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Purpose: The purpose of this study was to evaluate the capabilities of multiparametric magnetic resonance imaging (MRI) in differentiating between lipid-poor adrenal adenoma (LPAA) and adrenocortical carcinoma (ACC)., Materials and Methods: Patients of two centers who underwent surgical resection of LPAA or ACC after multiparametric MRI were retrospectively included. A training cohort was used to build a diagnostic algorithm obtained through recursive partitioning based on multiparametric MRI variables, including apparent diffusion coefficient and chemical shift signal ratio (i.e., tumor signal intensity index). The diagnostic performances of the multiparametric MRI-based algorithm were evaluated using a validation cohort, alone first and then in association with adrenal tumor size using a cut-off of 4 cm. Performances of the diagnostic algorithm for the diagnosis of ACC vs. LPAA were calculated using pathology as the reference standard., Results: Fifty-four patients (27 with LPAA and 27 with ACC; 37 women; mean age, 48.5 ± 13.3 [standard deviation (SD)] years) were used as the training cohort and 61 patients (24 with LPAA and 37 with ACC; 47 women; mean age, 49 ± 11.7 [SD] years) were used as the validation cohort. In the validation cohort, the diagnostic algorithm yielded best accuracy for the diagnosis of ACC vs. LPAA (75%; 46/61; 95% CI: 55-88) when used without lesion size. Best sensitivity was obtained with the association of the diagnostic algorithm with tumor size (96%; 23/24; 95% CI: 80-99). Best specificity was obtained with the diagnostic algorithm used alone (76%; 28/37; 95% CI: 60-87)., Conclusion: A multiparametric MRI-based diagnostic algorithm that includes apparent diffusion coefficient and tumor signal intensity index helps discriminate between ACC and LPAA with high degrees of specificity and accuracy. The association of the multiparametric MRI-based diagnostic algorithm with adrenal lesion size helps maximize the sensitivity of multiparametric MRI for the diagnosis of ACC., Competing Interests: Declaration of competing interest The authors declare no actual or potential conflict of interest related to this study., (Copyright © 2024. Published by Elsevier Masson SAS.)

Since the first description of Nelson syndrome 60 years ago, 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 speeds (CTPS) can be observed. Using simple metrics in patients with CTP, CTPS was reported to vary from a few millimeters to up to 40 mm per year. Rapid CTPS/ Nelson's syndrome was associated with more severe Cushing's disease, higher adrenocorticotropic hormone (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 with intermediate morning ACTH measurements probably provide a reliable way to detect early and manage fast-growing tumors and, therefore, limit the complications., Competing Interests: We declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported., (Thieme. All rights reserved.)

Context: Outcome of craniopharyngioma is related to its locoregional extension, which impacts resectability and the risk of surgical complications. To maximize resection and minimize complications, optic tract localization, temporal lobe extension, and hypothalamic involvement are essential factors for surgical management., Objective: To assess the outcome of craniopharyngiomas depending on their relation to the hypothalamus location., Methods: We conducted a retrospective analysis of 79 patients with a craniopharyngioma who underwent surgery from 2007 to 2022. Craniopharyngiomas were classified in 3 groups, depending on the type of hypothalamus involvement assessed by preoperative magnetic resonance imaging: infra-hypothalamic (type A, n = 33); perforating the hypothalamus (type B, n = 40); and supra-hypothalamic (type C, n = 6). Surgical strategy was guided by the type of hypothalamic involvement, favoring endonasal approaches for type A and type B, and transcranial approaches for type C., Results: Long-term disease control was achieved in 33/33 (100%), 37/40 (92%), and 5/6 (83%) patients in type A, B, and C, respectively. In type B, vision was improved in 32/36 (89%) patients, while hypothalamic function was improved, stable, or worsened in 6/40 (15%), 32/40 (80%), and 2/40 (5%) patients, respectively. Papillary craniopharyngiomas were found in 5/33 (15%), 9/40 (22%), and 3/6 (50%) patients in types A, B, and C, respectively. In 4 patients, BRAF/MEK inhibitors were used, with significant tumor shrinkage in all cases., Conclusion: Craniopharyngiomas located below the hypothalamus or perforating it can be safely treated by transsphenoidal surgery. For supra-hypothalamic craniopharyngiomas, postoperative results are less favorable, and documenting a BRAF mutation may improve outcome, if targeted therapy was efficient enough to replace surgical debulking., (© The Author(s) 2024. Published by Oxford University Press on behalf of the Endocrine Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Objective: Cushing's syndrome is characterized by high morbidity and mortality with high interindividual variability. Easily measurable biomarkers, in addition to the hormone assays currently used for diagnosis, could reflect the individual biological impact of glucocorticoids. The aim of this study is to identify such biomarkers through the analysis of whole blood transcriptome., Design: Whole blood transcriptome was evaluated in 57 samples from patients with overt Cushing's syndrome, mild Cushing's syndrome, eucortisolism, and adrenal insufficiency. Samples were randomly split into a training cohort to set up a Cushing's transcriptomic signature and a validation cohort to assess this signature., Methods: Total RNA was obtained from whole blood samples and sequenced on a NovaSeq 6000 System (Illumina). Both unsupervised (principal component analysis) and supervised (Limma) methods were used to explore the transcriptome profile. Ridge regression was used to build a Cushing's transcriptome predictor., Results: The transcriptomic profile discriminated samples with overt Cushing's syndrome. Genes mostly associated with overt Cushing's syndrome were enriched in pathways related to immunity, particularly neutrophil activation. A prediction model of 1500 genes built on the training cohort demonstrated its discriminating value in the validation cohort (accuracy .82) and remained significant in a multivariate model including the neutrophil proportion (P = .002). Expression of FKBP5, a single gene both overexpressed in Cushing's syndrome and implied in the glucocorticoid receptor signaling, could also predict Cushing's syndrome (accuracy .76)., Conclusions: Whole blood transcriptome reflects the circulating levels of glucocorticoids. FKBP5 expression could be a nonhormonal marker of Cushing's syndrome., Competing Interests: Conflict of interest: G.A. is on the editorial board of EJE. G.A. was not involved in the review or editorial process for this paper, on which he is listed as an author., (© The Author(s) 2024. Published by Oxford University Press on behalf of European Society of Endocrinology.)

Cushing's syndrome is due to overproduction of cortisol, leading to abnormal and prolonged exposure to cortisol. The most common etiology is Cushing disease, while adrenal causes are rarer. Knowledge of the genetics of Cushing's syndrome, and particularly the adrenal causes, has improved considerably over the last 10 years, thanks in particular to technical advances in high-throughput sequencing. The present study, by a group of experts from the French Society of Endocrinology and the French Society of Pediatric Endocrinology and Diabetology, reviewed the literature on germline genetic alterations leading to a predisposition to develop Cushing's syndrome. The review led to a consensus statement on genetic screening for Cushing disease and adrenal Cushing's syndrome., (Copyright © 2024 The Author(s). Published by Elsevier Masson SAS.. All rights reserved.)

Background: During pituitary surgery, CSF leaks are often treated by intrasellar packing, using muscle or fat grafts. However, this strategy may interfere with the interpretation of postoperative MRI and may impact the quality of resection in cases of second surgery, due to the existence of additional fibrous tissue. We present an alternative technique, using a diaphragm reconstruction with a heterologous sponge combining fibrinogen and thrombin (TachoSil), applied in selected patients with low-flow CSF leaks. This study investigates the surgical outcome of patients treated with this strategy., Methods: From a cohort of 2231 patients treated from June 2011 to June 2023 by endoscopic endonasal approach for pituitary surgery, the surgical technique of diaphragm repair with TachoSil patch performed in 55 patients (2.6%) was detailed, and the rate of closure failure was analyzed at 6 months postoperatively. No intrasellar packing was used and sellar floor reconstruction was performed whenever possible. The rate of postoperative CSF leak was compared with that reported in three previous publications that also used the TachoSil patch technique., Results: Patients were mostly women (F/M ratio: 1.2) with a median age of 53.6 years. Surgery was indicated for non-functioning adenomas, Cushing's disease, acromegaly, and Rathke's cleft cysts in 38/55 (69.1%), 6/55 (10.9%), 5/55 (9.1%) and 6/55 (10.9%) patients respectively. The rate of postoperative CSF leak was 1.8% (n = 1/55), which was not significantly different from that reported in the three cohorts from the literature (2.8%, p > 0.05). No postoperative meningitis was recorded., Conclusions: In highly selected patients with low-flow CSF leaks related to small focal diaphragm defects, diaphragm reconstruction using a TachoSil patch can be a safe and valuable alternative to intrasellar packing., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Austria, part of Springer Nature.)

