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  1. Abstract Background Cushing’s disease (CD) is rare in pediatric patients. It is characterized by elevated plasma adrenocorticotropic hormone (ACTH) from pituitary adenomas, with damage to multiple systems and development. In recent years, genetic studies have shed light on the etiology and several mutations have been identified in patients with CD. Case presentation A girl presented at the age of 10 years and 9 months with facial plethora, hirsutism and acne. Her vision and eye movements were impaired. A quick weight gain and slow growth were also observed. Physical examination revealed central obesity, moon face, buffalo hump, supra-clavicular fat pads and bruising. Her plasma ACTH level ranged between 118 and 151 pg/ml, and sella enhanced MRI showed a giant pituitary tumor of 51.8 × 29.3 × 14.0 mm. Transsphenoidal pituitary debulk adenomectomy was performed and immunohistochemical staining confirmed an ACTH-secreting adenoma. Genetic analysis identified a novel germline GPR101 (p.G169R) and a somatic USP8 (p. S719del) mutation. They were hypothesized to impact tumor growth and function, respectively. Conclusions We reported a rare case of pediatric giant pituitary ACTH adenoma and pointed out that unusual concurrent mutations might contribute to its early onset and large volume. Peer Review reports Background Cushing’s disease (CD) is caused by the overproduction of adrenocorticotropic hormone (ATCH) by pituitary adenomas (PAs). It is rare in children and accounts for approximately 75% of pediatric Cushing’s syndrome from 7 to 17 years of age [1]. Weight gain and facial changes are more common in children than in adults [2]. Growth retardation is also a characteristic of children with hypercortisolemia [3]. Genetic alterations such as somatic USP8, RASD1, TP53 mutations, and germline AIP, MEN1, and CABLES1 mutations have been identified in CD patients [4]. Here we report a case of pediatric invasive pituitary ACTH macroadenoma associated with a novel germline GPR101 (p. G169R) and a somatic USP8 (p. S719del) mutation. Case presentation The girl was born at full term with a length of 48 cm and a weight of 2900 g. Her neuromotor and cognitive development was comparable to those of children of the same age. At the age of 9 years and 4 months she developed plethora, hirsutism, facial acne, rapid weight gain, and increased abdominal circumference. Her skin darkened, and purple striae appeared on thighs and in the armpits. She became dull and less talkative, as indicated by her parents. At 10 years and 3 months, the patient complained of pain around the left orbit with an intensity of 4–5 points on a numerical rating scale (NRS). Five months later bilateral blepharoptosis appeared, with significantly impaired vision of the left eye. Soon both eyes failed to rotate in all directions. On admission the patient was 10 years and 9 months, with a height of 144 cm (90–97th percentile) and a weight of 48 kg (25–50th percentile). Her weight gain was 20 kg, while the height increased by only 2–3 cm in 18 months. Her blood pressure was 115/76mmHg, and her heart rate was 80 bpm. Apart from the signs mentioned above, physical examination revealed central obesity (BMI 23.1 kg/m2), moon face, buffalo hump, supra-clavicular fat pads and bruising at the left fossa cubitalis. Her pupils were 7 mm in diameter and barely reacted to light. There was a fan-shaped visual field defect in the left eye. Her breasts were Tanner stage III and pubic hair was Tanner stage II, although menarche had not yet occurred. The parents and her younger brother at 6 years of age did not have symptoms related to Cushing syndrome, acromegaly or gigantism. There was no family history of pituitary tumor or other endocrine tumors. She had increased midnight serum cortisol (24.35 µg/dL, normal range < 1.8 µg/mL) and 24-hour urine free cortisol (24hUFC) (308.0 µg, normal range 12.3–103.5). The plasma ACTH level ranged from 118 to 151 pg/mL (< 46pg/mL). The 24hUFC was not suppressed (79.2 µg) after 48 h low-dose dexamethasone suppression test (LDDST), but suppressed to 32.8 µg (suppression rate 89.4%) after 48 h high-dose dexamethasone. Sella enhanced MRI showed a giant pituitary tumor measured 51.8 × 29.3 × 14.0 mm with heterogeneous density (Fig. 1). The mass compressed the optic chiasma and surrounded the bilateral cavernous sinus (Knosp 4). Therefore, an invasive giant pituitary ACTH adenoma was clinically diagnosed. The morning growth hormone (GH) was 1.0ng/ml (< 2 ng/ml) and insulin-like growth factor 1 416 ng/ml (88–452 ng/ml). The prolactin (PRL), luteinizing hormone (LH), follicle-stimulating hormone (FSH) and thyroid stimulating hormone (TSH) were all in normal ranges, as well as serum sodium, potassium, blood glucose and urine osmolality. Abdominal ultrasonography revealed a fatty liver. Tests concerning type 1 multiple endocrine neoplasia included serum calcium, phosphate, parathyroid hormone, gastrin and glucagon, which were all unremarkable (Table 1). Fig. 1 Contrast-enhanced coronal (A) and sagittal (B) T1-weighted MRI on admission. The sellar mass measured 51.8 × 29.3 × 14.0 cm (TD × VD × APD) with a heterogeneous density in the enhanced scan. The diaphragma sellea was dramatically elevated, with optic chiasm compressed. The sellar floor was sunken and bilateral cavernous sinus was surrounded (Knosp 4) Full size image Table 1 Laboratory data on admission Full size table Transsphenoidal pituitary debulk adenomectomy was performed immediately due to multiple cranial nerve involvement and the negative results of Sandostatin loading test. A decompression resection was done. The plasma ACTH level declined to 77 pg/ml and serum cortisol 30.2 µg/dl three days after the operation. Vision, pupil dilation, eye movements and blepharoptosis also partially improved. Histopathology and immunohistochemical staining confirmed a densely–granulated corticotroph adenoma (Fig. 2, NanoZoomer S360 digital slide scanner and NDP.view 2.9.25 software, Hamamatsu, Japan). Neither necrosis nor mitotic activity was observed. The immunostaining for somatostatin receptor SSTR2A was positive with a cytoplasmic pattern, while GH, PRL, TSH, FSH, LH and PIT were all negative. The Ki 67 index was found to be 10%. One month after the operation the ACTH level increased to 132 pg/mL again, and the parents agreed to refer their child for radiotherapy to control the residual tumor. Fig. 2 Histopathology and immunohistochemistry staining results of the pituitary tumor. By light microscopy, the tumor cells were mostly basophilic and arranged in papillary architecture. Neither necrosis nor mitotic activity was observed (A hematoxylin-eosin, ×200). Immunohistochemistry staining was positive for ACTH (B immunoperoxidase, ×200) and transcription factor T-PIT (C immunoperoxidase, ×200). Cytoplasmic staining of SSTR2A was observed in around 1/3 tumor cells besides the strong staining of endothelial cells (D immunoperoxidase, ×200). The Ki-67 index was 10% (E immunoperoxidase, ×200). Cytokeratin CAM5.2 was diffusely positive in the cytoplasm (F immunoperoxidase, ×200). The positive control for ACTH and T-PIT was the human anterior pituitary gland, and for SSRT2, Ki-67 and CAM5.2 were cerebral cortex, tonsil and colonic mucosa, respectively Full size image The early onset and invasive behavior of this tumor led to the consideration of whether there was a genetic defect. Genetic studies were recommended for the families and they all agreed and signed the written informed consent forms. Whole exome sequencing (WES) was performed on the patient’s blood sample using an Illumina HiSeq sequencer to an average read depth of at least 90 times per individual. Raw sequence files were mapped to the GRCH37 human reference genome and analyzed using the Sentieon software. The results revealed a germline heterozygous GPR101 gene mutation c.505G > C (p.Gly169Arg), which was subsequently confirmed to be of maternal origin by Sanger sequencing. Meanwhile WES of the tumor tissue identified an additional somatic heterozygous c.2155_2157delTCC (p.S719del) mutation of the USP8 gene . Discussion and conclusions In this report, we described an extremely giant and invasive pituitary ACTH adenoma in a 10-year-old girl. According to Trouillas et al., invasive and proliferative pituitary tumors have a poor prognosis [5]. CD is rare among children, and the fast-growing and invasive nature of the tumor in this case led to the investigation of genetic causes. The somatic USP8 gene mutation has been recently reported to be associated with the pathogenesis of CD [6, 7]. This gene encodes ubiquitin-specific protease 8 (USP8). S718, S719 and P720 are hotspots in different studies [6,7,8,9,10,11,12,13,14]. They are located at the 14-3-3 binding motif, and the mutations disrupt the binding between USP8 and 14-3-3 protein, which leads to increased deubiquitination and EGFR signaling. High levels of EGFR consequently trigger proopiomelanocortin (POMC) transcription and ACTH secretion [6, 7]. The p.S719del mutation has been previously reported and its pathogenicity has been confirmed [7]. Thus, we speculate the p.S719del mutation plays a role in this patient with CD. It is noteworthy that in our case, the pituitary corticotrophin adenoma was extremely giant and bilaterally invasive. USP8 mutations have been found in 31% of pediatric CD patients [10]. It is well known that microadenomas are most common in adult and pediatric CD patients. Previously, the Chinese and Japanese cohorts observed smaller sizes of USP8-mutated PAs than wild-type PAs [7, 9]. The Chinese cohort also reported a lower rate of invasive adenomas in USP8-mutated PAs [7]. This may be explained by the finding that UPS8 mutations did not significantly promote cell proliferation more than the wild-type ones [6]. Other cohorts suggested no difference in tumor size or invasiveness between USP8-mutated and wild-type PAs [8, 10, 12,13,14], which may be partially explained by the differences in sample sizes and ethnic backgrounds. Owing to the lack of evidence of USP8 mutations significantly contributing to tumor growth and invasiveness, additional pathogenesis should be investigated in this case. The p.Gly169Arg mutation of the GPR101 gene has not been reported in patients with pituitary tumors. In silico predictions were performed using Polyphen-2, Mutation Taster and PROVEAN, and all of the programs reported it to be pathogenic. The GPR101 gene encodes an orphan G protein-coupled receptor (GPCR) and microduplication encompassing the gene has been proven to be the cause of X-linked acrogigantism (XLAG) [15]. XLAG is characterized by the early onset of pituitary GH-secreting macroadenomas. Point mutations of