Intraadrenal corticotropin in bilateral macronodular adrenal hyperplasia.

BACKGROUND: Bilateral macronodular adrenal hyperplasia is a rare cause of primary adrenal Cushing's syndrome. In this form of hyperplasia, hypersecretion of cortisol suppresses the release of corticotropin by pituitary corticotrophs, which results in low plasma corticotropin levels. Thus, the disease has been termed corticotropin-independent macronodular adrenal hyperplasia. We examined the abnormal production of corticotropin in these hyperplastic adrenal glands. METHODS: We obtained specimens of hyperplastic macronodular adrenal tissue from 30 patients with primary adrenal disease. The corticotropin precursor proopiomelanocortin and corticotropin expression were assessed by means of a polymerase-chain-reaction assay and immunohistochemical analysis. The production of corticotropin and cortisol was assessed in 11 specimens with the use of incubated explants and cell cultures coupled with hormone assays. Corticotropin levels were measured in adrenal and peripheral venous blood samples from 2 patients. RESULTS: The expression of proopiomelanocortin messenger RNA (mRNA) was detected in all samples of hyperplastic adrenal tissue. Corticotropin was detected in steroidogenic cells arranged in clusters that were disseminated throughout the adrenal specimens. Adrenal corticotropin levels were higher in adrenal venous blood samples than in peripheral venous samples, a finding that was consistent with local production of the peptide within the hyperplastic adrenals. The release of adrenal corticotropin was stimulated by ligands of aberrant membrane receptors but not by corticotropin-releasing hormone or dexamethasone. A semiquantitative score for corticotropin immunostaining in the samples correlated with basal plasma cortisol levels. Corticotropin-receptor antagonists significantly inhibited in vitro cortisol secretion. CONCLUSIONS: Cortisol secretion by the adrenals in patients with macronodular hyperplasia and Cushing's syndrome appears to be regulated by corticotropin, which is produced by a subpopulation of steroidogenic cells in the hyperplastic adrenals. Thus, the hypercortisolism associated with bilateral macronodular adrenal hyperplasia appears to be corticotropin-dependent. (Funded by the Agence Nationale de la Recherche and others.).

ARMC5 mutations in macronodular adrenal hyperplasia with Cushing's syndrome.

BACKGROUND: Corticotropin-independent macronodular adrenal hyperplasia may be an incidental finding or it may be identified during evaluation for Cushing's syndrome. Reports of familial cases and the involvement of both adrenal glands suggest a genetic origin of this condition. METHODS: We genotyped blood and tumor DNA obtained from 33 patients with corticotropin-independent macronodular adrenal hyperplasia (12 men and 21 women who were 30 to 73 years of age), using single-nucleotide polymorphism arrays, microsatellite markers, and whole-genome and Sanger sequencing. The effects of armadillo repeat containing 5 (ARMC5) inactivation and overexpression were tested in cell-culture models. RESULTS: The most frequent somatic chromosome alteration was loss of heterozygosity at 16p (in 8 of 33 patients for whom data were available [24%]). The most frequent mutation identified by means of whole-genome sequencing was in ARMC5, located at 16p11.2. ARMC5 mutations were detected in tumors obtained from 18 of 33 patients (55%). In all cases, both alleles of ARMC5 carried mutations: one germline and the other somatic. In 4 patients with a germline ARMC5 mutation, different nodules from the affected adrenals harbored different secondary ARMC5 alterations. Transcriptome-based classification of corticotropin-independent macronodular adrenal hyperplasia indicated that ARMC5 mutations influenced gene expression, since all cases with mutations clustered together. ARMC5 inactivation decreased steroidogenesis in vitro, and its overexpression altered cell survival. CONCLUSIONS: Some cases of corticotropin-independent macronodular adrenal hyperplasia appear to be genetic, most often with inactivating mutations of ARMC5, a putative tumor-suppressor gene. Genetic testing for this condition, which often has a long and insidious prediagnostic course, might result in earlier identification and better management. (Funded by Agence Nationale de la Recherche and others.).

Osilodrostat, a potent oral 11ß-hydroxylase inhibitor: 22-week, prospective, Phase II study in Cushing's disease.