COVID-19 is associated with heterogeneous outcome. Early identification of a severe progression of the disease is essential to properly manage the patients and improve their outcome. Biomarkers reflecting an increased inflammatory response, as well as individual features including advanced age, male gender, and pre-existing comorbidities, are risk factors of severe COVID-19. Yet, these features show limited accuracy for outcome prediction. The aim was to evaluate the prognostic value of whole blood transcriptome at an early stage of the disease. Blood transcriptome of patients with mild pneumonia was profiled. Patients with subsequent severe COVID-19 were compared to those with favourable outcome, and a molecular predictor based on gene expression was built. Unsupervised classification discriminated patients who would later develop a COVID-19-related severe pneumonia. The corresponding gene expression signature reflected the immune response to the viral infection dominated by a prominent type I interferon, with IFI27 among the most over-expressed genes. A 48-genes transcriptome signature predicting the risk of severe COVID-19 was built on a training cohort, then validated on an external independent cohort, showing an accuracy of 81% for predicting severe outcome. These results identify an early transcriptome signature of severe COVID-19 pneumonia, with a possible relevance to improve COVID-19 patient management., (© 2024. The Author(s).)

In endocrinology, the types and quantity of digital data are increasing rapidly. Computing capabilities are also developing at an incredible rate, as illustrated by the recent expansion in the use of popular generative artificial intelligence (AI) applications. Numerous diagnostic and therapeutic devices using AI have already entered routine endocrine practice, and developments in this field are expected to continue to accelerate. Endocrinologists will need to be supported in managing AI applications. Beyond technological training, interdisciplinary vision is needed to encompass the ethical and legal aspects of AI, to manage the profound impact of AI on patient/provider relationships, and to maintain an optimal balance between human input and AI in endocrinology., (© The Author(s) 2024. Published by Oxford University Press on behalf of the Endocrine Society. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Objective: Carney complex (CNC) is a rare genetic syndrome, mostly due to germline loss-of-function pathogenic variants in PRKAR1A. Carney complex includes pigmented skin lesions, cardiac myxomas, primary pigmented nodular adrenocortical dysplasia, and various breast benign tumors., Design: The present study was designed to describe the characteristics of breast lesions in CNC patients and their association with other manifestations of CNC and PRKAR1A genotype., Methods: A 3-year follow-up multicenter French prospective study of CNC patients included 50 women who were analyzed for CNC manifestations and particularly breast lesions, with breast imaging, genotyping, and hormonal settings., Results: Among the 38 women with breast imaging, 14 (39%) had breast lesions, half of them bilateral. Ten women (26%) presented with benign lesions and six with breast carcinomas (16%): one had ductal carcinoma in situ at 54, and five had invasive cancer before 50 years old, whom one with contralateral breast cancer during follow-up. The occurrence of breast cancer was more frequent in women with PRKAR1A pathogenic variant odds ratio = 6.34 (1.63-17.91) than in general population of same age. The mean age at breast cancer diagnosis was 44.7 years old: 17 years younger than in the general population. Breast cancer patients had good prognosis factors. All breast carcinomas occurred in individuals with familial CNC and PRKAR1A pathogenic variants. Loss of heterozygosity at the PRKAR1A locus in the 2 invasive breast carcinomas analyzed suggested a driver role of this tumor suppressor gene., Conclusions: As CNC could predispose to breast carcinoma, an adequate screening strategy and follow-up should be discussed in affected women., Clinical Trial Registration: ClinicalTrial.gov NCT00668291., Competing Interests: Conflict of interest: The authors have no conflict of interest to declare. Co-authors G.R. and G.A. are on the editorial board of EJE. They were not involved in the review or editorial process for this paper, on which they are listed as authors., (© The Author(s) 2024. Published by Oxford University Press on behalf of European Society of Endocrinology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Background: As the population ages, the number of elderly patients with an indication for pituitary surgery is rising. Information on the outcome of patients aged over 75 is limited. This study reports a large series assessing the feasibility of surgical resection in this specific age range, focusing on surgical complications and postoperative results., Methods: A retrospective cohort study of patients with pituitary adenomas and Rathke's cleft cysts was conducted. All patients were aged 75 years or over and treated by a single expert neurosurgical team. A control population included 2379 younger adult patients operated by the same surgeons during the same period., Results: Between 2008 and 2022, 155 patients underwent surgery. Indication was based on vision impairment in most patients (79%). Median follow-up was 13 months (range: 3-96). The first surgery was performed with an endoscopic transsellar approach, an extended endonasal transtuberculum approach and a microscopic transcranial approach in 96%, 3%, and 1% of patients, respectively. Single surgery was sufficient to obtain volume control in 97% of patients. From Kaplan-Meier estimates, 2-year and 5-year disease control with a single surgery were 97.3% and 86.2%, respectively. Resection higher than 80% was achieved in 77% of patients. No vision worsening occurred. In acromegaly and Cushing's disease, endocrine remission was obtained in 90% of non-invasive adenomas. Surgical complications were noted in 5% of patients, with 30-day mortality, hematoma, cerebrospinal fluid leak, meningitis, and epistaxis occurring in 0.6%, 0.6%, 1.9%, 0.6%, and 1.3% respectively. New endocrine anterior deficits occurred in only 5%, while no persistent diabetes insipidus was noted. Compared with younger patients, the complication rate was not statistically different., Conclusions: Surgery beyond the age of 75, mainly relying on an endoscopic endonasal transsellar approach, is effective and safe, provided that patients are managed in tertiary centers., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Austria, part of Springer Nature.)

Gastrointestinal stromal tumors (GISTs) are mesenchymal neoplasms, believed to originate from the interstitial cells of Cajal (ICC), often caused by overexpression of tyrosine kinase receptors (TKR) KIT or PDGFRA. Here, we present evidence that the embryonic stem cell factor FOXD3, first identified as 'Genesis' and involved in both gastrointestinal and neural crest cell development, is implicated in GIST pathogenesis; its involvement is investigated both in vitro and in zebrafish and a mouse model of FOXD3 deficiency. Samples from a total of 58 patients with wild-type GISTs were used for molecular analyses, including Sanger sequencing, comparative genomic hybridization, and methylation analysis. Immunohistochemistry and western blot evaluation were used to assess FOXD3 expression. Additionally, we conducted in vitro functional studies in tissue samples and in transfected cells to confirm the pathogenicity of the identified genetic variants. Germline partially inactivating FOXD3 sequence variants (p.R54H and p.Ala88_Gly91del) were found in patients with isolated GISTs. Chromosome 1p loss was the most frequent chromosomal abnormality identified in tumors. In vitro experiments demonstrate the impairment of FOXD3 in the presence of those variants. Animal studies showed disruption of the GI neural network and changes in the number and distribution in the ICC. FOXD3 suppresses KIT expression in human cells; its inactivation led to an increase in ICC in zebrafish, as well as mice, providing evidence for a functional link between FOXD3 defects and KIT overexpression leading to GIST formation.

Background: Adrenocortical carcinoma is rare and aggressive endocrine cancer of the adrenal gland. Within adrenocortical carcinoma, a recently described subtype characterized by a CpG island methylator phenotype (CIMP) has been associated with an especially poor prognosis. However, the drivers of CIMP remain unknown. Furthermore, the functional relation between CIMP and poor clinical outcomes of patients with adrenocortical carcinoma stays elusive., Results: Here, we show that CIMP in adrenocortical carcinoma is linked to the increased expression of DNA methyltransferases DNMT1 and DNMT3A driven by a gain of gene copy number and cell hyperproliferation. Importantly, we demonstrate that CIMP contributes to tumor aggressiveness by favoring tumor immune escape. This effect could be at least partially reversed by treatment with the demethylating agent 5-azacytidine., Conclusions: In sum, our findings suggest that co-treatment with demethylating agents might enhance the efficacy of immunotherapy and could represent a novel therapeutic approach for patients with high CIMP adrenocortical carcinoma., (© 2023. The Author(s).)

