Primary Pigmented Nodular Adrenocortical Disease Research Articles

MTOR pathway is activated by PKA in adrenocortical cells and participates in vivo to apoptosis resistance in primary pigmented nodular adrenocortical disease (PPNAD).

Primary pigmented nodular adrenocortical disease (PPNAD) is associated with inactivating mutations of the PRKAR1A tumor suppressor gene that encodes the regulatory subunit R1α of the cAMP-dependent protein kinase (PKA). In human and mouse adrenocortical cells, these mutations lead to increased PKA activity, which results in increased resistance to apoptosis that contributes to the tumorigenic process. We used in vitro and in vivo models to investigate the possibility of a crosstalk between PKA and mammalian target of rapamycin (mTOR) pathways in adrenocortical cells and its possible involvement in apoptosis resistance. Impact of PKA signaling on activation of the mTOR pathway and apoptosis was measured in a mouse model of PPNAD (AdKO mice), in human and mouse adrenocortical cell lines in response to pharmacological inhibitors and in PPNAD tissues by immunohistochemistry. AdKO mice showed increased mTOR complex 1 (mTORC1) pathway activity. Inhibition of mTORC1 by rapamycin restored sensitivity of adrenocortical cells to apoptosis in AdKO but not in wild-type mice. In both cell lines and mouse adrenals, rapid phosphorylation of mTORC1 targets including BAD proapoptotic protein was observed in response to PKA activation. Accordingly, BAD hyperphosphorylation, which inhibits its proapoptotic activity, was increased in both AdKO mouse adrenals and human PPNAD tissues. In conclusion, mTORC1 pathway is activated by PKA signaling in human and mouse adrenocortical cells, leading to increased cell survival, which is correlated with BAD hyperphosphorylation. These alterations could be causative of tumor formation.

In Silico Analyses Reveals Nuclear Asymmetry of Spongiocytes and Compact Cells of Adrenocorticotrophic Hormone-Independent Macronodular Adrenocortical Hyperplasia

BackgroundLittle information is available about the risk of progression of seemingly benign adrenocortical hyperplasias to carcinomatous conditions. Using in silico approaches of digitally archived tissue sections, the nuclear morphometric parameters were compared to assess nuclear asymmetry as an index for nuclear atypia. MethodsFour groups of nuclei were used for the current study: spongiocytes and compact cells obtained from adrenocorticotropic hormone (ACTH)-independent macronodular hyperplasia, which were hypothesized to be high risk for nuclear asymmetry, and primary pigmented nodular adrenocortical disease and micronodular adrenocortical hyperplasia samples were used as internal controls. ResultsAnalyses reveal high nuclear irregularity index of spongiocytes and shape factor abnormalities of both spongiocytes and compact cells of ACTH-independent macronodular adrenal hyperplasia compared with the other 2 groups (high F values and very low P values after analyses of variances), thus confirming the hypothesis that ACTH-independent macronodular adrenal hyperplasia present with subtle morphometric features of nuclear atypia. ConclusionsThis probably puts this class of adrenocortical tumors at risk of dysplastic progression, and more studies are needed to test the hypothesis.

Carney complex presenting with a unilateral adrenocortical nodule: a case report

IntroductionCarney complex is an autosomal dominant syndrome with multiple neoplasms in different sites, including myxomas, endocrine tumors and lentigines lesions. To the best of our knowledge, this is the first report of Carney complex presenting with a unilateral adrenal adenoma associated with a pituitary incidentaloma.Case presentationA 27-year-old Iranian woman was referred to our endocrinology clinic with amenorrhea and hirsutism, further confirming a diagnosis of adrenocorticotropic hormone-independent Cushing’s syndrome. The cause was believed to be a right adrenocortical adenoma based on a computed tomography scan. Our patient underwent a right laparoscopic adrenalectomy and pathological examination revealed pigmented micronodular adrenal hyperplasia. Pituitary magnetic resonance imaging also documented a microadenoma that was considered to be an incidentaloma based on normal pituitary function tests. Recurrence of hypercortisolism led to a left laparoscopic adrenalectomy, providing further evidence for the diagnosis of primary pigmented nodular adrenocortical disease. Carney complex was established in light of her history of cardiac myxomas.ConclusionWe present what we believe to be the first case of Carney complex presenting with a unilateral adrenocortical adenoma in association with a pituitary incidentaloma. Although primary pigmented nodular adrenocortical disease is rare as a component of Carney complex, it should be considered in the differential diagnosis of Cushing's syndrome. Rarely, adrenal and pituitary imaging can be misleading.

