In the cohort of 122 ACCs studied by Assié and colleagues,78 homozygous deletion of the gene was found for 19 tumors, and heterozygous mutations for 7 tumors including 5 with a LOH of the wild type allele. ZNRF3 and CTNNB1 mutations were mutually exclusive (see Fig. 4). Transcriptome of tumors with alterations of ZNFR3 shows activation of B-catenin targets but milder than the level observed in CTNNB1-mutated ACC. ZNFR3 also constitutes a new tumor suppressor gene. By the sum of the CTNNB1- and ZNRF3-altered ACC, activation of the Wnt/B-catenin pathway could be present in 39% of ACC.78
Insulin-like growth factor 2 (IGF2)
Beckwith-Wiedemann syndrome Beckwith-Wiedemann syndrome (BWS) (OMIM No. 130650) is a pediatric disease leading to visceromegaly (macroglossia, hemihyperpla- sia), malformations (wall defect, umbilical hernia), and predisposition to embryonal malignancies. Fetal adrenocortical cytomegaly is a specific feature. ACCs are one of the several malignant tumors to which patients are predisposed.94 Dysregulation of the locus 11p15.5 by genetic and epigenetic alterations is responsible for the dis- ease. This locus includes the genes H19, IGF2, and CDKN1C (p57kip2). H19, a 2,3 kb-long noncoding RNA linked with several cancers, is expressed from the maternal allele, IGF2, from the paternal. Both are regulated by an imprinting center (IC1) usually methylated on the paternal chromosome and unmethylated on the maternal. The
maternal expression of CDKN1C is under the control of another imprinting center (IC2). In BWS, gain of methylation at IC1 (5% of patients) or loss of methylation of IC2 (50% of patients) leads to overexpression of IGF2 by biallelic expression of the gene and reduced expression of CDKN1C and H19. These methylation changes occur with different genomic alterations as DNA methylation changes, uniparental disomy, copy numbers variations, and mutation of CDKN1C.94,95 Beyond these genetic/epige- netic alterations, the physiopathology of BWS is not yet well understood. BWS related to CDKN1C mutations or loss of maternal methylation have a lower risk of tumors in comparison with the gain of methylation of IC1,94 suggesting that overexpression of IGF2 or downregulation of H19 participates in the process of tumorigenesis.
Alteration of the 11p15 locus and insulin-like growth factor 2 overexpression in sporadic adrenocortical cancer IGF2 is a growth factor that stimulates proliferation and inhibits apoptosis in several cell types through the MAPK and PI3K signaling path- ways. Overexpression of IGF2 was one of the first molecular events characterized in about 90% of ACCs. Like in BWS, Gicquel and colleagues4 described a loss of maternal allele methylation, or duplication of the paternal allele, leading to overexpres- sion of IGF2. Several transcriptome studies have confirmed that IGF2 overexpression is observed in more than 85% of ACC and that this gene is the top upregulated gene in ACC as compared with normal adrenal or ACA.96-99 Overexpression of the IGF-I receptor (IGF1-R), upregulation of IGFBP2, and downregulation of the IGF2 receptors have also been described.100 An early clinical trial with an inhibitor of the IGF1-R demonstrated partial responses in some patients.101 In pediatric cases, an overex- pression of IGF2 is also found in adenomas; but expression of IGF1-R is higher in ACC and associated with a worse prognosis.102 However, in adult ACC, overexpres- sion of IGF2 is not associated with a worse prognosis. 103 Furthermore inhibition of the IGF receptors to treat adrenal cancer has not yet proved to be very efficient. A tran- scriptome study did not show a different profile of gene expression between ACC with or without IGF2 overexpression.89 Surprisingly, mice models with overexpression of IGF2 fail to show a strong oncogenic potential even in association with B-catenin activation.100,104 The tumorigenic potential of IGF2 is also misunderstood. Because of the genetic alterations at the 11p15 locus, a possible involvement of H19 or the microRNA miR-483 (see later discussion) located at 11p15 is suggested. Mutations of CDKN1C are not commonly found in ACC.105 H19 is a 2,3 kb-long noncoding RNA linked with several cancers. However, it seems to play a dual role independent of the tumor, either tumor suppressor or oncogene. For example, it could be upregu- lated by the cMyc transcription factor or downregulated by p53 in function of the tumor models. 106 In ACC, H19 expression is low,98,107 suggesting a potential tumor suppressor role.
The other genetic alterations
Others syndromes The MEN1 is an autosomal dominant syndrome caused by a germ- line mutation of the MEN1 gene (11q13) (OMIM No. 131100). It includes primary hyper- parathyroidism (95%), endocrine pancreatic tumors (50%), pituitary adenomas (40%), and thymic carcinoid.108 ACT and/or hyperplasia are reported in 9% to 55% of the patients. These tumors are mainly benign and nonfunctional tumors, although some malignant lesions and CPA have been described.45,108 Somatic mutations of MEN1 were not clearly reported initially in sporadic ACC.109,110 However, a recent WES revealed somatic MEN1 mutations in 7% of screened ACC.78
The Lynch syndrome or hereditary nonpolyposis colorectal cancer syndrome (OMIM No. 120435) is an autosomal dominant disease secondary to mutations in
DNA mismatch-repair genes. Several other malignant tumors are associated with the disease, including ACC.111,112 In a recent retrospective cohort of ACC, 3.2% of pa- tients had a Lynch syndrome. 112
The occurrence of ACC has been reported in patients presenting other syndromes as the CNC but the contribution of the molecular defects to the malignancy is unclear. 113-115
Other somatic alterations Mutations in TERT, DAXX, and ATRX have been recently found in a European cohort of ACC by WES and SNP analysis.78 These genes are involved in telomere length maintenance. TERT codes for telomerase, and its muta- tions are involved in several cancers. DAXX and ATRX mutations have been associ- ated with the maintenance of telomere length by a telomerase-independent mechanism.78 The specific role of these genes in adrenocortical tumorigenesis war- rants further investigation.