The classification of tumours of the pituitary gland has recently been revised in the 2021 5th edition World Health Organization (WHO) Classification of Central Nervous System Tumours (CNS5) and 2022 5th edition WHO Classification of Endocrine and Neuroendocrine Tumours (ENDO5). This brief review aims to appraise the most relevant changes and updates introduced in the two classifications. A new nomenclature has been introduced in CNS5 and ENDO5 to align adenohypophyseal tumours with the classification framework of neuroendocrine neoplasia. The term pituitary neuroendocrine tumour (PitNET) with subtype information has therefore been adopted and preferred to adenoma. Pituitary carcinoma has been replaced by metastatic PitNET. The ICD-O coding has been changed from benign to malignant in line with NETs from other organs. Histological typing and subtyping based on immunohistochemistry for lineage-restricted pituitary transcription factors are regarded as the cornerstone for accurate classification. Such an approach does not fully reflect the complexity and dynamics of pituitary tumorigenesis and the variability of transcription factors expression. ENDO5 does not support a grading and/or staging system and argues that histological typing and subtyping are more robust than proliferation rate and invasiveness to stratify tumours with low or high risk of recurrence. However, the prognostic and predictive relevance of histotype is not fully validated. Recent studies suggest the existence of clinically relevant molecular subgroups and emphasize the need for a standardized, histo-molecular integrated approach to the diagnosis of PitNETs to further our understanding of their biology and overcome the unsolved issue of grading and/or staging system.

Purpose of the Report: Adrenocortical carcinoma (ACC) is an extremely rare endocrine malignancy, which cannot always be diagnosed during conventional radiology and hormonal investigations. 18 F-FDG PET could help predict malignancy, but more data are necessary to support future guidelines., Methods: A cohort of 63 patients with histologically proven ACC (n = 55) or metastatic ACC with steroid oversecretion (n = 8) was assembled. All patients underwent an 18 F-FDG PET, and the SUV max and the adrenal-to-liver SUV max ratio were calculated. The 18 F-FDG PET parameters were compared with clinical, pathological, and outcome data., Results: Fifty-six of 63 patients (89%) had an ACC with an adrenal-to-liver SUV max ratio >1.45, which was a previously defined cutoff value to predict malignancy with 100% sensitivity. Seven ACCs (11%) had a lower uptake (adrenal-to-liver SUV max <1.45), most of them with a proliferation marker Ki-67 expression level <10%. A positive correlation between 18 F-FDG PET parameters (SUV max and adrenal-to-liver SUV max ratio) and tumor size, ENSAT (European Network for the Study of Adrenal Tumors) staging, total Weiss score, and the Ki-67 was found. The strong correlation between SUV max and Ki-67 ( r = 0.47, P = 0.0009) suggests a relationship between 18 F-FDG uptake levels and tumor proliferation. No statistically significant associations between outcome parameters (progression-free or overall survival) and 18 F-FDG PET parameters were found., Conclusions: This large cohort study shows that most cases of ACC demonstrate high 18 F-FDG uptake. However, the positive correlation observed between SUV max and Ki-67 expression levels seems to explain the possibility of identifying some ACC with a low or inexistent 18 F-FDG uptake. These findings have practical implications for the management of patients with an adrenal mass., Competing Interests: Conflicts of interest and sources of funding: none declared., (Copyright © 2023 Wolters Kluwer Health, Inc. All rights reserved.)

Introduction: Diagnosis of endogenous hyperinsulinism relies on the occurrence of a hypoglycemia, concomitant with inadequate high insulin and C-peptide levels. However, diagnostic cutoffs are not consensual among the different learned societies. The objective of this work was to propose optimized cutoffs for these three parameters for the diagnosis of endogenous hyperinsulinism., Methods: All the patients having performed a fasting trial in Cochin Hospital Endocrinology Department between February 2012 and August 2022 were included. The results of glycemia, insulin and C-peptide levels during fasting trial were collected and analyzed., Results: One hundred and fifty-nine patients were included: 26 with endogenous hyperinsulinism and 133 without endogenous hyperinsulinism. ROC analysis of glycemia nadir during fasting trial identified the value of 2.3 mmol/L as the optimal cutoff, ensuring a sensitivity of 100% associated with a specificity of 81%. ROC analysis of insulin and C-peptide levels concomitant with hypoglycemia <2.3 mmol/L showed very good diagnostic performances of both parameters with respective cutoffs of 3.1 mUI/L (=21.5 pmol/L; sensitivity = 96%; specificity = 92%) and 0.30 nmol/L (sensitivity = 96%; specificity = 100%). Insulin to glycemia ratio as well as C-peptide to glycemia ratio (in pmol/mmol) at the time of glycemia nadir did not show better diagnostic performances than C-peptide alone., Conclusion: A C-peptide level 0.3 nmol/L concomitant with a hypoglycemia <2.3 mmol/L appears as the best criterion to make the diagnosis of endogenous hyperinsulinism. Insulin level can be underestimated on hemolyzed blood samples, frequently observed in fasting trial, and thus shows lower diagnostic performances., (© The Author(s) 2023. Published by Oxford University Press on behalf of (ESE) European Society of Endocrinology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

In the elective field of adrenal imaging, artificial intelligence (AI) can be used for adrenal lesion detection, characterization, hypersecreting syndrome management and patient follow-up. Although a perfect AI tool that includes all required steps from detection to analysis does not exist yet, multiple AI algorithms have been developed and tested with encouraging results. However, AI in this setting is still at an early stage. In this regard, most published studies about AI in adrenal gland imaging report preliminary results that do not have yet daily applications in clinical practice. In this review, recent developments and current results of AI in the field of adrenal imaging are presented. Limitations and future perspectives of AI are discussed., Competing Interests: Declaration of Competing Interest The authors declare that they have no competing interest in relation with this article., (Copyright © 2022. Published by Elsevier Masson SAS.)

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.

Background: Arterial hypertension represents a worldwide health burden and a major risk factor for cardiovascular morbidity and mortality. Hypertension can be primary (primary hypertension, PHT), or secondary to endocrine disorders (endocrine hypertension, EHT), such as Cushing's syndrome (CS), primary aldosteronism (PA), and pheochromocytoma/paraganglioma (PPGL). Diagnosis of EHT is currently based on hormone assays. Efficient detection remains challenging, but is crucial to properly orientate patients for diagnostic confirmation and specific treatment. More accurate biomarkers would help in the diagnostic pathway. We hypothesized that each type of endocrine hypertension could be associated with a specific blood DNA methylation signature, which could be used for disease discrimination. To identify such markers, we aimed at exploring the methylome profiles in a cohort of 255 patients with hypertension, either PHT (n = 42) or EHT (n = 213), and at identifying specific discriminating signatures using machine learning approaches., Results: Unsupervised classification of samples showed discrimination of PHT from EHT. CS patients clustered separately from all other patients, whereas PA and PPGL showed an overall overlap. Global methylation was decreased in the CS group compared to PHT. Supervised comparison with PHT identified differentially methylated CpG sites for each type of endocrine hypertension, showing a diffuse genomic location. Among the most differentially methylated genes, FKBP5 was identified in the CS group. Using four different machine learning methods-Lasso (Least Absolute Shrinkage and Selection Operator), Logistic Regression, Random Forest, and Support Vector Machine-predictive models for each type of endocrine hypertension were built on training cohorts (80% of samples for each hypertension type) and estimated on validation cohorts (20% of samples for each hypertension type). Balanced accuracies ranged from 0.55 to 0.74 for predicting EHT, 0.85 to 0.95 for predicting CS, 0.66 to 0.88 for predicting PA, and 0.70 to 0.83 for predicting PPGL., Conclusions: The blood DNA methylome can discriminate endocrine hypertension, with methylation signatures for each type of endocrine disorder., (© 2022. The Author(s).)

