The spectrum of growth hormone excess in Carney complex and genotype-phenotype correlations.

CONTEXT: Carney complex (CNC) is a familial neoplasia syndrome associated with growth hormone (GH) excess (GHE). OBJECTIVE: To describe the frequency of GHE in a large cohort of patients with CNC, and to identify genotype-phenotype correlations. METHODS: Patients with CNC with at least one biochemical evaluation of GH secretion at our center from 1995-2021 (n=140) were included in the study. Diagnosis of GHE was based on levels of insulin-like growth factor-1 (IGF-1), GH suppression during oral glucose tolerance test (OGTT), GH stimulation after thyrotropin (TRH) administration and overnight GH secretion. RESULTS: Fifty patients (35.7%) had GHE and 28 subjects (20%) had symptomatic acromegaly, with median age at diagnosis of 25.3 and 26.1 years respectively. Most of the patients (99.3%) had a PRKAR1A gene defect. There was a higher risk of GHE in patients harboring a variant that led to no expression of the affected allele [Hazard risk (HR): 3.06, 95% Confidence Intervals (CI): 1.2-7.8] and for patients harboring the hotspot variant c.491_492delTG (HR: 2.10, 95%CI: 1.1-4.1). Almost half of patients with CNC had an abnormal finding on pituitary imaging. CNC patients with an abnormal pituitary imaging had higher risk of GHE (HR: 2.94, 95%CI: 1.5-5.8), especially when single or multiple adenoma-like lesions were identified. Management of patients with symptomatic acromegaly involved surgical and medical approaches. CONCLUSION: Dysregulation of GH secretion is a common finding in CNC. The clinical spectrum of this disorder and its association with genetic and imaging characteristics of the patient make prompt diagnosis and management more successful.

Spatial Transcriptomic Analysis of Pituitary Corticotroph Tumors.

Context: Spatial transcriptomic (ST) analysis of tumors provides a novel approach to studying gene expression along with the localization of tumor cells in their environment to uncover spatial interactions. Design: We present ST analysis of corticotroph pituitary neuroendocrine tumors (PitNETs) from formalin-fixed, paraffin-embedded tissues. ST data were compared to immunohistochemistry results. Gene expression profiles were reviewed for cluster annotations, and differentially expressed genes were used for pathway analysis. Results: Seven tumors were used for ST analysis. In situ annotation of tumor tissue was inferred from the gene expression profiles and was in concordance with the annotation made by a pathologist. Furthermore, relative gene expression in the tumor corresponded to common protein staining used in the evaluation of PitNETs, such as reticulin and Ki-67 index. Finally, we identified intratumor heterogeneity; clusters within the same tumor may present with different transcriptomic profiles, unveiling potential intratumor cell variability. Conclusion: Together, our results provide the first attempt to clarify the spatial cell profile in PitNETs.

The utility of IGF1 in the evaluation of pediatric patients with endogenous hypercortisolemia.

BACKGROUND: Cushing Disease (CD) is a rare endocrine disorder associated with impaired growth hormone (GH) and short stature. Insulin-like growth factor-1 (IGF-1) is a marker of GH secretion. METHODS: Patients with young onset CD (<21 years old) and available IGF-1 levels at diagnosis and/or follow-up were studied (total = 194, diagnosis = 174, follow-up = 104). IGF-1 was reported as z-score (IGF1z). RESULTS: IGF1z was lower than expected in the general population (median IGF1z: -0.92 [-1.54, 0.07], p < 0.0001) at diagnosis and remained low at follow-up (median: -1.13 [-1.78, -0.66], p < 0.0001). There was no correlation of IGF1z at diagnosis with BMI; there was a weak correlation with height (rs = 0.19, p = 0.035). IGF1z was inversely correlated with markers of hypercortisolemia, including morning (rs = -0.31, p < 0.0001) and midnight cortisol (rs = -0.30, p < 0.0001), and with insulin resistance (Homeostatic Model Assessment for Insulin Resistance, HOMA-IR, rs = -0.27, p < 0.01). CONCLUSIONS: IGF-1 levels in CS are on the lower side of the normal range during active disease and remain low at one year after treatment. IGF-1 levels correlated mainly with markers of hypercortisolemia rather than the short stature of patients and should not be used in the assessment of growth in this population. IMPACT: We report that IGF-1 levels in childhood during active hypercortisolemia and up to 1 year after resolution are on the lower side of the normal range. Our results demonstrate that IGF-1 levels during active hypercortisolemia correlate mainly with markers of Cushing syndrome. This report adds data to the current literature where reports of IGF-1 in Cushing syndrome have shown variable results. Understanding the lack of utility of IGF-1 in assessing growth parameters in the pediatric Cushing syndrome population is important for physicians caring for these patients who should not use IGF-1 for diagnostic or treatment decisions.