Chromosomal Alterations
Some chromosomal alterations can be found by the histologic examination of the tu- mor. ACC presents with a high mitotic grade associated with abnormal mitotic figures, aneuploidy, or polyploidy.116 Chromosomal alterations in ACC have been well charac- terized by different approaches: by conventional CGH, 117-119 by CGH array, 120-122 and, recently, by SNP array.78,123 Alterations in copy numbers in ACCs are consider- ably higher in comparison with benign tumors.120 If the alteration of certain loci is inconstant, copy number gains involving chromosomes 5, 7, 12, and 20 and losses in chromosome 22 were more often reported (see Fig. 4). Copy-neutral LOH detected by SNP array occurs in 30% to 90% of ACC.78,123 SNP array allows the identification of gene amplification or deletion. In the European cohort of ACC, these alterations have been found in the locus of TERT, CD4, CDKN2A, RB1, and ZNFR3 (see earlier discussion) and in the locus 3q13.1 and 4q34.3 where the long noncoding RNA LOC285194 and LINC00290 are respectively located. An implication of the involve- ment of these 2 noncoding RNA has been suggested.78 The diagnostic and prognostic potentials of these chromosomal alterations need to be confirmed.6,120,123 Indeed, ACA presents with LOH in about 25% of cases123 and can have a gain of material, especially in larger adenomas. 117,118,120
Epigenetics Alterations in Adrenocortical Cancer
Methylation
DNA methylation is the most characterized epigenetic mechanism of regulation of transcription. This methylation occurs in the cytosine of CpG dinucleotides that are particularly enriched in specific regions of the promoter called CpG islands.124 Beyond the abnormalities described at the IGF2 locus, a global alteration of methylation pattern has been described in ACC by 3 studies done at the genome-wide level. 125-127 In these studies, the ACC presented a hypomethylation of intergenic regions127 and a global hypermethylation of promoter regions. 125-127 The profile of methylation of ACT could discriminate ACC from ACA.127 Additionally, the levels of methylation of CpG islands distinguish 2 groups of ACC: one, named non-CIMP (CpG island methylator phenotype), is slightly hypermethylated compared with ACA and another hypermethy- lated named CIMP. Within the CIMP group, 2 further subgroups were delineated: CIMP-low and CIMP-high, referring to the levels of hypermethylation (see Fig. 4). The prognosis was worse for the CIMP carcinomas than the non-CIMP and worse for the CIMP-high than the CIMP-low.125 The CIMP-high corresponded to the group C1Ax (poor prognosis without mutations) and C1A-p53 (poor prognosis with TP53
mutations) and the non-CIMP corresponded to the C1A/B-catenin and the C1B group (good prognosis) as determined by transcriptome analysis (see Fig. 4).78,81,125 The hypermethylated genes included genes involved in cell cycle and apoptosis regula- tion126 or in cell proliferation and immune response.125 Their levels of expression are inversely correlated to the level of methylation as expected. 124,126
microRNAs
MicroRNAs (miRNAs) are small RNAs (approximately 22 nucleotides). They play an important role in the posttranslational regulation of gene expression by targeting mRNAs for cleavage or translational repression. A deregulation of the expression of the miRNAs is involved in several cancers through activation of oncogenes or silencing of suppressor gene tumors. Their profile of expression has been proposed as diagnostic or prognostic markers, achievable in practice because they can be detected in blood.128 In ACC, a deregulation of several miRNAs has been observed. The determination of their expression profiles in adrenocortical tumors was the aim of several recent studies. The expression of miRNAs seems to differ significantly between ACAs and ACCs. Several miRNAs were particularly highlighted. The difference between the delta cycle threshold of the high-expressed miR-503 and low-expressed miR-511 was proposed to distinguish benign tumor from malignant tumors.129 A high expression of the miR-483-5p in ACC130-133 and a low expression of miR-195 and miR-335130-134 have been well demonstrated. The level of circu- lating miR-483-5p allows for the distinction between ACC and benign tumors. 133,135 In childhood adrenocortical tumors, a set of miRNAs harbors a differential expres- sion in comparison with normal adrenal tissue. These miRNAs are mainly downregu- lated (miR-99a, miR-100); however, an upregulation of miR-483 is found in childhood ACC similar to that observed in adult ACC. The profile of miRNA expression shows 3 clusters in ACC associated with different prognoses.132 In a European cohort of ACC,78 3 clusters of miRNA have been differentiated: Mi1, Mi2, and Mi3. Mi1 pre- sents the largest difference of miRNA expression in comparison with normal adrenal samples and is associated to the poor prognosis group C1A (see Fig. 4). High levels of miR-483-5p and low levels of miR-195 in tumor tissue130 or in the blood133 are associated with a worse prognosis. The miRNA expression pattern could differ for certain specific ACC variants, such as oncocytic ACC.136 In vitro studies have shown that the level of miR-483-5p and miR-195 affects cell proliferation and death.132 In particular, the miR-483-3p is inversely correlated with the expression of the proapoptotic protein PUMA (p53 upregulated modulator of apoptosis) sug- gesting a role for this miR in apoptosis regulation.132 The gene encoding miR-483- 5p is located in the IGF2 locus, and the level of IGF2 expression is directly correlated with this miRNA. 131,135 The miR-99a and miR-100 participate in the regulation of mTOR signaling, a pathway involved in cellular proliferation in ACC.137
SUMMARY
Recent advances in the discovery of the genetic alterations of adrenocortical tumors have been possible with genomics methods. However, the genetic defects associated with some adrenocortical tumors, including non-PRKACA CPA or non-ARMC5 PBMAH, are still unknown and represent the new challenge for these whole- genome studies. Large-scale clinical studies are now needed to determine the precise role of the molecular tools derived from these pan-genomic studies in the diagnosis and the prognostication of adrenocortical tumors. The elucidation of the molecular ge- netics underlying the pathogenesis of adrenocortical tumors paves the way toward personalized medicine. The identification of new genes involved in adrenal cortical
tumorigenesis, such as ARMC5 and ZNRF3, opens new questions about their physi- ologic and pathologic functions. A greater understanding of the role of these new genes is essential in order to develop innovative, targeted therapies for tumors of the adrenal cortex.