Pathology of the Adrenal Cortex: a Reappraisal of the Past 25 Years Focusing on Adrenal Cortical Tumors

A reappraisal of the major advances in the diagnostic pathology of adrenal cortical lesions and tumors in the last 25 years is presented, with special reference to the definition of malignancy in primary adrenal cancer and its variants. Slightly more than 25 years ago, Weiss proposed his diagnostic scoring system for adrenal cortical carcinoma. This represented a milestone for adrenal pathologists and the starting point for further modifications of the system, either through minor changes in the scoring procedure itself or concentrating on some particular Weiss criterion such as mitotic index, integrated into alternative scoring schemes or algorithms that are currently under validation. Improvements in diagnostic immunohistochemistry have led to the identification of markers of cortical origin, such as Melan-A, alpha-inhibin, and SF-1 and of prognostic factors in carcinoma, such as the Ki-67 proliferation index and SF-1 itself. With regard to hyperplastic conditions, genetic investigations have allowed the association of the majority of cases of primary pigmented nodular adrenocortical disease (PPNAD) in Carney complex to mutations in the gene encoding the regulatory subunit 1A of protein kinase A (PRKAR1A). Other hereditary conditions are also associated with adrenal cortical tumors, including the Li-Fraumeni, Beckwith-Wiedemann, Gardner, multiple endocrine neoplasia type 1, and neurofibromatosis type 1 syndromes. Moreover, several advances have been made in the knowledge of the molecular background of sporadic tumors, and a number of molecules/genes are of particular interest as potential diagnostic and prognostic biomarkers.

Does Somatostatin Have a Role in the Regulation of Cortisol Secretion in Primary Pigmented Nodular Adrenocortical Disease (PPNAD)? A Clinical and in Vitro Investigation

Somatostatin (SST) receptors (SSTRs) are expressed in a number of tissues, including the adrenal cortex, but their role in cortisol secretion has not been well characterized. The objective of the study was to investigate the expression of SSTRs in the adrenal cortex and cultured adrenocortical cells from primary pigmented nodular adrenocortical disease (PPNAD) tissues and to test the effect of a single injection of 100 μg of the SST analog octreotide on cortisol secretion in patients with PPNAD. The study was conducted at an academic research laboratory and clinical research center. Expression of SSTRs was examined in 26 PPNAD tissues and the immortalized PPNAD cell line CAR47. Ten subjects with PPNAD underwent a randomized, single-blind, crossover study of their cortisol secretion every 30 minutes over 12 hours (6:00 pm to 6:00 am) before and after the midnight administration of octreotide 100 μg sc. SSTRs expression was investigated by quantitative PCR and immunohistochemistry. The CAR47 and primary cell lines were studied in vitro. The data of the 10 patients were analyzed before and after the administration of octreotide. All SSTRs, especially SSTR1-3, were expressed in PPNAD at significantly higher levels than in normal adrenal. SST was found to differentially regulate expression of its own receptors in the CAR47 cell line. However, the administration of octreotide to patients with PPNAD did not significantly affect cortisol secretion. SSTRs are overexpressed in PPNAD tissues in comparison with normal adrenal cortex. Octreotide did not exert any significant effect on cortisol secretion in a short clinical pilot study in a small number of patients with PPNAD, but long-acting SST analogs targeting multiple SSTRs may be worth investigating in this condition.

Heterogeneous Clinical Manifestations of Cushing’s Syndrome in a Family with Primary Pigmented Nodular Adrenocortical Disease