Paediatric Cushing syndrome: a prospective, multisite, observational cohort study.

BACKGROUND: Paediatric endogenous Cushing syndrome is a rare condition with variable signs and symptoms of presentation. We studied a large cohort of paediatric patients with endogenous Cushing syndrome with the aim of describing anthropometric, clinical, and biochemical characteristics as well as associated complications and outcomes to aid diagnosis, treatment, and management. METHODS: In this prospective, multisite cohort study, we studied children and adolescents (≤18 years at time of presentation) with a diagnosis of Cushing syndrome. Patients had either received their initial diagnosis and evaluation at the Eunice Kennedy Shriver National Institute of Child Health and Human Development (Bethesda, MD, USA) or been referred from other centres in the USA or outside the USA. We collected participants' clinical, biochemical, and imaging findings and recorded their post-operative course until their latest appointment. FINDINGS: Of 342 paediatric patients with a diagnosis of Cushing syndrome, 193 (56%) were female and 149 (44%) male. 261 (76%) patients had corticotroph pituitary neuroendocrine tumours (Cushing disease), 74 (22%) had adrenal-associated Cushing syndrome, and seven (2%) had ectopic Cushing syndrome. Patients were diagnosed at a median of 2 years (IQR 1·0-3·0) after the first concerning sign or symptom, and patients with adrenal-associated Cushing syndrome were the youngest at diagnosis (median 10·4 years [IQR 7·4-13·6] vs 13·0 years [10·5-15·3] for Cushing disease vs 13·4 years [11·0-13·7] for ectopic Cushing syndrome; p<0·0001). Body-mass index z-scores did not differ between the diagnostic groups (1·90 [1·19-2·34] for adrenal-associated Cushing syndrome vs 2·18 [1·60-2·56] for Cushing disease vs 2·22 [1·42-2·35] for ectopic Cushing syndrome; p=0·26). Baseline biochemical screening for cortisol and adrenocorticotropin at diagnosis showed overlapping results between subtypes, and especially between Cushing disease and ectopic Cushing syndrome. However, patients with ectopic Cushing syndrome had higher urinary free cortisol (fold change in median cortisol concentration from upper limit of normal: 15·5 [IQR 12·7-18·0]) than patients with adrenal-associated Cushing syndrome (1·5 [0·6-5·7]) or Cushing disease (3·9 [2·3-6·9]; p<0·0001). Common complications of endogenous Cushing syndrome were hypertension (147 [52%] of 281 patients), hyperglycaemia (78 [30%] of 260 patients), elevated alanine transaminase (145 [64%] of 227 patients), and dyslipidaemia (105 [48%] of 219 patients). Long-term recurrence was noted in at least 16 (8%) of 195 patients with Cushing disease. INTERPRETATION: This extensive description of a unique cohort of paediatric patients with Cushing syndrome has the potential to inform diagnostic workup, preventative actions, and follow-up of children with this rare endocrine condition. FUNDING: Intramural Research Program, Eunice Kennedy Shriver National Institute of Child Health & Human Development, National Institutes of Health.

FAF1 Gene Involvement in Pituitary Corticotroph Tumors.