Corticotroph macroadenomas are rare but difficult to manage intracranial neoplasms. Mutations in the two Cushing's disease mutational hotspots USP8 and USP48 are less frequent in corticotroph macroadenomas and invasive tumors. There is evidence that TP53 mutations are not as rare as previously thought in these tumors. The aim of this study was to determine the prevalence of TP53 mutations in corticotroph tumors, with emphasis on macroadenomas, and their possible association with clinical and tumor characteristics. To this end, the entire TP53 coding region was sequenced in 86 functional corticotroph tumors (61 USP8 wild type; 66 macroadenomas) and the clinical characteristics of patients with TP53 mutant tumors were compared with TP53/USP8 wild type and USP8 mutant tumors. We found pathogenic TP53 variants in 9 corticotroph tumors (all macroadenomas and USP8 wild type). TP53 mutant tumors represented 14% of all functional corticotroph macroadenomas and 24% of all invasive tumors, were significantly larger and invasive, and had higher Ki67 indices and Knosp grades compared to wild type tumors. Patients with TP53 mutant tumors had undergone more therapeutic interventions, including radiation and bilateral adrenalectomy. In conclusion, pathogenic TP53 variants are more frequent than expected, representing a relevant amount of functional corticotroph macroadenomas and invasive tumors. TP53 mutations associated with more aggressive tumor features and difficult to manage disease., (© 2022. The Author(s).)

Objective: The management of giant pituitary tumors is complex, with few publications and recommendations. Consequently, patient's care mainly relies on clinical experience. We report here a first large series of patients with giant pituitary tumors managed by a multidisciplinary expert team, focusing on treatments and outcome., Methods: A retrospective cohort study was conducted. Giant pituitary tumors were defined by a main diameter > 40mm. Macroprolactinomas sensitive to dopamine agonists were excluded. All patients were operated by a single neurosurgical team. After surgery, multimodal management was proposed, including hormone replacement, radiotherapy and anti-tumor medical therapies. Outcome was modeled using Kaplan-Meyer representation. A logistic regression model was built to identify the risk factors associated with surgical complications., Results: 63 consecutive patients presented a giant adenoma, most often with visual defects. Patients were operated once, twice or three times in 59%, 40% and 1% of cases respectively, mainly through endoscopic endonasal approach. Giant adenomas included gonadotroph, corticotroph, somatotroph, lactotroph and mixed GH-PRL subtypes in 67%, 14%, 11%, 6% and 2% of patients respectively. Vision improved in 89% of patients with prior visual defects. Severe surgical complications occurred in 11% of patients, mainly for tumors > 50 mm requiring microscopic transcranial approach. Additional radiotherapy was needed for 29% of patients, 3 to 56 months after first surgery. For 6% of patients, Temozolomide treatment was required, 19 to 66 months after first surgery., Conclusions: Giant pituitary tumors require multimodal management, with a central role of surgery. Most often, tumor control can be achieved by expert multidisciplinary teams., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Gaillard, Adeniran, Villa, Jouinot, Raffin-Sanson, Feuvret, Verrelle, Bonnet, Dohan, Bertherat, Assié and Baussart.)

Hypertension is a major global health problem with high prevalence and complex associated health risks. Primary hypertension (PHT) is most common and the reasons behind primary hypertension are largely unknown. Endocrine hypertension (EHT) is another complex form of hypertension with an estimated prevalence varying from 3 to 20% depending on the population studied. It occurs due to underlying conditions associated with hormonal excess mainly related to adrenal tumours and sub-categorised: primary aldosteronism (PA), Cushing's syndrome (CS), pheochromocytoma or functional paraganglioma (PPGL). Endocrine hypertension is often misdiagnosed as primary hypertension, causing delays in treatment for the underlying condition, reduced quality of life, and costly antihypertensive treatment that is often ineffective. This study systematically used targeted metabolomics and high-throughput machine learning methods to predict the key biomarkers in classifying and distinguishing the various subtypes of endocrine and primary hypertension. The trained models successfully classified CS from PHT and EHT from PHT with 92% specificity on the test set. The most prominent targeted metabolites and metabolite ratios for hypertension identification for different disease comparisons were C18:1, C18:2, and Orn/Arg. Sex was identified as an important feature in CS vs. PHT classification.

Objective: Large response of steroid precursors, including 17-hydroxyprogesterone, to adrenocorticotropic hormone (ACTH) has been described in adrenocortical tumors, suggesting the existence of intra-tumoral enzymatic deficiencies. This study aimed to compare steroidogenesis enzymes activity in unilateral and bilateral benign tumors using serum steroid profiling in liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) in the basal state and after ACTH 1-24 stimulation., Design and Methods: A serum profile of seven consecutive adrenal steroids was determined in LC-MS/MS in the basal state (T0) and after ACTH 1-24 stimulation (T60) in 35 patients with bilateral adrenocortical tumors (BL), 38 patients with unilateral tumors (UL) and 37 control subjects (CT). Response amplitude of each individual steroid was evaluated by T60/T0 ratio, whereas enzymatic activity was assessed by the downstream/upstream steroid ratio. Adrenal volume was quantified by a semi-automatic segmentation method., Results: For the seven steroids assayed, the amplitude of response to ACTH was higher in BL than in UL and in CT. The difference between BL and UL persisted even after matching patients on adrenal volume. On glucocorticoids pathway, enzymatic activity of CYP11B1 was significantly decreased in BL (78.3 (43.1-199.4)) in comparison to both UL (122.7 (13.8-228.4), P = 0.0002) and CT (186.8 (42.1-1236.3), P < 0.0001). On mineralocorticoids and androgens pathways, the enzymatic activity of CYP11B2 and CYP17A1-17,20 lyase was also lower in BL than UL and CT., Conclusions: Decreased activity of distal steroidogenesis enzymes CYP11B1, CYP11B2 and CYP17A1-17,20 lyase, responsible for an explosive response to ACTH of upstream precursors in bilateral tumors, limits the synthesis of bioactive steroids, in particular cortisol, despite the increase in adrenal mass., Significance Statement: Activity of distal steroidogenesis enzymes (CYP11B1, CYP11B2 and CYP17A1 on glucocorticoids, mineralocorticoids and androgens pathways, respectively) is decreased in adrenocortical benign tumors. This decrease is more pronounced in bilateral lesions and seems to depend more on the nature of the lesion than on the increase in adrenal volume. It is responsible for the explosive response to ACTH of steroid precursors located upstream of these enzymes. It probably allows bioactive steroids, particularly cortisol, to stay in the normal range for a long time despite the increase in adrenal mass.

Introduction: Osilodrostat is a new 11β-hydroxylase inhibitor with a mode of action analogous to Metyrapone. The objective of this study was to compare steroidogenic profiles in patients treated with either Osilodrostat or Metyrapone for adrenocorticotrophic hormone (ACTH)-dependent Cushing's syndrome (CS)., Methods: Patients followed up at Cochin hospital Endocrinology department between March 2019 and December 2021 for an ACTH-dependent CS, controlled by either Osilodrostat or Metyrapone, were included. A serum profile of five steroids (cortisol, 11-deoxycortisol, 17-hydroxyprogesterone, androstenedione and testosterone) was determined using UPLC- tandem mass spectrometry (UPLC-MS/MS)., Results: Nineteen patients treated with Osilodrostat, eight patients treated with Metyrapone and six patients treated with consecutive Metyrapone then Osilodrostat were included. Hypocortisolism (basal cortisol <100 nmol/L) was found in 48% of patients treated with Osilodrostat and 7% of patients treated with Metyrapone. 11-deoxycortisol and androstenedione levels were higher in patients treated with Metyrapone (80.9 (2.2-688.4) and 14.9 (2.5-54.3) nmol/L, respectively) than in patients treated with Osilodrostat (10.3 (0.5-71.9) and 4.0 (0.3-13.3) nmol/L) (P = 0.0009 and P = 0.0005). Testosterone level in women was also higher in Metyrapone group (3.3 (0.93-4.82) nmol/L vs 1.31(0.13-5.09) nmol/L, P = 0.0146). CYP11B1 activity (11-deoxycortisol/cortisol) was not significantly different between the two groups. CYP21A2 activity (17OHprogesterone/11-deoxycortisol) and CYP17A1 activity (17OHprogesterone/androstenedione) were significantly decreased in Osilodrostat group (P < 0.0001)., Conclusion: In patients with ACTH-dependent CS, the use of CYP11B1 inhibitors in routine care suggests that Osilodrostat has a less specific effect on the inhibition of steroidogenic enzymes than Metyrapone. This might explain a smaller increase in 11-deoxycortisol and androgen levels in patients treated with Osilodrostat.