PURPOSE: In a 10-week proof-of-concept study (LINC 1), the potent oral 11ß-hydroxylase inhibitor osilodrostat (LCI699) normalized urinary free cortisol (UFC) in 11/12 patients with Cushing's disease. The current 22-week study (LINC 2; NCT01331239) further evaluated osilodrostat in patients with Cushing's disease. METHODS: Phase II, open-label, prospective study of two patient cohorts. Follow-up cohort: 4/12 patients previously enrolled in LINC 1, offered re-enrollment if baseline mean UFC was above ULN. Expansion cohort: 15 newly enrolled patients with baseline UFC > 1.5 × ULN. In the follow-up cohort, patients initiated osilodrostat twice daily at the penultimate efficacious/tolerable dose in LINC 1; dose was adjusted as needed. In the expansion cohort, osilodrostat was initiated at 4 mg/day (10 mg/day if baseline UFC > 3 × ULN), with dose escalated every 2 weeks to 10, 20, 40, and 60 mg/day until UFC ≤ ULN. Main efficacy endpoint was the proportion of responders (UFC ≤ ULN or ≥50% decrease from baseline) at weeks 10 and 22. RESULTS: Overall response rate was 89.5% (n/N = 17/19) at 10 weeks and 78.9% (n/N = 15/19) at 22 weeks; at week 22, all responding patients had UFC ≤ ULN. The most common AEs observed during osilodrostat treatment were nausea, diarrhea, asthenia, and adrenal insufficiency (n = 6 for each). New or worsening hirsutism (n = 2) and/or acne (n = 3) were reported among four female patients, all of whom had increased testosterone levels. CONCLUSIONS: Osilodrostat treatment reduced UFC in all patients; 78.9% (n/N = 15/19) had normal UFC at week 22. Treatment with osilodrostat was generally well tolerated.

A genome-wide scan identifies mutations in the gene encoding phosphodiesterase 11A4 (PDE11A) in individuals with adrenocortical hyperplasia.

Phosphodiesterases (PDEs) regulate cyclic nucleotide levels. Increased cyclic AMP (cAMP) signaling has been associated with PRKAR1A or GNAS mutations and leads to adrenocortical tumors and Cushing syndrome. We investigated the genetic source of Cushing syndrome in individuals with adrenocortical hyperplasia that was not caused by known defects. We performed genome-wide SNP genotyping, including the adrenocortical tumor DNA. The region with the highest probability to harbor a susceptibility gene by loss of heterozygosity (LOH) and other analyses was 2q31-2q35. We identified mutations disrupting the expression of the PDE11A isoform-4 gene (PDE11A) in three kindreds. Tumor tissues showed 2q31-2q35 LOH, decreased protein expression and high cyclic nucleotide levels and cAMP-responsive element binding protein (CREB) phosphorylation. PDE11A codes for a dual-specificity PDE that is expressed in adrenal cortex and is partially inhibited by tadalafil and other PDE inhibitors; its germline inactivation is associated with adrenocortical hyperplasia, suggesting another means by which dysregulation of cAMP signaling causes endocrine tumors.

From Nelson's syndrome to Corticotroph Tumor Progression speed: an update.

Since the first description 60 years ago of Nelson syndrome, the way to consider corticotroph pituitary neuroendocrine tumors (PitNETs) after bilateral adrenalectomy has evolved. Today, it is globally acknowledged that only a subset of corticotroph PitNETs is aggressive. After adrenalectomy, corticotroph tumor progression (CTP) occurs in about 30 to 40% of patients during a median follow-up of 10 years. When CTP occurs, various CTP (CTPS) speeds can be obsersed. Using a simple metrics in patients with CTP, CTPS was reported to vary from few millimeters per year to up to 40 mm per year. Rapid CTPS/Nelson's syndrome was associated with more severe Cushing, higher ACTH in the year following adrenalectomy and higher Ki67 on pituitary pathology. Complications such as apoplexy, cavernous syndrome and visual defects were associated with higher CTPS. During follow-up, early morning ACTH absolute variations properly reflected CTPS. Finally, CTPS was not higher after than before adrenalectomy, suggesting that cortisol deprivation after adrenalectomy does not impact CTPS in a majority of patients. Taken together, rapid CTPS/Nelson's syndrome probably reflects the intrinsic aggressiveness of some corticotroph PitNETs. The precise molecular mechanisms related to corticotroph PitNET aggressiveness remain to be deciphered. Regular MRIs combined to intermediate morning ACTH measurements probably provide a reliable way to detect early and manage fast growing tumors, and therefore to limit the complications.

Testicular impairment in Primary Adrenal Insufficiency caused by Nicotinamide Nucleotide Transhydrogenase (NNT) deficiency - a case report: implication of oxidative stress and importance of fertility preservation.