REFERENCES
1. Libé R, Bertherat J. Molecular genetics of adrenocortical tumours, from familial to sporadic diseases. Eur J Endocrinol 2005; 153:477-87.
2. Bertherat J, Groussin L, Sandrini F, et al. Molecular and functional analysis of PRKAR1A and its locus (17q22-24) in sporadic adrenocortical tumors: 17q los- ses, somatic mutations, and protein kinase A expression and activity. Cancer Res 2003;63:5308-19.
3. Gicquel C, Raffin-Sanson ML, Gaston V, et al. Structural and functional abnor- malities at 11p15 are associated with the malignant phenotype in sporadic adre- nocortical tumors: study on a series of 82 tumors. J Clin Endocrinol Metab 1997; 82:2559-65.
4. Gicquel C, Bertagna X, Schneid H, et al. Rearrangements at the 11p15 locus and overexpression of insulin-like growth factor-II gene in sporadic adrenocor- tical tumors. J Clin Endocrinol Metab 1994;78:1444-53.
5. Reincke M, Karl M, Travis WH, et al. p53 mutations in human adrenocortical neo- plasms: immunohistochemical and molecular studies. J Clin Endocrinol Metab 1994;78:790-4.
6. Assié G, Jouinot A, Bertherat J. The ‘omics’ of adrenocortical tumours for personalized medicine. Nat Rev Endocrinol 2014;10:215-28.
7. Lacroix A. Heredity and cortisol regulation in bilateral macronodular adrenal hy- perplasia. N Engl J Med 2013;369:2147-9.
8. Lacroix A. ACTH-independent macronodular adrenal hyperplasia. Best Pract Res Clin Endocrinol Metab 2009;23:245-59.
9. Louiset E, Duparc C, Young J, et al. Intraadrenal corticotropin in bilateral macro- nodular adrenal hyperplasia. N Engl J Med 2013;369:2115-25.
10. Bertherat J, Horvath A, Groussin L, et al. Mutations in regulatory subunit type 1A of cyclic adenosine 5’-monophosphate-dependent protein kinase (PRKAR1A): phenotype analysis in 353 patients and 80 different genotypes. J Clin Endocri- nol Metab 2009;94:2085-91.
11. Stratakis CA, Kirschner LS, Carney JA. Clinical and molecular features of the Carney complex: diagnostic criteria and recommendations for patient evaluation. J Clin Endocrinol Metab 2001;86:4041-6.
12. Bertherat J. Carney complex (CNC). Orphanet J Rare Dis 2006;1:21.
13. Espiard S, Bertherat J. Carney complex. Front Horm Res 2013;41:50-62.
14. Matyakhina L, Pack S, Kirschner LS, et al. Chromosome 2 (2p16) abnormalities in Carney complex tumours. J Med Genet 2003;40:268-77.
15. Casey M, Vaughan CJ, He J, et al. Mutations in the protein kinase A R1alpha regulatory subunit cause familial cardiac myxomas and Carney complex. J Clin Invest 2000; 106:R31-8.
16. Kirschner LS, Carney JA, Pack SD, et al. Mutations of the gene encoding the protein kinase A type I-alpha regulatory subunit in patients with the Carney complex. Nat Genet 2000;26:89-92.
17. Horvath A, Bertherat J, Groussin L, et al. Mutations and polymorphisms in the gene encoding regulatory subunit type 1-alpha of protein kinase A (PRKAR1A): an update. Hum Mutat 2010;31:369-79.
18. Groussin L, Kirschner LS, Vincent-Dejean C, et al. Molecular analysis of the cy- clic AMP-dependent protein kinase A (PKA) regulatory subunit 1A (PRKAR1A) gene in patients with Carney complex and primary pigmented nodular adreno- cortical disease (PPNAD) reveals novel mutations and clues for pathophysi- ology: augmented PKA signaling is associated with adrenal tumorigenesis in PPNAD. Am J Hum Genet 2002;71:1433-42.
