Advances in the management of paediatric Cushing's disease

Cushing's disease (CD) is rare in the paediatric age range, but may present a difficult therapeutic challenge. Most paediatric endocrinologists have limited experience managing children or adolescents with CD and thus benefit from close consultation with adult colleagues. Prior to definitive treatment, a diagnostic protocol for investigation is required which broadly follows the model for adult patients. Treatment strategies for CD are described and critically appraised. The management of paediatric CD patients after cure also presents challenges for optimizing growth, bone health, reproduction and body composition from childhood into and during adult life.     

The discriminatory value of the low-dose dexamethasone suppression test in the investigation of paediatric Cushing's syndrome

BACKGROUND: Low- and high-dose dexamethasone suppression tests (LDDST, HDDST) are used in the investigation of Cushing's syndrome (CS). In adults with Cushing's disease (CD), cortisol suppression during LDDST predicts suppression during the HDDST. METHODS: We reviewed the results of the LDDST (0.5 mg 6 hourly x 48 h), HDDST (2.0 mg 6 hourly x 48 h) and corticotrophin-releasing hormone (CRH) test in 32 paediatric patients with CS: 24 had CD, 1 ectopic ACTH syndrome, 5 nodular adrenal hyperplasia and 2 adrenocortical tumours. Results: In CD, LDDST suppressed cortisol from 590.7 +/- 168.8 (mean +/- SD) to 333.7 +/- 104.0 nmol/l after 48 h (0 vs. 48 h, p < 0.05; mean suppression, 45.1%; CI (30.8, 59.4%); 16/24 (66%) suppressed >30%; mean suppression 68.1%, CI (58.1, 77.9%)). The HDDST suppressed cortisol from 596.3 +/- 174.5 to 47.1 +/- 94.8 nmol/l after 48 h (0 vs. 48 h, p < 0.05; mean suppression, 93.5%; CI (88.2, 98.8%) with 17/24 (71%) suppressing to <50 nmol/l and 100% to <50% of baseline). In the LDDST, suppression correlated with that during the HDDST (r = +0.45, p < 0.05) with >30% suppression predicting that in the HDDST and hence CD. CRH increased cortisol by +100.3% (CI 62, 138.5%), 22/24 (91.7%) showing a >20% increase. In the other CS pathologies (n = 8) the LDDST induced no significant decrease in cortisol. CONCLUSION: The LDDST was of diagnostic value by discriminating between CD and other CS aetiologies. In our view the HDDST is redundant in the investigation of paediatric CS.     

Course of pregnancies in women with Cushing's disease treated by gamma-knife

Data concerning pregnancy in women with Cushing's disease treated by gamma-knife (GK) are scanty. We present and discuss the course and outcome of five pregnancies in two women with Cushing's disease (CD), the first of whom was treated only by GK, and the second one treated by surgery, GK and ketoconazole. In the first patient, pregnancy was uneventful and full-term. During gestation, plasma ACTH, serum cortisol and 24-h urinary free cortisol (UFC) levels were steady, and always in the normal range for healthy non-pregnant individuals. The newborn was healthy and normal-weight. In the second woman, two pregnancies, occurring 3 years after GK and few months after ketoconazole withdrawal, were interrupted by spontaneous abortion or placental disruption despite normal cortisol levels. This patient became again pregnant 3 years later and delivered vaginally a healthy full-term infant. Seven months after the delivery, the patient became pregnant again and at the 39th week of gestation delivered vaginally a healthy male. Hypoprolactinemia and/or central hypothyroidism occurred in both cases. In women with CD treated by GK, pregnancy can occur. However, pregnancy is at risk even when ACTH and cortisol levels are normalized by treatment. After GK, evaluation of pituitary function is mandatory due to the risk of hypopituitarism.     

Growth in paediatric Crohn's disease

Growth failure (GF) is one of the major complications affecting children with inflammatory bowel disease. The faltering is temporary in 40-50% of cases and prolonged in 10-20% in Crohn's disease (CD). Such failure is rare in children with ulcerative colitis (5%). This complication is often associated with retarded bone development and delayed onset of sexual maturation. The delayed linear growth has a variety of causes including insufficient intake due to anorexia and the inflammatory process with increased energy and protein expenditure. Other factors are increased intestinal loss, secondary hypopituitarism and treatment with steroids. Therapeutic strategies of CD in children have changed this last decade by introducing new therapeutic agents such as topic steroids, immunosuppressors, anti-TNF (antibody and notably in children enteral nutrition which has shown its efficacy in inducing remissions of active CD, restoring nutritional status and stimulation of linear growth. The results of a recent prospective multicentric study over 2 years in 82 CD show that severe GF (-2 SD) is initially present in 15% (n = 12), among them 11 remain < -2SD after 2 years of follow-up. Six patients who were on the normal range initially increased their GF during the follow-up (< -2SD) (total 21% < -2SD (n = 17) at 2 years). At inclusion in this group there was no difference in growth velocity, used of steroids, enteral nutrition or severity of CD as compared to the group with no GF. It suggests that new treatment strategy should be developed in the future for this specific complication of paediatric CD.     