Cushing's disease (CD) is caused by rare pituitary corticotroph tumors that lead to corticotropin (ACTH) excess. Variants in FAF1, a pro-apoptotic protein involved in FAS-induced cell death, have been implicated in malignant disorders but the involvement of FAF1 in pituitary tumors has not been studied. Genetic data from patients with CD were reviewed for variants in FAF1 gene. Knockout mice (KO) were followed to assess the development of any pituitary disorder or cortisol excess. AtT-20 cells were used to study the effects of the variants of interest on ACTH secretion and cell proliferation. Three variants of interest were identified in 5 unique patients, two of which had rare allele frequency in genomic databases and were predicted to be likely pathogenic. KO mice were followed over time and no difference in their length/weight was noted. Additionally, KO mice did not develop any pituitary lesions and retained similar corticosterone secretion with wild type. AtT-20 cells transfected with FAF1 variants of interest or WT expression plasmids showed no significant difference in cell death or Pomc gene expression. However, in silico prediction models suggested significant differences in secondary structures of the produced proteins. In conclusion, we identified two FAF1 variants in patients diagnosed with CD with a potential pathogenic effect on the protein function and structure. Our in vitro and in vivo studies did not reveal an association of FAF1 defects with pituitary tumorigenesis and further studies may be needed to understand any association.

False Negative Inferior Petrosal Sinus Sampling in Young-Onset Cushing Disease: What Happens Next.

INTRODUCTION: False negative results during inferior petrosal sinus sampling (IPSS) may complicate the diagnostic evaluation of patients with ACTH-dependent Cushing syndrome (CS). The management of these patients can be confusing for clinicians and lead to delayed management. METHODS: We studied patients with young-onset (<21yo) CD who underwent IPSS during their diagnostic evaluation. For all patients, diagnosis of CD was eventually confirmed based on histologic evaluation of a resected pituitary tumor or remission after transsphenoidal surgery (TSS). RESULTS: We recorded a rare incidence of false negative IPSS results in 5 out of the 142 IPSS procedures (3.5%), performed in 4 unique patients. Patients with negative IPSS did not differ in demographic (age and sex) or biochemical (diurnal ACTH/cortisol or 24hour urinary free cortisol) data from the remaining. Additional workup was performed in three of the four patients including evaluation for ectopic sources of CS and repeat IPSS. Two of these patients also received medical treatment for suppression of cortisol production. One patient decided to proceed with pituitary exploration without additional evaluation. All patients finally underwent surgery and achieved remission. DISCUSSION/CONCLUSION: In patients with CD, IPSS may rarely lead to false negative results. Management of these patients usually includes screening for ectopic sources of ACTH/CRH secretion, repeating IPSS if ectopic workup is negative, and considering medical management until final diagnosis of the source of hypercortisolism is made.

Spatial Transcriptomic Analysis of Pituitary Corticotroph Tumors Unveils Intratumor Heterogeneity.

Spatial transcriptomic (ST) analysis of tumors provides a novel approach on studying gene expression along with the localization of tumor cells in their environment to uncover spatial interactions. Herein, we present ST analysis of corticotroph pituitary neuroendocrine tumors (PitNETs) from formalin-fixed, paraffin-embedded (FFPE) tissues. We report that the in situ annotation of tumor tissue can be inferred from the gene expression profiles and is in concordance with the annotation made by a pathologist. Furthermore, relative gene expression in the tumor corresponds to common protein staining used in the evaluation of PitNETs, such as reticulin and Ki-67 index. Finally, we identify intratumor heterogeneity; clusters within the same tumor may present with different secretory capacity and transcriptomic profiles, unveiling potential intratumor cell variability with possible therapeutic interest. Together, our results provide the first attempt to clarify the spatial cell profile in PitNETs.

Diurnal Range and Intra-patient Variability of ACTH Is Restored With Remission in Cushing's Disease.

