The Many Faces of Primary Aldosteronism and Cushing Syndrome: A Reflection of Adrenocortical Tumor Heterogeneity

Adrenal cortical tumors constitute a heterogeneous group of neoplasms with distinct clinical, morphological, and molecular features. Recent discoveries of specific genotype–phenotype correlations in adrenal cortical adenomas have transformed our understanding of their respective endocrine syndromes. Indeed, a proportion of patients with primary aldosteronism are now known to harbor adrenal cortical adenomas with heterogeneous molecular alterations (KCNJ5, ATP1A1, ATP2B3, and CACNA1D) involving the calcium/calmodulin kinase signaling pathway. Several lines of evidence suggest that KCNJ5-mutant aldosterone-producing adenomas have distinct clinicopathological phenotype compared to those harboring ATP1A1, ATP2B3, and CACNA1D mutations. Benign adrenal cortical tumors presenting with Cushing syndrome often have diverse mutations (PRKACA, PRKAR1A, GNAS, PDE11A, and PDE8B) involving the cyclic AMP signaling pathway. In addition to cortisol-producing adenomas, bilateral micronodular adrenocortical disease and primary bilateral macronodular adrenal hyperplasia (PBMAH) have also expanded the spectrum of benign neoplasms causing adrenal Cushing disease. The recent discovery of inactivating ARMC5 germline mutations in PBMAH has challenged the old belief that this disorder is mainly a sporadic disease. Emerging evidence suggests that PBMAH harbors multiple distinct clonal proliferations, reflecting the heterogeneous genomic landscape of this disease. Although most solitary adrenal cortical tumors are sporadic, there is an increasing recognition that inherited susceptibility syndromes may also play a role in their pathogenesis. This review highlights the molecular and morphological heterogeneity of benign adrenal cortical neoplasms, reflected in the diverse presentations of primary aldosteronism and adrenal Cushing syndrome.

clinical manifestations with heterogeneous molecular and histopathologic features, as well as distinctive proliferative biology, biomarker expression, and prognostic cluster profiles. While the distinction of carcinomas from adenomas is critical, the recent discovery of specific genotype-phenotype correlations in aldosterone-and glucocorticoid-producing benign adrenal cortical tumors has significant implications for patient management. This review provides an update on the newly described molecular histophenotypic correlates of benign adrenal cortical tumors, resulting in heterogeneous presentations of hyperaldosteronism and hypercortisolism.
HeTeROgeNeiTY iN ALDOSTeRONe-PRODUCiNg BeNigN ADReNAL CORTiCAL NeOPLASMS

Clinical and Histopathological Heterogeneity
Since the first description by Conn in 1955, much has been learned regarding the clinical, pathological, and molecular properties of primary aldosteronism (1)(2)(3)(4). Inappropriate or autonomous aldosterone secretion, often detected through serum aldosterone-to-renin ratio, is attributed to a group of adrenal cortical proliferations that includes adrenal cortical hyperplasia, adenoma, and rare carcinomas (5,6). Currently, bilateral adrenal cortical hyperplasia is regarded as the most common cause of primary aldosteronism, often diagnosed in older patients with a slight male predilection (1)(2)(3)(4)7). Affected individuals with bilateral disease often present with less severe hypertension and more frequent normokalemia (1)(2)(3)(4)7). In contrast, those with unilateral disease (i.e., aldosterone-producing adenoma) generally have more severe hypertension and more frequent hypokalemia (1)(2)(3)(4)7). Adrenal venous sampling has emerged as an important diagnostic tool to help localize the source of aldosterone excess and to distinguish unilateral from bilateral adrenal disease. Since the latter is treated medically, aldosterone-producing tumors are much more commonly seen in adrenalectomy specimens taken for primary aldosteronism. Most aldosterone-producing adenomas are composed of tumor cells rich in lipid contents. As a result, adenomas typically exhibit lower attenuation (<10 Hounsfield units) on unenhanced CT imaging studies (8,9), and their cut surface often appear golden yellow (5,6). The application of CYP11B2 (aldosterone synthase) immunohistochemistry has enhanced our understanding of CT-detected adrenal nodules, as these may not necessarily represent the source of aldosterone excess, even when ipsilateral lateralization is detected by adrenal vein sampling, likely because additional smaller/microscopic cortical proliferations may be responsible for the hormonal overproduction (10)(11)(12). To complicate this conundrum, zona glomerulosa (ZG) hyperplasia or multiple functional micronodules have also been described in patients with primary aldosteronism (11,13,14).