INTRODUCTION: Nicotinamide nucleotide transhydrogenase (NNT) gene deficiency has recently been shown to be involved in Primary Adrenal Insufficiency (PAI). NNT encodes an inner mitochondrial membrane protein that produces large amounts of NADPH. NADPH is used in several biosynthesis pathways and the oxidoreduction of free radicals by the glutathione and thioredoxin systems in mitochondria. Patients with PAI due to NNT deficiency may also exhibit extra-adrenal manifestations, usually including gonadal impairment. CASE REPORT: We present the case of a 35-year-old patient referred to our center for primary infertility with non-obstructive azoospermia, in a context of PAI and obesity. PAI genetic exploration carried out at the age of thirty revealed NNT deficiency due to the presence of two deleterious mutations (one on each allele) in the NNT gene. Scrotal ultrasound revealed a right Testicular Adrenal Rest Tumor (TART). Intensification of glucocorticoid therapy over the course of 8 months failed to reduce the TART volume or improve sperm production and endocrine function. No spermatozoa were found after surgical exploration of both testes, and subsequent histopathological analysis revealed bilateral Sertoli cell-only syndrome. A retrospective review of the hypothalamic-pituitary-gonadic axis hormonal assessment over 20 years showed progressive impairment of testicular function, accelerated during adulthood, leading to hypergonadotropic hypogonadism and non-obstructive azoospermia when the patient reached his thirties, while the PAI remained controlled over the same period. CONCLUSION: This case report provides, for the first time, direct evidence of complete germ line loss in an azoospermic man with NNT deficiency. Additional data further support the hypothesis of a determinant role of oxidative cellular damage due to reactive oxygen species (ROS) imbalance in the severe gonadal impairment observed in this NNT-deficient patient. Early and regular evaluation of gonadal function should be performed in patients with PAI, especially with NNT deficiency, as soon as the patients reach puberty. Fertility preservation options should then be provided in early adulthood for these patients.

RéSUMé: INTRODUCTION: Le gène Nicotinamide Nucleotide Transhydrogenase (NNT) a été récemment impliqué dans l'Insuffisance Surrénalienne Primaire (ISP). Il code pour une protéine de la membrane mitochondriale interne qui produit de fortes quantités de NADPH. Le NADPH est utilisé par plusieurs voies de biosynthèse et dans l'oxydo-réduction de radicaux libres par les voies de signalisation impliquant le glutathion et la thioredoxine dans la mitochondrie. Les patients avec une ISP, en lien avec un déficit du gène NNT, peuvent également présenter des manifestations extra-surrénaliennes, dont une altération gonadique. CAS CLINIQUE: Nous présentons le cas clinique d'un homme de 35 ans adressé à notre centre d'Assistance Médicale à la Procréation pour infertilité primaire avec azoospermie non obstructive, dans un contexte d'ISP et d'obésité. L'exploration génétique effectuée à l'âge de 30 ans a identifié un déficit complet de la protéine NNT dû à la présence de deux mutations hétérozygotes (une sur chaque allèle), délétères. L'échographie scrotale a montré une tumeur testiculaire d'origine surrénalienne à droite. L'intensification du traitement par glucocorticoides pendant 8 mois n'a pas réduit le volume de la tumeur ni amélioré la production spermatique ou la fonction testiculaire endocrine. Aucun spermatozoïde n'a été retrouvé après exploration chirurgicale testiculaire bilatérale, en lien avec un syndrome de Cellules de Sertoli Seules. L'étude rétrospective de l'axe hypothalamo-hypophysaire-gonadique monte une altération progressive de la fonction testiculaire, accélérée à l'âge adulte, aboutissant à un hypogonadisme hypergonadotrope et une azoospermie non-obstructive à 30 ans, alors que l'ISP était contrôlée pendant cette période. CONCLUSION: Ce cas clinique met en évidence pour la première fois une disparition complète de la lignée germinale chez un patient avec un déficit en NNT. Il avance des arguments en faveur de l'hypothèse d'un rôle déterminant des dommages cellulaires, dus à un excès de radicaux oxygénés dans cette atteinte régulière de la fonction gonadique. Cette dernière devrait être suivie à partir de la puberté chez les patients ISP et plus particulièrement ceux ayant un déficit en NNT. Une préservation de la fertilité pourrait leur être proposée lorsqu'ils deviennent adultes.

Corticotroph tumor progression speed after adrenalectomy.

Objectives: After bilateral adrenalectomy in Cushing's disease, corticotroph tumor progression occurs in one-third to half of patients. However, progression speed is variable, ranging from slow to rapid. The aim was to explore corticotroph progression speed, its consequences and its risk factors. Design: A retrospective single-center observational study. Methods: In total,103 patients with Cushing's disease who underwent bilateral adrenalectomy between 1990 and 2020 were included. Clinical, biological, histological and MRI features were collected. Median duration of follow-up after bilateral adrenalectomy was 9.31 years. Results: In total,44 patients progressed (43%). Corticotroph tumor progression speed ranged from 1 to 40.7 mm per year. Progression speed was not different before and after bilateral adrenalectomy (P = 0.29). In univariate analyses, predictive factors for rapid corticotroph tumor progression included the severity of Cushing's disease before adrenalectomy as the cause of adrenalectomy, high ACTH in the year following adrenalectomy and high Ki67 immunopositivity in the tumor. During follow-up, early morning ACTH absolute variation was associated with corticotroph tumor progression speed (P-value = 0.001). ACTH measurement after dynamic testing did not improve this association. Conclusion: After adrenalectomy, corticotroph progression speed is highly variable and manageable with MRI and ACTH surveillance. Progression speed does not seem related to bilateral adrenalectomy but rather to intrinsic properties of highly proliferative and secreting tumors.