19. Patronas Y, Horvath A, Greene E, et al. In vitro studies of novel PRKAR1A mutants that extend the predicted Rla protein sequence into the 3’-untrans- lated open reading frame: proteasomal degradation leads to RIa haploin- sufficiency and Carney complex. J Clin Endocrinol Metab 2012;97: E496-502.
20. Groussin L, Horvath A, Jullian E, et al. A PRKAR1A mutation associated with pri- mary pigmented nodular adrenocortical disease in 12 kindreds. J Clin Endocri- nol Metab 2006;91:1943-9.
21. Sahut-Barnola I, de Joussineau C, Val P, et al. Cushing’s syndrome and fetal fea- tures resurgence in adrenal cortex-specific Prkar1a knockout mice. PLoS Genet 2010;6:e 1000980.
22. Yu B, Ragazzon B, Rizk-Rabin M, et al. Protein kinase A alterations in endocrine tumors. Horm Metab Res 2012;44:741-8.
23. Gaujoux S, Tissier F, Groussin L, et al. Wnt/beta-catenin and 3’,5’-cyclic adeno- sine 5’-monophosphate/protein kinase A signaling pathways alterations and so- matic beta-catenin gene mutations in the progression of adrenocortical tumors. J Clin Endocrinol Metab 2008;93:4135-40.
24. Tadjine M, Lampron A, Ouadi L, et al. Detection of somatic beta-catenin muta- tions in primary pigmented nodular adrenocortical disease (PPNAD). Clin Endo- crinol (Oxf) 2008;69:367-73.
25. Horvath A, Mathyakina L, Vong Q, et al. Serial analysis of gene expression in adrenocortical hyperplasia caused by a germline PRKAR1A mutation. J Clin En- docrinol Metab 2006;91:584-96.
26. Horvath A, Boikos S, Giatzakis C, et al. A genome-wide scan identifies mutations in the gene encoding phosphodiesterase 11A4 (PDE11A) in individuals with adrenocortical hyperplasia. Nat Genet 2006;38:794-800.
27. Libé R, Horvath A, Vezzosi D, et al. Frequent phosphodiesterase 11A gene (PDE11A) defects in patients with Carney complex (CNC) caused by PRKAR1A mutations: PDE11A may contribute to adrenal and testicular tumors in CNC as a modifier of the phenotype. J Clin Endocrinol Metab 2011;96:E208-14.
28. Libé R, Fratticci A, Coste J, et al. Phosphodiesterase 11A (PDE11A) and genetic predisposition to adrenocortical tumors. Clin Cancer Res 2008;14:4016-24.
29. Vezzosi D, Libé R, Baudry C, et al. Phosphodiesterase 11A (PDE11A) gene de- fects in patients with acth-independent macronodular adrenal hyperplasia (AI- MAH): functional variants may contribute to genetic susceptibility of bilateral adrenal tumors. J Clin Endocrinol Metab 2012;97:E2063-9.
30. Horvath A, Mericq V, Stratakis CA. Mutation in PDE8B, a cyclic AMP-specific phosphodiesterase in adrenal hyperplasia. N Engl J Med 2008;358:750-2.
31. Rothenbuhler A, Horvath A, Libé R, et al. Identification of novel genetic variants in phosphodiesterase 8B (PDE8B), a cAMP-specific phosphodiesterase highly expressed in the adrenal cortex, in a cohort of patients with adrenal tumours. Clin Endocrinol (Oxf) 2012;77:195-9.
32. Tsai LC, Shimizu-Albergine M, Beavo JA. The high-affinity cAMP-specific phos- phodiesterase 8B controls steroidogenesis in the mouse adrenal gland. Mol Pharmacol 2011;79:639-48.
33. Beuschlein F, Fassnacht M, Assié G, et al. Constitutive activation of PKA cata- lytic subunit in adrenal Cushing’s syndrome. N Engl J Med 2014;370:1019-28.
34. Forlino A, Vetro A, Garavelli L, et al. PRKACB and Carney complex. N Engl J Med 2014;370:1065-7.
35. Gagliardi L, Hotu C, Casey G, et al. Familial vasopressin-sensitive ACTH-inde- pendent macronodular adrenal hyperplasia (VPs-AIMAH): clinical studies of three kindreds. Clin Endocrinol (Oxf) 2009;70:883-91.
36. Imöhl M, Köditz R, Stachon A, et al. Catecholamine-dependent hereditary Cush- ing’s syndrome - follow-up after unilateral adrenalectomy. Med Klin (Munich) 1983;2002(97):747-53 [in Germany].
37. Lee S, Hwang R, Lee J, et al. Ectopic expression of vasopressin V1b and V2 re- ceptors in the adrenal glands of familial ACTH-independent macronodular adre- nal hyperplasia. Clin Endocrinol (Oxf) 2005;63:625-30.
38. Miyamura N, Taguchi T, Murata Y, et al. Inherited adrenocorticotropin- independent macronodular adrenal hyperplasia with abnormal cortisol secretion by vasopressin and catecholamines: detection of the aberrant hormone recep- tors on adrenal gland. Endocrine 2002; 19:319-26.
39. Vezzosi D, Cartier D, Régnier C, et al. Familial adrenocorticotropin-independent macronodular adrenal hyperplasia with aberrant serotonin and vasopressin ad- renal receptors. Eur J Endocrinol 2007; 156:21-31.
