Introduction

Benign adrenocortical tumors (ACT) are a heterogeneous group of lesions of the adrenal cortex. Somatic and germline mutations in key molecular pathways including cyclic AMP (cAMP) and Wntsignaling pathways have been shown to play pivotal roles in the development of ACT. Even in cases where there are no known mutations⁽¹⁾, the cAMP pathway in particular appears to be involved. The discovery of mutations in GNAS, which encodes the alpha subunit (Gsα) of the stimulatory guanine nucleotide-binding protein, first reported in benign cortisol-producing adenomas (CPA) in patients with McCune-Albright syndrome (MAS), paved the way for identifying the cAMP-signaling pathway as the most important one in the pathogenesis of benign cortisol-producing ACTs. This was rapidly followed by the discovery of mutations in the regulatory subunit type 1-α (RIα) of protein kinase A (PKA, PRKAR1A gene) and phosphodiesterase-11A and -8B (the PDE11A and PDE8B gene respectively) in Carney complex (CNC) and isolated adrenal hyperplasia, and the recently identified germline mutations in the tumor suppressor gene ARMC5 (armadillo repeat containing 5) and somatic mutations in KCNJ5, which have been implicated in the majority of primary bilateral macronodular adrenocortical hyperplasia (PBMAH) ^(2,3) and aldosterone producing adenomas (APA) respectively. Other syndromes with increased predisposition to benign ACT include Carney triad (CT), Carney-Stratakis syndrome (CSS), familial adenomatous polyposis (FAP), and hereditary leiomyomatosis and renal cancer syndrome (HLRCS).

Classification of Benign Adrenocortical Tumors (ACT)

The first comprehensive classification of ACT was proposed in 2007⁽³⁾. Broadly, ACT can be divided into adrenocortical adenomas (ACA), adrenocortical hyperplasia, and adrenocortical cancer (ACC)⁽³⁾. These lesions can be unilateral or bilateral. ACA are classified on the basis of their radiographic and biochemical characteristics as being either functional or nonfunctional and benign or malignant. In a postmortem series, ACA were present in 5% of cases, while adrenocortical hyperplasia was present in 36%⁽⁴⁾. Conversely, PBMAH is estimated to affect 10% and 15% of Cushing syndrome (CS) patients in young adulthood and childhood respectively⁽³⁾, with figures likely to be higher in subclinical CS. Approximately 75-90% of ACT leading to CS are due to a unilateral and benign CPA, with the remainder of the majority are primary pigmented nodular adrenocortical disease (PPNAD), isolated massive adrenocortical disease (iMAD), and PBMAH⁽⁵⁾.

PBMAH was first described in 1964⁽⁶⁾, and was previously called massive macronodular adrenocortical disease (MMAD), bilateral macronodular adrenal hyperplasia (BMAH), or ACTH-independent macronodular adrenocortical hyperplasia (AIMAH). The term PBMAH was proposed given the recent discovery of a local intraadrenal secretion of adrenocorticotropic hormone (ACTH) with an autocrine/paracrine effect on cortisol secretion through aberrant G-protein-coupled receptors (GPCR)^{(7, 8)}. Asynchronous involvement of only one adrenal gland in PBMAH is rare, but could pose a diagnostic challenge for clinicians. Secondary bilateral adrenocortical hyperplasia or adenomatous formation due to excess ACTH stimulation of the adrenal glands from either ectopic ACTH secretion or Cushing disease (dysregulated cortisol production from a pituitary ACTH-secreting tumor), which may become autonomously functioning, should be differentiated from primary ACT due to genetic causes as the workup and management differs.

Cortisol-producing bilateral adrenocortical hyperplasias (BAH) are divided into micronodular (<1 cm in diameter) or macrocronodular (>1cm in diameter)⁽³⁾. An additional feature is pigmentation (i.e.: mainly lipofuscin) within the lesion or the surrounding adrenal cortex, which characterizes a particular type of BAH called PPNAD. A careful histologic examination of the adrenal tissue by an experienced pathologist is therefore a critical step in subtyping the various types of BAH.

The micronodular subtypes are usually diagnosed in children and young adults, and are either pigmented (c-PPNAD, familial as seen in CNC, or isolated, i-PPNAD) or not pigmented (iMAD)⁽³⁾. The macronodular subtypes, which are usually diagnosed in adults over the age of 50, may be sporadic or familial. Syndromic forms are seen with mutations in ARMC5, APC, MEN1, FH and the CT, CSS and HLRCS. Other subtypes of macronodular PBMAH include primary bimorphic adrenocortical disease (PBAD), as seen in MAS, and lesions with GPCR that produce excess cortisol only in response to certain endogenous factors (e.g. gastrointestinal inhibitory polypeptide, GIP), as seen with fooddependent Cushing syndrome (FDCS).