Most aldosterone-producing tumors are predominantly composed of clear cells with high lipid content. The neoplastic cells contain mitochondria with lamellar type or plate-like cristae. It should be noted that the cytomorphological details of an individual tumor can be heterogeneous due to variation in four distinct types of tumor cells that have been characterized in aldosterone-producing adenomas: (i) zona fasciculata (ZF)-like clear cells, (ii) ZG-like cells, (iii) zona-reticularis-like compact cells, and (iv) compact cells displaying overlapping cytomorphological features of both ZG-and fasciculata-like cells (5,6) (Figure 1). Recent evidence suggests that the heterogeneous cytomorphological features of an aldosterone-producing adenoma may be related to its underlying genotype, reflecting different CYP11B2 (aldosterone synthase) and CYP11B1 (11β-hydroxylase that converts 11-deoxycortisol to cortisol) expression profile in the tumor (see below) (14)(15)(16)(17)(18). The presence of oncocytic cytomorphology also expands the histopathologic heterogeneity of aldosterone-producing adrenal cortical adenomas, including those with intracytoplasmic globular inclusions due to degenerate mitochondria (19). 2 | Molecular alterations in aldosterone-producing benign adrenal cortical neoplasms. In normal physiology, aldosterone production by adrenal zona glomerulosa (ZG) cells is mediated by the calcium/calmodulin signaling pathway. In the resting state, ZG cells are hyperpolarized, a process mainly mediated by K + channels. Activation of the renin-angiotensin system results in the production of angiotensin II, which binds to type 1 angiotensin II receptor (AT2R) causing inhibition of K + currents and depolarization of cell membrane. The latter activates voltage-gated Ca 2+ channel leading to influx of Ca 2+ and activation of the calmodulin kinase pathway, which enables aldosterone synthesis. In primary aldosteronism, mutations implicating the K + channels (KCNJ5), ATPases (ATP1A1, ATP2B3), and Ca 2+ channels (CACNA1D) have been identified in ~60% of sporadic aldosterone-producing adenomas. These molecular alterations are thought to result in autonomous aldosterone production and cellular proliferation through aberrant activation of the calcium/calmodulin kinase signaling pathway.
The non-tumorous adrenal cortex often exhibits aldosterone-producing cell clusters (APCCs) and paradoxical ZG layer hyperplasia to justify the unilateral source of primary aldosteronism in the absence of other CYP11B2-expressing adrenal cortical proliferation (11,18). APCCs typically display a mixture of ZG-and ZF-like cells with both components showing a transcriptome profile similar to that of the ZG layer but with higher CY11B2 expression than that paradoxical ZG layer hyperplasia in a significant proportion of aldosterone-producing adenomas (5,6,11,22) (Figures 1C and 3). The paradoxical ZG layer hyperplasia is typically diagnosed when a continuous ZG layer is identified (5, 6) (Figure 3). In some situations, small microscopic proliferations originating from the ZG layer may extend into the ZF layer, in the setting of a continuous ZG layer (paradoxical hyperplasia with linear growth). There has been recent debate on the biological significance of APCCs, which are generally defined by subcapsular CYP11B2-expressing adrenal cortical cell clusters extending into the ZF (23,24) (Figure 3). Traditionally, pathologists considered these areas to be part of the spectrum of paradoxical hyperplasia; however, variations in the definition of these microscopic findings persist (25). In some studies, regions of APCCs have been described in the normal adrenals glands of patients without evidence of primary aldosteronism (24). In other studies, APCCs were seen in the normal cortex (24). Using microarray sequencing and NGS approach, the identification of somatic mutations in calcium channels and ATPases in APCCs suggested a potential precursor role in the pathogenesis of primary aldosteronism (24). The lack of somatic KCNJ5 mutations in unilateral hyperplasia and in the paradoxical ZG hyperplasia surrounding KCNJ5-mutant aldosterone-producing adenomas is of significance (22). Further studies are required to establish the precursor role of APCCs and its relationship to ZG paradoxical hyperplasia with linear growth in primary aldosteronism.