40. Hofland J, Hofland LJ, van Koetsveld PM, et al. ACTH-independent macronod- ular adrenocortical hyperplasia reveals prevalent aberrant in vivo and in vitro re- sponses to hormonal stimuli and coupling of arginine-vasopressin type 1a receptor to 11ß-hydroxylase. Orphanet J Rare Dis 2013;8:142.
41. Libé R, Coste J, Guignat L, et al. Aberrant cortisol regulations in bilateral macro- nodular adrenal hyperplasia: a frequent finding in a prospective study of 32 pa- tients with overt or subclinical Cushing’s syndrome. Eur J Endocrinol 2010;163: 129-38.
42. Swords FM, Noon LA, King PJ, et al. Constitutive activation of the human ACTH receptor resulting from a synergistic interaction between two naturally occurring missense mutations in the MC2R gene. Mol Cell Endocrinol 2004;213:149-54.
43. Fragoso MC, Domenice S, Latronico AC, et al. Cushing’s syndrome secondary to adrenocorticotropin-independent macronodular adrenocortical hyperplasia due to activating mutations of GNAS1 gene. J Clin Endocrinol Metab 2003;88: 2147-51.
44. Weinstein LS, Shenker A, Gejman PV, et al. Activating mutations of the stimula- tory G protein in the McCune-Albright syndrome. N Engl J Med 1991;325: 1688-95.
45. Gatta-Cherifi B, Chabre O, Murat A, et al. Adrenal involvement in MEN1. Anal- ysis of 715 cases from the Groupe d’etude des Tumeurs Endocrines database. Eur J Endocrinol 2012; 166:269-79.
46. Gaujoux S, Grabar S, Fassnacht M, et al. ß-catenin activation is associated with specific clinical and pathologic characteristics and a poor outcome in adreno- cortical carcinoma. Clin Cancer Res 2011;17:328-36.
47. Hsiao HP, Kirschner LS, Bourdeau I, et al. Clinical and genetic heterogeneity, overlap with other tumor syndromes, and atypical glucocorticoid hormone secretion in adrenocorticotropin-independent macronodular adrenal hyperpla- sia compared with other adrenocortical tumors. J Clin Endocrinol Metab 2009; 94:2930-7.
48. Matyakhina L, Freedman RJ, Bourdeau I, et al. Hereditary leiomyomatosis associated with bilateral, massive, macronodular adrenocortical disease and
atypical Cushing syndrome: a clinical and molecular genetic investigation. J Clin Endocrinol Metab 2005;90:3773-9.
49. Shuch B, Ricketts CJ, Vocke CD, et al. Adrenal nodular hyperplasia in hereditary leiomyomatosis and renal cell cancer. J Urol 2013; 189:430-5.
50. Assié G, Libé R, Espiard S, et al. ARMC5 mutations in macronodular adrenal hy- perplasia with Cushing’s syndrome. N Engl J Med 2013;369:2105-14.
51. Faucz FR, Zilbermint M, Lodish MB, et al. Macronodular adrenal hyperplasia due to mutations in an armadillo repeat containing 5 (ARMC5) gene: a clinical and genetic investigation. J Clin Endocrinol Metab 2014;99:E1113-9.
52. Alencar GA, Lerario AM, Nishi MY, et al. ARMC5 mutations are a frequent cause of primary macronodular adrenal hyperplasia. J Clin Endocrinol Metab 2014;99: E1501-9.
53. Elbelt U, Trovato A, Kloth M, et al. Molecular and clinical evidence for an ARMC5 tumor syndrome: concurrent inactivating germline and somatic mutations are associated with both primary macronodular adrenal hyperplasia and meningi- oma. J Clin Endocrinol Metab 2015;100(1):E119-28.
54. Gagliardi L, Schreiber AW, Hahn CN, et al. ARMC5 mutations are common in fa- milial bilateral macronodular adrenal hyperplasia. J Clin Endocrinol Metab 2014; 99:E1784-92.
55. Cao Y, He M, Gao Z, et al. Activating hotspot L205R mutation in PRKACA and adrenal Cushing’s syndrome. Science 2014;344:913-7.
56. Zhu J, Cui L, Wang W, et al. Whole exome sequencing identifies mutation of EDNRA involved in ACTH-independent macronodular adrenal hyperplasia. Fam Cancer 2013; 12:657-67.
57. Dall’Asta C, Ballarè E, Mantovani G, et al. Assessing the presence of abnormal regulation of cortisol secretion by membrane hormone receptors: in vivo and in vitro studies in patients with functioning and non-functioning adrenal ade- noma. Horm Metab Res 2004;36:578-83.
58. Goh G, Scholl UI, Healy JM, et al. Recurrent activating mutation in PRKACA in cortisol-producing adrenal tumors. Nat Genet 2014;46(6):613-7.
59. Kobayashi H, Usui T, Fukata J, et al. Mutation analysis of Gsalpha, adrenocorti- cotropin receptor and p53 genes in Japanese patients with adrenocortical neo- plasms: including a case of Gsalpha mutation. Endocr J 2000;47:461-6.
60. Libé R, Mantovani G, Bondioni S, et al. Mutational analysis of PRKAR1A and Gs(alpha) in sporadic adrenocortical tumors. Exp Clin Endocrinol Diabetes 2005; 113:248-51.