Mutations and polymorphisms in the gene encoding regulatory subunit type 1-alpha of protein kinase A (PRKAR1A): an update.

PRKAR1A encodes the regulatory subunit type 1-alpha (RIalpha) of the cyclic adenosine monophosphate (cAMP)-dependent protein kinase (PKA). Inactivating PRKAR1A mutations are known to be responsible for the multiple neoplasia and lentiginosis syndrome Carney complex (CNC). To date, at least 117 pathogenic variants in PRKAR1A have been identified (online database: http://prkar1a.nichd.nih.gov). The majority are subject to nonsense mediated mRNA decay (NMD), leading to RIalpha haploinsufficiency and, as a result, activated cAMP signaling. Recently, it became apparent that CNC may be caused not only by RIalpha haploinsufficiency, but also by the expression of altered RIalpha protein, as proven by analysis of expressed mutations in the gene, consisting of amino acid substitutions and in-frame genetic alterations. In addition, a new subgroup of mutations that potentially escape NMD and result in CNC through altered (rather than missing) protein has been analyzed-these are frame-shifts in the 3' end of the coding sequence that shift the stop codon downstream of the normal one. The mutation detection rate in CNC patients is recently estimated at above 60%; PRKAR1A mutation-negative CNC patients are characterized by significant phenotypic heterogeneity. In this report, we present a comprehensive analysis of all presently known PRKAR1A sequence variations and discuss their molecular context and clinical phenotype.

Thoracoscopic removal of mediastinal parathyroid glands: a critical appraisal of an emerging technique.

OBJECTIVE: To retrospectively evaluate the feasibility of thoracoscopic removal of mediastinal parathyroids. SUMMARY BACKGROUND DATA: Mediastinal exploration to resect ectopic parathyroid(s) is needed in approximately 2% of cases in hyperparathyroidism. Recent advances in thoracoscopic surgery allow for a minimally invasive treatment. METHODS: From 1999 through 2007, 13 patients affected by primary hyperparathyroidism (11 females, mean age 60 years, range: 22-88) underwent thoracoscopic removal of mediastinal parathyroids. Scintigraphy produced positive results in 11 of 13 cases, computed tomography scan in 9 of 10, parathyroid hormone venous sampling in 10 of 10 patients, and magnetic resonance imaging in 5 of 7. Right thoracoscopic access was used in 9 patients, left in 4. Postoperative outcome was analyzed. RESULTS: Thoracoscopy enabled retrieval of mediastinal parathyroids in 10 of 13 (78%) cases. Mean operating time was 92 minutes (range: 50-240). One procedure (8%) was converted. No perioperative deaths/major complications occurred. Mild complications occurred in 2 of 13 (15%) patients (pneumothorax/pneumonia, transient recurrent nerve palsy). Mean hospital stay was 4.7 days (range: 2-15). At a mean follow-up of 73 months (range: 16-105), parathyroid hormone and calcium venous concentrations were high in 3 patients. Unsuccessful procedures were related to doubtful or non-concordant preoperative localization. CONCLUSIONS: The thoracoscopic approach for mediastinal parathyroidectomy is feasible and safe. An accurate preoperative work-up should be standardized to avoid useless procedures. In case of negative preoperative localization of the abnormal gland, thoracoscopy should not be adopted as a diagnostic tool.

Unexpected diagnosis for an adrenal tumor: synovial sarcoma.

A 39-year-old woman presented with an incidentally discovered mass of the left adrenal fossa. Computed tomography and magnetic resonance imaging did not show any other lesion. Histologically, this mass was composed of a dense proliferation of spindle cells with a fibrosarcomatous-like pattern. Immunohistochemistry using anticytokeratin showed some epithelial cells within the tumor. The diagnosis of primitive synovial sarcoma of the left adrenal fossa was confirmed by the presence of the characteristic t(X;18) translocation. Despite radiotherapy, several chemotherapies, and 2 other surgical resections, the patient died 30 months after the initial diagnosis. To our knowledge, this report constitutes the first described case of synovial sarcoma arising in the adrenal gland.

Efficacy and safety of osilodrostat in patients with Cushing's disease (LINC 3): a multicentre phase III study with a double-blind, randomised withdrawal phase.