Objective: Primary bilateral macronodular adrenal hyperplasia (PBMAH) is a heterogeneous disease characterized by adrenal macronodules and variable levels of cortisol excess, with not clearly established clinical diagnostic criteria. It can be caused by ARMC5 germline pathogenic variants. In this study, we aimed to identify predictive criteria for ARMC5 variants., Methods: We included 352 consecutive index patients from 12 European centers, sequenced for germline ARMC5 alteration. Clinical, biological and imaging data were collected retrospectively., Results: 52 patients (14.8%) carried ARMC5 germline pathogenic variants and showed a more distinct phenotype than non-mutated patients for cortisol excess (24-h urinary free cortisol 2.32 vs 1.11-fold ULN, respectively, P < 0.001) and adrenal morphology (maximal adrenal diameter 104 vs 83 mm, respectively, P < 0.001) and were more often surgically or medically treated (67.9 vs 36.8%, respectively, P < 0.001). ARMC5-mutated patients showed a constant, bilateral adrenal involvement and at least a possible autonomous cortisol secretion (defined by a plasma cortisol after 1 mg dexamethasone suppression above 50 nmol/L), while these criteria were not systematic in WT patients (78.3%). The association of these two criteria holds a 100% sensitivity and a 100% negative predictive value for ARMC5 pathogenic variant., Conclusion: We report the largest series of index patients investigated for ARMC5 and confirm that ARMC5 pathogenic variants are associated with a more severe phenotype in most cases. To minimize negative ARMC5 screening, genotyping should be limited to clear bilateral adrenal involvement and autonomous cortisol secretion, with an optimum sensitivity for routine clinical practice. These findings will also help to better define PBMAH diagnostic criteria.

Purpose: This study aimed to investigate the genetic cause of food-dependent Cushing syndrome (FDCS) observed in patients with primary bilateral macronodular adrenal hyperplasia (PBMAH) and adrenal ectopic expression of the glucose-dependent insulinotropic polypeptide receptor. Germline ARMC5 alterations have been reported in about 25% of PBMAH index cases but are absent in patients with FDCS., Methods: A multiomics analysis of PBMAH tissues from 36 patients treated by adrenalectomy was performed (RNA sequencing, single-nucleotide variant array, methylome, miRNome, exome sequencing)., Results: The integrative analysis revealed 3 molecular groups with different clinical features, namely G1, comprising 16 patients with ARMC5 inactivating variants; G2, comprising 6 patients with FDCS with glucose-dependent insulinotropic polypeptide receptor ectopic expression; and G3, comprising 14 patients with a less severe phenotype. Exome sequencing revealed germline truncating variants of KDM1A in 5 G2 patients, constantly associated with a somatic loss of the KDM1A wild-type allele on 1p, leading to a loss of KDM1A expression both at messenger RNA and protein levels (P = 1.2 × 10 -12 and P < .01, respectively). Subsequently, KDM1A pathogenic variants were identified in 4 of 4 additional index cases with FDCS., Conclusion: KDM1A inactivation explains about 90% of FDCS PBMAH. Genetic screening for ARMC5 and KDM1A can now be offered for most PBMAH operated patients and their families, opening the way to earlier diagnosis and improved management., Competing Interests: Conflict of Interest The authors declare no conflicts of interest., (Copyright © 2021 American College of Medical Genetics and Genomics. All rights reserved.)

Detection and characterization of adrenal lesions have evolved during the past two decades. Although the role of imaging in adrenal lesions associated with hormonal secretion is usually straightforward, characterization of non-functioning adrenal lesions may be challenging to confidently identify those that need to be resected. Although many adrenal lesions can be readily diagnosed when they display typical imaging features, the diagnosis may be challenging for atypical lesions. Computed tomography (CT) remains the cornerstone of adrenal imaging, but other morphological or functional modalities can be used in combination to reach a diagnosis and avoid useless biopsy or surgery. Early- and delayed-phase contrast-enhanced CT images are essential for diagnosing lipid-poor adenoma. Ongoing studies are evaluating the capabilities of dual-energy CT to provide valid virtual non-contrast attenuation and iodine density measurements from contrast-enhanced examinations. Adrenal lesions with attenuation values between 10 and 30 Hounsfield units (HU) on unenhanced CT can be characterized by MRI when iodinated contrast material injection cannot be performed. 18 F-FDG PET/CT helps differentiate between atypical benign and malignant adrenal lesions, with the adrenal-to-liver maximum standardized uptake value ratio being the most discriminative variable. Recent studies evaluating the capabilities of radiomics and artificial intelligence have shown encouraging results.

Objective: Cushing's syndrome represents a state of excessive glucocorticoids related to glucocorticoid treatments or to endogenous hypercortisolism. Cushing's syndrome is associated with high morbidity, with significant inter-individual variability. Likewise, adrenal insufficiency is a life-threatening condition of cortisol deprivation. Currently, hormone assays contribute to identify Cushing's syndrome or adrenal insufficiency. However, no biomarker directly quantifies the biological glucocorticoid action. The aim of this study was to identify such markers., Design: We evaluated whole blood DNA methylome in 94 samples obtained from patients with different glucocorticoid states (Cushing's syndrome, eucortisolism, adrenal insufficiency). We used an independent cohort of 91 samples for validation., Methods: Leukocyte DNA was obtained from whole blood samples. Methylome was determined using the Illumina methylation chip array (~850 000 CpG sites). Both unsupervised (principal component analysis) and supervised (Limma) methods were used to explore methylome profiles. A Lasso-penalized regression was used to select optimal discriminating features., Results: Whole blood methylation profile was able to discriminate samples by their glucocorticoid status: glucocorticoid excess was associated with DNA hypomethylation, recovering within months after Cushing's syndrome correction. In Cushing's syndrome, an enrichment in hypomethylated CpG sites was observed in the region of FKBP5 gene locus. A methylation predictor of glucocorticoid excess was built on a training cohort and validated on two independent cohorts. Potential CpG sites associated with the risk for specific complications, such as glucocorticoid-related hypertension or osteoporosis, were identified, needing now to be confirmed on independent cohorts., Conclusions: Whole blood DNA methylome is dynamically impacted by glucocorticoids. This biomarker could contribute to better assessment of glucocorticoid action beyond hormone assays.

Objective: Microprolactinomas are currently treated with dopamine agonists. Outcome information on microprolactinoma patients treated by surgery is limited. This study reports the first large series of consecutive non-invasive microprolactinoma patients treated by pituitary surgery and evaluates the efficiency and safety of this treatment., Design: Follow-up of a cohort of consecutive patients treated by surgery., Methods: Between January 2008 and October 2020, 114 adult patients with pure microprolactinomas were operated on in a single tertiary expert neurosurgical department, using an endoscopic endonasal transsphenoidal approach. Eligible patients presented with a microprolactinoma with no obvious cavernous invasion on MRI. Prolactin was assayed before and after surgery. Disease-free survival was modeled using Kaplan-Meier representation. A cox regression model was used to predict remission., Results: Median follow-up was 18.2 months (range: 2.8-155). In this cohort, 14/114 (12%) patients were not cured by surgery, including ten early surgical failures and four late relapses occurring 37.4 months (33-41.8) after surgery. From Kaplan-Meier estimates, 1-year and 5-year disease free survival was 90.9% (95% CI: 85.6-96.4%) and 81% (95% CI: 71.2-92.1%) respectively. The preoperative prolactinemia was the only significant preoperative predictive factor for remission (P < 0.05). No severe complication was reported, with no anterior pituitary deficiency after surgery, one diabetes insipidus, and one postoperative cerebrospinal fluid leakage properly treated by muscle plasty., Conclusions: In well-selected microprolactinoma patients, pituitary surgery performed by an expert neurosurgical team is a valid first-line alternative treatment to dopamine agonists.

Forty percent of somatotroph tumors harbor recurrent activating GNAS mutations, historically called the gsp oncogene. In gsp -negative somatotroph tumors, GNAS expression itself is highly variable; those with GNAS overexpression most resemble phenotypically those carrying the gsp oncogene. GNAS is monoallelically expressed in the normal pituitary due to methylation-based imprinting. We hypothesize that changes in GNAS imprinting of gsp -negative tumors affect GNAS expression levels and tumorigenesis. We characterized the GNAS locus in two independent somatotroph tumor cohorts: one of 23 tumors previously published (PMID: 31883967) and classified by pan-genomic analysis, and a second with 82 tumors. Multi-omics analysis of the first cohort identified a significant difference between gsp -negative and gsp -positive tumors in the methylation index at the known differentially methylated region (DMR) of the GNAS A/B transcript promoter, which was confirmed in the larger series of 82 tumors. GNAS allelic expression was analyzed using a polymorphic Fok1 cleavage site in 32 heterozygous gsp -negative tumors. GNAS expression was significantly reduced in the 14 tumors with relaxed GNAS imprinting and biallelic expression, compared to 18 tumors with monoallelic expression. Tumors with relaxed GNAS imprinting showed significantly lower SSTR2 and AIP expression levels. Altered A/B DMR methylation was found exclusively in gsp -negative somatotroph tumors. 43% of gsp -negative tumors showed GNAS imprinting relaxation, which correlated with lower GNAS , SSTR2 and AIP expression, indicating lower sensitivity to somatostatin analogues and potentially aggressive behavior.