Molecular Pathways in Benign Adrenocortical Tumors

Two major molecular pathways in adrenocortical development have been implicated in the formation of ACT, and include the cAMP and Wnt-singling pathways. Briefly, GPCR (e.g. melanocortin 2 receptor, MC2R) undergo conformational changes in response to a variety of extracellular stimuli, including catecholamines, ACTH, or neurotransmitters. In the case of glucocorticoids, ACTH activates MC2R, a seven-transmembrane receptor, which leads to activation of adenyl cyclase (AC) through a Gsα subunit (encoded by GNAS). This step exchanges GDP for GTP, which in turn converts ATP into cAMP, activating protein kinase A (PKA). PKA is a holoenzyme that consists of a tetramer of two homo- or heterodimer regulatory subunits (R1α, R1β, R2α and R2β), and catalytic subunits (Cα, Cβ, Cγ and PRKX) that are encoded by several genes⁽⁹⁾. Their dissociation in the presence of cAMP enables phosphorylation of PKA targets, including gene expression to mediate cell growth, differentiation and hormone production (e.g.: cortisol). The major function of the regulatory subunits of PKA is to keep the catalytic subunits inactive in the absence of cAMP⁽⁹⁾.

Alterations in any of these complex steps in the cAMP-dependent signaling pathway may predispose to the formation of ACT. The first alterations were reported in GNAS as seen in patients with MAS, followed by CNC through the inactivating mutations in PRKAR1A of PKA. This leads to constitutive activation of the pathway by increasing the availability of the PKA catalytic subunits. However, PKA signaling may either inhibit or stimulate cell proliferation depending on its specific role in the cell cycle^(10,11). The variability in PKA’s cell growth control may partially explain why some biochemically active ACT are small and difficult to detect clinically.

The Wnt-signaling pathway consists of two major pathways; one is β-catenin-dependent and one is β-catenin-independent. The Wnt/β-catenin signaling pathway consists of binding of the ligand to a series of Frizzled family receptors, such as LRP6, which activates phosphoproteins that inhibit the phosphorylation of β-catenin. The nuclear accumulation of β-catenin leads to the transcription of important genes such as WISP2, CTNNB1, and GSK3B, as seen in PBMAH and PPNAD⁽¹²⁾. Both signaling pathways share the downstream activation of certain oncogenic signals, but differ substantially in their effects on others depending on the adrenocortical lesion⁽¹³⁾. This differential effect may partially explain why some somatic activating or inactivating mutations in the same signaling pathway lead to different types of ACT.

Carney Complex (CNC)

Carney complex (CNC) is a hereditary multiple neoplasia syndrome with an autosomal dominant (AD) inheritance due to alterations in PRKAR1A (17q22-24, CNC1 locus), an apparent tumor suppressor gene which encodes for the R1α subunit of PKA⁽¹⁴⁾. CNC is less commonly due to alterations of a yet-unidentified gene on chromosome 2p16 (CNC2) or PRKACB amplification. Over one hundred germline inactivating mutations in R1α of PKA spread along the whole coding sequence, with the majority leading to a premature stop codon by nonsense or frame shift, have been described in approximately 80% of CNC patients^(15,16). The overall penetrance of CNC among PRKAR1A mutations’ carriers is over 95% by the age of 50.

The clinical manifestations of CNC are broad. The main manifestation is CS from PPNAD in approximately 60% of patients. Other tumors include cardiac myxomas, pigmented skin lesions (lentiginosis and blue nevi), somatotroph-pituitary adenomas, benign large cell calcifying Sertoli cell tumor (LCCSCT) of the testis, benign thyroid nodules, differentiated thyroid cancer, and melanocytic schwannomas. In-frame deletion of exon 3 and the c.708 +1G>T mutation appears to confer a more severe CNC phenotype, while the splice variant c.709(-7-2)del6 and the initiationalternating substitution c.1A>G/p.M1Vp lead to an incomplete penetrance of CNC⁽¹⁷⁾. The hotspot c.491-492delTG mutation is more closely associated with lentigines, cardiac myxoma, and thyroid tumors than all other PRKAR1A mutations. Expressed RIα mutant proteins usually present with a more severe and aggressive CNC-phenotype. Conversely, CNC2 occurs later in life with a lower frequency of myxomas, schwannomas, thyroid tumors and LCCSCT. The clinical diagnosis of CNC is established if two or more of the aforementioned major manifestations are present. However, if a patient presents with a family history of CNC and one or more of these manifestations, genetic testing will help establish the diagnosis.

Some patients may present with mild disease with i-PPNAD, with or without accompanied lentiginosis. This “subtype” of CNC is usually diagnosed before 8 years of age and may be due to pathogenic mutations in PRKAR1A, particularly c.709 (-7-2) del6 or c.1A>G/p.M1V, in approximately 50% of cases. Mutations in PDE11A, or PDE8B, have also been described and are detailed later. The diagnosis of i-PPNAD should be considered after a thorough exclusion of CNC, with close surveillance of the other possible manifestations of CNC as they may evolve with time.

Conclusions

The identification of several genetic alterations leading to the formation of benign ACT has paved our understanding of adrenocortical development and disease. Altered genes in the cAMP and Wntsignaling pathways have uncovered new molecular pathways, which have been implicated in the proliferation of adrenocortical cells, with potential therapeutic implications using novel therapies. However, large-scale clinical and molecular studies are underway and needed to further expand our understanding of the genotype-phenotypebiochemical correlations and genetic counseling for affected individuals and their families.

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