61. Almeida MQ, Azevedo MF, Xekouki P, et al. Activation of cyclic AMP signaling leads to different pathway alterations in lesions of the adrenal cortex caused by germline PRKAR1A defects versus those due to somatic GNAS mutations. J Clin Endocrinol Metab 2012;97:E687-93.
62. Sato Y, Maekawa S, Ishii R, et al. Recurrent somatic mutations underlie corticotropin-independent Cushing’s syndrome. Science 2014;344:917-20.
63. Espiard S, Ragazzon B, Bertherat J. Protein kinase A alterations in adrenocor- tical tumors. Horm Metab Res 2014;46(12):869-75.
64. Berthon A, Martinez A, Bertherat J, et al. Wnt/B-catenin signalling in adrenal physiology and tumour development. Mol Cell Endocrinol 2012;351:87-95.
65. Tissier F, Cavard C, Groussin L, et al. Mutations of beta-catenin in adrenocortical tumors: activation of the Wnt signaling pathway is a frequent event in both benign and malignant adrenocortical tumors. Cancer Res 2005;65:7622-7.
66. Bonnet S, Gaujoux S, Launay P, et al. Wnt/B-catenin pathway activation in adre- nocortical adenomas is frequently due to somatic CTNNB1-activating mutations,
which are associated with larger and nonsecreting tumors: a study in cortisol- secreting and -nonsecreting tumors. J Clin Endocrinol Metab 2011;96:E419-26. 67. Libè R, Fratticci A, Bertherat J. Adrenocortical cancer: pathophysiology and clinical management. Endocr Relat Cancer 2007; 14:13-28.
68. Bougeard G, Sesboué R, Baert-Desurmont S, et al. Molecular basis of the Li- Fraumeni syndrome: an update from the French LFS families. J Med Genet 2008;45:535-8.
69. Malkin D, Li FP, Strong LC, et al. Germ line p53 mutations in a familial syndrome of breast cancer, sarcomas, and other neoplasms. Science 1990;250:1233-8.
70. Ruijs MW, Verhoef S, Rookus MA, et al. TP53 germline mutation testing in 180 families suspected of Li-Fraumeni syndrome: mutation detection rate and rela- tive frequency of cancers in different familial phenotypes. J Med Genet 2010; 47:421-8.
71. Srivastava S, Zou ZQ, Pirollo K, et al. Germ-line transmission of a mutated p53 gene in a cancer-prone family with Li-Fraumeni syndrome. Nature 1990;348:747-9.
72. Faria AM, Almeida MQ. Differences in the molecular mechanisms of adrenocor- tical tumorigenesis between children and adults. Mol Cell Endocrinol 2012;351: 52-7.
73. Custódio G, Parise GA, Kiesel Filho N, et al. Impact of neonatal screening and surveillance for the TP53 R337H mutation on early detection of childhood adre- nocortical tumors. J Clin Oncol 2013;31:2619-26.
74. Herrmann LJ, Heinze B, Fassnacht M, et al. TP53 germline mutations in adult patients with adrenocortical carcinoma. J Clin Endocrinol Metab 2012;97: E476-85.
75. Raymond VM, Else T, Everett JN, et al. Prevalence of germline TP53 mutations in a prospective series of unselected patients with adrenocortical carcinoma. J Clin Endocrinol Metab 2013;98:E119-25.
76. Wasserman JD, Zambetti GP, Malkin D. Towards an understanding of the role of p53 in adrenocortical carcinogenesis. Mol Cell Endocrinol 2012;351:101-10.
77. Heinze B, Herrmann LJ, Fassnacht M, et al. Less common genotype variants of TP53 polymorphisms are associated with poor outcome in adult patients with adrenocortical carcinoma. Eur J Endocrinol 2014;170:707-17.
78. Assié G, Letouzé E, Fassnacht M, et al. Integrated genomic characterization of adrenocortical carcinoma. Nat Genet 2014;46:607-12.
79. Libè R, Groussin L, Tissier F, et al. Somatic TP53 mutations are relatively rare among adrenocortical cancers with the frequent 17p13 loss of heterozygosity. Clin Cancer Res 2007; 13:844-50.
80. Gicquel C, Bertagna X, Gaston V, et al. Molecular markers and long-term recur- rences in a large cohort of patients with sporadic adrenocortical tumors. Cancer Res 2001;61:6762-7.
81. Ragazzon B, Libé R, Gaujoux S, et al. Transcriptome analysis reveals that p53 and {beta}-catenin alterations occur in a group of aggressive adrenocortical cancers. Cancer Res 2010;70:8276-81.
82. Demeure MJ, Coan KE, Grant CS, et al. PTTG1 overexpression in adrenocortical cancer is associated with poor survival and represents a potential therapeutic target. Surgery 2013;154:1405-16 [discussion: 1416].
83. Ragazzon B, Libé R, Assié G, et al. Mass-array screening of frequent mutations in cancers reveals RB1 alterations in aggressive adrenocortical carcinomas. Eur J Endocrinol 2014;170:385-91.
84. Half E, Bercovich D, Rozen P. Familial adenomatous polyposis. Orphanet J Rare Dis 2009;4:22.
85. Beuschlein F, Reincke M, Königer M, et al. Cortisol producing adrenal ade- noma-a new manifestation of Gardner’s syndrome. Endocr Res 2000;26: 783-90.
