Long-term outcomes of children treated for Cushing's disease: a single center experience.

PURPOSE: Pediatric Cushing's disease (CD) is rare and there are limited data on the long-term outcomes. We assessed CD recurrence, body composition, pituitary function and psychiatric comorbidity in a cohort of pediatric CD patients. METHODS: Retrospective review of 21 CD patients, mean age at diagnosis 12.1 years (5.7-17.8), managed in our center between 1986 and 2010. Mean follow-up from definitive treatment was 10.6 years (2.9-27.2). RESULTS: Fifteen patients were in remission following transsphenoidal surgery (TSS) and 5 were in remission following TSS + external pituitary radiotherapy (RT). One patient underwent bilateral adrenalectomy (BA). CD recurrence occurred in 3 (14.3 %) patients: 2 at 2 and 6 years after TSS and 1 7.6 years post-RT. The BA patient developed Nelson's syndrome requiring pituitary RT 0.6 years post-surgery. Short-term growth hormone deficiency (GHD) was present in 14 patients (81 % patients tested) (11 following TSS and 3 after RT) and 4 (44 % of tested) had long-term GHD. Gonadotropin deficiency caused impaired pubertal development in 9 patients (43 %), 4 requiring sex steroid replacement post-puberty. Four patients (19 %) had more than one pituitary hormone deficiency, 3 after TSS and 1 post-RT. Five patients (24 %) had long-term psychiatric co-morbidities (cognitive dysfunction or mood disturbance). There were significant long-term improvements in growth, weight and bone density but not complete reversal to normal in all patients. CONCLUSIONS: The long-term consequences of the diagnosis and treatment of CD in children is broadly similar to that seen in adults, with recurrence of CD after successful treatment uncommon but still seen. Pituitary hormone deficiencies occurred in the majority of patients after remission, and assessment and appropriate treatment of GHD is essential. However, while many parameters improve, some children may still have mild but persistent defects.

Investigation for Paediatric Cushing's Syndrome Using Twenty-Four-Hour Urinary Free Cortisol Determination.

OBJECTIVE: Paediatric Cushing's syndrome (CS) remains a challenge to diagnose and exclude. We assessed the accuracy of 24-hour urinary free cortisol (UFC) determination in children referred for suspected CS. DESIGN: We conducted a retrospective study of paediatric patients referred to our centre with suspected CS between 1982 and 2014. PATIENTS: Of 66 subjects (mean age 12.9 years; range 4.4-16.9), there were 47 cases of CS (29 males), which included Cushing's disease (CD; 39 patients, 25 males), primary pigmented nodular adrenocortical disease (8 patients, 4 males) and 19 'controls' (6 males) in whom the diagnosis of CS was excluded. MEASUREMENTS: The subjects had between one and five 24-hour UFC collections analysed by radioimmunoassay, chemiluminescent immunoassay or liquid chromatography-mass spectrometry. The data were normalised, corrected for body surface area (m2) and assessed using receiver operating characteristic analysis and an independent two-tailed t test. RESULTS: The diagnostic accuracy of 24-hour UFC for CS was excellent (area under the curve 0.98, 95% CI 0.946-1.00, sensitivity 89%, specificity 100%). CONCLUSIONS: Twenty-four-hour UFC is a reliable and practical investigation with high diagnostic accuracy for paediatric CS. However, further investigations may be required if the UFC is normal but there is a high diagnostic suspicion of CS.

Diagnosing unilateral primary aldosteronism - comparison of a clinical prediction score, computed tomography and adrenal venous sampling.

CONTEXT: In patients with primary aldosteronism (PA), adrenalectomy is potentially curative for those correctly identified as having unilateral excessive aldosterone production. It has been suggested that a recently developed and published clinical prediction score (CPS) may correctly identify some patients as having unilateral disease, without recourse to adrenal venous sampling. OBJECTIVE: We have applied the CPS to a large cohort of PA patients with defined and documented outcomes. We also incorporated a minor modification to the CPS and a radiological grading score (RGS) into our analysis to assess whether its performance could be augmented. RESULTS: A total of 75 patients with a robust diagnosis following bilateral adrenal venous cannulation and/or strictly defined surgical outcome were analysed. Applying the CPS to this group of patients produced a sensitivity of 38·8% and a specificity of 88·5% of correctly identifying unilateral aldosterone production. Using a suggested modification to the CPS, in which different levels of hypokalaemia were given different weightings, the sensitivity rose to 40·8%, with an identical specificity. Using the RGS alone improved sensitivity to 91·7%, but specificity was reduced to 62·5%. CONCLUSION: Applying the recently developed CPS to this cohort of patients, it was not possible to reproduce the 100% specificity reported in the original publication. Using the modified score or incorporating the RGS did not improve its performance. In this cohort, we were unable to show superiority of the CPS over an imaging-based strategy. CPS may have a role in guiding clinical decision-making, especially in those whose adrenal venous sampling (AVS) has been unsuccessful.

