Xavier Bertagna

Treatment of Adrenocorticotropin-Dependent Cushing’s Syndrome: A Consensus Statement

OBJECTIVE Our objective was to evaluate the published literature and reach a consensus on the treatment of patients with ACTH-dependent Cushings syndrome, because there is no recent consensus on the management of this rare disorder. PARTICIPANTS Thirty-two leading endocrinologists, clinicians, and neurosurgeons with specific expertise in the management of ACTH-dependent Cushings syndrome representing nine countries were chosen to address 1) criteria for cure and remission of this disorder, 2) surgical treatment of Cushings disease, 3) therapeutic options in the event of persistent disease after transsphenoidal surgery, 4) medical therapy of Cushings disease, and 5) management of ectopic ACTH syndrome, Nelsons syndrome, and special patient populations. EVIDENCE Participants presented published scientific data, which formed the basis of the recommendations. Opinion shared by a majority of experts was used where strong evidence was lacking. CONSENSUS PROCESS Participants met for 2 d, during which there were four chaired sessions of presentations, followed by general discussion where a consensus was reached. The consensus statement was prepared by a steering committee and was then reviewed by all authors, with suggestions incorporated if agreed upon by the majority. CONCLUSIONS ACTH-dependent Cushings syndrome is a heterogeneous disorder requiring a multidisciplinary and individualized approach to patient management. Generally, the treatment of choice for ACTH-dependent Cushings syndrome is curative surgery with selective pituitary or ectopic corticotroph tumor resection. Second-line treatments include more radical surgery, radiation therapy (for Cushings disease), medical therapy, and bilateral adrenalectomy. Because of the significant morbidity of Cushings syndrome, early diagnosis and prompt therapy are warranted.

Urine Steroid Metabolomics as a Biomarker Tool for Detecting Malignancy in Adrenal Tumors

Context: Adrenal tumors have a prevalence of around 2% in the general population. Adrenocortical carcinoma (ACC) is rare but accounts for 2–11% of incidentally discovered adrenal masses. Differentiating ACC from adrenocortical adenoma (ACA) represents a diagnostic challenge in patients with adrenal incidentalomas, with tumor size, imaging, and even histology all providing unsatisfactory predictive values. Objective: Here we developed a novel steroid metabolomic approach, mass spectrometry-based steroid profiling followed by machine learning analysis, and examined its diagnostic value for the detection of adrenal malignancy. Design: Quantification of 32 distinct adrenal derived steroids was carried out by gas chromatography/mass spectrometry in 24-h urine samples from 102 ACA patients (age range 19–84 yr) and 45 ACC patients (20–80 yr). Underlying diagnosis was ascertained by histology and metastasis in ACC and by clinical follow-up [median duration 52 (range 26–201) months] without evidence of metastasis in ACA. Steroid excretion data were subjected to generalized matrix learning vector quantization (GMLVQ) to identify the most discriminative steroids. Results: Steroid profiling revealed a pattern of predominantly immature, early-stage steroidogenesis in ACC. GMLVQ analysis identified a subset of nine steroids that performed best in differentiating ACA from ACC. Receiver-operating characteristics analysis of GMLVQ results demonstrated sensitivity = specificity = 90% (area under the curve = 0.97) employing all 32 steroids and sensitivity = specificity = 88% (area under the curve = 0.96) when using only the nine most differentiating markers. Conclusions: Urine steroid metabolomics is a novel, highly sensitive, and specific biomarker tool for discriminating benign from malignant adrenal tumors, with obvious promise for the diagnostic work-up of patients with adrenal incidentalomas.