Purpose: To evaluate the clinical manifestations of Cushing’s Syndrome (CS) and associated genetic mutation in patients with Primary Pigmented Nodular Adrenocortical Disease (PPNAD). Methods: Seven family members were screened for mutations of the PRKAR1A gene. Gene mutation screening used genomic DNA (from peripheral blood leukocytes and, in some cases, adrenal gland tissue) and subsequent DNA sequencing. The five patients showing genetic mutation were assessed clinically for baseline cortisol and adrenocorticotropic hormone and adrenal imaging studies (abdominal computed tomography and adrenal scintigraphy). Low-dose and high-low dexamethasone suppression tests were performed in these five patients. Results: PRKAR1A gene mutation was detected in five of the seven family members. Four of the five gene mutation-positive patients presented with overt CS due to pathology-proven PPNAD. After unilateral adrenalectomy in these four patients, overt CS persisted for two patients and CS symptoms abated for the other two. The remission period of CS was >11 years in one instance (case III-2) and continues for >12 years in the other (case II-4), with no observable disease of the contralateral non-resected adrenal gland. For each of the two remission patients, one adrenal gland had been larger (by abdominal computed tomography) and had had stronger function (by 131I-6β- iodomethyl-19-norcholesterol scintigraphy) than the other. In one (case III-2) of the two remission patients, one adrenal displayed focal PPNAD while the other displayed diffuse PPNAD. The clinical manifestations of CS did not resolve after unilateral adrenalectomy in case II-2 and III-7. The fifth gene mutation-positive patient (case III-4) is still in the latent stage of CS. Conclusions: The clinical manifestations of CS and adrenal image features in patients with PPNAD are heterogeneous. Detailed adrenal imaging of these patients is necessary to assist with the decision to perform unilateral adrenalectomy.

Zaburzenia psychiczne jako dominujący objaw hiperkortyzolemii u 17-letniej pacjentki z pierwotną pigmentową chorobą guzkową nadnerczy

Zaburzenia psychiczne jako dominujący objaw hiperkortyzolemii u 17-letniej pacjentki z pierwotną pigmentową chorobą guzkową nadnerczy

Differences in Adiposity in Cushing Syndrome Caused byPRKAR1AMutations: Clues for the Role of Cyclic AMP Signaling in Obesity and Diagnostic Implications

The cAMP signaling pathway is implicated in bilateral adrenocortical hyperplasias. Bilateral adrenocortical hyperplasia is often associated with ACTH-independent Cushing syndrome (CS) and may be caused by mutations in genes such as PRKAR1A, which is responsible for primary pigmented nodular adrenocortical disease (PPNAD). PRKAR1A regulates cAMP-dependent protein kinase (PKA), an essential enzyme in the regulation of adiposity. Although CS is invariably associated with obesity, its different forms, including those associated with PKA defects, have not been compared. The purpose of this study was to characterize the phenotypic and molecular differences in periadrenal adipose tissue (PAT) between patients with CS with and without PRKAR1A mutations. Samples from adrenalectomies of 51 patients were studied: patients with CS with (n = 13) and without (n = 32) PRKAR1A mutations and a comparison group with aldosterone-producing adenomas (APAs) (n = 6). In addition, clinical data from a larger group of patients with Cushing disease (n = 89) and hyperaldosteronism (n = 26) were used for comparison. Body mass index (BMI), abdominal computed tomography scans, and cortisol data were collected preoperatively. PAT was assayed for PKA activity, cAMP levels, and PKA subunit expression. BMI was lower in adult patients with CS with PRKAR1A mutations. cAMP and active PKA levels in PAT were elevated in patients with CS with PRKAR1A mutations. Increased PKA signaling in PAT was associated with lower BMI in CS. Differences in fat distribution may contribute to phenotypic differences between patients with CS with and without PRKAR1A mutations. The observed differences are in agreement with the known roles of cAMP signaling in regulating adiposity, but this is the first time that germline defects of PKA are linked to variable obesity phenotypes in humans.

Systematic screening for PRKAR1A gene rearrangement in Carney complex: identification and functional characterization of a new in-frame deletion

Point mutations of the PRKAR1A gene are a genetic cause of Carney complex (CNC) and primary pigmented nodular adrenocortical disease (PPNAD), but in 30% of the patients no mutation is detected. Set up a routine-based technique for systematic detection of large deletions or duplications of this gene and functionally characterize these mutations. Multiplex ligation-dependent probe amplification (MLPA) of the 12 exons of the PRKAR1A gene was validated and used to detect large rearrangements in 13 typical CNC and 39 confirmed or putative PPNAD without any mutations of the gene. An in-frame deletion was characterized by western blot and bioluminescence resonant energy transfer technique for its interaction with the catalytic subunit. MLPA allowed identification of exons 3-6 deletion in three patients of a family with typical CNC. The truncated protein is expressed, but rapidly degraded, and does not interact with the protein kinase A catalytic subunit. MLPA is a powerful technique that may be used following the lack of mutations detected by direct sequencing in patients with bona fide CNC or PPNAD. We report here one such new deletion, as an example. However, these gene defects are not a frequent cause of CNC or PPNAD.