CONTEXT: Single ACTH measurements have limited ability to distinguish patients with Cushing's disease (CD) from those in remission or with other conditions. OBJECTIVE: To investigate the changes in ACTH levels before and after transsphenoidal surgery (TSS) to identify trends that could confirm remission from CD and help establish ACTH cutoffs for targeted clinical trials in CD. DESIGN: Retrospective analysis of CD patients who underwent TSS from 2005 to -2019. SETTING: Referral center. PATIENTS: CD patients (n = 253) with ACTH measurements before and after TSS. INTERVENTIONS: TSS for CD. MAIN OUTCOME MEASURES: Remission after TSS. RESULTS: Remission was observed in 223 patients after TSS. Those in remission had higher ACTH variability at AM (P = .02) and PM (P < .001) time points compared to nonremission. The nonremission group had a significantly narrower diurnal range compared to the remission group (P = <.0001). A decrease in plasma ACTH of ≥50% from mean preoperative levels predicted CD remission after TSS, especially when using PM values. The absolute plasma ACTH concentration and ratio of preoperative to postoperative values were significantly associated with nonremission after multivariable logistic regression (adj P < .001 and .001, respectively). CONCLUSIONS: Our findings suggest that ACTH variability is suppressed in CD, and remission from CD is associated with the restoration of this variability. Furthermore, a decrease in plasma ACTH by 50% or more may serve as a predictor of remission post-TSS. These insights could guide clinicians in developing rational outcome measures for interventions targeting CD adenomas.

Germline loss-of-function PAM variants are enriched in subjects with pituitary hypersecretion.

Introduction: Pituitary adenomas (PAs) are common, usually benign tumors of the anterior pituitary gland which, for the most part, have no known genetic cause. PAs are associated with major clinical effects due to hormonal dysregulation and tumoral impingement on vital brain structures. PAM encodes a multifunctional protein responsible for the essential C-terminal amidation of secreted peptides. Methods: Following the identification of a loss-of-function variant (p.Arg703Gln) in the peptidylglycine a-amidating monooxygenase (PAM) gene in a family with pituitary gigantism, we investigated 299 individuals with sporadic PAs and 17 familial isolated PA kindreds for PAM variants. Genetic screening was performed by germline and tumor sequencing and germline copy number variation (CNV) analysis. Results: In germline DNA, we detected seven heterozygous, likely pathogenic missense, truncating, and regulatory SNVs. These SNVs were found in sporadic subjects with growth hormone excess (p.Gly552Arg and p.Phe759Ser), pediatric Cushing disease (c.-133T>C and p.His778fs), or different types of PAs (c.-361G>A, p.Ser539Trp, and p.Asp563Gly). The SNVs were functionally tested in vitro for protein expression and trafficking by Western blotting, splicing by minigene assays, and amidation activity in cell lysates and serum samples. These analyses confirmed a deleterious effect on protein expression and/or function. By interrogating 200,000 exomes from the UK Biobank, we confirmed a significant association of the PAM gene and rare PAM SNVs with diagnoses linked to pituitary gland hyperfunction. Conclusion: The identification of PAM as a candidate gene associated with pituitary hypersecretion opens the possibility of developing novel therapeutics based on altering PAM function.

Exploratory Study of the Association of Genetic Factors With Recovery of Adrenal Function in Cushing Disease.

Successful treatment of endogenous Cushing disease (CD) is often followed by a period of adrenal insufficiency (AI). We performed an exploratory study on genetic factors potentially involved in the hypothalamic-pituitary-adrenal (HPA) axis recovery in patients with CD after remission. We identified 90 patients who achieved remission after surgery and had a minimum of 3 months follow-up. Variants in a selected panel of genes that were rare in the general population and predicted as damaging in silico were retrieved from whole exome sequencing analysis. We did not identify any variant with significant correlation with recovery time after adjusting for multiple comparisons. On gene-specific analysis the BAG1 gene showed a correlation with shorter duration of postsurgical AI, but both patients with BAG1 variants later experienced a recurrence. After excluding patients with recurrence, no statistical association was recorded. To conclude, we did not identify a strong genetic modifier of HPA recovery in this exploratory study.

Cortisol and ACTH Measurements at Extubation From Pituitary Surgery Predicts Hypothalamic-Pituitary-Adrenal Axis Function.