Role of the PKA‐Regulated Transcription Factor CREB in Development and Tumorigenesis of Endocrine Tissues

Abstract: The cAMP pathway plays a major role in the development of endocrine tissues and various molecular defects of key components of this pathway (G protein, receptors, PKA, etc.) have been observed in endocrine tumors. The ubiquitous transcription factor CREB (cAMP‐response element binding protein) binds to the cAMP response element (CRE) and stimulates transcription after phosphorylation on Ser133 by PKA. The CREB family of transcription factors contains three members: CREB, CREM, and ATF‐1. Targeted expression of dominant‐negative mutants of CREB in transgenic mice leads to somatotrophs or thyroid hypoplasia. GH‐secreting adenomas are benign secreting tumors expressing an activated mutant Gαs protein (Gsp) in about 40% of cases. In GH‐secreting adenomas CREB is always expressed and often highly phosphorylated. The CREM isoform ICER is stimulated by cAMP, and its expression is increased in Gsp‐harboring tumors. After transfection in pituitary somatotroph cells, activating mutations of Gs protein (Gsp) and overexpression of wild‐type GαS stimulate transcription of various CRE‐containing promoters via CREB in a Ser133‐specific‐dependent manner. Activation of the cAMP pathway by ACTH is required for adrenal cortex (AdCx) maintenance and steroidogenesis. CREB is expressed in normal AdCx. Alterations of CRE binding proteins with loss of CREB expression and compensatory overexpression of CREMτ is observed in the human adrenocortical cancer cell line H295R. Similar alterations are found at the protein level in human malignant adrenocortical tumors. In conclusion, the CREB family of transcription factors plays an important role in the development, differentiation, and proliferation of endocrine tissues. Various alterations of the CREB family of transciption factors can be observed in endocrine tumors.

LCI699, a potent 11β-hydroxylase inhibitor, normalizes urinary cortisol in patients with Cushing's disease: results from a multicenter, proof-of-concept study.

INTRODUCTION The clinical features and increased mortality associated with Cushings syndrome result from a chronic excess of circulating cortisol. As LCI699 potently inhibits 11β-hydroxylase, which catalyzes the final step of cortisol synthesis, it is a potential new treatment for Cushings disease, the most common cause of endogenous Cushings syndrome. METHODS Adult patients with moderate-to-severe Cushings disease (urinary free cortisol [UFC] levels >1.5 × ULN [upper limit of normal]) received oral LCI699 for 10 weeks in this proof-of-concept study. LCI699 was initiated at 4 mg/d in two equal doses; the dose was escalated every 14 days to 10, 20, 40, and 100 mg/d until UFC normalized, whereupon the dose was maintained until treatment ended (day 70). The primary endpoint was UFC ≤ ULN or a ≥50% decrease from baseline at day 70. RESULTS Twelve patients were enrolled and completed the study. Baseline UFC ranged over 1.6-17.0 × ULN. All 12 patients achieved UFC ≤ULN or a ≥50% decrease from baseline at day 70; 11 (92%) had normal UFC levels at that time. After treatment discontinuation (day 84), UFC was >ULN in 10 patients with available measurements. Mean 11-deoxycortisol, 11-deoxycorticosterone, and adrenocorticotropic hormone levels increased during treatment and declined after discontinuation. Mean systolic and diastolic blood pressure decreased from baseline by 10.0 and 6.0 mmHg, respectively. LCI699 was generally well tolerated; most adverse events (AEs) were mild or moderate. The most common AEs included fatigue (7/12), nausea (5/12), and headache (3/12). No serious drug-related AEs were reported. CONCLUSIONS LCI699 was efficacious and well tolerated in patients with Cushings disease enrolled in this proof-of-concept study.