Several studies reported that KCNJ5-mutant aldosteroneproducing adenomas are associated with younger age, female gender, and larger tumor size than KCNJ5-wild-type aldosteroneproducing adenomas (16-18, 22, 29). In contrast, KCNJ5-wildtype adenomas were reported to be more common in older men and smaller in size (16). In keeping with the correlates of KCNJ5wild-type adenomas, Azizan et al. showed that aldosteroneproducing adenomas with CACNA1D or ATP1A1 mutations were generally smaller than 1 cm (17). Furthermore, KCNJ5-mutant aldosterone-producing adenomas tend to be either solitary tumors or dominant tumors (22). Together, these findings may explain why larger solitary nodules are more frequently observed in female patients, whereas multinodular disease appears to be more commonly found in male patients (18). In the study by Dekkers et al., KCNJ5, ATP1A1, and CACNA1D were variably identified in adrenalectomy specimens of patients with solitary or multiple nodules; interestingly, in multinodular glands, only a single nodule was usually found to harbor mutations (11). In that series, no ATP2B3 mutation was noted in adrenals with multiple nodules (11); however, a subsequent series identified a case with ATP2B3 mutation in one nodule and KCNJ5 and ATP2B3 mutation in another distinct nodule within the same adrenal gland (14). A recent meta-analysis on correlates of KCNJ5-mutant tumors also revealed more pronounced hyperaldosteronism in affected patients (29), whereas no significant differences in blood pressure and serum potassium levels were observed (29).
Several lines of evidence suggest that KCNJ5-mutant tumors are indeed different from those harboring mutations in ATPases and CACNA1D. This distinction stems from differences in both cytomorphological features and expression levels for biomarkers of ZG-(CYP11B2) and ZF-(CYP11B1 and CYP17) like phenotypes (11,(14)(15)(16)(17)(18)29). For instance, aldosterone-producing adenomas with KCNJ5 mutations are enriched in tumors that are predominantly composed of ZF-like clear cells (Figures 1A,B) that show higher CYP17A1 and CYP11B1 expression profiles and significantly lower expression profiles for CYP11B2 (11,(15)(16)(17)(18). In contrast, tumors that are composed predominantly of ZG-like compact cells (Figures 1C,D) are typically enriched in ATP1A1-, ATP2B3-, and CACNA1D-mutant aldosterone-producing adenomas that show increased and strong CYP11B2 expression and predominantly negative CYP11B1 or CYP17A1 expression profiles (11,(15)(16)(17)(18). The series by Chin and colleagues demonstrated that tumors with ZG-like phenotype had an average of 50% compact cells (range, 20-90%) (15). In addition, Monticone et al. demonstrated inverse correlation between tumor size and CYP11B2 expression (18). In fact, this finding is also consistent with the correlates of KCNJ5-mutant tumors. The significant differences between KCNJ5 mutant and wild-type tumors initiated a scientific discussion on the possibility that tumor formation and hyperfunctionality may represent independent processes (14). While these findings question the ZG origin of KCNJ5-mutant adenomas, it also raises the possibility that KCNJ5-mutant tumors may arise from existing adrenal cortical nodules that undergo functional dysregulation, a hypothesis that requires further investigation (27,28). A recent series reported significantly higher Ki-67 proliferation indices (although all groups had Ki-67 labeling indices <5%) in CACNA1D-and ATP1A1-mutant tumors than in those harboring KCNJ5 mutations (15).