The Role of Unilateral Adrenalectomy in Corticotropin‐Independent Bilateral Adrenocortical Hyperplasias

The objective of the present study was twofold: to demonstrate our experience with unilateral adrenalectomy in the treatment of adrenocorticotropic hormone (ACTH)-independent Cushing syndrome (CS) caused by bilateral adrenocortical hyperplasias, and to evaluate the long-term results as evidenced by the main laboratory and clinical findings. From February 2000 to August 2009, unilateral adrenalectomy was performed on 27 patients with ACTH-independent CS and bilateral adrenocortical hyperplasias, including 14 patients with ACTH-independent macronodular adrenal hyperplasia (AIMAH) and 13 patients with primary pigmented nodular adrenocortical disease (PPNAD). Signs and symptoms of CS, endocrine examinations, and radiographic imaging were evaluated preoperatively and postoperatively. At a median follow-up of 69 months (range: 23-120 months) for AIMAH and 47 months (range: 16-113 months) for PPNAD, 25 patients were cured by unilateral adrenalectomy. Serum cortisol level, daily urinary free cortisol (UFC), and plasma ACTH regained the normal range in both AIMAH and PPNAD patients at monthly follow-up visits; the circadian serum cortisol rhythm returned to normal, and a normal responsiveness to overnight low-dose dexamethasone administration (LDDST) became obvious. Both systolic and diastolic blood pressure (BP) levels were significantly reduced: 85 % of patients recovered normal BP levels, and the remaining patients need antihypertensive drugs, but at a reduced dose. No surgery-related morbidity occurred, and there was no sign of further enlargement of the residual adrenal gland after successful unilateral adrenalectomy. One patient with PPNAD and another patient with AIMAH with similar weights and sizes of the bilateral adrenals needed contralateral adrenalectomy. Unilateral adrenalectomy may be the suitable treatment for selected patients with AIMAH and PPNAD. It can achieve long-term remission of CS and improve glycemic control and BP values.

The complex of myxomas, spotty skin pigmentation and endocrine overactivity (Carney complex): imaging findings with clinical and pathological correlation

The complex of myxomas, spotty skin pigmentation and endocrine overactivity, or Carney complex (CNC), is a familial multiple endocrine neoplasia and lentiginosis syndrome. CNC is inherited in an autosomal dominant manner and is genetically heterogeneous. Its features overlap those of McCune-Albright syndrome and other multiple endocrine neoplasia (MEN) syndromes. Spotty skin pigmentation is the major clinical manifestation of the syndrome, followed by multicentric heart myxomas, which occur at a young age and are the lethal component of the disease. Myxomas may also occur on the skin (eyelid, external ear canal and nipple) and the breast. Breast myxomas, when present, are multiple and bilateral among female CNC patients, an entity which is also described as “breast-myxomatosis” and is a characteristic feature of the syndrome. Affected CNC patients often have tumours of two or more endocrine glands, including primary pigmented nodular adrenocortical disease (PPNAD), an adrenocorticotropin hormone (ACTH)-independent cause of Cushing’s syndrome, growth hormone (GH)-secreting and prolactin (PRL)-secreting pituitary adenomas, thyroid adenomas or carcinomas, testicular neoplasms (large-cell calcifying Sertoli cell tumours [LCCSCT]) and ovarian lesions (cysts and cancinomas). Additional infrequent but characteristic manifestations of CNC are psammomatous melanotic schwannomas (PMS), breast ductal adenomas (DAs) with tubular features, and osteochondromyxomas or “Carney bone tumour”. Teaching Points • Almost 60 % of the known CNC kindreds have a germline inactivating mutations in the PRKAR1A gene. • Spotty skin pigmentation is the major clinical manifestation of CNC, followed by heart myxomas. • Indicative imaging signs of PPNAD are contour abnormality and hypodense spots within the gland. • Two breast tumours may present in CNC: myxoid fibroadenomas (breast myxomatosis) and ductal adenomas. • Additional findings of CNC are psammomatous melanotic schwannomas (PMSs) and osteochondromyxomas.