GPR101 have been found in patients with PAs that are mostly GH-secreting [15,16,17]. Although their prevalence is very low, an in vitro study supported the pathogenic role of p.E308D, the most common mutation of GPR101. This led to increased cell proliferation and GH production in rat pituitary GH3 cells [15]. Rare cases of PRL, ACTH or TSH-secreting PAs with GPR101 variants were also documented [16, 18]. To date, there have been five cases of ACTH-secreting PAs with four different germline GPR101 mutations: two cases of p.E308D, p.I122T, p.T293I and p.G31S, although in silico predictions and in vitro evaluations using AtT-20 cells have respectively determined the latter two mutations to be non-pathogenic [16, 18]. These patients were mainly children and young adults. Unlike pituitary GH-secreting tumors, the role of GPR101 mutations in the pathophysiology of CD is still questionable. Trivellin et al. demonstrated no statistically significant difference in GPR101 expression between corticotropinomas and normal human pituitaries. No significant correlation between GPR101 and POMC expression levels was found neither [18]. Given the evidences above, we hypothesize that the somatic USP8 mutation is responsible for the overexpression of ACTH in this CD girl while the germline GPR101 mutation contributes to the early onset and fast-growing nature of the tumor. Similarly, a 27-year-old woman with Nelson’s syndrome originally considered to be associated with a germline AIP variant (p.Arg304Gln) was recently reported to have a somatic USP8 mutation. The patient progressed rapidly and underwent multiple transsphenoidal surgeries [19]. Since germline AIP mutations are more commonly seen in GH-secreting PAs [20], the authors proposed that the USP8 mutation might have shifted the tumor towards ACTH-secreting [19]. Further investigations into the pathogenicity of GPR101 p.Gly169Arg and AIP p.Arg304Gln mutations are required to support the hypothesis. In summary, we report a novel germline GPR101 and somatic USP8 mutation in a girl with an extremely giant pituitary ACTH adenoma. The concurrent mutations may lead to the growth and function of the tumor, respectively. Further investigations should be carried out to verify the role of the concurrent mutations in the pathogenesis of pediatric CD. Availability of data and materials The WES data of the blood sample of the patient is available in the NGDC repository (https://ngdc.cncb.ac.cn/gsa-human/) and the accession number is HRA002396. Any additional information is available from the authors upon reasonable request. Abbreviations CD: Cushing’s disease ACTH: adrenocorticotropic hormone PA: pituitary adenoma NRS: numerical rating scale 24hUFC: 24-hour urine free cortisol LDDST: low-dose dexamethasone suppression test USP8: ubiquitin-specific protease 8 POMC: proopiomelanocortin GPCR: G protein-coupled receptor XLAG: X-linked acrogigantism References Weber A, Trainer PJ, Grossman AB, Afshar F, Medbak S, Perry LA, et al. Investigation, management and therapeutic outcome in 12 cases of childhood and adolescent Cushing’s syndrome. Clin Endocrinol (Oxf). 1995;43(1):19–28. CAS Article Google Scholar Storr HL, Alexandraki KI, Martin L, Isidori AM, Kaltsas GA, Monson JP, et al. Comparisons in the epidemiology, diagnostic features and cure rate by transsphenoidal surgery between paediatric and adult-onset Cushing’s disease. Eur J Endocrinol. 2011;164(5):667–74. CAS Article Google Scholar Magiakou MA, Mastorakos G, Oldfield EH, Gomez MT, Doppman JL, Cutler GB Jr, et al. Cushing’s syndrome in children and adolescents. Presentation, diagnosis, and therapy. N Engl J Med. 1994;331(10):629–36. CAS Article Google Scholar Hernández-Ramírez LC, Stratakis CA. Genetics of Cushing’s Syndrome. Endocrinol Metab Clin North Am. 2018;47(2):275–97. Article Google Scholar Trouillas J, Roy P, Sturm N, Dantony E, Cortet-Rudelli C, Viennet G, et al. A new prognostic clinicopathological classification of pituitary adenomas: a multicentric case-control study of 410 patients with 8 years post-operative follow-up. Acta Neuropathol. 2013;126(1):123–35. Article Google Scholar Reincke M, Sbiera S, Hayakawa A, Theodoropoulou M, Osswald A, Beuschlein F, et al. Mutations in the deubiquitinase gene USP8 cause Cushing’s disease. Nat Genet. 2015;47(1):31–8. CAS Article Google Scholar Ma Z-Y, Song Z-J, Chen J-H, Wang Y-F, Li S-Q, Zhou L-F, et al. Recurrent gain-of-function USP8 mutations in Cushing’s disease. Cell Res. 2015;25(3):306–17. CAS Article Google Scholar Perez-Rivas LG, Theodoropoulou M, Ferrau F, Nusser C, Kawaguchi K, Stratakis CA, et al. The Gene of the Ubiquitin-Specific Protease 8 Is Frequently Mutated in Adenomas Causing Cushing’s Disease. J Clin Endocrinol Metab. 2015;100(7):E997–1004. CAS Article Google Scholar Hayashi K, Inoshita N, Kawaguchi K, Ibrahim Ardisasmita A, Suzuki H, Fukuhara N, et al. The USP8 mutational status may predict drug susceptibility in corticotroph adenomas of Cushing’s disease. Eur J Endocrinol. 2016;174(2):213–26. CAS Article Google Scholar Faucz FR, Tirosh A, Tatsi C, Berthon A, Hernandez-Ramirez LC, Settas N, et al. Somatic USP8 Gene Mutations Are a Common Cause of Pediatric Cushing Disease. J Clin Endocrinol Metab. 2017;102(8):2836–43. Article Google Scholar Albani A, Perez-Rivas LG, Dimopoulou C, Zopp S, Colon-Bolea P, Roeber S, et al. The USP8 mutational status may predict long-term remission in patients with Cushing’s disease. Clin Endocrinol (Oxf). 2018;89:454–8. CAS Article Google Scholar Ballmann C, Thiel A, Korah HE, Reis AC, Saeger W, Stepanow S, et al. USP8 Mutations in Pituitary Cushing Adenomas-Targeted Analysis by Next-Generation Sequencing. J Endocr Soc. 2018;2(3):266–78. CAS Article Google Scholar Losa M, Mortini P, Pagnano A, Detomas M, Cassarino MF, Pecori Giraldi F. Clinical characteristics and surgical outcome in USP8-mutated human adrenocorticotropic hormone-secreting pituitary adenomas. Endocrine. 2019;63(2):240–6. CAS Article Google Scholar Weigand I, Knobloch L, Flitsch J, Saeger W, Monoranu CM, Hofner K, et al. Impact of USP8 Gene Mutations on Protein Deregulation in Cushing Disease. J Clin Endocrinol Metab. 2019;104(7):2535–46. Article Google Scholar Trivellin G, Daly AF, Faucz FR, Yuan B, Rostomyan L, Larco DO, et al. Gigantism and acromegaly due to Xq26 microduplications and GPR101 mutation. N Engl J Med. 2014;371(25):2363–74. CAS Article Google Scholar Lecoq AL, Bouligand J, Hage M, Cazabat L, Salenave S, Linglart A, et al. Very low frequency of germline GPR101 genetic variation and no biallelic defects with AIP in a large cohort of patients with sporadic pituitary adenomas. Eur J Endocrinol. 2016;174(4):523–30. CAS Article Google Scholar Iacovazzo D, Caswell R, Bunce B, Jose S, Yuan B, Hernández-Ramírez LC, et al. Germline or somatic GPR101 duplication leads to X-linked acrogigantism: a clinico-pathological and genetic study. Acta Neuropathol Commun. 2016;4(1):56. Article Google Scholar Trivellin G, Correa RR, Batsis M, Faucz FR, Chittiboina P, Bjelobaba I, et al. Screening for GPR101 defects in pediatric pituitary corticotropinomas. Endocr Relat Cancer. 2016;23(5):357–65. CAS Article Google Scholar Perez-Rivas LG, Theodoropoulou M, Puar TH, Fazel J, Stieg MR, Ferrau F, et al. Somatic USP8 mutations are frequent events in corticotroph tumor progression causing Nelson’s tumor. Eur J Endocrinol. 2018;178(1):57–63. CAS Article Google Scholar Tatsi C, Stratakis CA. The Genetics of Pituitary Adenomas. J Clin Med. 2019;9(1). Download references Acknowledgements We thanked Dr. Xiaohua Shi and Dr. Yu Xiao from the Department of Pathology, Peking Union Medical College Hospital for their expertise in pituitary pathology and critical help in accomplishment of our manuscript. Funding This research was supported by “The National Key Research and Development Program of China” (No. 2016YFC0901501), “CAMS Innovation Fund for Medical Science” (CAMS-2017-I2M–1–011). They mainly covered the fees for genetic analysis and publications. Author information Authors and Affiliations Department of Pediatrics, Peking Union Medical College Hospital, Chinese Academy of Medical Science and Peking Union Medical College, Beijing, 100730, China Xu-dong Bao Department of Endocrinology, Key Laboratory of Endocrinology of National Health Commission, Peking Union Medical College Hospital, Chinese Academy of Medical Science and Peking Union Medical College, Beijing, 100730, China Lin Lu, Hui-juan Zhu, Xiao Zhai, Yong Fu, Feng-ying Gong & Zhao-lin Lu Department of Neurosurgery, Peking Union Medical College Hospital, Chinese Academy of Medical Science and Peking Union Medical College, Beijing, 100730, China Yong Yao, Ming Feng & Ren-zhi Wang Contributions XB and LL contributed to the study design and manuscript writing. HZ and FG performed genetic analysis. XZ and YF collected the clinical data. YY, MF and RW provided the tumor tissue and histopathology data. ZL revised the manuscript. All authors have read and approved the final manuscript. Corresponding author Correspondence to Lin Lu. Ethics declarations Ethics approval and consent to participate This study was approved by the Ethics Committee of Peking Union Medical College Hospital. The parents of the patient provided written informed consent for research participation. Consent for publication The parents of the patient provided written informed consent for the publication of indirectly identifiable data in this research. Competing interests The authors declare that they have no competing interests. Additional information Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. 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  2. Abstract Cushing's disease causes numerous metabolic disorders, cognitive decline, and sarcopenia, leading to deterioration of the general health in older individuals. Cushing's disease can be treated with transsphenoidal surgery, but thus far, surgery has often been avoided in older patients. We herein report an older woman with Cushing's disease whose cognitive impairment and sarcopenia improved after transsphenoidal surgery. Although cognitive impairment and sarcopenia in most older patients show resistance to treatment, our case indicates that normalization of the cortisol level by transsphenoidal surgery can be effective in improving the cognitive impairment and muscle mass loss caused by Cushing's disease. References (27) 1. Lindholm J, Juul S, Jorgensen JO, et al. Incidence and late prognosis of Cushing's syndrome: a population-based study. J Clin Endocrinol Metab 86: 117-123, 2001. 2. Starkman MN. 