Activation of the Wnt/beta-catenin signaling pathway has also been reported in 60-70% of aldosterone-producing adenomas (30). CTNNB1 mutations were reported in ~5% of these tumors (31,32). Several studies have confirmed the tumorigenic role of this mutation as it was mutually exclusive to ion channel-related (KCNJ5, ATP1A1, ATP2B3, and CACNA1D) mutations in primary aldosteronism (14,31,32). CTNNB1-mutant aldosteroneproducing adenomas appear more prevalent in female (31,32) and older patients with a shorter duration of hypertension (31). Aldosterone-producing adenomas with CTNNB1 mutations have been reported to have higher CYP11B2 mRNA and protein (by immunohistochemistry) expression levels compared to those harboring KCNJ5 mutations (32). These tumors were no different than KCNJ5-mutant adenomas with respect to their tumor size, aldosterone levels, and age at the time of diagnosis (32). In addition, Åkerström et al. did not identify specific cytomorphological correlate of CTNNB1-harboring aldosterone-producing adenomas (32). These adenomas exhibited cytoplasmic and/or nuclear beta-catenin expression (32) as well as a variable expression of CYP11B1 (typically expressed in ZF layer) and CYP11B2 (typically expressed in ZG layer) by immunohistochemistry. In some tumors, diffuse CYP11B2 expression with concomitantly low CYP11B1 expression was seen, whereas in other tumors, heterogeneous expression for both CYP11B1 and CYP11B2 was observed (31,32). Some tumors also showed CYP11B2positive and negative regions (14) or diffuse CYP11B1 positivity with low CYP11B2 expression (31,32). Several researchers have hypothesized that CTNNB1 mutations likely play a role in tumorigenesis rather than in aldosterone production (31); however, others have proposed that CTNNB1 mutations play a role in aldosterone overproduction through aberrant activation of beta-catenin that can result in overexpression of AT1 receptor, as well as certain nuclear receptors (e.g., NURR1 and NURR7) and conversion of progesterone into 11β-deoxycorticosterone (26). The MAPK and PI3K/AKT signaling pathways were also reported to be involved in a proportion of sporadic aldosteroneproducing adenomas (27).
Although most aldosterone-producing adrenocortical proliferations are sporadic, three hereditary forms of hyperaldosteronism have been described, accounting for approximately 5% of primary aldosteronism cases (5,6). Of these, type 3 familial hyperaldosteronism is associated with germline KCNJ5 mutations; therefore, a small proportion of seemingly sporadic aldosterone-producing adenomas with KCNJ5 mutations may in fact be a harbinger of this condition. The other two forms of familial hyperaldosteronism are attributed to gene rearrangements involving CYP11B1/CYP11B2 (type I familial hyperaldosteronism) and potential alterations at 7p22 (type II familial hyperaldosteronism). The identification of a novel germline CACNA1H (M1549V) mutation encoding the low voltage activated T-type calcium channel (Cav3.2) has broadened the genomic landscape of familial hyperaldosteronism (33); the adrenalectomy specimen of one affected individual showed ZG layer hyperplasia (33).
Interestingly, primary aldosteronism was also recently described in African American patients harboring germline mutations in the armadillo repeat containing 5 (ARMC5) (34); however, a more recent study of predominantly Caucasian patients with primary aldosteronism did not identify a pathogenetic ARMC5 mutation (35).

Clinical and Histopathological Heterogeneity
Since the first description in 1932, our understanding of endogenous Cushing syndrome has evolved substantially (36). Similar to primary aldosteronism, the causes of adrenal Cushing syndrome encompass a wide spectrum of adrenal cortical proliferations that exhibit clinical, morphological, and molecular heterogeneity. Adrenal Cushing syndrome can present in patients of all ages, including children (36)(37)(38)(39)(40)(41). The clinical presentations range from characteristic biochemical and phenotypical signs and symptoms, as seen in florid Cushing syndrome to much milder or even asymptomatic "subclinical" Cushing syndrome (36)(37)(38)(39)(40).