Somatic TP53 Mutations Are Relatively Rare among Adrenocortical Cancers with the Frequent 17p13 Loss of Heterozygosity

Purpose: Allelic losses [loss of heterozygosity (LOH)] at the 17p13 locus are frequent (85%) in adrenocortical cancers. The tumor suppressor gene TP53 is located at 17p13. The aim of the study was to determine the frequency of TP53 somatic inactivating mutations in adrenocortical tumors with 17p13 LOH and their clinico-biological correlations. Experimental Design:TP53 somatic mutations, intragenic LOH (VNTR1 marker), and p53 overexpression were studied in 36 adrenocortical tumors with 17p13 LOH determined by Southern blot. Results:TP53 mutations were detected in 33% of the tumors, and VNTR1 LOH was present in 44% of the cases and did not always correlate with the presence of a TP53 mutation. Only the TP53-mutant tumors exhibit a strong nuclear immunoreactivity. TP53-mutant tumors were significantly larger than wild-type TP53 tumors (median tumor weight: 640 versus 185 g; P = 0.02), were associated with a more advanced stage of tumor progression (MacFarlane stage IV; P = 0.01), and had a shorter disease-free survival (P = 0.03). Conclusions: The finding that only a minority of adrenocortical tumors with 17p13 LOH had either a VNTR1 LOH or a TP53 mutation indicates that TP53 might not be the only or major tumor suppressor gene at 17p13 involved in adrenocortical cancer progression. We suggest that a genetic instability of the 17p13 region, occurring early in adrenocortical cancer development, involves various genes located in this region. TP53 might be only one of them, and its alteration by the occurrence of inactivating mutation is associated with the development of more aggressive tumors.

Efficiency and Tolerance of mitotane in Cushing's Disease in 76 patients from a single center

CONTEXT Alternatives to transsphenoidal pituitary surgery may be required in Cushings disease (CD) as a first- or second-line treatment. Mitotane is a potent anti-cortisolic drug but has been rarely investigated in the treatment of CD. OBJECTIVE Evaluation of the efficacy and tolerance of mitotane in CD patients. DESIGN AND SETTING Retrospective analysis of 76 patients treated with mitotane from 219 patients diagnosed with CD between 1993 and 2009 in a single center. MAIN OUTCOME MEASURE Remission was defined as normalization of 24-h urinary free cortisol (24-h-UFC). RESULTS Remission was achieved in 48 (72%) of the 67 long-term treated patients, after a median time of 6.7 (5.2-8.2) months. Mean plasma mitotane concentration at the time of remission was 10.5 ± 8.9 mg/l, with a mean daily dose of 2.6 ± 1.1 g. A negative linear relationship was observed between plasma mitotane concentration and 24-h-UFC (P<0.0001). Seventeen of 24 (71%) patients with durable remission subsequently experienced recurrence, after a median time of 13.2 (5.0-67.9) months. At the time of treatment discontinuation, ACTH concentration was statistically associated with a lower recurrence probability (hazard ratios 0.57 (0.32-1.00), P=0.05). Intolerance leading to treatment discontinuation occurred in 19 patients (29%). A pituitary adenoma became identifiable during mitotane treatment in 12 (25%) of the 48 patients with initial negative pituitary imaging allowing subsequent transsphenoidal surgery. CONCLUSION Mitotane is useful at different stages of CD. Mitotane dose adjustment based on plasma concentration monitoring and side effects could control hypercortisolism in the majority of CD patients.

Frequent Phosphodiesterase 11A Gene (PDE11A) Defects in Patients with Carney Complex (CNC) Caused by PRKAR1A Mutations: PDE11A May Contribute to Adrenal and Testicular Tumors in CNC as a Modifier of the Phenotype