Context: Early prediction of hypothalamic-pituitary-adrenal (HPA) axis function following transsphenoidal surgery (TSS) can improve patient safety and reduce costs. Objective: Systematic measurement of ACTH and cortisol at extubation following anesthesia to predict remission from Cushing's disease (CD) and HPA axis preservation following non-CD surgery. Design: Retrospective analysis of clinical data between August 2015 and May 2022. Setting: Referral center. Patients: Consecutive patients (n = 129) undergoing TSS who had perioperative ACTH and cortisol measurements. Interventions: ACTH and cortisol measurement at extubation. Further serial 6-hourly measurements in CD patients. Main outcome measures: Prediction of future HPA axis status based on ACTH/cortisol at extubation. Results: ACTH and cortisol increased sharply in all patients at extubation. CD patients (n = 101) had lower ACTH values than non-CD patients (110.1 vs 293.1 pg/mL; P < 0.01). In non-CD patients, lower plasma ACTH at extubation predicted the need for eventual corticosteroid replacement (105.8 vs 449.1 pg/mL, P < 0.01). In CD patients, the peak post-extubation cortisol at 6 hours was a robust predictor for nonremission (60.7 vs 219.2 µg/dL, P = 0.03). However, normalized early postoperative value (NEPV; the post-extubation values minus the peak preoperative CRH or desmopressin test values) of cortisol reliably distinguished nonremission earlier, at the time of extubation (-6.1 vs 5.9, P = 0.01), and later. Conclusions: We found that at extubation following TSS, ACTH can predict the need for eventual steroid replacement in non-Cushing's patients. In patients with CD, we found a robust prediction of nonremission with NEPV cortisol at extubation and later.

Germline loss-of-function PAM variants are enriched in subjects with pituitary hypersecretion.

Pituitary adenomas (PAs) are common, usually benign tumors of the anterior pituitary gland which, for the most part, have no known genetic cause. PAs are associated with major clinical effects due to hormonal dysregulation and tumoral impingement on vital brain structures. Following the identification of a loss-of-function variant (p.Arg703Gln) in the PAM gene in a family with pituitary gigantism, we investigated 299 individuals with sporadic PAs and 17 familial isolated pituitary adenomas kindreds for PAM variants. PAM encodes a multifunctional protein responsible for the essential C-terminal amidation of secreted peptides. Genetic screening was performed by germline and tumor sequencing and germline copy number variation (CNV) analysis. No germline CNVs or somatic single nucleotide variants (SNVs) were identified. We detected seven likely pathogenic heterozygous missense, truncating, and regulatory SNVs. These SNVs were found in sporadic subjects with GH excess (p.Gly552Arg and p.Phe759Ser), pediatric Cushing disease (c.-133T>C and p.His778fs), or with different types of PAs (c.-361G>A, p.Ser539Trp, and p.Asp563Gly). The SNVs were functionally tested in vitro for protein expression and trafficking by Western blotting, for splicing by minigene assays, and for amidation activity in cell lysates and serum samples. These analyses confirmed a deleterious effect on protein expression and/or function. By interrogating 200,000 exomes from the UK Biobank, we confirmed a significant association of the PAM gene and rare PAM SNVs to diagnoses linked to pituitary gland hyperfunction. Identification of PAM as a candidate gene associated with pituitary hypersecretion opens the possibility of developing novel therapeutics based on altering PAM function.

Subclinical Hemorrhage of ACTH-secreting Pituitary Adenomas in Children and Adolescents Changes Their Biochemical Profile.