The histopathologic manifestations of adrenal Cushing syndrome include adrenal cortical hyperplasia, adenoma, and carcinoma (36). With the exception of ACTH-dependent bilateral adrenal cortical hyperplasia, which is due to a pituitary corticotroph tumor or ectopic ACTH-secreting neoplasm, the concept of primary adrenal cortical hyperplasia is a misnomer. Indeed, this is further supported by the fact that hyperplasia typically represents a reversible cell proliferation that is driven by a known stimulus. In fact, when the stimulus stops, the hyperplastic cellular proliferation often regresses if no clonal events are superimposed. Unlike hyperplastic processes, neoplastic proliferations are considered to represent cellular proliferations originating from genetically transformed cells. Therefore, in adrenal Cushing syndrome, diagnostic entities falling into the spectrum of primary bilateral nodular (micronodular or macronodular) adrenal cortical hyperplasia do not represent true hyperplastic phenomenon because these nodular proliferations generally include genetically transformed cells (6,36,(42)(43)(44)(45). Consequently, these lesions should be regarded as multiple benign cortical neoplasms. Given the frequent genetic predisposition of micronodular hyperplasia (nodules < 1 cm), the term "primary bilateral micronodular adrenocortical disease" has been used rather than hyperplasia. However, those presenting with bilateral multiple macronodular cortical proliferations (nodules exceeding 1 cm) have been traditionally referred to as "primary bilateral macronodular adrenal hyperplasia" (PBMAH) despite accumulating evidence indicating the diverse clonal nature of the nodules (42,44).
Primary bilateral micronodular adrenocortical disease frequently presents at a younger age and results in Cushing syndrome. Traditionally, it is divided into two subgroups: (i) a pigmented  subtype, which is also referred to as primary pigmented nodular adrenocortical disease (PPNAD), and (ii) a non-pigmented or scarcely pigmented subtype, which is also known as isolated micronodular adrenocortical disease (i-MAD) (36,(45)(46)(47)(48)(49)(50). The association between inherited genetic predisposition and the pathogenesis of primary bilateral micronodular adrenocortical disease has been well characterized (see Molecular Characteristics and Pathogenesis) (36,45). Consequently, PPNAD is further subdivided based on its association with Carney complex: (i) c-PPNAD for those associated with Carney complex, and (ii) i-PPNAD for isolated cases with may be either familial (due to germline mutations) or sporadic (due to somatic alterations) (36,45,50). Unlike PBMAH, primary bilateral micronodular adrenocortical disease presents with normal size or slightly enlarged adrenal glands (36,(45)(46)(47)(48). Grossly, PPNAD reveals multiple small dark brown nodules typically confined to the adrenal cortex. Histologically, compact cell-rich nodules (with lipofuscin pigment deposition in PPNAD), ranging from 1 to 4 mm, are identified in the deep ZF reticularis (45,48) (Figure 4). In c-PPNAD, the multiple small pigmented nodules may be associated with variable degree of cortical atrophy and mild intracapsular and epicapsular adrenocortical cell extension (45). A recent retrospective review of patients with Carney complex from the Mayo Clinic also described an attenuated form of PPNAD, which was referred as c-PPNAD variant (45). This c-PPNAD variant was characterized by scattered pigmented micronodules instead of numerous pigmented micronodules, and there was accompanying prominent ZF with intracapsular and epicapsular cortical proliferations (45). These observations highlight the heterogeneity of histophenotypic manifestations in c-PPNAD.
Primary bilateral macronodular adrenal hyperplasia is rare and accounts for less than 1% of cases of endogenous Cushing syndrome (51,52). In contrast to the micronodular form, most PBMAH cases are diagnosed in adults aged between 40 and 70 years; however, pediatric forms of PBMAH also exist (36,45,53,54). Affected individuals do not always present with overt Cushing syndrome, as milder forms with subclinical Cushing syndrome have also been described (54). For several years, PBMAH was thought to be primarily a sporadic disease. However, recent advances in our understanding of its molecular basis have highlighted hereditary predisposition mechanisms (see Molecular Characteristics and Pathogenesis). The degree of bilateral adrenal enlargement is also heterogeneous in patients with this disease (54). Grossly, the adrenal glands exhibit a lobulated or bosselated appearance due to multiple irregular yellow nodules, each exceeding 1.0 cm in size (6,36,48). Histologically, multiple unencapsulated irregular cortical nodular proliferations, composed of lipid-rich clear cells or mixed clear-and-compact cells, are noted ( Figure 5). There is significant heterogeneity in both the clinical and histophenotypic manifestations associated with this condition. For instance, patients with McCune-Albright syndrome can also present with PBMAH causing Cushing syndrome in the first year of life. In addition to PBMAH, bilateral primary bimorphic adrenocortical disease and rare examples of adenomas can also occur in the adrenal glands of patients with McCune-Albright syndrome (55).