BACKGROUND Carney complex (CNC) is an autosomal dominant multiple neoplasia, caused mostly by inactivating mutations of the regulatory subunit 1A of the protein kinase A (PRKAR1A). Primary pigmented nodular adrenocortical disease (PPNAD) is the most frequent endocrine manifestation of CNC with a great inter-individual variability. Germline, protein-truncating mutations of phosphodiesterase type 11A (PDE11A) have been described to predispose to a variety of endocrine tumors, including adrenal and testicular tumors. OBJECTIVES Our objective was to investigate the role of PDE11A as a possible gene modifier of the phenotype in a series of 150 patients with CNC. RESULTS A higher frequency of PDE11A variants in patients with CNC compared with healthy controls was found (25.3 vs. 6.8%, P < 0.0001). Among CNC patients, those with PPNAD were significantly more frequently carriers of PDE11A variants compared with patients without PPNAD (30.8 vs. 13%, P = 0.025). Furthermore, men with PPNAD were significantly more frequently carriers of PDE11A sequence variants (40.7%) than women with PPNAD (27.3%) (P < 0.001). A higher frequency of PDE11A sequence variants was also found in patients with large-cell calcifying Sertoli cell tumors (LCCSCT) compared with those without LCCSCT (50 vs. 10%, P = 0.0056). PDE11A variants were significantly associated with the copresence of PPNAD and LCCSCT in men: 81 vs. 20%, P < 0.004). The simultaneous inactivation of PRKAR1A and PDE11A by small inhibitory RNA led to an increase in cAMP-regulatory element-mediated transcriptional activity under basal conditions and after stimulation by forskolin. CONCLUSIONS We demonstrate, in a large cohort of CNC patients, a high frequency of PDE11A variants, suggesting that PDE11A is a genetic modifying factor for the development of testicular and adrenal tumors in patients with germline PRKAR1A mutation.

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 Cushings 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 Cushings 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.

Approach to the Cushing's Disease Patient With Persistent/Recurrent Hypercortisolism After Pituitary Surgery

Although it is the ideal treatment, pituitary surgery is not always successful, and success is not always lasting. Close surveillance, clinical and biological, will detect immediate failure or late recurrence. The reason must be thoroughly explored with the somewhat dogmatic rule that the patient should be offered the best surgery in expert hands, and a repeat surgical attempt must be systematically discussed. When repeat pituitary surgery is not indicated or has failed, then comes the difficult task to choose between a number of options directed toward different targets: directly suppress tumor ACTH by pituitary radiotherapy (conventional or stereotaxic) or with medications (somatostatin analog such as pasireotide, or dopaminergic drug such as cabergoline), directly suppress adrenocortical activity with medications (inhibitors of adrenal steroidogenesis such as ketoconazole or metyrapone, or the adrenolytic Lysodren), or by surgery (bilateral adrenalectomy), and finally oppose peripheral cortisol action with the antiglucocorticoid mifepristone. No single option is ideal, able to provide at the same time a high success rate and a rapid onset of action, to restore a normal pituitary adrenal axis, and to have good tolerability. Close follow-up and thorough evaluation of the cortisolic status will eventually dictate a switch in treatment options and/or combination strategies over time. The tumor status and its possible oncogenic threat, the severity of the hypercortisolism, and the patient perspectives (wish of fertility) are among the major parameters that can help a multidisciplinary approach toward the best option.

Pathogenesis of adrenocortical cancer

The study of the clonality of adrenocortical tumours (ACTs) has shown that adrenocortical cancers (ACCs) are of monoclonal origin. Numerous chromosomal alterations have been observed in ACCs, and they are much more frequent than in adrenocortical adenomas. Progress in the genetics of familial syndromes associated with ACTs helped to identify significant somatic molecular alterations in sporadic adult ACCs. Somatic mutations of the tumour suppressor gene TP53 are observed in a third of ACCs. Interestingly, allelic losses (LOH) at the TP53 locus (17p13) are very frequent, observed in more than 85% of ACCs. The insulin-like growth factor II (IGF-II) locus (11p15) is imprinted. IGF-II is over-expressed in 90% of ACCs. Transcriptome studies have identified an IGF-II cluster of genes significantly over-expressed in ACCs. Transcriptome analysis suggests also that the Wnt/beta-catenin signalling pathway is activated in ACT. About a third of ACCs harbours somatic activating mutations of the beta-catenin gene. This recent progress in the molecular genetics of ACC has led to the development of new molecular markers for the diagnosis of malignancy; these might also help to identify prognostic markers of ACC and may ultimately lead to novel therapeutic approaches.