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  3. Abstract Introduction Stress-related brain disorders can be associated with glucocorticoid disturbance and hippocampal alteration. However, it remains largely unknown how cortisol affects the structure and function of hippocampus. Cushing's disease (CD) provides a unique “hyperexpression model” to explore the effects of excessive cortisol on hippocampus as well as the relation between these effects and neuropsychological deficits. Methods We acquired high-resolution T1-weighted and resting-state functional magnetic resonance imaging in 47 CD patients and 53 healthy controls. We obtained the volume and functional connectivity of the hippocampal rostral and caudal subregions in both groups. Relationships between hippocampal alterations, neuroendocrine, and neuropsychological assessments were identified. Results Relative to control subjects, the CD patients had smaller volumes of all four hippocampal subregions. Furthermore, whole brain resting-state functional connectivity analyses with these four different hippocampal regions as seeds revealed altered hippocampal functional connectivity with high-order networks, involving the DMN, frontoparietal, and limbic networks in CD patients. The intrinsic hippocampal functional connectivity was associated with the quality of life of the CD patients. Conclusions Our findings elucidate the cumulative effect of excess cortisol on the morphology and function of hippocampus and reinforce the need for effective interventions in stress-related brain disease to halt potential hippocampal damage. 1 INTRODUCTION Converging evidence has pointed to a strong linkage between the cortisol and human brain and stress-related neuropsychiatry disorders, such as major depression disorder and posttraumatic stress disorder (de Kloet et al., 2005). However, it remains to be established how this stress hormone influences specific brain structures and functions, particularly in humans, which is of particular importance for both treatment of stress-related disorders and research on cortisol effects in the brain. Cushing's disease (CD) is caused by an adrenocorticotropic hormone pituitary adenoma and characterized by chronic hypercortisolism. This condition is therefore a unique and natural “hyperexpression model” to investigate the chronic effects of cortisol on brain physiology and cognition (Zhang et al., 2021). By applying multimodal neuroimaging techniques to CD patients, previous studies have observed that chronic hypercortisolism could cause a number of abnormalities in various brain phenotypes. Among these neural changes of CD patients, hippocampal anomalies are the most replicated findings. Studies on CD patients report hippocampal changes that converge with morphologic alterations such as reduction in volume (Burkhardt et al., 2015; Toffanin et al., 2011). Moreover, abnormal cerebral blood flow and glucose metabolism in hippocampus have also been found in CD patients. Both structural and functional alterations in the hippocampus might contribute to the psychotic symptoms in CD patients (Frimodt-Møller et al., 2019). However, it is well established that psychosis is better described as a brain connectional diaschisis rather than isolated regional dysfunctions (Matthews & Hampshire, 2016). These current hippocampus-related findings were mainly obtained by voxel-based or regional analyses of brain volume or metabolism properties, and researchers have not determined whether the organizational patterns of hippocampal functional connectivity are disrupted in CD patients. The hippocampus is easily targeted by long-term hypercortisolism because this area is a part of the stress response system and is abundant in mineralocorticoid receptors and glucocorticoid receptors (McEwen et al., 2016). Also recently, studies on macaques and humans have observed that hippocampus is an anatomically and functionally heterogeneous region along the rostral/caudal-dorsal/ventral axis (Schultz & Engelhardt, 2014). Specifically, the rostral hippocampus has connections with prefrontal regions and relates to stress, emotion, and affect. In contrast, the caudal hippocampus mainly connects to sensory cortical areas and performs primarily cognitive functions (Fanselow & Dong, 2010). Therefore, the hippocampus should be studied in a set of separate structures with rostral and caudal hippocampus. Whether the hippocampal subregions exhibit differentially altered connectivity patterns responding to chronic hypercortisolism remains largely unknown. The present study further extends this work by examining the relationship between hippocampal subregions and resting-state functional connectivity in large-scale brain networks, as measured by resting-state fMRI (rs-fMRI) (Park & Friston, 2013). We focus on default mode network (DMN), frontoparietal, and limbic networks, given their involvement in stress related psychiatric illnesses. The first is the DMN, which supports self-related cognitive functions. Complementing the DMN is the frontoparietal network, which supports the cognitive regulation of behavior and emotion. Finally, the limbic networks play a key role in emotion regulation. In this study, first, to explore the structural changes of hippocampal subregions in CD patients, we performed a volumetric MRI analysis of the four subregions (left rostral hippocampus, left caudal hippocampus, right rostral hippocampus, and right caudal hippocampus). Given the known direct neurotoxic effects of cortisol on hippocampus, we predicted that chronic hypercortisolism caused smaller hippocampal volumes in CD patients. Second, we used these four subregions as seed regions separately and mapped whole-brain functional connectivity patterns associated with each subregion to examine alterations in hippocampal functional connectivity in CD patients. Considering the psychiatric symptoms in CD patients, it is reasonable to expect the presence of altered hippocampal functional connectivity with high-order networks. 2 MATERIAL AND METHODS 2.1 Participants A total of 47 participants with a diagnosis of CD and 53 healthy control (HC) subjects were included in this study. The CD patients underwent transsphenoidal surgery at the Department of Neurosurgery, The First Medical Center of Chinese People's Liberation Army (PLA) General Hospital between May 2017 and November 2019. According to the clinical practice guideline (Nieman et al., 2015), CD was diagnosed by experienced endocrinologists and confirmed by postsurgical pathology. The detailed preoperative assessments of diagnostic criteria have been reported in our previous study. HCs were recruited from the local community and were controlled for any history of psychopathology abnormalities. All participants were right-handed and had normal vision and auditory sensation. The study was approved by the local ethics committee of the Chinese PLA General Hospital and written informed consent was obtained from each participant. The data of these 47 CD and 53 HC subjects have been partially used in our previous studies (Wang et al., 2019; Zhang et al., 2021). 2.2 Neuroendocrine and neuropsychological assessment All participants underwent biochemical evaluation to assess their cortisol level. We quantified the levels of 24-h urinary free cortisol (24hUFC, nmol/24h); serum cortisol (nmol/L) at 0:00, 8:00, and 16:00. Cortisol was detected with an ADVIA Centaur Analyzer (Siemens Healthcare Diagnostics, Tarrytown, NY, USA). Cortisol levels at 8:00 as well as 24hUFC were also measured in 51 HC subjects. All participants underwent a comprehensive neuropsychological assessment with an expert psychiatrist, including Self-Rating Depression Scale (SDS), Self-Rating Anxiety Scale (SAS), Mini-mental State Examination (MMSE), and Montreal Cognitive Assessment (MoCA). Moreover, health-related quality of life and neuropsychiatric symptoms of CD patients were evaluated with the Cushing's Quality-of-Life (CushingQoL) questionnaire (Nelson et al., 2013) and Chinese version of the neuropsychiatric inventory (CNPI) (Leung et al., 2001), respectively. 2.3 Image acquisition Structural and functional MRI data were acquired on a 3.0-Tesla MR system (Discovery MR750, General Electric) with an 8-channel head coil. High-resolution structural 3D T1-weighted images were conducted using a sagittal Fast Spoiled Gradient-Echo (FSPGR) sequence with the following parameters: repetition time = 6.7 ms, echo time = 2.9 ms, flip angle = 7°, field of view = 250 × 250 mm2, number of slices = 192, voxel size = 1 × 1 × 1 mm3 with no gap. The functional images were acquired using an echo-planar imaging (EPI) sequence with repetition time = 2000 ms, echo time = 30 ms, flip angle = 90°, thickness/gap = 3.5 mm/0.5 mm, slices = 36, field of view = 224 × 224 mm2, voxel size = 3.5 × 3.5 × 3.5 mm3, number of total volumes = 240. Soft earplugs were used to attenuate scanner noise and head motion was restrained with foam padding. During functional scanning, all participants were requested to keep their eyes closed and stay awake. 2.4 rs-fMRI data preprocessing Preprocessing of the rs-fMRI images was conducted using SPM12 and Data Processing Assistant for Resting-State fMRI (DPABI, http://www.restfmri.net/forum/DPARSF). The first 10 volume of the functional images were removed to avoid initial steady-state problems. Then functional images were spatially realigned to the first image for motion correction, and reslicing for acquisition temporal delay. The head motion of all participants in this study had no more than 2-mm translation or 2° rotation in any direction. Next, functional images were coregistered to each participant's segmented gray matter T1 image, and then spatially normalized to the MNI space, resampled to 3-mm isotropic voxels. Subsequently, the global signal, white matter signal, cerebrospinal fluid signal and 24-motion vectors were regressed from the data. Finally, linear detrending and bandpass filter (0.01−0.08 Hz) were carried out to reduce the effects of low-frequency drift and high-frequency physiological noise. 2.5 Hippocampal functional connectivity The hippocampus has been functionally parcellated into four subregions (left rostral hippocampus, left caudal hippocampus, right rostral hippocampus, and right caudal hippocampus) based on Human Brainnetome Atlas (Fan et al., 2016). On each hippocampal subregion, we performed seed-based functional connectivity analysis. Briefly, hippocampal functional connectivity maps were obtained by computing the Pearson correlation coefficient for each voxel's time course with the average time course inside the region of interest. Notably, the computation was constrained within a gray-matter mask which was generated by thresholding (a threshold of 0.2) a prior gray-matter probability map in SPM12. The resulting correlation coefficients were further converted to z scores using Fisher's r-to-z transform to improve normality. For each subject, we obtained 4 z-score maps indicative of the intrinsic functional connectivity patterns of the four hippocampal subregions. To exclude the possible confounding effect of hippocampal volume in CD patients, we performed a voxel-based morphometry analysis on structural MRI images and took the volume of hippocampal subregions as a covariate in the functional connectivity statistical analyses. 