BACKGROUND: Cushing's disease is a rare endocrine disorder characterised by cortisol overproduction with severe complications. Therapies for cortisol reduction are often necessary. Here we report the outcomes from the pivotal phase III study of osilodrostat (a potent oral inhibitor of cytochrome P450 11B1, mitochondrial [11ß-hydroxylase]; Novartis Pharma AG, Basel, Switzerland) in patients with Cushing's disease. METHODS: LINC 3 was a prospective, multicentre, open-label, phase III study with a double-blind randomised withdrawal period, that comprised four periods. Patients aged 18-75 years, with confirmed persistent or recurrent Cushing's disease (defined as mean 24-h urinary free cortisol [UFC] concentration >1·5 times the upper limit of normal [ULN] and morning plasma adrenocorticotropic hormone above the lower limit of normal) who had previously had pituitary surgery or irradiation, or were newly diagnosed and who refused surgery or were not surgical candidates, were recruited from 66 hospital sites and private clinical practices in 19 countries. In period 1, open-label osilodrostat was initiated in all participants and adjusted every 2 weeks (1-30 mg twice daily; film-coated tablets for oral administration) on the basis of mean 24-h UFC concentration and safety until week 12. In period 2, weeks 13-24, osilodrostat was continued at the therapeutic dose determined during period 1. In period 3, beginning at week 26, participants who had a mean 24-h UFC concentration of less than or equal to the ULN at week 24, without up-titration after week 12, were randomly assigned (1:1), via an interactive-response technology, stratified by osilodrostat dose at week 24 and history of pituitary irradiation, to continue osilodrostat or switch to placebo for 8 weeks. Participants and investigators were masked to treatment assignment. Ineligible participants continued open-label osilodrostat. In period 4, weeks 35-48, all participants were given open-label osilodrostat until core-study end. The primary objective was to compare the efficacy of osilodrostat versus placebo at the end of period 3. The primary endpoint was the proportion of participants who had been randomly assigned to treatment or placebo with a complete response (ie, mean 24-h UFC concentration of ≤ULN) at the end of the randomised withdrawal period (week 34), without up-titration during this period. The key secondary endpoint was the proportion of participants with a complete response at the end of the single-arm, open-label period (ie, period 2, week 24) without up-titration during weeks 13-24. Analysis was by intention-to-treat for all patients who received at least one dose of osilodrostat (full analysis set; key secondary endpoint) or randomised treatment (randomised analysis set; primary endpoint) and safety was assessed in all enrolled patients who received at least one dose of osilodrostat and had at least one post-baseline safety assessment. LINC 3 is registered with ClinicalTrials.gov, NCT02180217, and is now complete. FINDINGS: Between Nov 12, 2014, and March 22, 2017, 202 patients were screened and 137 were enrolled. The median age was 40·0 years (31·0-49·0) and 106 (77%) participants were female. 72 (53%) participants were eligible for randomisation during the withdrawal phase, of whom 36 were assigned to continue osilodrostat and 35 were assigned to placebo; one patient was not randomly assigned due to investigator decision and continued open-label osilodrostat. More patients maintained a complete response with osilodrostat versus with placebo at week 34 (31 [86%] vs ten [29%]; odds ratio 13·7 [95% CI 3·7-53·4]; p<0·0001). At week 24, 72 (53%; 95% CI 43·9-61·1) of 137 patients maintained a complete response without up-titration after week 12. Most common adverse events (ie, occurred in >25% of participants) were nausea (57 [42%]), headache (46 [34%]), fatigue (39 [28%]), and adrenal insufficiency (38 [28%]). Hypocortisolism occurred in 70 (51%) patients and adverse events related to adrenal hormone precursors occurred in 58 (42%) patients. One patient died, unrelated to study drug, after the core study phase. INTERPRETATION: Twice-daily osilodrostat rapidly reduced mean 24-h UFC and sustained this reduction alongside improvements in clinical signs of hypercortisolism; it was also generally well tolerated. Osilodrostat is an effective new treatment option that is approved in Europe for the treatment of endogenous Cushing's syndrome and in the USA for Cushing's disease. FUNDING: Novartis Pharma AG.

Pangenomic Classification of Pituitary Neuroendocrine Tumors.

Pituitary neuroendocrine tumors (PitNETs) are common, with five main histological subtypes: lactotroph, somatotroph, and thyrotroph (POU1F1/PIT1 lineage); corticotroph (TBX19/TPIT lineage); and gonadotroph (NR5A1/SF1 lineage). We report a comprehensive pangenomic classification of PitNETs. PitNETs from POU1F1/PIT1 lineage showed an epigenetic signature of diffuse DNA hypomethylation, with transposable elements expression and chromosomal instability (except for GNAS-mutated somatotrophs). In TPIT lineage, corticotrophs were divided into three classes: the USP8-mutated with overt secretion, the USP8-wild-type with increased invasiveness and increased epithelial-mesenchymal transition, and the large silent tumors with gonadotroph transdifferentiation. Unexpected expression of gonadotroph markers was also found in GNAS-wild-type somatotrophs (SF1 expression), challenging the current definition of SF1/gonadotroph lineage. This classification improves our understanding and affects the clinical stratification of patients with PitNETs.