Duodenopancreatic neuroendocrine tumors (DPNETs) aggressiveness is heterogeneous. Tumor grade and extension are commonly used for prognostic determination. Yet, grade classes are empirically defined, with regular updates changing the definition of classes. Genomic screening may provide more objective classes and reflect tumor biology. The aim of this study was to provide a transcriptome classification of DPNETs. We included 66 DPNETs, covering the entire clinical spectrum of the disease in terms of secretion, grade, and stage. Three distinct molecular groups were identified, associated with distinct outcomes (log-rank P < 0.01): (i) better-outcome DPNETs with pancreatic beta-cell signature. This group was mainly composed of well-differentiated, grade 1 insulinomas; (ii) poor-outcome DPNETs with pancreatic alpha-cell and hepatic signature. This group included all neuroendocrine carcinomas and grade 3 DPNETs, but also some grade 1 and grade 2 DPNETs and (iii) intermediate-outcome DPNETs with pancreatic exocrine and progenitor signature. This group included grade 1 and grade 2 DPNETs, with some insulinomas. Fibrinogen gene FGA expression was one of the topmost expressed liver genes. FGA expression was associated with disease-free survival (HR = 1.13, P = 0.005) and could be validated on two independent cohorts. This original pathophysiologic insight provides new prognostic classification perspectives.

The major prognosis factor of adrenocortical carcinoma (ACC) is the completeness of surgery. The aim of our study was to identify preoperative imaging features associated with direct liver involvement (DLI) by right-sided ACC. Two radiologists, blinded to the outcome, independently reviewed preoperative CT and MRI examinations for eight signs of DLI, in patients operated for right-sided ACC and retrospectively included from November 2007 to January 2020. DLI was confirmed using surgical and histopathological findings. Kappa values were calculated. Univariable and multivariable analyses were performed by using a logistic regression model. Receiver operating characteristic (ROC) curves were built for CT and MRI. Twenty-nine patients were included. Seven patients had DLI requiring en bloc resection. At multivariable analysis, focal ACC bulge was the single independent sign associated with DLI on CT (OR: 60.00; 95% CI: 4.60-782.40; p < 0.001), and ACC contour disruption was the single independent sign associated with DLI on MRI (OR: 126.00; 95% CI: 6.82-2328.21; p < 0.001). Both signs were highly reproducible, with respective kappa values of 0.85 and 0.91. The areas under ROC curves of MRI and CT models were not different ( p = 0.838). Focal ACC bulge on CT and ACC contour disruption on MRI are independent and highly reproducible signs, strongly associated with DLI by right-sided ACC on preoperative imaging. MRI does not improve the preoperative assessment of DLI by comparison with CT.

Context: Identification of patients with endocrine forms of hypertension (EHT) (primary hyperaldosteronism [PA], pheochromocytoma/paraganglioma [PPGL], and Cushing syndrome [CS]) provides the basis to implement individualized therapeutic strategies. Targeted metabolomics (TM) have revealed promising results in profiling cardiovascular diseases and endocrine conditions associated with hypertension., Objective: Use TM to identify distinct metabolic patterns between primary hypertension (PHT) and EHT and test its discriminating ability., Methods: Retrospective analyses of PHT and EHT patients from a European multicenter study (ENSAT-HT). TM was performed on stored blood samples using liquid chromatography mass spectrometry. To identify discriminating metabolites a "classical approach" (CA) (performing a series of univariate and multivariate analyses) and a "machine learning approach" (MLA) (using random forest) were used.The study included 282 adult patients (52% female; mean age 49 years) with proven PHT (n = 59) and EHT (n = 223 with 40 CS, 107 PA, and 76 PPGL), respectively., Results: From 155 metabolites eligible for statistical analyses, 31 were identified discriminating between PHT and EHT using the CA and 27 using the MLA, of which 16 metabolites (C9, C16, C16:1, C18:1, C18:2, arginine, aspartate, glutamate, ornithine, spermidine, lysoPCaC16:0, lysoPCaC20:4, lysoPCaC24:0, PCaeC42:0, SM C18:1, SM C20:2) were found by both approaches. The receiver operating characteristic curve built on the top 15 metabolites from the CA provided an area under the curve (AUC) of 0.86, which was similar to the performance of the 15 metabolites from MLA (AUC 0.83)., Conclusion: TM identifies distinct metabolic pattern between PHT and EHT providing promising discriminating performance., (© The Author(s) 2020. Published by Oxford University Press on behalf of the Endocrine Society.)

Adrenocortical carcinoma (ACC) is an orphan disease lacking effective systemic treatment options. The low incidence of the disease and high cost of clinical trials are major obstacles in the search for improved treatment strategies. As a novel approach, registry-based clinical trials have been introduced in clinical research, so allowing for significant cost reduction, but without compromising scientific benefit. Herein, we describe how the European Network for the Study of Adrenal Tumours (ENSAT) could transform its current registry into one fit for a clinical trial infrastructure. The rationale to perform randomized registry-based trials in ACC is outlined including an analysis of relevant limitations and challenges. We summarize a survey on this concept among ENSAT members who expressed a strong interest in the concept and rated its scientific potential as high. Legal aspects, including ethical approval of registry-based randomization were identified as potential obstacles. Finally, we describe three potential randomized registry-based clinical trials in an adjuvant setting and for advanced disease with a high potential to be executed within the framework of an advanced ENSAT registry. Thus we, therefore, provide the basis for future registry-based trials for ACC patients. This could ultimately provide proof-of-principle of how to perform more effective randomized trials for an orphan disease.

Benign adrenal tumors cover a spectrum of lesions with distinct morphology and steroid secretion. Current classification is empirical. Beyond a few driver mutations, pathophysiology is not well understood. Here, a pangenomic characterization of benign adrenocortical tumors is proposed, aiming at unbiased classification and new pathophysiological insights. Benign adrenocortical tumors (n = 146) were analyzed by transcriptome, methylome, miRNome, chromosomal alterations and mutational status, using expression arrays, methylation arrays, miRNA sequencing, SNP arrays, and exome or targeted next-generation sequencing respectively. Pathological and hormonal data were collected for all tumors. Pangenomic analysis identifies four distinct molecular categories: (1) tumors responsible for overt Cushing, gathering distinct tumor types, sharing a common cAMP/PKA pathway activation by distinct mechanisms; (2) adenomas with mild autonomous cortisol excess and non-functioning adenomas, associated with beta-catenin mutations; (3) primary macronodular hyperplasia with ARMC5 mutations, showing an ovarian expression signature; (4) aldosterone-producing adrenocortical adenomas, apart from other benign tumors. Epigenetic alterations and steroidogenesis seem associated, including CpG island hypomethylation in tumors with no or mild cortisol secretion, miRNA patterns defining specific molecular groups, and direct regulation of steroidogenic enzyme expression by methylation. Chromosomal alterations and somatic mutations are subclonal, found in less than 2/3 of cells. New pathophysiological insights, including distinct molecular signatures supporting the difference between mild autonomous cortisol excess and overt Cushing, ARMC5 implication into the adreno-gonadal differentiation faith, and the subclonal nature of driver alterations in benign tumors, will orient future research. This first genomic classification provides a large amount of data as a starting point.