2.6 Statistical analysis All demographic and clinical variables including neuroendocrine and neuropsychological scores were compared by two-sample t-tests. Sex composition of the two groups was compared using a Pearson's chi-square test (two-tailed). To explore differences in hippocampal functional connectivity between CD patients and HCs, general linear models were performed in a voxel-wise fashion. To exclude the possible confounding effects of age, gender, education level, and volume of hippocampal subregions, we used these measures as covariates in the general linear models. Multiple comparison correction was performed using a FDR of 0.05 within the grey matter mask. In CD patients group, a linear regression analysis was further performed to explore the relationship between functional connectivity of the clusters showing significant group differences and neuropsychological scores as well as the endocrinological indicators (cortisol and 24hUFC). Multiple comparisons were also corrected using the FDR method with a corrected threshold of q < 0.05. 3 RESULTS 3.1 Demographic, endocrinological, and neuropsychological results Table 1 shows the demographic characteristics of the CD patients and the HCs. There were no significant differences in terms of age, sex distribution, and years of education between groups. Compared with HCs, CD patients had significantly lower MoCA scores and higher SDS and SAS scores (Table 1). As expected, the CD patients had significantly higher levels of serum cortisol and 24hUFC (p < .001). Moreover, we calculated the volumes of the four hippocampal subregions and found smaller volumes of all four hippocampal subregions in the CD patients. TABLE 1. Participant characteristics CDs (n = 47) HCs (n = 53) p Value Age (years) 37.38 ± 10.61 (20–59) 34.79 ± 10.72 (21–63) .113 Gender (male/female) 4/43 4/49 .859 Education (years) 11.00 ± 4.11 11.74 ± 3.10 .311 Illness duration (months) 41.62 ± 53.71 — — Neuropsychological tests MoCA 22.47 ± 3.98 (n = 45) 27.72 ± 2.00 <.001 SDS 40.18 ± 9.96 (n = 45) 27.13 ± 4.42 <.001 SAS 38.27 ± 7.90 (n = 45) 26.98 ± 4.47 <.001 CNPI 11.93 ± 9.68 (n = 45) — — Cushing QOL 37.76 ± 8.29 (n = 45) — — Endocrinological tests Serum cortisol (nmol/L) 0:00 am 633.81 ± 237.59 (n = 46) — — 8:00 am 735.34 ± 279.44 (n = 47) 358.51 ± 107.43 (n = 51) <.001 16:00 pm 671.05 ± 273.56 (n = 47) — — 24hUFC (nmol/24 h) 2381.59 ± 1653.16 (n = 41) 252.03 ± 119.47 (n = 47) <.001 Volume of hippocampal subregions (mm3) Left rostral hippocampus 343.75 ± 39.15 (257.18–423.27) 365.69 ± 27.19 (313.21–442.06) .001 Left caudal hippocampus 272.69 ± 32.74 (206.63–339.04) 296.39 ± 23.13 (249.62–347.61) <.001 Right rostral hippocampus 305.10 ± 33.71 (229.67–396.89) 336.76 ± 25.98 (274.95–415.16) <.001 Right caudal hippocampus 320.42 ± 32.60 (238.16–396.58) 347.87 ± 27.16 (294.00–415.80) <.001 Abbreviations: 24hUFC, 24-h urinary free cortisol.; CDs, Cushing's disease patients; CNPI, Chinese version of neuropsychiatric inventory; Cushing QOL, Cushing Quality of Life Scale; HCs, healthy controls; MoCA, Montreal Cognitive Assessment; SAS, Self-Rating Anxiety Scale; SDS, Self-Rating Depression Scale. Note: All values are expressed as mean ± SD. Group differences in sex between CDs and HCs were examined using chi-square test. Group differences in the other demographic and clinical characteristics between CDs and HCs were examined using two-sample t-tests (two-tailed). 3.2 Spatial distribution of hippocampal functional connectivity The hippocampal functional connectivity maps of both CD patients and HCs are presented in Figure 1. Visually, the spatial distributions of hippocampal functional connectivity were highly similar between groups, in spite of some differences in strength. We observed that the brain regions significantly positively connecting to hippocampus were primarily distributed in several limbic network regions (the orbital frontal cortex, bilateral medial temporal regions, and temporal pole) and DMN regions (bilateral medial frontal cortex, posterior cingulate gyrus/precuneus, and anterior cingulate cortex). Brain regions with negative connectivity to hippocampus were chiefly distributed in the frontoparietal network regions (dorsolateral prefrontal cortex, supramarginal gyrus, and angular gyrus). FIGURE 1 Open in figure viewerPowerPoint Between-group differences in functional connectivity of the hippocampal subregions. The first column shows the hippocampal functional connectivity subregions. The second and third columns show the hippocampal functional connectivity maps within CD and HC groups, respectively. Further between-group comparisons showed that CD patients had significantly altered hippocampal functional connectivities relative to HCs, with a corrected statistical threshold of p < .05. ROI1, left rostral hippocampus; ROI2, left caudal hippocampus; ROI3, right rostral hippocampus; ROI4, right caudal hippocampus; ROI, region of interest; CD, Cushing's disease; HC, healthy control 3.3 Altered hippocampal functional connectivity in CD patients The significant differences in functional connectivity with each hippocampal subregion between the CD patients and HCs groups are illustrated in third column of Figure 1. Both the right and left rostral hippocampus exhibited significantly decreased functional connectivity with the superior parietal lobe (SPL), a component of the frontoparietal network. Moreover, right rostral hippocampus exhibited additional increased functional connectivity with right inferior frontal gyrus, a component of DMN. For the left caudal hippocampus, significantly altered functional connectivity was found to the DMN regions, including (bilateral medial frontal cortex, angular gyrus, anterior, and posterior cingulate cortex). We also observed decreased functional connectivity between the right caudal hippocampus and anterior cingulate cortex. Additionally, the right caudal hippocampus exhibited increased functional connectivity with some limbic regions including the right orbital frontal cortex and temporal pole (Table 2). TABLE 2. Brain regions showing changed RSFC between CDs and HCs groups Peak MNI coordinate Brain regions BA Cluster size (voxels) x y z Peak T ROI-based RSFC ROI1 R IFG 48 219 57 21 —3 4.598 L angular 39 423 −27 −72 51 −5.530 RIO2 R thalamus − 114 9 −6 3 −5.905 L angular 39 195 −27 −72 54 −4.830 R angular 39 384 36 −66 48 −5.607 ROI3 R MTG 20 633 39 6 −21 4.410 L angular 39 195 −27 −72 54 −4.830 R angular 39 384 36 −66 48 −5.607 MFG/ACC 10/32 572 −3 42 −3 −4.033 PCC/PreCUN 26/23 709 12 −45 27 −4.502 ROI4 MFG/ACC 32 465 3 48 6 −4.670 R MTG/OFC 48/21 747 30 3 −21 4.208 Note: Statistical threshold was set at p < .05, corrected. Abbreviations: CDs, Cushing's disease patients; HCs, healthy controls; ROI, regions of interest; BA, Brodmann areas; MNI, Montreal Neurological Institute; RSFC, resting-state functional connectivity; SFG, superior frontal gyrus; MFG, middle frontal gyrus; dMFG, dorsal medial frontal gyrus; IPL, inferior parietal lobule; AG, angular gyrus; ROL, rolandic operculum; Ins, insular; PrCG, precentral gyrus; L, left; R, right; ROI1, left rostral hippocampus; ROI2, left caudal hippocampus; ROI3, right rostral hippocampus; ROI4, right caudal hippocampus. 3.4 Brain–behavior relationships in the CD patients In the correlation analyses of CD patients, the mean values of the functional connectivity between the left caudal hippocampus and anterior cingulate cortex correlated positively with the Cushing's QoL scores (r = .327, p < .05) (Figure 2). No other correlations were found for volumes and functional connectivity of the four hippocampal subregions with neuroendocrine and neuropsychological assessment in the CD patients. FIGURE 2 Open in figure viewerPowerPoint Significant correlations between left hippocampal functional connectivity and the quality of life in CD patients. CD, Cushing's disease; Hip, hippocampus; ACC, anterior cingulate cortex 4 DISCUSSION Using a cohort of CD patients and HCs, the present study performed a comprehensive investigation to reveal how the chronic hypercortisolism affects the morphology and connectivity of hippocampal subregions and their relationships with neuroendocrine and neuropsychological assessment. Compared with the HCs, the CD patients had smaller volumes of all four hippocampal subregions. Furthermore, CD patients exhibited differential patterns of altered hippocampal functional connectivity with high-order networks, involving the DMN, frontoparietal, and limbic networks. The intrinsic hippocampal functional connectivity was associated with the quality of life of the CD patients. Together, these findings elucidate the cumulative effect of cortisol on the morphology and function of hippocampus and provide important information to further understand the role of hippocampus in stress-related brain disease. Cortisol, the end product of the hypothalamic–pituitary–adrenal axis, plays a critical role in the body's response to stress and maintenance of homeostasis (Sapolsky et al., 2000); however, chronic hypercortisolism is known to impair neurons in the hippocampus. CD patients naturally demonstrate chronic excessive amounts of cortisol; therefore these patients serve as a natural “hyperexpression model” to investigate the chronic effects of cortisol on human hippocampus. Importantly, we showed the CD patients are associated with smaller hippocampal volumes in all four subregions. In line with our study, previous structural imaging studies have shown hippocampal volume decreases in CD patients (Frimodt-Møller et al., 2019; Toffanin et al., 2011). Furthermore, Brown et al. found that healthy volunteers were associated with a significant reduction in hippocampal volume following only 3-day stress doses of corticosteroid administration, strongly suggesting the effects of cortisol on hippocampal size. It is important to note that chronic hypercortisolism can affect the hippocampus in at least two ways: by direct neurotoxic effects on the hippocampus (Lupien et al., 2018; Uno et al., 1994) and by reduction in hippocampal neurogenesis (Saaltink & Vreugdenhil, 2014). Moreover, cortisol stimulates the release of excitatory amino acids glutamate on hippocampal cells (de Kloet et al., 2005). On the other hand, chronic elevations of cortisol also reduce neurotrophic factors that includes nerve growth factor and