Phosphodiesterase 11A (PDE11A) and genetic predisposition to adrenocortical tumors.

PURPOSE: We have reported previously nonsense inactivating mutations of the phosphodiesterase 11A (PDE11A) gene in patients with micronodular adrenocortical hyperplasia and Cushing syndrome. The aim of this study is to investigate the presence of somatic or germ-line PDE11A mutations in various types of adrenocortical tumors: ACTH-independent macronodular adrenocortical hyperplasia (AIMAH), adrenocortical adenoma (ACA), and adrenocortical cancer (ACC). EXPERIMENTAL DESIGN: PDE11A was sequenced in 117 adrenocortical tumors and 192 controls subjects; immunohistochemistry for PDE11A and tumor cyclic AMP levels were studied in a subgroup of adrenocortical tumors. RESULTS: One PDE11A inactivating mutation (R307X) was found in one ACA, 22 germ-line missense variants (18.8%) were found in adrenocortical tumors, and only 11 missense variants (5.7%) were found in controls. By comparing the common mutations, a higher frequency of mutations in adrenocortical tumors than in age/sex-matched controls were observed [16% versus 10% in ACC, 19% versus 10% in ACA, and 24% versus 9% in AIMAH; odds ratio (OR), 3.53; P = 0.05]. Somatic DNA from adrenocortical tumors with missense variants showed a wild-type allelic loss. A significant difference between ACC and controls was observed for a polymorphism in exon 6 (E421E; OR, 2.1; P = 0.03) and three associated polymorphisms located in intron 10-exon 11-intron 11 (OR, 0.5; P = 0.01). In AIMAH/ACA, cyclic AMP levels were higher than in normal adrenals and decreased PDE11A immunostaining was present in adrenocortical tumors with PDE11A variants. CONCLUSIONS: The present investigation of a large cohort of adrenocortical tumors suggests that PDE11A sequence defects predispose to a variety of lesions (beyond micronodular adrenocortical hyperplasia) and may contribute to the development of these tumors in the general population.

Value of Molecular Classification for Prognostic Assessment of Adrenocortical Carcinoma.

IMPORTANCE: The risk stratification of adrenocortical carcinoma (ACC) based on tumor proliferation index and stage is limited. Adjuvant therapy after surgery is recommended for most patients. Pan-genomic studies have identified distinct molecular groups closely associated with outcome. OBJECTIVE: To compare the molecular classification for prognostic assessment of ACC with other known prognostic factors. DESIGN, SETTING, AND PARTICIPANTS: In this retrospective biomarker analysis, ACC tumor samples from 368 patients who had undergone surgical tumor removal were collected from March 1, 2005, to September 30, 2015 (144 in the training cohort and 224 in the validation cohort) at 21 referral centers with a median follow-up of 35 months (interquartile range, 18-74 months). Data were analyzed from March 2016 to March 2018. EXPOSURES: Meta-analysis of pan-genomic studies (transcriptome, methylome, chromosome alteration, and mutational profiles) was performed on the training cohort. Targeted biomarker analysis, including targeted gene expression (BUB1B and PINK1), targeted methylation (PAX5, GSTP1, PYCARD, and PAX6), and targeted next-generation sequencing, was performed on the training and validation cohorts. MAIN OUTCOMES AND MEASURES: Disease-free survival. Cox proportional hazards regression and C indexes were used to assess the prognostic value of each model. RESULTS: Of the 368 patients (mean [SD] age, 49 [16] years), 144 were in the training cohort (100 [69.4%] female) and 224 were in the validation cohort (142 [63.4%] female). In the training cohort, pan-genomic measures classified ACC into 3 molecular groups (A1, A2, and A3-B), with 5-year survival of 9% for group A1, 45% for group A2, and 82% for group A3-B (log-rank P < .001). Molecular class was an independent prognostic factor of recurrence in stage I to III ACC after complete surgery (hazard ratio, 55.91; 95% CI, 8.55-365.40; P < .001). The combination of European Network for the Study of Adrenal Tumors (ENSAT) stage, tumor proliferation index, and molecular class provided the most discriminant prognostic model (C index, 0.88). In the validation cohort, the molecular classification, determined by targeted biomarker measures, was confirmed as an independent prognostic factor of recurrence (hazard ratio, 5.96 [95% CI, 1.81-19.58], P = .003 for the targeted classifier combining expression, methylation, and chromosome alterations; and 2.61 [95% CI, 1.31-5.19], P = .006 for the targeted classifier combining methylation, chromosome alterations, and mutational profile). The prognostic value of the molecular markers was limited for patients with stage IV ACC. CONCLUSIONS AND RELEVANCE: The findings suggest that in localized ACC, targeted classifiers may be used as independent markers of recurrence. The determination of molecular class may improve individual prognostic assessment and thus may spare unnecessary adjuvant treatment.