Familial primary pigmented nodular adrenocortical disease without Carney complex (CNC): A case report and review of literature

Primary pigmented nodular adrenocortical disease (PPNAD) is a rare cause of familial Cushing's syndrome. It is characterized by bilateral adrenocortical hyperplasia with small to normal-sized adrenal glands containing multiple small adrenal cortical pigmented nodules [1,2]. PPNAD may occur in an isolated form or as familial PPNAD. Familial cases of PPNAD are usually associated with Carney complex in which Cushing’s syndrome is the most common endocrine manifestation [3]. Familial cases of PPNAD without associated Carney complex are very rare. Only a few cases of familial isolated PPNAD have been reported in the literature, mostly in females [4]. Isolated familial PPNAD has got a better prognosis than familial PPNAD associated with Carney Complex. This observation has important consequences for clinical management, follow-up and genetic counselling of such patients. Familial cases of PPNAD are rare and mostly present in females with associated Carney complex. We herein report a case of familial Cushing’s syndrome in male siblings due to PPNAD without associated Carney complex.

Adrenal Nodular Hyperplasia in Hereditary Leiomyomatosis and Renal Cell Cancer

The condition hereditary leiomyomatosis and renal cell carcinoma is characterized by cutaneous leiomyomas, uterine fibroids and aggressive papillary renal cell carcinoma. A number of our patients with hereditary leiomyomatosis and renal cell carcinoma had atypical adrenal nodules, which were further evaluated to determine whether these nodules were associated with hereditary leiomyomatosis and renal cell carcinoma. Patients with hereditary leiomyomatosis and renal cell carcinoma underwent a comprehensive clinical and genetic evaluation. We reviewed the clinical presentation, anatomical and functional imaging, endocrine evaluation, pathological examination and germline mutation testing results. Of 255 patients with hereditary leiomyomatosis and renal cell carcinoma 20 (7.8%) had primary adrenal lesions, including 4 with bilateral adrenal lesions and 4 with multiple nodules. Two patients had adrenocorticotropic hormone independent hypercortisolism. A total of 27 adrenal lesions were evaluated. The imaging characteristics of 5 of these lesions (18.5%) were not consistent with adenoma by noncontrast computerized tomography criteria. Positron emission tomography was positive in 7 of 10 cases (70%). A total of 12 nodules were surgically resected from 10 adrenal glands. Pathological examination revealed macronodular adrenal hyperplasia in all specimens. Unilateral and bilateral adrenal nodular hyperplasia was detected in a subset of patients with hereditary leiomyomatosis and renal cell carcinoma. A functional endocrine evaluation is recommended when an adrenal lesion is discovered. Imaging frequently reveals lesions that are not typical of adenomas and positron emission tomography may be positive. To date no patient has had adrenal malignancy, and active surveillance of hereditary leiomyomatosis and renal cell carcinoma adrenal nodules appears justified.

Targeted Inhibition of c-KIT Activation Sensitizes Adrenocortical Tumor Cells to Apoptosis