brain-derived neurotrophic factor (McEwen et al., 2015). The different patterns of functional connectivity in rostral hippocampus versus caudal hippocampus might be associated to the specific cytoarchitecture along the rostral/caudal hippocampus. Accumulated evidence from both animal and human studies suggests that different parts of the hippocampus display distinctive gene expression and anatomical projections patterns (Fanselow & Dong, 2010). In detail, gene expression in the rostral hippocampus correlates with regions involved in emotion and stress (amygdala and hypothalamus). Moreover, the rostral hippocampus has connections with prefrontal regions, exerts strong regulatory control of the hypothalamic–pituitary–adrenal axis with a negative feedback (Toffanin et al., 2011). Accordingly, as demonstrated in this study, chronic hypercortisolism predominantly disrupted the functional connectivity in rostral hippocampus. Another major finding in this study was altered hippocampal functional connectivity with DMN, frontoparietal, and limbic networks in CD individuals relative to that in HCs. Emerging evidence proposes that interactions within and between these large-scale brain networks play important roles on brain functions and may be affected in multiple psychiatric disorders (Menon, 2011; Sha et al., 2019). Among these brain networks, the DMN is anchored in the medial prefrontal cortex and posterior cingulate cortex and is implicated in internally directed attention and self-referential processing (Raichle, 2015), while the frontoparietal and limbic networks support the cognitive regulation of emotion, attention, and behavior (Buhle et al., 2014; Kohn et al., 2014). The engagement of these high-level functional networks may suggest the linkage of abnormal stress hormone cortisol to cognitive deficits in CD patients. In line with our study, previous studies have shown stress-induced cortisol increase was associated with altered connectivity within the major brain networks (Zhang et al., 2019, 2020, 2020). Meanwhile, structural and functional alterations in these brain systems are also found in CD patients. For example, many functional imaging studies have consistently demonstrated altered brain activities and functional connectivity involving in DMN, frontoparietal, and limbic networks (Jiang et al., 2017; Wang et al., 2019; Zhang et al., 2021), even in the patients with long-term remission of CD (van der Werff et al., 2015). Importantly, previous studies have shown that the CD patients had widespread reductions of white matter integrity, which provide further evidence for the structural substrate for the persistence of these functional deficits (Pires et al., 2015; van der Werff et al., 2014). Here, we propose that by altering hippocampal processes via the abundant glucocorticoid and mineralocorticoid receptors, exposure to hypercortisolism disrupts the interactions with DMN, frontoparietal, and limbic networks in CD patients, thus engender vulnerability for emotional and cognitive problems. In line with this view is evidence that altered hippocampal functional connectivity is associated with the quality of life in CD patients. Because impaired quality of life is a persistent complaint from CD patients (Webb et al., 2018), it is important to accurately assess which aspects of QoL are affected in order to better understand the severity of hypercortisolism on patients and the potential efficacy of treatment. CushingQoL questionnaire has proven to be a valuable resource for assessing health-related QoL in CD patients, based on the combination of psychosocial issues and physical problems (Nelson et al., 2013). A better understanding of the neuroplasticity and continuing quality of life change may in turn facilitate advances in management and intervention. Several issues need to be addressed further. First, although the sample size of this study was relatively large, the findings still need to be further replicated in an independent sample. Second, the cross-sectional, observational nature of our study design precludes any causal conclusions. Therefore, studies tracking dynamic changes in hippocampal functional connectivity following the remission of hypercortisolism are needed. We are currently following up participants as part of a longitudinal study. Finally, a combined analysis of multimodal imaging including structural and metabolic data would provide integrated information on the effect of cortisol excess on human brain. In short, we demonstrate that CD patients present atypical morphology and functional connectivity of hippocampus. Here we observed the chronic hypercortisolism caused smaller volumes of all hippocampal subregions. This volume change was in line with the preclinical research that excess cortisol cause dendritic shrinkage and loss of spines in the hippocampus. Functionally, CD patients demonstrated altered hippocampal connectivity whose nodes include key components of the DMN, frontoparietal, and limbic networks. These multimodal results reinforce the need for effective therapeutic interventions in stress-related brain disease to halt possible hippocampal damage. ACKNOWLEDGMENTS This study was supported by the National Natural Science Foundation of China (No. 82001798 and No. 81871087), Military Young Scholar Medical Research Fund of Chinese PLA General Hospital (No. QNF19071), and Medical Big Data and Artificial Intelligence Development Fund of Chinese PLA general Hospital (No. 2019MBD-039). CONFLICT OF INTEREST The authors report no biomedical financial interests or potential conflicts of interest. Read more, including references, at https://onlinelibrary.wiley.com/doi/10.1002/brb3.2507
  4. Published: May 15, 2022 (see history) DOI: 10.7759/cureus.25017 Cite this article as: Fernandez C, Bhatia S, Rucker A, et al. (May 15, 2022) Intermittent Blurry Vision: An Unexpected Presentation of Cushing’s Syndrome Due to Primary Bilateral Macronodular Adrenal Hyperplasia (PBMAH). Cureus 14(5): e25017. doi:10.7759/cureus.25017 Abstract Cushing’s syndrome (CS) is an uncommon endocrine disorder resulting from prolonged exposure to elevated glucocorticoids, with 10-15 million annual cases per the American Association of Neurological Surgeons. Exogenous and endogenous causes can further be divided into adrenocorticotropic hormone (ACTH) dependent (i.e Cushing’s Disease) or ACTH independent. ACTH-independent CS can be caused by primary bilateral macronodular adrenal hyperplasia (PBMAH) representing less than 1% cases of CS. We report a case of a woman presenting with chronic resistant hypertension, episodic blurry vision, weight gain and wasting of extremities. She was diagnosed with Cushing’s syndrome due to PBMAH. Our patient’s presentation was unusual as she presented at 40 years old, 10 years earlier than expected for PBMAH; and primarily with complaints of episodic blurry vision. Her symptoms also progressed rapidly as signs and symptoms largely presented over the course of 12 months, however responded well to surgical resection. Introduction Cushing’s syndrome (CS) is an uncommon endocrine disorder caused by prolonged exposure to elevated glucocorticoids [1]. There are exogenous or endogenous causes. The National Institute of Health’s (NIH) Genetic and Rare Diseases Information Center (GARD) estimated the prevalence of endogenous CS to be 1 in 26,000 [2]. According to a large study, the annual incidence of CS in individuals less than 65 years old was nearly 49 cases per million [3]. Cushing’s disease (CD), which is defined as Cushing’s syndrome caused by an adrenocorticotropic hormone (ACTH)-secreting pituitary tumor, accounts for approximately 80% of patients with CS; whereas ACTH-independent CS accounts for the remaining 20% [4]. Among the causes of pituitary ACTH-independent CS is bilateral macronodular adrenal hyperplasia which is rare, comprising less than 1% of patients with CS [5]. Herein is a case of rapid onset Cushing’s syndrome due to PBMAH initially presenting as episodes of bilateral blurry vision. Case Presentation The patient is a 40-year-old female with a past medical history of resistant hypertension (on four agents), and recently diagnosed type 2 diabetes mellitus (started on insulin regimen). Patient was recently seen by her primary care provider, with complaints of intermittent episodes of blurry vision going on for months. As part of evaluation in December 2020, the patient underwent a renal ultrasound as part of evaluation by the primary physician for uncontrolled hypertension. The doppler incidentally showed an indeterminate hypoechoic mass on the right kidney and presumably located within the right adrenal gland, measuring 3.4 x 5.4 cm, without sonographic evidence of renal artery stenosis. The left kidney appeared normal. She was recommended to have further evaluation with contrast enhanced MR or CT with adrenal protocol. In January 2021, the patient was sent from her PCP’s office to the ED as the patient was having blurred vision. She had a plain CT scan of the brain that was unremarkable. The patient's systolic blood pressure was in the 160s-170s mm Hg upon arrival to ED compliance with home medications of 5mg of amlodipine daily, 25mg of metoprolol succinate daily, 100mg of losartan daily, and 25mg of hydrochlorothiazide daily. Physical exam reported obesity without evidence of abdominal striae. Blood work in the ED showed elevated blood glucose level over 600 (mg/dL) despite being on a regimen of lantus 60 units, metformin 1000mg twice a day, and semaglutide SQ weekly. Hemoglobin A1c was greater than 15.5%, and vitamin D was low (15.6 ng/mL). The morning ACTH was low (<5pg/mL) (nAM levels: 7.2 - 63.3 pg/mL), AM cortisol was high at 26.1 ug/ml (normal: 5.0 - 23.0 ug/mL), plasma aldosterone was normal at 4.2 ng/dL with a normal plasma renin at 1.96 (0.25 - 5.82 ng/mL/h). 