Context: Subclinical pituitary hemorrhage, necrosis, and/or cystic degeneration (SPH) presents mainly in large tumors and prolactinomas. The characteristics of patients with Cushing disease (CD) and SPH are not known. Objective: To determine if SPH affects the presentation and biochemical profile of young patients with CD. Methods: Pediatric and adolescent patients who were diagnosed with CD between 2005 and 2021 and available magnetic resonance imaging images were evaluated for SPH. The clinical and biochemical characteristics of patients with and without SPH were compared. Results: Evidence of possible SPH was present in 12 out of 170 imaging studies (7.1%). Patients with and without SPH had similar age at diagnosis and sex distribution but differed in disease duration (median duration: 1.0 year [1.0-2.0] in the SPH group vs 2.5 years [1.5-3.0] in the non-SPH group, P = .014). When comparing their biochemical evaluation, patients with SPH had higher levels of morning adrenocorticotropin (ACTH) (60.8 pg/mL [43.5-80.3]) compared to patients without SPH (39.4 pg/mL [28.2-53.2], P = .016) and the degree of cortisol reduction after overnight high dose (8 mg or weight-based equivalent) dexamethasone was lower (-58.0% [-85.4 to -49.7]) compared to patients without SPH (85.8 [-90.5 to -76.8], P = .035). The presence of SPH did not affect the odds of remission after surgery or the risk of recurrence after initial remission. Conclusion: SPH in ACTH-secreting pituitary adenomas may affect their biochemical response during endocrine evaluations. They may, for example, fail to suppress to dexamethasone which can complicate diagnosis. Thus, SPH should be mentioned on imaging and taken into consideration in the work up of pediatric patients with CD.

Copeptin Levels Before and After Transsphenoidal Surgery for Cushing Disease: A Potential Early Marker of Remission.

Context: Arginine-vasopressin and CRH act synergistically to stimulate secretion of ACTH. There is evidence that glucocorticoids act via negative feedback to suppress arginine-vasopressin secretion. Objective: Our hypothesis was that a postoperative increase in plasma copeptin may serve as a marker of remission of Cushing disease (CD). Design: Plasma copeptin was obtained in patients with CD before and daily on postoperative days 1 through 8 after transsphenoidal surgery. Peak postoperative copeptin levels and Δcopeptin values were compared among those in remission vs no remission. Results: Forty-four patients (64% female, aged 7-55 years) were included, and 19 developed neither diabetes insipidus (DI) or syndrome of inappropriate anti-diuresis (SIADH). Thirty-three had follow-up at least 3 months postoperatively. There was no difference in peak postoperative copeptin in remission (6.1 pmol/L [4.3-12.1]) vs no remission (7.3 pmol/L [5.4-8.4], P = 0.88). Excluding those who developed DI or SIADH, there was no difference in peak postoperative copeptin in remission (10.2 pmol/L [6.9-21.0]) vs no remission (5.4 pmol/L [4.6-7.3], P = 0.20). However, a higher peak postoperative copeptin level was found in those in remission (14.6 pmol/L [±10.9] vs 5.8 (±1.4), P = 0.03]) with parametric testing. There was no difference in the Δcopeptin by remission status. Conclusions: A difference in peak postoperative plasma copeptin as an early marker to predict remission of CD was not consistently present, although the data point to the need for a larger sample size to further evaluate this. However, the utility of this test may be limited to those who develop neither DI nor SIADH postoperatively.

Neurofibromatosis Type 1 Has a Wide Spectrum of Growth Hormone Excess.

Overgrowth due to growth hormone (GH) excess affects approximately 10% of patients with neurofibromatosis type 1 (NF1) and optic pathway glioma (OPG). Our aim is to describe the clinical, biochemical, pathological, and genetic features of GH excess in a retrospective case series of 10 children and adults with NF1 referred to a tertiary care clinical research center. Six children (median age = 4 years, range of 3−5 years), one 14-year-old adolescent, and three adults (median age = 42 years, range of 29−52 years) were diagnosed with NF1 and GH excess. GH excess was confirmed by the failure to suppress GH (<1 ng/mL) on oral glucose tolerance test (OGTT, n = 9) and frequent overnight sampling of GH levels (n = 6). Genetic testing was ascertained through targeted or whole-exome sequencing (n = 9). Five patients (all children) had an OPG without any pituitary abnormality, three patients (one adolescent and two adults) had a pituitary lesion (two tumors, one suggestive hyperplasia) without an OPG, and two patients (one child and one adult) had a pituitary lesion (a pituitary tumor and suggestive hyperplasia, respectively) with a concomitant OPG. The serial overnight sampling of GH levels in six patients revealed abnormal overnight GH profiling. Two adult patients had a voluminous pituitary gland on pituitary imaging. One pituitary tumor from an adolescent patient who harbored a germline heterozygous p.Gln514Pro NF1 variant stained positive for GH and prolactin. One child who harbored a heterozygous truncating variant in exon 46 of NF1 had an OPG that, when compared to normal optic nerves, stained strongly for GPR101, an orphan G protein-coupled receptor causing GH excess in X-linked acrogigantism. We describe a series of patients with GH excess and NF1. Our findings show the variability in patterns of serial overnight GH secretion, somatotroph tumor or hyperplasia in some cases of NF1 and GH excess. Further studies are required to ascertain the link between NF1, GH excess and GPR101, which may aid in the characterization of the molecular underpinning of GH excess in NF1.