Context: Catecholamines and adrenocortical steroids are important regulators of blood pressure. Bidirectional relationships between adrenal steroids and catecholamines have been established but whether this is relevant to patients with pheochromocytoma is unclear., Objective: This study addresses the hypothesis that patients with pheochromocytoma and paraganglioma (PPGL) have altered steroid production compared with patients with primary hypertension., Design: Multicenter cross-sectional study., Setting: Twelve European referral centers., Patients: Subjects included 182 patients with pheochromocytoma, 36 with paraganglioma and 270 patients with primary hypertension. Patients with primary aldosteronism (n = 461) and Cushing syndrome (n = 124) were included for additional comparisons., Intervention: In patients with PPGLs, surgical resection of tumors., Outcome Measures: Differences in mass spectrometry-based profiles of 15 adrenal steroids between groups and after surgical resection of PPGLs. Relationships of steroids to plasma and urinary metanephrines and urinary catecholamines., Results: Patients with pheochromocytoma had higher (P < .05) circulating concentrations of cortisol, 11-deoxycortisol, 11-deoxycorticosterone, and corticosterone than patients with primary hypertension. Concentrations of cortisol, 11-deoxycortisol, and corticosterone were also higher (P < .05) in patients with pheochromocytoma than with paraganglioma. These steroids correlated positively with plasma and urinary metanephrines and catecholamines in patients with pheochromocytoma, but not paraganglioma. After adrenalectomy, there were significant decreases in cortisol, 11-deoxycortisol, corticosterone, 11-deoxycorticosterone, aldosterone, and 18-oxocortisol., Conclusions: This is the first large study in patients with PPGLs that supports in a clinical setting the concept of adrenal cortical-medullary interactions involving an influence of catecholamines on adrenal steroids. These findings could have implications for the cardiovascular complications of PPGLs and the clinical management of patients with the tumors., (© Endocrine Society 2020.)

Background: The prognosis of adrenocortical carcinoma (ACC) is heterogeneous. Genomic studies have identified ACC subgroups characterized by specific molecular alterations, including features measured at DNA level (somatic mutations, chromosome alterations, DNA methylation), which are closely associated with outcome. The aim of this study was to evaluate intratumor heterogeneity of prognostic molecular markers at the DNA level., Methods: Two different tissue samples (primary tumor, local recurrence or metastasis) were analyzed in 26 patients who underwent surgery for primary or recurrent ACC. DNA-related biomarkers with prognostic role were investigated in frozen and paraffin-embedded samples. Somatic mutations of p53/Rb and Wnt/β-catenin pathways were assessed using next-generation sequencing (n = 26), chromosome alteration profiles were determined using SNP arrays (n = 14) and methylation profiles were determined using four-gene bisulfite pyrosequencing (n = 12)., Results: Somatic mutations for ZNRF3, TP53, CTNN1B and CDKN2A were found in 7, 6, 6 and 4 patients, respectively, with intratumor heterogeneity in 8/26 patients (31%). Chromosome alteration profiles were 'Noisy' (numerous and anarchic alterations) in 8/14 and 'Chromosomal' (extended patterns of loss of heterozygosity) in 5/14 of the study samples. For these profiles, no intratumor heterogeneity was observed. Methylation profiles were hypermethylated in 5/12 and non-hypermethylated in 7/12 of the study samples. Intratumor heterogeneity of methylation profiles was observed in 2/12 patients (17%)., Conclusions: Intratumor heterogeneity impacts DNA-related molecular markers. While somatic mutation can differ, prognostic DNA methylation and chromosome alteration profile seem rather stable and might be more robust for the prognostic assessment.

Introduction: Carney Complex (CNC) is a rare multiple endocrine and nonendocrine neoplasia syndrome. Manifestations and genotype-phenotype correlations have been described by retrospective studies, but no prospective study evaluating the occurrence of the different manifestations has been available so far., Methods: This multicenter national prospective study included patients with CNC, primary pigmented nodular adrenal disease (PPNAD), or a pathogenic PRKAR1A mutation; after a full initial workup, participants were followed for 3 years with annual standardized evaluation., Results: The cohort included 70 patients (50 female/20 male, mean age 35.4 ± 16.7 years, 81% carrying PRKAR1A mutation). The initial investigations allowed identification of several manifestations. At the end of the 3-year follow-up, the newly diagnosed manifestations of the disease were subclinical acromegaly in 6 patients, bilateral testicular calcifications in 1 patient, and cardiac myxomas in 2 patients. Recurrences of cardiac myxomas were diagnosed in 4 patients during the 3-year follow-up study period. Asymptomatic abnormalities of the corticotroph and somatotroph axis that did not meet criteria of PPNAD and acromegaly were observed in 11.4% and 30% of the patients, respectively. Patients carrying the PRKAR1A c.709-7del6 mutation had a mild phenotype., Conclusion: This study underlines the importance of a systematic follow-up of the CNC manifestations, especially a biannual screening for cardiac myxoma. By contrast, regular screening for the other manifestations after a first extensive workup could be spread out, leading to a lighter and more acceptable follow-up schedule for patients. These are important results for recommendations for long-term management of CNC patients., (© Endocrine Society 2020. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

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., (Copyright © 2019 Elsevier Inc. All rights reserved.)

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.

Benign adrenocortical adenomas and hyperplasia are relatively common and include a spectrum of distinct entities, which diagnosis depends on the macroscopic aspect and the secretion profile. Recent advances in genomics have proposed high-throughput molecular characterization of adrenal tumors, thereby improving our knowledge on the pathophysiology and tumorigenesis of these tumors. Genomic (exome and chromosome alteration profiles), epigenomic (micro-RNAs expression and methylation profiles) and transcriptomic (gene expression profiles) studies highlighted the major roles of intracellular calcium signaling in aldosterone-producing adenomas (APA), of protein kinase A (PKA)/cAMP pathway in cortisol-producing tumors, and of Wnt/beta-catenin pathway in non-secreting tumors. Exome sequencing revealed new major drivers in all tumor types, including KCNJ5, ATP1A1, ATP2B3, CACNA1D and CACNA1H mutations in APA, PRKACA mutations in cortisol-producing adenomas (CPA) and ARMC5 mutations in primary macronodular adrenocortical hyperplasia (PMAH). The clinical impact of these findings is just starting to evolve. The identification of genetic syndromes, such as germline ARMC5 mutations in PMAH, has allowed genetic counseling. Key molecular alterations could serve as a basis for the development of targeted medical treatments for benign adrenal tumors. The recent developments in genomics, including single-cell technologies, and in proteomics and metabolomics will probably offer new perspectives for characterizing benign adrenal tumorigenesis., (Copyright © 2019. Published by Elsevier Ltd.)

Background: EZH2 is overexpressed and associated with poor prognosis in adrenocortical carcinoma (ACC) and its inhibition reduces growth and aggressiveness of ACC cells in culture. Although EZH2 was identified as the methyltransferase that deposits the repressive H3K27me3 histone mark, it can cooperate with transcription factors to stimulate gene transcription., Methods: We used bioinformatics approaches on gene expression data from three cohorts of patients and a mouse model of EZH2 ablation, to identify targets and mode of action of EZH2 in ACC. This was followed by ChIP and functional assays to evaluate contribution of identified targets to ACC pathogenesis., Results: We show that EZH2 mostly works as a transcriptional inducer in ACC, through cooperation with the transcription factor E2F1 and identify three positive targets involved in cell cycle regulation and mitosis i.e., RRM2, PTTG1 and ASE1/PRC1. Overexpression of these genes is associated with poor prognosis, suggesting a potential role in acquisition of aggressive ACC features. Pharmacological and siRNA-mediated inhibition of RRM2 blocks cell proliferation, induces apoptosis and inhibits cell migration, suggesting that it may be an interesting target in ACC., Conclusions: Altogether, these data show an unexpected role of EZH2 and E2F1 in stimulating expression of genes associated with ACC aggressiveness.