Glucocorticoid-producing adenomas can occur at all ages, with a slight female predisposition (36,46,47). Most of these tumors are well-delineated cortical neoplasms with a yellow appearance, but pigmented variants (termed "black adenomas") have also been reported (6,48). The non-tumorous cortex is typically atrophic due to the negative feedback suppression of excess glucocorticoid on the hypothalamic-pituitary-adrenal axis. The tumor cells are composed mainly of clear cells that contain mitochondria with tubulovesicular cristae. Scattered compact cells  with variable oncocytic change can occur. While black adenomas are not specific to adrenal Cushing syndrome and can be seen in non-functioning adrenal cortical adenomas, these are composed predominantly of pigmented compact cells (Figure 6). Komiya and colleagues reported that glucocorticoid-producing black adenomas are more frequently associated with lower urinary 17-ketosteroid than glucocorticoid-producing conventional (clear cell rich) adenomas (56). The same study also showed that there are no differences in serum cortisol concentrations and response to dexamethasone suppression between glucocorticoidproducing conventional and black adenomas (56). Interestingly, aldosterone and glucocorticoid co-secreting adenomas have also been described (57,58). The radiological findings of patients with ACTH-independent Cushing syndrome vary substantially based on the underlying pathology. For instance, PBMAH typically shows asymmetric, lobulated and bilateral enlargement of adrenal glands due to multiple large nodules ranging from 1 to 5 cm (36,52). In contrast, those presenting with bilateral multiple micronodular proliferations (e.g., PPNAD) exhibit multiple pigmented hypodense micronodules in a background of slightly enlarged bilateral adrenal glands on CT (59)(60)(61). Glucocorticoid-producing conventional adrenal cortical adenomas are often small (<4.0 cm), solitary, and well demarcated with low attenuation values on CT (8,9,36). The predominance of compact cells in black adenomas can result in higher density on CT when comparing with conventional lipid-rich adenomas (56). Rare examples of bilateral cortisol-secreting adenomas have also been reported (62).

Molecular Characteristics and Pathogenesis
Alterations in the cyclic adenosine monophosphate (cAMP) or protein kinase A (PKA) signaling pathway have been linked to the pathogenesis of glucocorticoid-producing adrenal cortical neoplasms and bilateral adrenocortical micronodular disease (6,36,43,58,63) (Figure 7). In normal physiological conditions, cellular proliferation and functional differentiation of glucocorticoid-producing cells require conformational changes of G-protein-coupled receptors due to binding of ACTH to melanocortin-2 receptors (36). G-protein alpha stimulatory subunit is involved in the activation of adenylyl cyclase to generate cAMP from ATP. An increased cytoplasmic level of cAMP activates PKA, by releasing its catalytic subunits (6,36,63). These free catalytic subunits phosphorylate downstream elements resulting in gene transcription and enabling cortisol synthesis (63). The PKA has a tetrameric structure consisting of two regulatory and two catalytic subunits (63). Phosphodiesterases (PDEs) regulate this process by hydrolyzing cyclic nucleotides including cAMP. Activating mutations in the stimulatory G-protein alpha subunit (GNAS) and the catalytic subunit of PKA (PRKACA), as well as inactivating mutations in the type 1 alpha regulatory subunit of PKA (PRKAR1A) and cAMP-hydrolyzing PDEs (PDE11A and PDE8B), have been linked with various morphological correlates of ACTH-independent adrenal Cushing syndrome (6,36,63,64). Mutations in CTNNB1 have also been described in glucocorticoidproducing adrenal cortical proliferations including adenomas (58,65). Aldosterone and glucocorticoid co-secreting adenomas Cortisol-producing adrenocortical adenomas -Usually unilateral solitary tumors measuring <5 cm -Heterogeneous cytomorphology, ranging from ZF-like clear cell composition ("yellow