24-hour urine free cortisol (UFC) was high at 1299.5 (4.0-50.0 mcg/24h). CT of the abdomen/pelvis with and without contrast showed low-attenuation masses (less than 5 Hounsfield units) present in both adrenal glands measuring 6.9 x 5.3 cm on the right and 4.5 x 3.9 cm on the left, and did not demonstrate significant arterial enhancement (Figure 1). MR imaging of the abdomen without and with contrast was also obtained and showed the same masses of the bilateral adrenal glands, with largest on the left measured 3.6 cm and largest on the right measured 3.7 cm, as well as mild fatty infiltration of the liver. General surgery and hematology/oncology were consulted and recommendations were made for outpatient follow-up with PCP and endocrinology. Figure 1: CT of the abdomen/pelvis with contrast showing low-attenuation masses present in both adrenal glands measuring 6.9 x 5.3 cm on the right (dark gray arrow) and 4.5 x 3.9 cm on the left (light gray arrow) In early February 2021, the patient again presented to the ED complaining of recurrent episodes of bilateral blurry vision. Examination was unremarkable, including an ophthalmological exam with slit lamp exam. Blurred vision was suspected to be due to osmotic swelling in the setting of severe hyperglycemia as the patient had persistently uncontrolled blood sugars. Recommendations were for tighter control of blood glucose, and follow-up with primary care and ophthalmology. Patient followed up with the endocrinologist in mid-February to which the patient reported first noticing a difference in her energy and changes to her weight around one year prior. She communicated a weight gain of 30 to 40 lbs over the past year. Patient had a reported history of gestational hypertension diagnosed five years ago when she gave birth to her daughter, which was steadily worsening over the past year. She reported intermittent myalgias and easy bruising. Patient had no family history or any apparent features to suggest multiple endocrine neoplasia (MEN) syndrome. Blood work revealed ACTH less than 1.5 pg/mL, AM cortisol was high at 24.5 mcg/dL, and normal aldosterone at 3.6 ng/dL, with normal renin and metanephrine levels. Physical examination revealed truncal obesity as well as a round face, cushingoid in appearance, and relatively thin extremities and abdominal striae. She was then referred to a surgical specialist, and it was decided that she would undergo laparoscopic bilateral adrenalectomy due to severe Cushing’s syndrome. The surgical pathology report revealed macro-nodular cortical hyperplasia of both left and right adrenal gland masses with random endocrine atypia. The largest nodule on the left measured 4.5 cm and the largest nodule on the right measured 6.6 cm. Post-operatively she was started on hydrocortisone 20 mg every morning and 10 mg every evening, and fludrocortisone 0.1 mg twice a day as part of her steroid replacement regimen. Eventually she changed to hydrocortisone 10 mg three times a day and fludrocortisone 0.1 mg once a day. For her diabetes, her insulin glargine decreased from 60 units to 20 units. Amlodipine and hydrochlorothiazide were discontinued from her antihypertensive medications; she continued losartan and metoprolol. Follow up blood work showed stable electrolytes with potassium 4.2 mmol/L (3.5-5.2 mmol/L), sodium 137 mmol/L (134-144mmol/L), chloride 100 mmol/L (96-106 mmol/L), and carbon dioxide 23 mmol/L (20-29mmol/L). Discussion ACTH-independent Cushing’s syndrome due to bilateral cortisol-secreting nodules is rare, accounting for 2% of CS cases. The majority of causes include primary bilateral macronodular adrenal hyperplasia (PBMAH), primary pigmented nodular adrenocortical disease (PPNAD), and bilateral adrenocortical adenomas (BAA). In PBMAH, typically patients are diagnosed within the fifth or sixth decade of life [4]. The usual age of onset for PPNAD is within the first to third decade of life, with median age in the pediatric population at age 15 years [6]. BAA is such a rare entity that there exists little epidemiological data with less than 40 reported cases until 2019 [7]. A small subset of patients present with overt clinical symptoms of CS, as hypercortisolism often follows an insidious course that can delay diagnosis from years to decades, with one series reporting a diagnostic delay of approximately eight years [8]. Serum and urine hormone screening in the right clinical setting can provide clues to these endocrine disorders, however diagnosis of ACTH-independent CS often occurs incidentally wherein a radiographic study was done for reasons other than to identify adrenal disease [9]. CT or MRI alone are not able to differentiate these disease entities, requiring pathological examination for final determination [7]. Adrenal venous sampling (AVS) and I-6B-iodomethyl-19-norcholesterol (I-NP-59) can aid in identifying hormone-secreting status of each adrenal lesion, however usefulness is debated among experts [10-12]. In all cases the end goal is to normalize adrenocortical hormones, and PBMAH primarily involves surgical resection with exogenous hormone replacement. Bilateral adrenalectomy is generally the treatment of choice with overt Cushing syndrome regardless of cortisol level. These patients require lifelong steroid administration [9,13]. Another approach is unilateral adrenalectomy of the larger or more metabolically active gland, which can be identified after AVS or I-NP-59 testing. This has been proposed in order to preserve some autonomous hormonal production and prevent adrenal crisis, however remission rates of Cushing syndrome as high as 84% have been reported with eventual need for bilateral adrenalectomy [7,8,14]. Steroid enzyme inhibition to control cortisol secretion has been used as an adjunct before surgery. In some patients with identified aberrant adrenal hormone receptors, targeted pharmacological inhibition remains an alternative medical approach [8]. Despite these alternatives to surgery, surgical resection remains the optimal approach [1]. Conclusions ACTH-independent Cushing’s syndrome due to PBMAH usually presents as an indolent course, with typical diagnosis in the fifth to sixth decade. As the use of imaging for other non-endocrine related investigations becomes more utilized, PBMAH being less of a rare entity. Clinical presentation usually dictates the timing of and type of surgical intervention. Although there are some reports of unilateral resection resulting in a cure, many of these cases eventually proceed to staged bilateral resection. Our patient’s presentation as her primary complaint was recurrent episodes of blurry vision that were suspected to be due to osmotic swelling because of her uncontrolled hyperglycemia. Her case was also unusual as she presented at 40 years old, an average of 10 years earlier than is typically diagnosed for PBMAH. Her symptoms also progressed rapidly over the course of 12 months with development of resistant hypertension and insulin-dependent diabetes requiring high basal insulin. Following surgical resection, her antihypertensive regimen was de-escalated and had significant reduction in insulin requirements, and was maintained on adrenocorticoid therapy. References Nieman LK: Recent updates on the diagnosis and management of Cushing's syndrome. Endocrinol Metab (Seoul). 2018, 33:139-46. 10.3803/EnM.2018.33.2.139 Rare Disease Database: Cushing Syndrome. (2021). Accessed: 12/17/2021: https://rarediseases.org/rare-diseases/cushing-syndrome/. Broder MS, Neary MP, Chang E, Cherepanov D, Ludlam WH: Incidence of Cushing's syndrome and Cushing's disease in commercially-insured patients <65 years old in the United States. Pituitary. 2015, 18:283-9. 10.1007/s11102-014-0569-6 Lacroix A, Feelders RA, Stratakis CA, Nieman LK: Cushing’s syndrome. Lancet. 2015, 386:913-27. 10.1016/S0140-6736(14)61375-1 Tokumoto M, Onoda N, Tauchi Y, et al.: A case of adrenocoricotrophic hormone -independent bilateral adrenocortical macronodular hyperplasia concomitant with primary aldosteronism. BMC Surg. 2017, 17:97. 10.1186/s12893-017-0293-z Stratakis CA: Cushing syndrome caused by adrenocortical tumors and hyperplasias (corticotropin- independent Cushing syndrome). Endocr Dev. 2008, 13:117-32. 10.1159/000134829 Gu YL, Gu WJ, Dou JT, et al.: Bilateral adrenocortical adenomas causing adrenocorticotropic hormone-independent Cushing's syndrome: a case report and review of the literature. World J Clin Cases. 2019, 7:961-71. 10.12998/wjcc.v7.i8.961 Lacroix A: ACTH-independent macronodular adrenal hyperplasia. Best Pract Res Clin Endocrinol Metab. 2009, 23:245-59. 10.1016/j.beem.2008.10.011 Sweeney AT, Srivoleti P, Blake MA: Management of the patient with incidental bilateral adrenal nodules. J Clin Transl Endocrinol Case Rep. 2021, 20:100082. 10.1016/j.jecr.2021.100082 Lumachi F, Zucchetta P, Marzola MC, Bui F, Casarrubea G, Angelini F, Favia G: Usefulness of CT scan, MRI and radiocholesterol scintigraphy for adrenal imaging in Cushing's syndrome. Nucl Med Commun. 2002, 23:469-73. 10.1097/00006231-200205000-00007 Builes-Montaño CE, Villa-Franco CA, Román-Gonzalez A, Velez-Hoyos A, Echeverri-Isaza S: Adrenal venous sampling in a patient with adrenal Cushing syndrome. Colomb Med (Cali). 2015, 46:84-7. Guo YW, Hwu CM, Won JG, Chu CH, Lin LY: A case of adrenal Cushing's syndrome with bilateral adrenal masses. Endocrinol Diabetes Metab Case Rep. 2016, 2016:150118. 10.1530/EDM-15-0118 Wei J, Li S, Liu Q, et al.: ACTH-independent Cushing's syndrome with bilateral cortisol-secreting adrenal adenomas: a case report and review of literatures. BMC Endocr Disord. 2018, 18:22. 10.1186/s12902-018-0250-6 Osswald A, Quinkler M, Di Dalmazi G, et al.: Long-term outcome of primary bilateral macronodular adrenocortical hyperplasia after unilateral adrenalectomy. J Clin Endocrinol Metab. 2019, 104:2985-93. 10.1210/jc.2018-02204 From https://www.cureus.com/articles/90069-intermittent-blurry-vision-an-unexpected-presentation-of-cushings-syndrome-due-to-primary-bilateral-macronodular-adrenal-hyperplasia-pbmah
  5. Abstract Corticotroph cells give rise to aggressive and rare pituitary neoplasms comprising ACTH-producing adenomas resulting in Cushing disease (CD), clinically silent ACTH adenomas (SCA), Crooke cell adenomas (CCA) and ACTH-producing carcinomas (CA). The molecular pathogenesis of these tumors is still poorly understood. To better understand the genomic landscape of all the lesions of the corticotroph lineage, we sequenced the whole exome of three SCA, one CCA, four ACTH-secreting PA causing CD, one corticotrophinoma occurring in a CD patient who developed Nelson syndrome after adrenalectomy and one patient with an ACTH-producing CA. The ACTH-producing CA was the lesion with the highest number of single nucleotide variants (SNV) in genes such as USP8, TP53, AURKA, EGFR, HSD3B1 and CDKN1A. The USP8 variant was found only in the ACTH-CA and in the corticotrophinoma occurring in a patient with Nelson syndrome. In CCA, SNV in TP53, EGFR, HSD3B1 and CDKN1A SNV were present. HSD3B1 and CDKN1A SNVs were present in all three SCA, whereas in two of these tumors SNV in TP53, AURKA and EGFR were found. None of the analyzed tumors showed SNV in USP48, BRAF, BRG1 or CABLES1. The amplification of 17q12 was found in all tumors, except for the ACTH-producing carcinoma. The four clinically functioning ACTH adenomas and the ACTH-CA shared the amplification of 10q11.22 and showed more copy-number variation (CNV) gains and single-nucleotide variations than the nonfunctioning tumors. Keywords: corticotroph; Cushing disease; ACTH-secreting carcinoma; single nucleotide variation; copy number variation; exome 1. Introduction The