Background The combination of mitotane and platinum-etoposide chemotherapy is a front-line treatment in metastatic adrenocortical carcinoma (ACC), although this regimen shows limited efficacy. Pharmacokinetic drug-drug interaction between mitotane, a strong CYP3A4 inducer, and etoposide, which is a substrate of CYP3A4, may contribute to chemoresistance. The aim of this pilot study was to assess the pharmacokinetic interaction between mitotane and etoposide in ACC patients. Methods Five consecutive ACC patients treated with platinum etoposide (120-150 mg/m2 day 1-2-3 at cycle 1), with or without concomitant mitotane, were included. In the absence of limiting toxicity, a dose escalation of etoposide was proposed since cycle 2. Plasma etoposide concentrations were measured using liquid chromatography at 0, 4 and 24 h after each infusion. Clearance and area under the curve (AUC) of etoposide were determined at each cycle. Results Patients received two to six chemotherapy cycles, in association with mitotane (N = 4) or after mitotane discontinuation (N = 1). Etoposide clearance was two-fold higher with concomitant mitotane (4.95 L/h) than after mitotane discontinuation (2.53 L/h, P = 0.014), and 2.5-fold higher than that in reference population not treated with mitotane (1.81 L/h). Etoposide dose escalation was performed in four patients under mitotane, resulting in two minor tumor responses and one severe toxicity (febrile aplasia) at dose of 300 mg/m2/day. Tumor response was associated with higher etoposide AUC (267.3 vs 188.8 mg.h/L, P = 0.04). Conclusion A drug-drug interaction between mitotane and etoposide may contribute to the low efficacy of platinum-etoposide chemotherapy. This pilot study suggests further a potential benefit of increasing etoposide dose in ACC patients receiving mitotane.

In the setting of Cushing syndrome, genomic analyses can be performed either in tumors responsible for endogenous Cushing, or in patients exposed to glucocorticoid excess. Genomics of tumors identified several new genes - including ZNRF3 in adrenocortical carcinomas, PRKACA in cortisol-producing adrenal adenomas, ARMC5 in primary macronodular adrenal hyperplasia and USP8 in pituitary corticotroph adenomas. These genes shed new lights on the mechanisms responsible for these tumors. Integrated genomic studies of adrenal carcinomas identified distinct molecular classes, with remarkably different prognostic outcome. Beyond the mechanistic novelties, a new generation of prognostic markers emerges, with potentially important impact on patients care. For the future, genomic efforts should be pursued, focusing on poorly characterized tumors responsible for Cushing syndrome - including endocrine tumors secreting ACTH. In addition, epigenomics is emerging as an outstanding set of tools for characterizing tumors, unraveling unprecedented aspects of tumorigenesis. Applying these tools to endocrine tumors responsible for Cushing syndrome may also lead to important discoveries. Genomics of patients exposed to glucocorticoid excess is an emerging research field. Proof of principle studies have been performed, identifying molecular markers of glucocorticoid excess in blood. Research efforts should now concentrate on markers of mild glucocorticoid excesses - endogenous or exogenous -, owing to their high prevalence in general population. In addition, markers of individual susceptibility to each type of glucocorticoid complication are needed. It remains to be determined whether genomics can identify such markers., (Copyright © 2018 Elsevier Masson SAS. All rights reserved.)

Background: Adrenal incidentalomas are increasingly diagnosed and include a wide spectrum of lesions from benign adenomas to secreting or malignant lesions. The aim of the present study is to report a large single-institution experience of patients undergoing surgery for adrenal incidentaloma with particular attention to their diagnosis and post-operative course and the evolution of surgical practice over time., Methods: From 1993 to 2013, 274 patients underwent adrenalectomy for incidentaloma. All patients underwent standardized clinical, hormonal and imaging assessments., Results: Patients were mainly female (63.1%; n = 173), and the median age of patients was 56.5 years. After a complete hormonal evaluation, 47.9% (n = 129) of incidentalomas were classified as secreting tumours, including 24.4% (n = 67) subclinical cortisol-secreting adenomas and 18.9% (n = 52) pheochromocytomas. Adrenocortical carcinomas represented 9.5% (n = 26) of incidentalomas, and the risk of malignancy was significantly correlated with tumour size. The conversion rate after laparoscopic adrenalectomy (90.9%; n = 249) was 3.2% (n = 8). The overall morbidity rate was 13.9%, which included a 4.4% rate of severe morbidity (Clavien-Dindo ≥3). From 2008 onwards, there was a significant decrease (P < 0.001) in the use of surgical approaches for non-secreting adenomas., Conclusion: After a complete work-up, half of the incidentalomas were classified as subclinical oversecreting adrenal lesions and 10% proved to be malignant adrenocortical carcinomas. The debatable use of surgical approaches for benign nonfunctioning adenomas significantly decreased over time., (© 2017 Royal Australasian College of Surgeons.)

Mutations in the gene encoding the protein kinase A (PKA) catalytic subunit α have been found to be responsible for cortisol-producing adenomas (CPAs). In this study, we identified by whole-exome sequencing the somatic mutation p.S54L in the PRKACB gene, encoding the catalytic subunit β (Cβ) of PKA, in a CPA from a patient with severe Cushing syndrome. Bioluminescence resonance energy transfer and surface plasmon resonance assays revealed that the mutation hampers formation of type I holoenzymes and that these holoenzymes were highly sensitive to cAMP. PKA activity, measured both in cell lysates and with recombinant proteins, based on phosphorylation of a synthetic substrate, was higher under basal conditions for the mutant enzyme compared with the WT, while maximal activity was lower. These data suggest that at baseline the PRKACB p.S54L mutant drove the adenoma cells to higher cAMP signaling activity, probably contributing to their autonomous growth. Although the role of PRKACB in tumorigenesis has been suggested, we demonstrated for the first time to our knowledge that a PRKACB mutation can lead to an adrenal tumor. Moreover, this observation describes another mechanism of PKA pathway activation in CPAs and highlights the particular role of residue Ser54 for the function of PKA.

Objective: Somatic mutations in the ubiquitin-specific protease 8 ( USP8 ) gene are frequent in corticotroph tumors causing Cushing's disease (CD). Corticotroph tumor progression, the so-called Nelson's syndrome (NS), is a potentially life-threatening complication of bilateral adrenalectomy in patients with refractory CD that is caused by the development of an ACTH-secreting tumor of the pituitary gland. Whether USP8 alterations are also present in progressive Nelson's tumors has not been studied in detail so far., Design and Methods: Retrospective, multicenter study involving tumors from 33 patients with progressive corticotroph tumors (29 females) and screening for somatic mutations on the mutational hotspot of the USP8 gene in the exon 14 with Sanger sequencing., Results: Fifteen out of 33 tumors (45%) presented with a mutation in the exon 14 of USP8 , with c.2159C>A (p.Pro720Gln) being the most frequent (9/33), followed by c.2155&#95;2157delTCC (p.Ser718del, 4/33) and c.2152T>C (p.Ser718Pro, 2/33). This prevalence is similar to that previously reported for CD. Mutations were found exclusively in females. Other variables, such as age at diagnosis with NS, body mass index, hyperpigmentation, visual field defects, adenoma size or mortality, did not significantly differ between patients with wild-type and mutant tumors. Patients with USP8 mutant tumors exhibited higher levels of plasma ACTH after surgery (median: 640 vs 112 pg/mL, P  = 0.03). No differences were observed in ACTH normalization (<50 pg/mL) and tumor control after surgery for Nelson's tumor., Conclusion: Somatic mutations in USP8 are common in Nelson's tumors, indicating that they do not drive the corticotroph tumor progression that leads to NS, and may be associated with a less favorable biochemical outcome after surgery for Nelson's tumor., (© 2018 European Society of Endocrinology.)

Pangenomic studies identified distinct molecular classes for many cancers, with major clinical applications. However, routine use requires cost-effective assays. We assessed whether targeted next-generation sequencing (NGS) could call chromosomal alterations and DNA methylation status. A training set of 77 tumors and a validation set of 449 (43 tumor types) were analyzed by targeted NGS and single-nucleotide polymorphism (SNP) arrays. Thirty-two tumors were analyzed by NGS after bisulfite conversion, and compared to methylation array or methylation-specific multiplex ligation-dependent probe amplification. Considering allelic ratios, correlation was strong between targeted NGS and SNP arrays (r = 0.88). In contrast, considering DNA copy number, for variations of one DNA copy, correlation was weaker between read counts and SNP array (r = 0.49). Thus, we generated TARGOMICs, optimized for detecting chromosome alterations by combining allelic ratios and read counts generated by targeted NGS. Sensitivity for calling normal, lost, and gained chromosomes was 89%, 72%, and 31%, respectively. Specificity was 81%, 93%, and 98%, respectively. These results were confirmed in the validation set. Finally, TARGOMICs could efficiently align and compute proportions of methylated cytosines from bisulfite-converted DNA from targeted NGS. In conclusion, beyond calling mutations, targeted NGS efficiently calls chromosome alterations and methylation status in tumors. A single run and minor design/protocol adaptations are sufficient. Optimizing targeted NGS should expand translation of genomics to clinical routine., (Copyright © 2017 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.)