adenomas") to pigmented compact cell composition ("black adenomas," less common) -Adjacent cortex tends to be atrophic Heterogeneous somatic alterations involving: -cAMP/PKA signaling pathway (PRKACA a , GNAS a , PRKAR1A) -Wnt/β-catenin signaling pathway (CTNNB1) Heterogeneous age of onset, degree of hypercortisolism and tumor size depending on the underlying genetic mutation: -PRKACA-mutant ACAs tend to have ↓ tumor size, earlier age of onset, and more pronounced hypercortisolism -CTNNB1-mutant ACAs tend to have ↑tumor size and less pronounced hypercortisolism Primary micronodular adrenocortical disease -Usually bilateral multifocal tumors measuring <1 cm each -Compact eosinophilic cell cytomorphology -May be pigmented due to lipofuscin storage ("PPNAD") or non-pigmented/scarcely pigmented ("i-MAD") -Adjacent cortex shows variable degree of atrophy Usually germline alterations involving the cAMP/PKA signaling pathway (PRKAR1A a , PDE11A, PDE8B, CNC2 locus, PRKACA) -Heterogeneous presentation depending on the underlying molecular alteration -Frequent association with Carney complex but may occur in isolation -Classic appearance of multiple small "bead-like" nodules on imaging and may be associated with paradoxical cortisol response on Liddle's test -Earlier age of onset and more pronounced hypercortisolism than PBMAH  have been reported to harbor KCNJ5 mutations (57,58). Given the genotype-phenotype correlations in KCNJ5-mutant adenomas (see previous sections), a subset of these tumors may harbor weak glucocorticoid function (21).
Somatic mosaicism of GNAS has been implicated in the pathogenesis of McCune-Albright syndrome-related adrenal Cushing syndrome manifesting as bilateral macronodular adrenocortical disease (e.g., PBMAH), as well as a bimorphic cortical nodular disease and rare adenomas (55). Somatic activating GNAS mutations and somatic allelic loses of PRKAR1A were seen in up to 17 and 23% of glucocorticoid-producing adenomas, respectively (95,96). Interestingly, glucocorticoid-producing adenomas with somatic PRKAR1A alterations were more frequently associated with smaller tumor size and paradoxical increase in urinary cortisol levels following dexamethasone suppression (97). The latter is also a pattern shared with Carney complex-associated primary pigmented adrenocortical nodular disease (c-PPNAD) due to enhanced glucocorticoid receptor expression in the lesional cells (97).
Bilateral micronodular adrenocortical disease is also associated with heterogeneous molecular, histophenotypic, and clinical manifestations. Mutations in PRKAR1A, PDE11A, and PDE8B have all been implicated in the pathogenesis of bilateral micronodular adrenocortical disease presenting with Cushing syndrome (36,43,63,98). Germline inactivating mutations involving PRKAR1A accounts for 80% of c-PPNAD (43,99). A subset of patients with PRKAR1A-wild-type c-PPNAD has been linked to the CNC2 locus at chromosome 2p16; however, little is known with respect to the gene(s) associated with this condition (36,43). Most of the CNC2 locus-related cases tend to present later in life (43). Germline or sporadic inactivating PRKAR1A, PDE11A, and PDE8B mutations have also been described in i-PPNAD causing adrenal Cushing syndrome (36,63). PDE11A and PDE8B are also described in the pathogenesis of i-MAD (36,63).
The role of PDE variants impacting the clinical manifestations of Carney complex patients has also been documented. For instance, a higher frequency of PDE11A variants was noted in male patients with Carney complex presenting with large cell calcifying Sertoli cell tumor (100). Germline PDE11A variant mutation has also been described with adrenal enlargement but without evidence of Cushing syndrome (98). The discovery of PDE11A variants in patients with Carney complex (63) and genomic duplication of the locus of PRKACB (encoding the catalytic C-beta subunit of PKA) in a patient with Carney complex but without evidence of Cushing syndrome support the existence of versatile genomic alterations causing PKA dysregulation (101).