Inflammatory biomarkers in the evaluation of pediatric endogenous Cushing syndrome.

OBJECTIVE: Inflammatory biomarkers, such as absolute neutrophil and lymphocyte counts, neutrophil-to-lymphocyte ratio (NLR), platelet (PLT)-to-lymphocyte ratio (PLR) and monocyte-to-lymphocyte ratio (MLR), are associated with the progression and development of several disorders. Although patients with Cushing syndrome (CS) have immunosuppression with altered leucocyte counts, the profile of the inflammatory biomarkers in these patients has not been extensively studied. DESIGN: We compared a panel of inflammatory biomarkers in patients with active endogenous CS (n of complete blood count (CBC) reports = 319) and eucortisolemic subjects of similar age, gender and BMI (n of CBC reports = 93). Patients were divided into two age groups (6-12 years at the time of CBC and >12 years at the time of CBC) based on age differences in normal reference ranges. RESULTS: Patients with CS had higher NLR vs controls (6-12 years: 2.47 (1.86, 3.32) vs 1.35 (1.11, 2.27), P < 0.0001; >12 years: 3.00 (2.23-4.17) vs 1.80 (1.23-2.31), P < 0.0001). Similarly, absolute neutrophil and lymphocyte counts, MLR and PLR differed between patients with CS and controls. The inflammatory biomarkers correlated with indices of cortisol secretion, such as midnight serum cortisol, 24-h urinary free cortisol and morning cortisol. On receiver operating characteristic analysis, NLR showed high area under the curve (AUC) (6-12 years: cutoff of 1.72 had AUC: 0.77, >12 years: cutoff of 2.35 had AUC: 0.81). CONCLUSIONS: We conclude that multiple inflammatory biomarkers differed between patients with CS and controls suggesting substantial effects of hypercortisolemia on the immune system.

Cushing syndrome and glucocorticoids: T-cell lymphopenia, apoptosis, and rescue by IL-21.

BACKGROUND: Pediatric endogenous Cushing syndrome (eCs) is mainly caused by pituitary corticotropin-producing adenomas, and most glucocorticoid-dependent effects progressively regress upon tumor removal. eCs reproduces long-term, high-dose glucocorticoid therapy, representing a clean, natural, and unbiased model in which to study glucocorticoid bona fide effects on immunity. OBJECTIVE: We performed extensive immunologic studies in otherwise healthy pediatric patients with eCs before and 6 to 13 months after tumor resection, as well as in in vitro glucocorticoid-treated control cells. METHODS: Flow cytometry, immunoblotting, enzyme-linked immunosorbent assay, real-time quantitative PCR, and RNA-Seq techniques were used to characterize patients' and in vitro glucocorticoid treated cells. RESULTS: Reduced thymic output, decreased naive T cells, diminished proliferation, and increased T-cell apoptosis were detected before surgery; all these defects eventually normalized after tumor removal in patients. In vitro studies also showed increased T-cell apoptosis, with correspondingly diminished NF-κB signaling and IL-21 levels. In this setting, IL-21 addition upregulated antiapoptotic BCL2 expression and rescued T-cell apoptosis in a PI3K pathway-dependent manner. Similar and reproducible findings were confirmed in eCs patient cells as well. CONCLUSIONS: We identified decreased thymic output and lymphocyte proliferation, together with increased apoptosis, as the underlying causes to T-cell lymphopenia in eCs patients. IL-21 was decreased in both natural and in vitro long-term, high-dose glucocorticoid environments, and in vitro addition of IL-21 counteracted the proapoptotic effects of glucocorticoid therapy. Thus, our results suggest that administration of IL-21 in patients receiving long-term, high-dose glucocorticoid therapy may contribute to ameliorate lymphopenia and the complications associated to it.