86. Gaujoux S, Pinson S, Gimenez-Roqueplo A-P, et al. Inactivation of the APC gene is constant in adrenocortical tumors from patients with familial adenomatous pol- yposis but not frequent in sporadic adrenocortical cancers. Clin Cancer Res 2010; 16:5133-41.
87. Chapman A, Durand J, Ouadi L, et al. Identification of genetic alterations of AXIN2 gene in adrenocortical tumors. J Clin Endocrinol Metab 2011;96: E1477-81.
88. Guimier A, Ragazzon B, Assié G, et al. AXIN genetic analysis in adrenocortical carcinomas updated. J Endocrinol Invest 2013;36:1000-3.
89. Assie G, Giordano TJ, Bertherat J. Gene expression profiling in adrenocortical neoplasia. Mol Cell Endocrinol 2012;351:111-7.
90. Berthon A, Sahut-Barnola I, Lambert-Langlais S, et al. Constitutive beta-catenin activation induces adrenal hyperplasia and promotes adrenal cancer develop- ment. Hum Mol Genet 2010; 19:1561-76.
91. Heaton JH, Wood MA, Kim AC, et al. Progression to adrenocortical tumorigen- esis in mice and humans through insulin-like growth factor 2 and B-catenin. Am J Pathol 2012;181:1017-33.
92. Leal LF, Mermejo LM, Ramalho LZ, et al. Wnt/beta-catenin pathway deregulation in childhood adrenocortical tumors. J Clin Endocrinol Metab 2011;96:3106-14.
93. Hao HX, Xie Y, Zhang Y, et al. ZNRF3 promotes Wnt receptor turnover in an R- spondin-sensitive manner. Nature 2012;485:195-200.
94. Weksberg R, Shuman C, Beckwith JB. Beckwith-Wiedemann syndrome. Eur J Hum Genet 2010; 18:8-14.
95. Baskin B, Choufani S, Chen YA, et al. High frequency of copy number variations (CNVs) in the chromosome 11p15 region in patients with Beckwith-Wiedemann syndrome. Hum Genet 2014; 133:321-30.
96. De Fraipont F, El Atifi M, Cherradi N, et al. Gene expression profiling of human adrenocortical tumors using complementary deoxyribonucleic Acid microarrays identifies several candidate genes as markers of malignancy. J Clin Endocrinol Metab 2005;90:1819-29.
97. De Reyniès A, Assié G, Rickman DS, et al. Gene expression profiling reveals a new classification of adrenocortical tumors and identifies molecular predictors of malignancy and survival. J Clin Oncol 2009;27:1108-15.
98. Giordano TJ, Kuick R, Else T, et al. Molecular classification and prognostication of adrenocortical tumors by transcriptome profiling. Clin Cancer Res 2009;15: 668-76.
99. Giordano TJ, Thomas DG, Kuick R, et al. Distinct transcriptional profiles of adre- nocortical tumors uncovered by DNA microarray analysis. Am J Pathol 2003; 162:521-31.
100. Ribeiro TC, Latronico AC. Insulin-like growth factor system on adrenocortical tumorigenesis. Mol Cell Endocrinol 2012;351:96-100.
101. Haluska P, Worden F, Olmos D, et al. Safety, tolerability, and pharmacoki- netics of the anti-IGF-1R monoclonal antibody figitumumab in patients with refractory adrenocortical carcinoma. Cancer Chemother Pharmacol 2010;65: 765-73.
102. Almeida MQ, Fragoso MC, Lotfi CF, et al. Expression of insulin-like growth factor- II and its receptor in pediatric and adult adrenocortical tumors. J Clin Endocrinol Metab 2008;93:3524-31.
103. Guillaud-Bataille M, Ragazzon B, de Reyniès A, et al. IGF2 promotes growth of adrenocortical carcinoma cells, but its overexpression does not modify pheno- typic and molecular features of adrenocortical carcinoma. PloS One 2014;9: e103744.
104. Drelon C, Berthon A, Val P. Adrenocortical cancer and IGF2: is the game over or our experimental models limited? J Clin Endocrinol Metab 2013;98:505-7.
105. Barzon L, Chilosi M, Fallo F, et al. Molecular analysis of CDKN1C and TP53 in sporadic adrenal tumors. Eur J Endocrinol 2001;145:207-12.
106. Guo G, Kang Q, Chen Q, et al. High expression of long non-coding RNA H19 is required for efficient tumorigenesis induced by Bcr-Abl oncogene. FEBS Lett 2014;588:1780-6.
107. Gao ZH, Suppola S, Liu J, et al. Association of H19 promoter methylation with the expression of H19 and IGF-II genes in adrenocortical tumors. J Clin Endo- crinol Metab 2002;87:1170-6.
108. Agarwal SK. Multiple endocrine neoplasia type 1. Front Horm Res 2013;41: 1-15.
109. Heppner C, Reincke M, Agarwal SK, et al. MEN1 gene analysis in sporadic adrenocortical neoplasms. J Clin Endocrinol Metab 1999;84:216-9.
110. Kjellman M, Roshani L, Teh BT, et al. Genotyping of adrenocortical tumors: very frequent deletions of the MEN1 locus in 11q13 and of a 1-centimorgan region in 2p16. J Clin Endocrinol Metab 1999;84:730-5.
111. Karamurzin Y, Zeng Z, Stadler ZK, et al. Unusual DNA mismatch repair-deficient tumors in Lynch syndrome: a report of new cases and review of the literature. Hum Pathol 2012;43:1677-87.