Metyrapone in long-term management of Cushing's disease.

Metyrapone was used in the long-term management of 13 patients with pituitary-dependent bilateral adrenal hyperplasia (Cushing''s disease). The total length of treatment ranged from two to 66 months, with a mean of 21 months. The clinical features of the disease rapidly improved on metyrapone and this improvement was maintained. Although plasma ACTH concentrations rose in all patients, the increase was insufficient to overcome the adrenal blockade induced by the drug. Eight of the 13 patients had additional external pituitary irradiation as definitive treatment of their disease and one underwent a transfrontal hypophysectomy. Radiotherapy cured one patient, and after three years metyrapone was withdrawn. Slight hirsuties was noted in four of the seven women who received the drug for six months or more. A fifth woman had more severe hirsuties and this led to bilateral adrenalectomy. Other than hirsuties, side effects were few and the routine use of metyrapone is recommended as an adjunct to more definitive treatment in all patients who present with Cushing''s syndrome, irrespective of aetiology.     

Chronic subclinical prion disease induced by low-dose inoculum

We have compared the transmission characteristics of the two mouse-adapted scrapie isolates, ME7 and Rocky Mountain Laboratory (RML), in tga20 mice. These mice express elevated levels of PrP protein compared to wild-type mice and display a relatively short disease incubation period following intracerebral prion inoculation. Terminal prion disease in tga20 mice induced by ME7 or RML was characterized by a distinct pattern of clinical signs and different incubation times. High-dose RML inoculated intracerebrally into tga20 mice induced the most rapid onset of clinical signs, with mice succumbing to terminal disease after only 58 +/- 3 days. In contrast, high-dose ME7 gave a mean time to terminal disease of 74 +/- 0 days. Histological examination of brain sections from prion-inoculated tga20 mice at terminal disease showed that ME7 gave rise to a more general and extensive pattern of vacuolation than RML. Low-dose inoculum failed to induce terminal disease but did cause preclinical symptoms, including the appearance of reversible clinical signs. Some mice oscillated between showing no clinical signs and early clinical signs for many months but never progressed to terminal disease. Brain tissue from these mice with chronic subclinical prion disease, sacrificed at >200 days postinoculation, contained high levels of infectivity and showed the presence of PrP(Sc). Parallel analysis of brain tissue from mice with terminal disease showed similar levels of infectivity and detectable PrP(Sc). These results show that high levels of infectivity and the presence of the abnormal isomer of PrP can be detected in mice with subclinical disease following low-dose prion inoculation.     

Recurrent gain-of-function USP8 mutations in Cushing's disease

Cushing''s disease, also known as adrenocorticotropic hormone (ACTH)-secreting pituitary adenomas (PAs) that cause excess cortisol production, accounts for up to 85% of corticotrophin-dependent Cushing''s syndrome cases. However, the genetic alterations in this disease are unclear. Here, we performed whole-exome sequencing of DNA derived from 12 ACTH-secreting PAs and matched blood samples, which revealed three types of somatic mutations in a candidate gene, USP8 (encoding ubiquitin-specific protease 8), exclusively in exon 14 in 8 of 12 ACTH-secreting PAs. We further evaluated somatic USP8 mutations in additional 258 PAs by Sanger sequencing. Targeted sequencing further identified a total of 17 types of USP8 variants in 67 of 108 ACTH-secreting PAs (62.04%). However, none of these mutations was detected in other types of PAs (n = 150). These mutations aggregate within the 14-3-3 binding motif of USP8 and disrupt the interaction between USP8 and 14-3-3 protein, resulting in an elevated capacity to protect EGFR from lysosomal degradation. Accordingly, PAs with mutated USP8 display a higher incidence of EGFR expression, elevated EGFR protein abundance and mRNA expression levels of POMC, which encodes the precursor of ACTH. PAs with mutated USP8 are significantly smaller in size and have higher ACTH production than wild-type PAs. In surgically resected primary USP8-mutated tumor cells, USP8 knockdown or blocking EGFR effectively attenuates ACTH secretion. Taken together, somatic gain-of-function USP8 mutations are common and contribute to ACTH overproduction in Cushing''s disease. Inhibition of USP8 or EGFR is promising for treating USP8-mutated corticotrophin adenoma. Our study highlights the potentially functional mutated gene in Cushing''s disease and provides insights into the therapeutics of this disease.     