Paediatric patients with Cushing disease and negative pituitary MRI have a higher risk of nonremission after transsphenoidal surgery.

OBJECTIVE: Diagnostic workup of Cushing disease (CD) involves imaging evaluation of the pituitary gland, but in many patients no tumour is visualised. The aim of this study is to describe the association of magnetic resonance imaging (MRI) findings with the postoperative course of paediatric and adolescent patients with CD. PATIENTS: Patients with a diagnosis of CD at less than 21 years of age with MRI evaluation of the pituitary before first transsphenoidal surgery were included. MEASUREMENTS: Clinical, imaging and biochemical data were analysed. RESULTS: One hundred and eighty-six patients with paediatric or adolescent-onset CD were included in the study. Of all patients, 127 (68.3%) had MRI findings consistent with pituitary adenoma, while the remaining had negative or inconclusive MRI. Patients with negative MRI were younger in age and had lower morning cortisol and adrenocorticotropin levels. Of 181 patients with data on postoperative course, patients with negative MRI had higher odds of not achieving remission after the first surgery (odds ratio = 2.6, 95% confidence intervals [CIs] = 1.1-6.0) compared to those with positive MRI. In patients with remission after first transsphenoidal surgery, long-term recurrence risk was not associated with the detection of a pituitary adenoma in the preoperative MRI (hazard risk = 2.1, 95% CI = 0.7-5.8). CONCLUSIONS: Up to one-third of paediatric and adolescent patients with CD do not have a pituitary tumour visualised in MRI. A negative MRI is associated with higher odds of nonremission after surgery; however, if remission is achieved, long-term risk for recurrence is not associated with the preoperative MRI findings.

Cushing Syndrome in a Pediatric Patient With a KCNJ5 Variant and Successful Treatment With Low-dose Ketoconazole.

CONTEXT: Pathogenic variants in KCNJ5, encoding the GIRK4 (Kir3.4) potassium channel, have been implicated in the pathogenesis of familial hyperaldosteronism type-III (FH-III) and sporadic primary aldosteronism (PA). In addition to aldosterone, glucocorticoids are often found elevated in PA in association with KCNJ5 pathogenic variants, albeit at subclinical levels. However, to date no GIRK4 defects have been linked to Cushing syndrome (CS). PATIENT: We present the case of a 10-year-old child who presented with CS at an early age due to bilateral adrenocortical hyperplasia (BAH). The patient was placed on low-dose ketoconazole (KZL), which controlled hypercortisolemia and CS-related signs. Discontinuation of KZL for even 6 weeks led to recurrent CS. RESULTS: Screening for known genes causing cortisol-producing BAHs (PRKAR1A, PRKACA, PRKACB, PDE11A, PDE8B, ARMC5) failed to identify any gene defects. Whole-exome sequencing showed a novel KCNJ5 pathogenic variant (c.506T>C, p.L169S) inherited from her father. In vitro studies showed that the p.L169S variant affects conductance of the Kir3.4 channel without affecting its expression or membrane localization. Although there were no effects on steroidogenesis in vitro, there were modest changes in protein kinase A activity. In silico analysis of the mutant channel proposed mechanisms for the altered conductance. CONCLUSION: We present a pediatric patient with CS due to BAH and a germline defect in KCNJ5. Molecular investigations of this KCNJ5 variant failed to show a definite cause of her CS. However, this KCNJ5 variant differed in its function from KCNJ5 defects leading to PA. We speculate that GIRK4 (Kir3.4) may play a role in early human adrenocortical development and zonation and participate in the pathogenesis of pediatric BAH.