Expression of progesterone and estradiol receptors in normal adrenal cortex, adrenocortical tumors, and primary pigmented nodular adrenocortical disease.

Adrenal tumors occur more frequently in women and are the leading cause of Cushing's syndrome during pregnancy. We aimed to evaluate the potential role of sex steroids in the susceptibility of women to adrenocortical tumors. We evaluated the presence of the progesterone receptor (PR), estradiol receptors (ERs), and aromatase in 5 patients with primary pigmented nodular adrenal disease (PPNAD), 15 adrenocortical adenomas (ACAs) and adjacent normal tissues, 12 adrenocortical carcinomas (ACCs), and 3 normal adrenal glands (NA). The expression of PR and ERalpha was evaluated by enzyme immunoassays, real-time RT-PCR, immunohistochemistry, and cytosol-based ligand-binding assays. ERbeta and aromatase levels were evaluated by real-time RT-PCR. ERalpha concentrations were low in NA, in adrenal tissues adjacent to ACA (51+/-33), in ACC (53+/-78), and lower in ACA (11+/-11 fmol/mg DNA). Conversely, PR concentrations were high in NA and adrenal tissues adjacent to ACA, at 307+/-216 fmol/mg DNA, and were even higher in tumors - 726+/-706 fmol/mg DNA in ACA and 1154+/-1586 fmol/mg DNA in ACC - and in isolated PPNAD nodules. Binding study results in four tumors were compatible with binding to a steroid receptor. In patients with PPNAD, a strong positive immunohistochemical signal was associated with the sole isolated nodular regions. ERbeta transcript levels were very high in all samples except those for two ACCs, whereas aromatase levels were low. PR and ERbeta are clearly present in normal adrenal glands and adrenal tumors. Further studies may shed light on the possible pathogenic role of these receptors in adrenal proliferation.

Midnight salivary cortisol determination for assessing the outcome of transsphenoidal surgery in Cushing's disease.

CONTEXT: Midnight salivary cortisol (MSC) is now recognized as a reliable index for Cushing's syndrome diagnosis but has to be validated for the follow-up of treated patients. OBJECTIVE: Our objective was to evaluate MSC for assessing the outcome of transsphenoidal surgery (TSS) in patients with Cushing's disease (CD). DESIGN: We conducted a retrospective cohort study in a single center. PATIENTS AND METHODS: Sixty-eight patients treated by TSS between 1996 and 2006 and followed for at least 6 months with postoperative MSC were included. Mean follow-up (+/- sd) was 45 +/- 31 months. Morning plasma cortisol was determined 5 d after TSS, and MSC and urinary cortisol (UC) were determined 6-12 months after surgery. The remission group included hypocortisolic (morning plasma cortisol < 50 ng/ml and/or insufficient response to cosyntropin) and eucortisolic (midnight plasma cortisol < 75 ng/ml and normal UC) patients. Patients in the treatment failure group had high midnight plasma cortisol and UC concentrations. RESULTS: Fifty patients (74%) were in remission. Mean MSC was 0.7 +/- 0.4 ng/ml (range, 0.4-2.1 ng/ml) and 6.5 +/- 6.5 ng/ml (range, 2.1-27.2 ng/ml) for the remission and treatment failure groups, respectively (P = 0.001). A cutoff of 2 ng/ml for MSC gave a sensitivity of 100% and a specificity of 98% for treatment failure diagnosis, whereas UC less than 90 microg/d had a sensitivity of 71% and specificity of 98%. Postsurgical morning plasma cortisol less than or equal to 18 ng/ml had a sensitivity of 93% and specificity of 74%. CONCLUSIONS: MSC is a simple, robust marker of remission after TSS for CD.

Wnt/beta-catenin and 3',5'-cyclic adenosine 5'-monophosphate/protein kinase A signaling pathways alterations and somatic beta-catenin gene mutations in the progression of adrenocortical tumors.