Lipocorticotropic peptides in Cushing's disease: in vitro studies

The immunologic patterns of 3 human pituitary adenomas of Cushing's disease have been studied after gel exclusion chromatography (Sephadex G-50). The immunologic characteristics were examined with three radioimmunoassays specific for human corticotropin (ACTH), lipotropin (LPH) and beta-endorphin (beta-End). In cell tumor extracts, chromatographic peaks corresponding to beta-LPH, gamma-LPH, beta-End and ACTH were identified. The ACTH/beta-LP-beta-End ratio was 1 in the 3 cases. Additionally, in the 3 cases, a chromatographic peak, partially cross-reacting in the beta-End assay, was eluted after beta-End, thus suggesting the presence of a fragment of the molecule. In 1 case, a peak of large molecular weight material with N- and C-terminal beta-LPH and ACTH immunoreactivity was observed, which corresponded to the precursor material. The release and the effects of various stimuli were studied on dispersed tumor cells in primary culture. The tumor cells had a biphasic basal secretion rate with a rapid increase of ACTH/beta-LPH-beta-End in the culture medium during the first 2 h. Then the release, studied during 2 days, was slower. Chromatographic studies showed that the beta-LPH/beta-End ratio was 0.8 in the cells and 0.3 in the medium, due essentially to the release of beta-End and beta-End-like materials. The cells released ACTH and beta-LPH-beta-End in equimolar ratio after stimulation with arginine vasopressin (AVP). The maximum effect was obtained with 10(-6) M AVP (D50 = 1 10(-9) M). Dibutyryl cyclic AMP (2. 10(-3) M) induced maximal release of ACTH/beta-LPH-beta-End. This stimulation was suppressed by a 48-hour preincubation with dexamethasone (10(-8)-10(-6) M). There was no effect of TRH and LH-RH on cell release. Dopamine (10(-6) M) specifically blocked the release of ACTH/beta-LPH-beta-End in 1 case. These data showed (a) heterogeneity of chromatographic profiles from case to case; (b) the presence of material in the tumor, cell extracts and culture medium corresponding to fragment(s) of beta-End; (c) culture studies demonstrated that tumor cells remain responsive to AVP stimulation and dexamethasone suppression, and (d) the dopamine inhibition of ACTH and beta-End release needs further investigation.     

External irradiation of the hypophysis for Cushing's disease

During the past 12 years 17 patients with Cushing''s disease (bilateral adrenal hyperplasia secondary to excessive pituitary adrenocorticotrophic hormone) have been treated initially with external pituitary irradiation. Of the 15 patients who have had adequate follow-up, nine showed complete biochemical remission, and one showed biochemical improvement. There were no complications. It is therefore recommended that the first mode of therapy for all patients with Cushing''s disease should be pituitary irradiation if the patient''s clinical condition permits.     

Effect of short-term intralipid infusion on the immune response during low-dose endotoxemia in humans

Novel anti-inflammatory effects of insulin have recently been described, and insulin therapy to maintain euglycemia suppresses the plasma levels of free fatty acids (FFA) and increases the survival of critically ill patients. We aimed to explore the effect of short-term high levels of plasma FFA on the inflammatory response to a low dose of endotoxin. Fourteen healthy male volunteers underwent the following two trials in a randomized crossover design: 1) continuous infusion of 20% Intralipid [0.7 ml.kg(-1).h(-1) (1.54 g/kg)] for 11 h, and 2) infusion of isotonic saline for 11 h (control). In each trial, heparin was given to activate lipoprotein lipase, and an intravenous bolus of endotoxin (0.1 ng/kg) was given after 6 h of Intralipid/saline infusion. Blood samples and muscle and fat biopsies were obtained before the Intralipid/saline infusion and before as well as after infusion of an endotoxin bolus. Plasma levels of FFA, triglycerides, and glycerol were markedly increased during the Intralipid infusion. Endotoxin exposure induced an increase in plasma levels of TNF-alpha, IL-6, and neutrophils and further stimulated gene expression of TNF-alpha and IL-6 in both skeletal muscle and adipose tissue. The systemic inflammatory response to endotoxin was significantly pronounced during Intralipid infusion. Short-term hyperlipidemia enhances the inflammatory response to endotoxin, and skeletal muscle and adipose tissue are capable of producing essential inflammatory mediators after endotoxin stimulation.     