Primary Pigmented Nodular Adrenocortical Disease (PPNAD), a type of micro nodular adrenal hyperplasia (MAH) that occurs mostly in the context of Carney Complex or rarely in an isolated form is caused by germline inactivating mutations in PRKAR1A, the main regulatory subunit of cAMP dependent Protein Kinase (PKA). In addition, defects in phosphodiesterase genes 11A (PDE11A), 8B (PDE8B) and abnormalities of cAMP signaling are known to occur in a small fraction of MAH and massive macronodular adrenocortical disease (MMAD) respectively, suggesting that dysregulation of PKA is a key player in the genesis of these lesions. The only current treatment option is adrenalectomy resulting in adrenal insufficiency. In search of therapeutic targets to develop alternative strategies to treat this disease, we identified for the first time tumor specific over expression and constitutive activation of c-KIT in PPNAD compared to cortisol-producing adenomas (CPA), MMAD and aldosterone-producing adenomas (APA). Next, we investigated the mutational status of the signature exons in KIT gene and found no mutations. Real-time PCR analysis confirmed up regulation of KIT mRNA. Interestingly, KIT over-expression was recapitulated in adrenals from Prkar1a knockout mice reminiscent of that seen in human patients suggesting PKA specific dysregulation of KIT expression. To better characterize these changes at the biochemical level we used an adrenocortical cell line (CAR 47.01) derived from a patient with mutant PRKAR1A and compared it to the H295R adrenocortical cancer line. Consistent with the alterations seen in tumors, CAR47.01 cells exhibited enhanced expression and activation of c-KIT which was further enhanced by the treatment with forskolin, a PKA activator. This up regulation was markedly rescued by reintroduction of PRKAR1A suggesting that PKA functions upstream of c-KIT and the alterations in c-Kit are secondary to changes in PKA. Knockdown of PKAC using siRNA specific to PKAC significantly reduced c-KIT protein levels. Furthermore, in vitro kinase assay demonstrated c-KIT to be a direct phosphorylation target of PKA. The biological and functional consequences of this phosphorylation are currently being explored in vivo. To explore whether over expressed c-KIT is a therapeutic target, the cells were treated with imatinib mesylate, a pharmacological KIT inhibitor resulted in reduced cell viability and induced growth arrest and apoptosis in a time and dose-dependent manner. In the CAR47.01 cells, imatinib treatment induced down regulation of phospho-c-KIT and phospho-Akt, an immediate downstream phosphorylation target of KIT without altering total c-KIT and Akt levels. Interestingly imatinib treatment at 100mg/kg for 7days in a mouse xenograft model induced tumor stasis. Taken together, our data provide novel insights on PKA specific regulation of KIT and indicate KIT as a promising potential target for therapeutic intervention in adrenocortical neoplasms.

Protein Kinase A Alterations in Endocrine Tumors

Various molecular and cellular alterations of the cyclic adenosine monophosphate (cAMP) pathway have been observed in endocrine tumors. Since protein kinase A (PKA) is a central key component of the cAMP pathway, studies of the alterations of PKA subunits in endocrine tumors reveal new aspects of the mechanisms of cAMP pathway alterations in human diseases. So far, most alterations have been observed for the regulatory subunits, mainly PRKAR1A and to a lower extent, PRKAR2B. One of the best examples of such alteration today is the multiple neoplasia syndrome Carney complex (CNC). The most common endocrine gland manifestations of CNC are pituitary GH-secreting adenomas, thyroid tumors, testicular tumors, and ACTH-independent Cushing's syndrome due to primary pigmented nodular adrenocortical disease (PPNAD). Heterozygous germline inactivating mutations of the PKA regulatory subunit RIα gene (PRKAR1A) are observed in about two-third of CNC patients, and also in patients with isolated PPNAD. PRKAR1A is considered as a tumor suppressor gene. Interestingly, these mutations can also be observed as somatic alterations in sporadic endocrine tumors. More than 120 different PRKAR1A mutations have been found today. Most of them lead to an unstable mutant mRNA, which will be degraded by nonsense mediated mRNA decay. In vitro and in vivo functional studies are in progress to understand the mechanisms of endocrine tumor development due to PKA regulatory subunits inactivation. PRKAR1A mutations stimulate in most models PKA activity, mimicking in some way cAMP pathway constitutive activation. Cross-talks with other signaling pathways summarized in this review have been described and might participate in endocrine tumorigenesis.

Novel Genetic Mutation in the Background of Carney Complex

Carney complex is a rare disease inherited in an autosomal dominant manner. It is mostly caused by inactivating mutations of the subunit of protein kinase A. Carney complex is associated with atrial myxoma, nevi or myxomas of the skin, breast tumor and endocrine overactivity. Primary pigmented nodular adrenocortical disease is the specific endocrine manifestation. The authors present the history of a 53-year-old female patient who had undergone surgery for atrial myxomas, thyroid tumor and breast cancer. She was also operated for an adrenal adenoma causing Cushing’s syndrome. Genetic study revealed a novel mutation in the regulatory subunit of protein kinase A (ivs2-1G>A splice mutation in intron 2). Her heterozygous twins were also genetically screened and one of them carried the same mutation. The authors emphasize that despite the absence of specific treatment for patients with Carney complex, confirmation of the diagnosis by genetic studies is important for the close follow-up of the patient and early identification of novel manifestations.

Adrenocortical Cancer in Carney Complex: A Paradigm of Endocrine Tumor Progression or an Association of Genetic Predisposing Factors?