pathological spectrum of the corticotroph includes ACTH (adrenocorticotropic hormone)-secreting pituitary adenomas (PA), causing Cushing disease (CD), silent corticotroph adenomas (SCA), Crooke cell adenomas (CCA) and the rare ACTH-secreting carcinoma (ACTH-CA). Pituitary carcinomas account for 0.1 to 0.2% of all pituitary tumors and are defined by the presence of craniospinal or distant metastasis [1,2,3]. Most pituitary carcinomas are of corticotroph or lactotrope differentiation [3]. Although a few cases present initially as CA, the majority develop over the course of several months or years from apparently benign lesions [3,4]. CCA are characterized by the presence of hyaline material in more than 50% of the cells of the lesion, and most of them arise from silent corticotroph adenomas (SCA) or CD-provoking ACTH-secreting adenomas [5]. SCA are pituitary tumors with positive immunostaining for ACTH but are not associated with clinical or biochemical evidence of cortisol excess; they are frequently invasive lesions and represent up to 19% of clinically non-functioning pituitary adenomas (NFPA) [6]. ACTH-secreting PA represents up to 6% of all pituitary tumors and causes eloquent Cushing disease (CD), which is characterized by symptoms and signs of cortisol hypersecretion, including a two- to fivefold increase in mortality [7,8]. The 2017 World Health Organization (WHO) classification of PA considers not only the hormones these tumors synthesize but also the transcription factors that determine their cell lineage [9]. TBX19 is the transcription factor responsible for the terminal differentiation of corticotrophs [9]. All tumor lesions of corticotroph differentiation are positive for both ACTH and TBX19. ACTH-secreting PA causing CD are among the best genetically characterized pituitary tumors, with USP8 somatic variants occurring in up to 25–35% of sporadic cases [9]. Yet, information regarding the molecular pathogenesis of the lesions conforming to the whole pathological spectrum of the corticotroph is scarce. The aim of the present study is to characterize the genomic landscape of pituitary tumors of corticotroph lineage. For this purpose, we performed whole exome sequencing to uncover the mutational burden (single-nucleotide variants, SNV) and copy-number variations (CNVs) of these lesions. 2. Results 2.1. Clinical and Demographic Characteristics of the Patients A total of 10 tumor samples from 10 patients were evaluated: 4 ACTH-secreting adenomas causing clinically evident CD, three non-functioning adenomas that proved to be SCA upon immunohistochemistry (IHC), one ACTH-secreting CA with a prepontine metastasis, one rapidly growing ACTH-secreting adenoma after bilateral adrenalectomy (Nelson syndrome) in a patient with CD and one non-functioning, ACTH-producing CCA (Table 1). All except one patient were female; the mean age was 38.8 ± 16.5 years (range 17–61) (Table 1). They all harbored macroadenomas with a mean maximum diameter of 31.9 ± 13 mm (range 18–51). Cavernous sinus invasion was evident on MRI in all but one of the patients (Table 1). Homonymous hemianopia was present in seven patients, whereas right optic nerve atrophy and amaurosis were evident in patient with the ACTH-CA, and in patient with CD and pituitary apoplexy (Table 1). Detailed clinical data are included in Supplementary Table S1. Death was documented in only the patient with pituitary apoplexy, and one patient was lost during follow-up, as of October 2018. Table 1. Clinical features of the tumors analyzed and SNV present in each tumor. 2.2. General Genomic Characteristics of Neoplasms of Corticotrophic Lineage Overall, approximately 18,000 variants were found, including missense, nonsense and splice-site variants as well as frameshift insertions and deletions. Of these alterations, the majority corresponded to single-nucleotide variants, followed by insertions and deletions. The three most common base changes were transitions C > T, T > C and C > G; most of the genetic changes were base transitions rather than transversions (Figure 1). There were several genes across the whole genome affected in more than one way, meaning that the same gene presented missense and nonsense variants, insertions, deletions and splice-site variants (Figure 2). Many of these variants are of unknown pathogenicity and require further investigation. Gains in genetic material were found in 44 cytogenetic regions, whereas 72 cytogenetic regions showed loss of genetic material in all corticotroph tumors. Figure 1. Panel (A) shows the gadolinium-enhanced magnetic resonance imaging of the patient with ACTH-CA, highlighting in red the metastatic lesion in the prepontine area. Panel (B) shows the hematoxylin and eosin staining displaying the hyaline structures in the perinuclear areas denoting a Crooke cell adenoma. Panel (C,D) depict a representative corticotroph tumor with positive ACTH and TBX19 immunohistochemistry, respectively. Panel (E) shows four graphics: variant classification, variant type, SNV class and transition (ti) or transversion (tv) describing the general results of exome sequencing of the corticotroph tumors. Figure 2. Representative rainfall plots showing the SNV alterations throughout the whole genome of corticotroph tumors (A) CCA, (B) SCA, (C) CD and (D) ACTH-CA, displaying all base changes, including transversions and transitions. No kataegis events were found. Alterations across the genome were seen in all corticotroph tumors. 2.3. ACTH-Secreting Carcinoma (Tumor 1) SNV missense variants were found in the genes encoding TP53 (c.215G > C [rs1042522], p.Pro72Arg); AURKA (c.91T > A [rs2273535], p.Phe31Ile); EGFR (epidermal growth factor receptor, c.1562G > A [rs2227983], p.Arg521Lys); HSD3B1 (3-ß-hydroxisteroid dehydrogenase, c.1100C > A [rs1047303], p.Thr367Asn); CDKN1A (cyclin-dependent kinase inhibitor 1A or p21, c.93C > A [rs1801270], p.Ser31Arg); and USP8 (c.2159C > G [rs672601311], p.Pro720Arg). Interestingly, the previously reported USP48, BRAF, BRG1 and CABLES1 variants in pituitary CA cases were not found in this patient’s tumor (Figure 3). All SNV detected in WES experiments were validated by Sanger sequencing. The variants described were selected due to their potential pathogenic participation in other tumors and the allelic-risk association with tumorigenesis. Hereafter, all the mentioned variants in other corticotroph tumors are referred to by these aforementioned variants. Even though these same genes presented other variants, currently the significance of those variants is unknown. Figure 3. Panel (A) shows the oncoplot from the missense variants of the selected genes and their clinical–pathological features. Panels (B–G) depict USP8, EGFR, TP53, AURKA, CDKN1A and HSD3B1 proteins, respectively, with the changes found in DNA impacting aminoacidic changes. In general, the pituitary CA presented more CNV alterations than the benign tumors, with 27 and 32 cytogenetic regions showing gains and losses of genetic material, respectively. The cytogenetic regions showing gains were 10q11.22, 15q11.2, 16p12.3, 1p13.2 and 20p, where genes SYT15, POTEB, ARL6IP1, HIPK1 and CJD6 are coded, respectively. By contrast, 8p21.2 was the cytogenetic region showing loss of genetic material. The previously reported amplification of 1p13.2 was also detected in this tumor (Figure 4) [10]. Figure 4. Hierarchical clustering of corticotroph tumors according to their gains and losses across the whole genome (somatic chromosomes only). High contrast was used to enhance potential CNV alterations; nevertheless, there were only 44 unique cytogenetic regions that showed gains in genetic material with statistical significance, whereas only 72 unique cytogenetic regions showed loss of genetic material with statistical significance. 2.4. Crooke Cell Adenoma (Tumor 2) The CCA showed SNV in the genes encoding TP53, EGFR, HSD3B1 and CDKN1A. However, neither the genes encoding AURKA and USP8 nor those encoding USP48, BRAF, BRG1 and CABLES were affected in this tumor. In CCA, only two and fifteen gains and losses were observed in copy-number variation, respectively. CNVs only showed gains in cytogenetic regions 17q12 and 10q11.22, harboring genes CCL3L1 and NPY4R, respectively, whereas losses were found in cytogenetic regions 18q21.1, 15q12 and 2q11.2, harboring genes KATNAL2, TUBGCP5 and ANKRD36. 2.5. Silent Corticotroph Adenomas (Tumors 3–5) The three SCA shared SNVs in the genes encoding HSD3B1 and CDKN1A. SCA 4 and 5 showed SNV in the genes encoding EGFR, whereas SNV in the genes encoding AURKA and TP53 were present in SCA 3 and 5. None of the SCA were found to have SNV in the genes encoding USP8, USP48, BRAF, BRG1 or CABLES1. The SCA presented only two and eighteen gains and losses (CNV), respectively. In regard to CNV, the these clinically silent tumors presented gains of genetic material in cytogenetic regions 17q22 and 10q11.22, which harbor genes encoding CCL3L1 and NPY4R. Eighteen losses were found distributed in cytogenetic regions 18q21.1, 15q12 and 2q11.2, encompassing the genes encoding KATNAL2, TUBGCP5 and ANKRD36. This CNV pattern closely resembles the one found in the CCA, which is somewhat expected if we consider that both neoplasms are clinically non-functioning 2.6. ACTH-Secreting Adenomas (Cushing Disease) (Tumors 6–9) SNV of the genes encoding TP53 and HSD3B1 were present in tumor samples from all four CD patients, whereas none of these patients harbored adenomas with SNV in the genes encoding USP8 or CDKN1A. An SNV in the gene encoding AURKA was identified in only one of these tumors (tumor 8). EGFR SNV were found in tumors 7 and 9. None of the CD-causing ACTH-secreting adenomas showed the previously reported SNV in the genes encoding USP48, BRAF, BRG1 and CABLES1. CNV analysis in this group of eloquent-area corticotroph tumors revealed 25 gains and 55 losses of genetic material. The gains occurred in cytogenetic regions 17q12, 2p12, 9p24 and 10q11.22, where genes CCL3L1, CTNNA2, FOXD4 and NPY4R are coded, respectively. The losses were localized in cytogenetic regions 21p12, 15q11.2, and 8p23, harboring genes USP16, KLF13 and DEF130A, respectively. We also detected the previously reported 20p13 amplification [10]. 