The effect of low-dose naloxone infusion on plasma ACTH and LH in patients with Cushing's and Addison's diseases

The ACTH, cortisol and LH responses to low dose (0.8 mg/h) naloxone 90 min infusion were investigated in seven patients with untreated Cushing's disease, six patients with Addison's disease and four control subjects. Naloxone had no effects on ACTH hypersecretion or normal ACTH levels. These data confirm that naloxone cannot provide additional diagnostic or therapeutic approaches in ACTH hypersecretion syndromes, mainly in Cushing's disease. The mean percentage LH levels did not significantly change during low dose naloxone in controls or patients with Cushing's and Addison's diseases. This suggests that increased endogenous opioid peptides in these diseases may not modify the LH responses to low dose of naloxone. However, since three of five adults with Cushing's disease had increased LH levels during naloxone, further studies may be indicated.     

Results of repeated transsphenoidal surgery in Cushing's disease. Long-term follow-up

ObjectiveTranssphenoidal surgery (TSS) is the treatment of choice for Cushing's disease (CD). However, the best treatment option when hypercortisolism persists or recurs remains unknown. The aim of this study was to analyze the short and long-term outcome of repeat TSS in this situation and to search for response predictors.Patients and methodsData from 26 patients with persistent (n = 11) or recurrent (n = 15) hypercortisolism who underwent repeat surgery by a single neurosurgeon between 1982 and 2009 were retrospectively analyzed. Remission was defined as normalization of urinary free cortisol (UFC) levels, and recurrence as presence of elevated UFC levels after having achieved remission. The following potential outcome predictors were analyzed: adrenal status (persistence or recurrence) after initial TSS, tumor identification in imaging tests, degree of hypercortisolism before repeat TSS, same/different surgeon in both TSS, and time to repeat surgery.ResultsImmediate postoperative remission was achieved in 12 patients (46.2%). Five of the 10 patients with available follow-up data relapsed after surgery (median time to recurrence, 13 months). New hormone deficiencies were seen in seven patients (37%), and two patients had cerebrospinal fluid leakage. No other major complications occurred. None of the preoperative factors analyzed was predictive of surgical outcome.ConclusionsWhen compared to initial surgery, repeat TSS for CD is associated to a lower remission rate and a higher risk of recurrence and complications. Further studies are needed to define outcome predictors.     

Desmopressin test in the diagnosis and follow-up of cyclical Cushing's disease

ObjectiveTo assess the utility of the desmopressin (DDAVP) test in the diagnosis and follow-up of a cyclical Cushing's disease (CCS) case.Material and methodsLaboratory tests included morning and midnight serum cortisol levels, 24 h urine free cortisol excretion, midnight salivary cortisol levels, serum cortisol levels after low (1 mg) and high (8 mg) dexamethasone, plasma ACTH and serum cortisol levels after DDAVP. Magnetic resonance imaging (MRI) was used to assess the presence of a pituitary adenoma. The resected tumor specimen was studied by histological, immunohistochemical and cell biology techniques.ResultsA patient was referred to our unit with a diagnosis of Cushing's syndrome (CS) for further evaluation and treatment. However, no biochemical evidence of hypercortisolism was observed in the follow-up evaluations. Furthermore, the typical features of CS fluctuated throughout this period. A consistent positive response to the DDAVP stimulation test was observed during the diagnostic work-up, even when overt clinical features of CS were not apparent, raising suspicion for CCS. After two years of follow-up a definitive diagnosis of hypercortisolism was established. An MRI scan revealed a pituitary adenoma, as the source of ACTH production. After transphenoidal surgery, clinical signs of CS resolved and the response to DDAVP became negative. DDAVP induced a significant increase in ACTH levels in cultured pituitary adenoma cells, consistent with the in vivo DDAVP test results.ConclusionsOur case illustrates the utility of the DDAVP test in the evaluation of patients with suspected CCS. The DDAVP test could facilitate the management of CCS by shortening the time of diagnosis.     

A model for short-term control of the bacterioplankton by substrate and grazing

Substrate supply and grazing are the factors with the greatest potential for short-term control of planktonic bacterial density and productivity. A model was developed based on Monod kinetics, where growth rates are limited by food supply in a saturation type equation. In the model, substrate, bacteria, heterotrophic flagellates and zooplankton are state variables linked by trophic transfer and expressed as carbon. The steady state assumption allows calculation of equations indicating the following: (l) bacterial density is determined primarily by the ratio of substrate input to grazing rate; (2) bacterial production is balanced by a combination of losses due to maintenance, death and grazing, and occurs at a rate determined by the rate of substrate input and the growth yield; (3) ambient substrate concentration is directly related to grazing rate. Sensitivity analysis of the model on a computer demonstrates some differences between grazer-controlled and substrate-controlled bacterial systems, and predictions of the model are listed for possible validation in natural systems. The model is potentially useful in evaluating the ‘link vs. sink’ question, as it provides a framework for investigating energy flow through the microbial food web as a function of controlling factors.