Carney complex (CNC) was described in 1985 as “the complex of myxomas, spotty pigmentation, and endocrine overactivity” (1). The most frequent manifestations of CNC are lentiginosis, myxomas, and ACTH-independent Cushing’s syndrome due to primary pigmented nodular adrenocortical disease (PPNAD). PPNAD is a unique cause of adrenal Cushing syndrome in which bilateral pigmented micronodules are typically visible at macroscopic examination in the adrenal cortex. It is mostly the investigation of this rare type of adrenal tumors that led to the identification of CNC by Dr. J. A. Carney at the Mayo Clinic (2). Since the first description of CNC, an autosomal dominant inheritance was postulated. In 2000, the identification of the CNC1 gene (PRKAR1A), located at 17q22-24, was reported by Stratakis and colleagues (3) at the National Institutes of Health. This gene encodes the regulatory subunit 1A of the protein kinase A, a central component of the cAMP signaling pathway. Because cAMP signaling is activated by various molecular and cellular defects in endocrine tumors, it appeared quickly that the identification of CNC1 was a major step for the understanding of endocrine tumorigenesis. Germline PRKAR1A mutations are heterozygous inactivating mutations. Allelic loss of the wild-type allele is frequently observed in tumors from CNC patients. Therefore, PRKAR1A is considered as a tumor suppressor gene. So far only PPNAD, which is a typical benign secreting tumor with a very slow growth, has been reported in adrenal tumors of CNC patients. In this issue of the JCEM, two cases of adrenocortical cancer (ACC) are reported in CNC patients with germline PRKAR1A mutations. The paper by Anselmo et al. (20) describes a large Azorean family with at least 10 CNC patients and a germline heterozygous PRKAR1A point mutation (S147G). The index case was a 28-yr-old female who presented with a right 12-cm ACC with liver metastasis arising in the background of bilateral PPNAD. Clinical and hormonal investigations showed cortisol and androgen excess. Three other family members presented with PPNAD and a typical paradoxical increased cortisol secretion after dexamethasone suppression test. The other frequent CNC manifestation in this family was lentiginosis (facial pigmented macules typically located on the lips and eyelids in CNC). The paper by Morin et al. (21) describes a 22-yr-old female with cardiac myxoma and lentiginosis. Her mother also suffered from cardiac myxoma at age 23 and had lentiginosis. A germline heterozygous PRKAR1A mutation that creates a premature stop codon in exon 2 of the gene (p.Lys32Argfs*12) was found in the index case. Mutant PRKAR1A mRNA with a premature stop codon are usually degraded by a mechanism of nonsense-mediated mRNA decay, inactivating the mutant allele. This applies to the majority (about 80%) of the PRKAR1A mutations that have been reported (4). This 22-yr-old female patient was diagnosed with a cortisoland androgen-secreting 8.5-cm tumor of the left adrenal that was removed by laparoscopy. Pigmented micronodules typical of PPNAD were observed at pathological examination in the adrenal adjacent to the ACC. Three months after surgery, local recurrence was observed. The patient was then reoperated and treated with mitotane. Twenty-nine months later, liver and pulmonary metastasis were progressing.

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

The overwhelming majority of benign lesions of the adrenal cortex leading to Cushing syndrome are linked to one or another abnormality of the cAMP or protein kinase pathway. PRKAR1A-inactivating mutations are responsible for primary pigmented nodular adrenocortical disease, whereas somatic GNAS activating mutations cause macronodular disease in the context of McCune-Albright syndrome, ACTH-independent macronodular hyperplasia, and, rarely, cortisol-producing adenomas. The whole-genome expression profile (WGEP) of normal (pooled) adrenals, PRKAR1A- (3) and GNAS-mutant (3) was studied. Quantitative RT-PCR and Western blot were used to validate WGEP findings. MAPK and p53 signaling pathways were highly overexpressed in all lesions against normal tissue. GNAS-mutant tissues were significantly enriched for extracellular matrix receptor interaction and focal adhesion pathways when compared with PRKAR1A-mutant (fold enrichment 3.5, P < 0.0001 and 2.1, P < 0.002, respectively). NFKB, NFKBIA, and TNFRSF1A were higher in GNAS-mutant tumors (P < 0.05). Genes related to the Wnt signaling pathway (CCND1, CTNNB1, LEF1, LRP5, WISP1, and WNT3) were overexpressed in PRKAR1A-mutant lesions. WGEP analysis revealed that not all cAMP activation is the same: adrenal lesions harboring PRKAR1A or GNAS mutations share the downstream activation of certain oncogenic signals (such as MAPK and some cell cycle genes) but differ substantially in their effects on others.