112. Raymond VM, Everett JN, Furtado LV, et al. Adrenocortical carcinoma is a lynch syndrome-associated cancer. J Clin Oncol 2013;31:3012-8.
113. Anselmo J, Medeiros S, Carneiro V, et al. A large family with Carney complex caused by the S147G PRKAR1A mutation shows a unique spectrum of disease including adrenocortical cancer. J Clin Endocrinol Metab 2012;97:351-9.
114. Bertherat J. Adrenocortical cancer in Carney complex: a paradigm of endocrine tumor progression or an association of genetic predisposing factors? J Clin En- docrinol Metab 2012;97:387-90.
115. Morin E, Mete O, Wasserman JD, et al. Carney complex with adrenal cortical carcinoma. J Clin Endocrinol Metab 2012;97:E202-6.
116. Cibas ES, Medeiros LJ, Weinberg DS, et al. Cellular DNA profiles of benign and malignant adrenocortical tumors. Am J Surg Pathol 1990; 14:948-55.
117. Dohna M, Reincke M, Mincheva A, et al. Adrenocortical carcinoma is character- ized by a high frequency of chromosomal gains and high-level amplifications. Genes Chromosomes Cancer 2000;28:145-52.
118. Kjellman M, Kallioniemi OP, Karhu R, et al. Genetic aberrations in adrenocortical tumors detected using comparative genomic hybridization correlate with tumor size and malignancy. Cancer Res 1996;56:4219-23.
119. Zhao J, Speel EJ, Muletta-Feurer S, et al. Analysis of genomic alterations in spo- radic adrenocortical lesions. Gain of chromosome 17 is an early event in adre- nocortical tumorigenesis. Am J Pathol 1999; 155:1039-45.
120. Barreau O, de Reynies A, Wilmot-Roussel H, et al. Clinical and pathophysiolog ical implications of chromosomal alterations in adrenocortical tumors: an inte- grated genomic approach. J Clin Endocrinol Metab 2012;97:E301-11.
121. Stephan EA, Chung TH, Grant CS, et al. Adrenocortical carcinoma survival rates correlated to genomic copy number variants. Mol Cancer Ther 2008; 7:425-31.
122. Szabó PM, Tamási V, Molnár V, et al. Meta-analysis of adrenocortical tumour ge- nomics data: novel pathogenic pathways revealed. Oncogene 2010;29: 3163-72.
123. Ronchi CL, Sbiera S, Leich E, et al. Single nucleotide polymorphism array profiling of adrenocortical tumors-evidence for an adenoma carcinoma sequence? PloS One 2013;8:e73959.
124. Kulis M, Esteller M. DNA methylation and cancer. Adv Genet 2010;70:27-56.
125. Barreau O, Assié G, Wilmot-Roussel H, et al. Identification of a CpG island meth- ylator phenotype in adrenocortical carcinomas. J Clin Endocrinol Metab 2013; 98:E174-84.
126. Fonseca AL, Kugelberg J, Starker LF, et al. Comprehensive DNA methylation analysis of benign and malignant adrenocortical tumors. Genes Chromosomes Cancer 2012;51:949-60.
127. Rechache NS, Wang Y, Stevenson HS, et al. DNA methylation profiling identifies global methylation differences and markers of adrenocortical tumors. J Clin En- docrinol Metab 2012;97:E1004-13.
128. Lujambio A, Lowe SW. The microcosmos of cancer. Nature 2012;482:347-55.
129. Tömböl Z, Szabó PM, Molnár V, et al. Integrative molecular bioinformatics study of human adrenocortical tumors: microRNA, tissue-specific target prediction, and pathway analysis. Endocr Relat Cancer 2009; 16:895-906.
130. Soon PS, Tacon LJ, Gill AJ, et al. miR-195 and miR-483-5p identified as predic- tors of poor prognosis in adrenocortical cancer. Clin Cancer Res 2009;15: 7684-92.
131. Patterson EE, Holloway AK, Weng J, et al. MicroRNA profiling of adrenocortical tumors reveals miR-483 as a marker of malignancy. Cancer 2011;117:1630-9.
132. Özata DM, Caramuta S, Velázquez-Fernández D, et al. The role of microRNA deregulation in the pathogenesis of adrenocortical carcinoma. Endocr Relat Cancer 2011;18:643-55.
133. Chabre O, Libé R, Assie G, et al. Serum miR-483-5p and miR-195 are predictive of recurrence risk in adrenocortical cancer patients. Endocr Relat Cancer 2013; 20:579-94.
134. Schmitz KJ, Helwig J, Bertram S, et al. Differential expression of microRNA-675, microRNA-139-3p and microRNA-335 in benign and malignant adrenocortical tumours. J Clin Pathol 2011;64:529-35.
135. Patel D, Boufragech M, Jain M, et al. MiR-34a and miR-483-5p are candidate serum biomarkers for adrenocortical tumors. Surgery 2013; 154:1224-8 [discus- sion: 1229].
136. Duregon E, Rapa I, Votta A, et al. MicroRNA expression patterns in adrenocor- tical carcinoma variants and clinical pathologic correlations. Hum Pathol 2014; 45:1555-62.
137. Doghman M, El Wakil A, Cardinaud B, et al. Regulation of insulin-like growth factor-mammalian target of rapamycin signaling by microRNA in childhood adrenocortical tumors. Cancer Res 2010;70:4666-75.