Changes in functional properties of the caffeine-sensitive Ca2+ store during differentiation of human SH-SY5Y neuroblastoma cells.

We have used single cell fluorescence imaging techniques to examine how functional properties of the caffeine-sensitive Ca(2+) store change during differentiation of a sub-population of caffeine-sensitive SH-SY5Y cells. Application of caffeine (30 mM) 1-10.5 min after a 'priming' depolarisation pulse of 55 mM K(+) revealed that the caffeine-sensitive store in undifferentiated cells remained replete, whereas that in 9-cis retinoic acid (9cRA)-differentiated cells spontaneously dissipated with a t(1/2) of 2.8 min, and was essentially completely depleted approximately 10 min after priming. In 9cRA-differentiated cells that were stimulated with methacholine (10 microM) 1 min after priming, the amplitude, rate of rise and propagation velocity of the Ca(2+) wave in the neurites were all constant, whereas these kinetic parameters all progressively decreased as the wave travelled along the neurites in cells that were stimulated 10 min after priming. Use-dependent block with ryanodine inhibited the global Ca(2+) signal in 9cRA-differentiated cells stimulated with methacholine 1 min after priming (71+/-8%) but not 10 min after priming. Depolarisation was more effective at priming the caffeine-sensitive Ca(2+) store in 9cRA-differentiated cells, which lack a functional store-operated Ca(2+) entry pathway. We conclude that differentiation of caffeine-sensitive SH-SY5Y cells is accompanied by an increase in lability of the caffeine-sensitive Ca(2+) store, and that spontaneous dissipation of Ca(2+) from the store limits the time course of its molecular 'memory' during which it can amplify the hormone-induced Ca(2+) signal by Ca(2+)-induced Ca(2+) release.

Release and sequestration of Ca2+ by a caffeine- and ryanodine-sensitive store in a sub-population of human SH-SY5Y neuroblastoma cells.

We have used single cell fluorescence imaging techniques to examine the role that ryanodine receptors play in the stimulus-induced Ca(2+) responses of SH-SY5Y cells. The muscarinic agonist methacholine (1mM) resulted in a Ca(2+) signal in 95% of all cells. Caffeine (30 mM) however stimulated a Ca(2+) signal in only 1-7% of N-type (neuroblastic) cells within any given field. The caffeine response was independent of extracellular Ca(2+), regenerative in nature, and abolished in a use-dependent fashion by ryanodine. In caffeine-responsive cells, the magnitude of the methacholine-induced Ca(2+) signal was inhibited by 75.07 +/- 5.51% by pretreatment with caffeine and ryanodine, suggesting that the caffeine-sensitive store may act as a Ca(2+) source after muscarinic stimulation. When these data were combined with equivalent data from non-caffeine-responsive cells, the degree of apparent inhibition was significantly reduced. In contrast, after store depletion by caffeine, the Ca(2+) signal induced by 55 mM K(+) was potentiated 2.5-fold in the presence of ryanodine, suggesting that the store may act a Ca(2+) sink after depolarisation. We conclude that a caffeine- and ryanodine-sensitive store can act as a Ca(2+) source and sink in SH-SY5Y cells, and that effects of the store can become obscured if data from caffeine-insensitive cells are not excluded.

Mechanistic and functional changes in Ca2+ entry after retinoic acid-induced differentiation of neuroblastoma cells.

We have investigated effects of neuronal differentiation on hormone-induced Ca2+ entry. Fura-2 fluorescence measurements of undifferentiated SH-SY5Y neuroblastoma cells, stimulated with methacholine, revealed the presence of voltage-operated Ca2+-permeable, Mn2+-impermeable entry pathways, and at least two voltage-independent Ca2+- and Mn2+-permeable entry pathways, all of which apparently contribute to both peak and plateau phases of the Ca2+ signal. Similar experiments using 9-cis retinoic acid-differentiated cells, however, revealed voltage-operated Ca2+-permeable, Mn2+-impermeable channels, and, more significantly, the absence or down-regulation of the most predominant of the voltage-independent entry pathways. This down-regulated pathway is probably due to CCE (capacitative Ca2+ entry), since thapsigargin also stimulated Ca2+ and Mn2+ entry in undifferentiated but not differentiated cells. The Ca2+ entry components remaining in methacholine-stimulated differentiated cells contributed to only the plateau phase of the Ca2+ signal. We conclude that differentiation of SH-SY5Y cells results in a mechanistic and functional change in hormone-stimulated Ca2+ entry. In undifferentiated cells, voltage-operated Ca2+ channels, CCE and NCCE (non-CCE) pathways are present. Of the voltage-independent pathways, the predominant one appears to be CCE. These pathways contribute to both peak and plateau phases of the Ca2+ signal. In differentiated cells, CCE is either absent or down-regulated, whereas voltage-operated entry and NCCE remain active and contribute to only the plateau phase of the Ca2+ signal.