An unusual presentation of Carney complex with diffuse primary pigmented nodular adrenocortical disease on one adrenal gland and a nonpigmented adrenocortical adenoma and focal primary pigmented nodular adrenocortical disease on the other

A 24-year-old female patient with cushingoid appearance was admitted in May 2000. The endocrine studies showed ACTH-independent Cushing's syndrome. A 2-day high-dose dexamethasone suppression test (HDDST) revealed paradoxical increase of 24 h urinary free cortisol (UFC). Abdominal computed tomography demonstrated a left adrenal nodule (3 x 2 cm in diameter). An adrenal scintigram with ¹³¹I-6β-iodomethyl-19-norcholesterol showed uptake of the isotope in the left adrenal gland and non-visualization in the right adrenal gland throughout the examination course. A retroperitoneoscopic left total adrenalectomy was performed in July 2000. The cut surface of the left adrenal was yellow-tan grossly. Microscopically, the left adrenal nodule contained a nonpigmented adrenocortical adenoma (NP) and another focal primary pigmented nodular adrenocortical disease (PPNAD, FP) mixed lesion. The immunohistochemical studies of CYP17 demonstrate positive in NP and FP of the left adrenal gland. Very low baseline morning plasma cortisol (0.97 μg/dL) and subnormal ACTH (8.16 pg/mL) levels were measured 1.5 months after left adrenalectomy. Right adrenal gland recovered its function 6 months after left adrenalectomy. Plasma cortisol could be suppressed to 3.47 μg/dL by overnight low-dose dexamethasone suppression test 65 months after left adrenalectomy. Cushingoid features still did not appear 122 months after left adrenalectomy. In May 2011, this patient was readmitted due to cushingoid characteristics. Paradoxical rise of 24-h UFC to 2-day HDDST was demonstrated. Ultrasonography of thyroid showed bilateral thyroid cysts. Subtotal right adrenalectomy about 80% of right adrenal was performed. Diffuse PPNAD of the right adrenal was proved pathologically. Immunohischemical stain for CYP17 is positive in the right adrenal gland but weaker positive than that in the left adrenal gland. The genetic study of the peripheral blood, left adrenocortical nodule, and right PPNAD all showed p.R16X (c.46C>T) mutation of the PRKAR1A gene.

A Large Family with Carney Complex Caused by the S147G PRKAR1A Mutation Shows a Unique Spectrum of Disease Including Adrenocortical Cancer

Most tumors in Carney complex (CNC) are benign, including primary pigmented nodular adrenocortical disease (PPNAD), the main endocrine tumor in CNC. Adrenocortical cancer (AC) has never been observed in the syndrome. Herein, we describe a large Azorean family with CNC caused by a point mutation in the PRKAR1A gene coding for type 1-α (RIα) regulatory subunit of the cAMP-dependent protein kinase A, in which the index patient presented with AC. We studied the genotype-phenotype correlation in CNC. We reported on case series and in vitro testing of the PRKAR1A mutation in a tertiary care referral center. Twenty-two members of a family were investigated for Cushing syndrome and other CNC components; their DNA was sequenced for PRKAR1A mutations. Cushing syndrome due to PPNAD occurred in four patients, including the proposita who presented with AC and three who had Cushing syndrome and/or PPNAD. Lentigines were found in six additional patients who did not have PPNAD. A base substitution (c.439A>G/p.S147G) in PRKAR1A was identified in the proposita, in the three others with PPNAD, in the proposita's twin daughters who had lentigines but no evidence of hypercortisolism, and in five other family members, including one without lentigines or evidence of hypercortisolism. Unlike in other RIα defects, loss of heterozygosity was not observed in AC. The S147G mutation was compared to other expressed PRKAR1A mutations; it led to decreased cAMP and catalytic subunit binding by RIα and increased protein kinase A activity in vitro. In a large family with CNC, one amino acid substitution caused a spectrum of adrenal disease that ranged from lack of manifestations to cancer. PPNAD and AC were the only manifestations of CNC in these patients, in addition to lentigines. These data have implications for counseling patients with CNC and are significant in documenting the first case of AC in the context of PPNAD.