A recent study has broadened our knowledge of pathogenic PRKACA alterations in bilateral micronodular adrenocortical disease presenting with Cushing syndrome (64). In this series, germline duplication of PRKACA was identified in 5 of 35 patients with bilateral nodular cortical disease lacking germline PRKAR1A, PDE11A, PDE8B, and somatic GNAS mutations (31 i-PPNAD, 2 i-MAD, and 2 macronodular type) (64). No significant phenotypic differences were reported between those carrying a germline PRKACA duplication and those lacking this molecular alteration (64). Subsequent review of the adrenocortical morphology of the five patients with germline PRKACA duplication revealed distinct histophenotypes characterized by PPNAD with cortical atrophy in three patients and non-pigmented nodular adrenocortical hyperplasia with extranodular hyperplasia in two patients (102). The variation in phenotypes has been linked to the extent of genomic alteration (103).
In addition to bilateral micronodular adrenocortical disease, PBMAH is also characterized by heterogeneous presentations. Despite significant adrenal gland enlargement, PBMAH is characterized by relatively inefficient and low cortisol overproduction in comparison to its micronodular counterpart, an observation that has attracted interest from the scientific community (36). Poorly developed smooth endoplasmic reticulum in lipid-rich cortical cells and weak reactivity for 3-beta-hydroxysteroid dehydrogenase and other steroidogenic enzymes have been described (36,48). The low steroidogenic capacity of PBMAH has been well documented on several platforms including gene arrays analyses (76,87). Significant downregulation of genes encoding enzymes implicated in steroidogenesis (CYP11A, CYP17, and CYP21A2) has also been observed and used to explain this phenomenon in PBMAH (87). Accordingly, the diseased cells favor proliferation over functionality. The heterogeneity of PBMAH is also reflected by the diverse molecular alterations of this disease. Almeida et al. reported that larger nodules often harbored increased expression of bcl-2, E2F-1, c-KIT, MYB, PRKCA, and CTTNB1 when compared with smaller nodules in PBMAH (104). Assié et al. characterized the ARMC5 heterogeneity of individual nodules in PBMAH by demonstrating distinctive secondary somatic mutations arising in the background of germline susceptibility in individual macronodular proliferations (42). This observation combined with the putative tumor suppressor function of ARMC5 may explain the extensive genetic variance of ARCM5driven PBMAH (44).
The discovery of frequent germline ARCM5 alterations in patients with adrenal Cushing syndrome has advanced our understanding of this disease, especially with regards to its inherent genotype-phenotype correlations. Espiard et al. demonstrated that patients with ARMC5-driven PBMAH presented more frequently with overt Cushing syndrome, higher midnight plasma cortisol, urinary free cortisol, and cortisol after dexamethasone suppression test than those with ARMC5-wild-type disease (54). The same study also demonstrated that ARMC5-wild-type tumors are more frequently associated with subclinical Cushing syndrome and non-functioning status (54). In addition, adrenals with ARMC5driven PBMAH were significantly bigger and had more nodules than those with ARMC5-wild-type presentations (54,85).

Conclusion
Aldosterone-and cortisol-producing adrenal cortical tumors encompass a diverse group of neoplasms with distinct genetic and morphological features. The heterogeneous spectrum of molecular alterations seen in these lesions is increasingly reflected in their distinctive demographic, hormonal, cytomorphologic, and immunophenotypic profiles. Although most cases occur sporadically, an increasing number of tumors arise in the setting of familial syndromes with potential implications for gene testing and counseling. As the future of molecular endocrinology becomes more complex, it is anticipated that modern technologies will allow a deeper understanding of the structure, function, and prognosis of these tumors, which will hopefully translate into more precise therapeutic strategies to improve the management of patients with adrenal cortical disease.

AUTHOR CONTRiBUTiONS
Literature search, writing, and figures: OM and KD. Concept, design, and critical reviews: OM.