BACKGROUND: The Wnt/beta-catenin and cAMP signaling pathways play an important role in adrenal cortex tumorigenesis. Somatic activating mutations of the beta-catenin gene (CTNNB1) are the most frequent genetic defects identified both in adrenocortical adenomas (ACAs) and adrenocortical cancers (ACCs). PRKAR1A mutations leading to cAMP pathway dysregulation are observed in primary pigmented nodular adrenocortical diseases (PPNADs) and some sporadic ACAs. OBJECTIVE: The objective of the investigation was to study Wnt/beta-catenin dysregulation in adrenocortical tumors (ACTs) with cAMP pathway genetic alteration and search for secondary CTNNB1 somatic mutations in heterogeneous tumors. PATIENTS AND METHODS: Nine PPNADs, including five with macronodules, three ACAs with PRKAR1A somatic mutations, and one heterogeneous tumor with ACC developed within an ACA, were studied by immunohistochemistry and DNA sequencing. RESULTS: beta-Catenin accumulation was observed in all PPNADs, ACAs with PRKAR1A mutations, and the ACC component of the heterogeneous tumor. CTNNB1 somatic activating mutations were found in the macronodule of two of the five macronodular PPNADs, in one ACA with a PRKAR1A somatic mutation, and in the malignant part of the heterogeneous ACT. CONCLUSIONS: The Wnt/beta-catenin pathway is activated in PPNADs and ACAs with PRKAR1A mutations, suggesting a cross talk between the cAMP and Wnt/beta-catenin pathways in ACT development. In addition, the occurrence as an additional hit of a CTNNB1 somatic mutation is associated with larger or more aggressive ACTs. This underlines the importance of the Wnt/beta-catenin pathway in adrenal cortex tumorigenesis and the importance of genetic accumulation in the progression of ACTs.

Differential expression of parathyroid hormone-related protein in adrenocortical tumors: autocrine/paracrine effects on the growth and signaling pathways in H295R cells.

Adrenocortical tumors (ACT) are rare and heterogeneous, but their pathogenesis is unclear. The oncoprotein parathyroid hormone-related protein (PTHrP), found in many common tumors, can regulate their growth in an autocrine/paracrine fashion through the PTH-R1 receptor. Little is known about the role of PTHrP in ACT. We monitored the synthesis of PTHrP and PTH-R1 in a series of 25 ACT: 12 adrenocortical carcinomas (ACC) and 13 adrenocortical adenomas (ACA), and investigated the effects of PTHrP(1-34) on H295R cells derived from an ACC. PTH-R1 mRNA and proteins were detected by real-time PCR and Western blotting in all the ACT samples and in H295R cells. Their concentrations did not differ significantly from one ACT to another. PTHrP mRNA was assayed by quantitative real-time PCR. It was detected in 90% of ACC, and in 10% of ACA. There was a positive correlation with the prognostic factors, McFarlane stage and Weiss score. Tissue-specific PTHrP protein processing was shown by Western blotting. Immunohistochemical staining revealed numerous, dense foci of PTHrP-containing cells in ACC, but few positive cells in ACA or normal tissue. PTHrP stimulated the growth of H295R cells, whereas a specific anti-PTHrP antibody and a PTHrP-R1 antagonist both enhanced their apoptosis. PTHrP activated both adenylate cyclase/protein kinase A and the intracellular calcium/protein kinase C pathways via PTHrP-R1. The active synthesis of PTHrP is linked to poor prognosis in ACC, in which it may act as an autocrine/paracrine factor in tumor growth and malignancy.

Rapidly reversible myocardial edema in patients with acromegaly: assessment with ultrafast T2 mapping in a single-breath-hold MRI sequence.

OBJECTIVE: The purpose of this study was to use a single-breath-hold T2-mapping MRI sequence to evaluate the reversibility of myocardial edema in patients treated for acromegaly. SUBJECTS AND METHODS: Before and after treatment, 15 patients with acromegaly underwent myocardial T2 mapping with an experimental single-breath-hold black-blood fast spin-echo sequence. Myocardial T2 mapping with both a multiple-breath-hold fast spinecho sequence and the experimental sequence also was performed on 14 volunteers. T2 relaxation times were calculated with a standard linear least-squares fit applied to myocardial signal intensity. The T2 relaxation times of patients were compared with those of volunteers and correlated with levels of serum growth hormone and insulinlike growth factor 1. Left ventricular function and mass index were determined with cine MRI. RESULTS: T2 values before treatment were higher in patients (71 +/- 12 milliseconds) than in volunteers (55.9 +/- 3.6 milliseconds) (p = 0.0003). These T2 values in patients decreased soon after treatment (57.6 +/- 6.6 milliseconds, p = 0.0007). This reduction correlates with successful reduction of levels of serum growth hormone and insulinlike growth factor 1. In volunteers, myocardial T2 values did not vary significantly between the single-breath-hold sequence and the multiple-breath-hold fast spin-echo sequence. In patients, myocardial mass and left ventricular function did not differ significantly before and after treatment. CONCLUSION: Patients with acromegaly have increased myocardial T2 values, which decrease soon after treatment, reflecting reversible myocardial edema. T2 value is more sensitive than left ventricular mass index in the detection of early reversal of acromegalic cardiomyopathy. These results highlight the potential role of MRI in direct assessment of the tissular effects of growth hormone and insulinlike growth factor 1 and in evaluation of the efficacy of treatment.