2.7. ACTH-Secreting Adenoma Causing Nelson Syndrome (Tumor 10) This patient’s tumor showed SNV in the genes encoding USP8, TP53, HSD3B1 and CDKN1A but no alterations were found in the genes encoding EGFR and AURKA. This tumor and the ACTH-CA were the only two neoplasms that harbored a USP8 variant. No SNV were identified in the genes encoding USP48, BRAF, BRG1 and CABLES1. Interestingly, CNV analysis revealed the same gains and losses of genetic material found in tumors from other patients with CD. 2.8. Tumor Phylogenic Analysis We performed a phylogenetic inference analysis to unravel a hypothetical sequential step transformation from an SCA to a functioning ACTH-secreting adenoma and finally to an ACTH-CA. The theoretical evolutive development of the ACTH CA, departing from the SCA, shows two main clades, with the smallest one comprising two of the three SCA and two of the five ACTH-adenomas causing CD. Since these four tumors have the same SNV profile, we can assume that they harbor the genes that must be altered to make possible the transition from a silent to a clinically eloquent adenoma; the gene encoding ATF7IP (c.1589A > G [rs3213764], p.K529R) characterizes this clade. The second and largest clade includes the CCA, the ACTH-CA, one of the three SCA and three of the five most aggressive ACTH adenomas causing CD, including the adenoma of the patient with Nelson syndrome. This clade represents the molecular alterations required to evolve from a CD-causing ACTH-adenoma to a more aggressive tumor, or even to a CA and is characterized by the gene encoding MSH3 (c.235A > G [rs1650697], p.I79V) (Figure 5). Figure 5. Phylogenetic analysis of the corticotroph tumors. The theoretical evolutive development of the ACTH-CA, departing from the SCA shows two main clades. The first clade, characterized by ATF7IP gene, comprises 2 of the 3 SCA and 2 of the 5 ACTH-adenomas causing CD. The second clade is characterized by the gene encoding MSH3 and includes the CCA, the ACTH-CA, one of the 3 SCA and 3 of the 5 most aggressive ACTH adenomas causing CD, including the adenoma of the patient with Nelson syndrome. Red dots represent the Cushing Disease provoking adenomas, green dots represent the silent corticotroph tumors, brown dot represent the Crooke cell adenoma and the blue dot represent the corticotroph carcinoma. 2.9. Correlation between Gene Variants and Clinicopathological Features The USP8 variant positively correlated with increased tumor mass (p = 0.019). The CDKN1A variant was significantly associated with silent tumors (p = 0.036). The rest of the genetic variants did not correlate with any of the clinicopathological features tested. The presence of the EGFR variant was not distinctly associated with any of the clinical parameters and was equally present in functional as well as non-functional tumors (p = 0.392). AURKA SNV did not correlate with any of the features, including recurrence (p = 0.524). Detailed statistical results are presented in Supplementary Table S2. 3. Discussion Corticotrophs are highly specialized cells of the anterior pituitary that synthesize and secrete hormones that are essential for the maintenance of homeostasis. In this study, we sequenced the exome of 10 corticotroph tumors, including three SCA, four ACTH adenomas causing CD, an ACTH adenoma in a patient with Nelson syndrome, a CCA and an ACTH-CA in total, representing the broad pathological spectrum of this cell. Our results portray the genomic landscape of all the neoplasms that are known to affect the corticotroph. The neoplasm with the highest number of genomic abnormalities, including SNV and CNV, was the ACTH-CA, followed by the CCA and the CD tissues. Of all the genes harboring SNVs, six were found to be present in at least two of our tumor samples: HSD3B1, TP53, CDKN1A, EGFR, AURKA and USP8. The HSD3B1 gene encodes a rate-limiting enzyme required for all pathways of dihydrotestosterone synthesis and is abundantly expressed in adrenal tumors. Gain of function of this HSD3B1 variant, which has a global allelic prevalence of 0.69678 [11], results in resistance to proteasomal degradation with the consequent accumulation of the enzyme and has been associated with a poor prognosis in patients with prostate cancer [12]. Nine of the ten corticotroph tumors in our cohort harbored an SNV of the tumor suppressor gene TP53. The TP53 variant described in our cohort has been reported to be present in 80% of non-functioning pituitary adenomas and is apparently associated with a younger age at presentation and with cavernous sinus invasion [13]. Furthermore, this TP53 variant results in a reduced expression of CDKN1A and an increased expression of vascular endothelial growth factor (VEGF) as well as an increased cellular proliferation rate [13]. CDKN1A (also known as p21) is a cyclin-dependent kinase inhibitor regulating cell cycle progression. The SNV described in our study was reported to alter DNA binding ability and expression and has a global allelic frequency of 0.086945 [14]. This cyclin-dependent kinase inhibitor SNV was found to be associated with breast carcinoma [15] and lung cancer [16]. The presence of this SNV has not been previously explored in pituitary adenomas, although CDKN1A is downregulated in clinically non-functioning pituitary adenomas of gonadotrophic lineage but not in hormone-secreting tumors [17]. EGFR encodes a transmembrane tyrosine kinase receptor, activation of which leads to mitogenic signaling [18]. This gene is upregulated in several cancers and represents a target for molecular therapies [19]. The EGFR SNV described in our corticotroph tumor series was found to be associated with the response to neoadjuvant chemotherapy in patients with breast and lung cancer [18]. EGFR is normally expressed in corticotrophs, where it participates in the regulation of POMC (proopiomelanocortin) gene transcription and cellular proliferation [20]. The EGFR rs2227983 has a 0.264334 global allelic frequency [21]. AURKA is a cell-cycle regulatory serine/threonine kinase that promotes cell cycle progression by the establishment of the mitotic spindle and centrosome separation [22]. Alterations of these gene are related to centrosomal amplification, dysfunction of cytokinesis and aneuploidy [22]; it has a global allelic frequency of 0.18078 [23]. This same SNV has been associated with overall cancer risk, particularly breast, gastric, colorectal, liver and endometrial carcinomas, but it has never been formally studied in pituitary tumors [22]. Activating somatic variants of the gene encoding USP8 were recently found in 25–40% of ACTH-secreting adenomas causing CD [24,25]. Patients harboring these variants are usually younger, more frequently females and were found to have higher long-term recurrence rates in some but not all studies [26,27]. USP8 mediates the deubiquitination of EGFR by inhibiting its interaction with protein 14-3-3, which in turn prevents its proteosomal degradation. Signaling through the recycled deubiquitinated EGFR is increased, leading to increased POMC transcription and cellular proliferation. Most activating USP8 variants are located within its 14-3-3 binding motif [24,25]. Recently, USP8 and TP53 SNV were described in corticotroph tumors as drivers of aggressive lesions [28]. To our knowledge, USP8 variants have not been evaluated in patients with pituitary carcinomas, and none of the previously mentioned studies have included patients with Nelson syndrome. In our cohort, neither the CCA nor the SCA showed variants in USP8, in concordance with previously published studies [25,29], or in the genes USP48, BRAF, BRG1 and CABLES1 [9], and none of them were present in our cohort. Genetic structural variations in the human genome can be present in many forms, from SNV to large chromosomal aberrance [30]. CNV are structurally variant regions, including unbalanced deletions, duplications and amplifications of DNA segments ranging from a dozen to several hundred base pairs, in which copy-number differences have been observed between two or more genomes [31,32]. CNV are involved in the development and progression of many tumors and occur frequently in PA [30,33]. Hormone-secreting pituitary tumors show more CNV than non-functioning tumors [34]. Accordingly, our non-functioning SCA and CCA had considerably fewer chromosomal gains and losses than the CD-causing adenomas and the ACTH-CA. Expectedly, the ACTH-CA had significantly more cytogenetic abnormalities than any other tumor in our series. Interestingly, the ACTH-adenomas causing CD, the SCA and the CCA shared the gain of genetic material in 17q12, highlighting their benign nature. The 17q12 amplification has been described in gastric neoplasms [35]. The only cytogenetic abnormality shared by all types of corticotroph tumors was the gain of genetic material in 10q11.22. Amplification of 10q11.22 was previously described in Li–Fraumeni cancer predisposition syndrome [36]. The ACTH-CA, the CCA and one SCA clustered together showing a related CNV pattern; this CNV profile could be reflective of the aggressive nature of these neoplasms, since both CCA and SCA can follow a clinically aggressive course [5,6]. Our results show that all lesions conforming to the pathological spectrum of the corticotroph share some of the SNV and CNV profiles. These genomic changes are consistent with the potential existence of a continuum, whereby silent tumors can transform into a clinically eloquent tumor and finally to carcinoma, or at least a more aggressive tumor. It can also be interpreted as the common SNV shared by aggressive tumors. It is known that silent corticotroph adenomas may switch into a hormone-secreting tumor [37] and are considered a marker for aggressiveness and a risk factor for malignancy since most of the carcinomas are derived from functioning hormone-secreting adenomas. Our phylogenetic inference analysis showed that the genes ATF7IP and MSH3 could participate in a tumor transition ending in aggressive entities or even carcinomas. ATF7IP is a multifunctional nuclear protein mediating heterochromatin formation and gene regulation in several contexts [38], while MSH3 is a mismatch-repair gene [39]. Events related to heterochromatin remodeling and maintenance have been related to aggressive pituitary adenomas and carcinomas [40]. Additionally, alterations in mismatch-repair genes are related to pituitary tumor aggressiveness and resistance to pharmacologic treatment [41,42]. The variants described in ATF7IP and MSH3 are related to prostate and colorectal cancer, respectively [43,44]. There is evidence suggesting that the ATF7IP variant could be deleterious because it leads to a negative regulation of transcription [45]. Thus, these events could be biologically relevant to corticotroph tumorigenesis, although more research is needed. 4. Conclusions We have shown genomic evidence that within the tumoral spectrum of the corticotroph, functioning ACTH-secreting lesions harbor more SNV and CNV than non-functioning ACTH adenomas. The ACTH-secreting CA shows more genomic abnormalities than the other lesions, underscoring its more aggressive biological behavior. Phylogenetic inference analysis of our data reveals that silent corticotroph lesions may transform into functioning tumors, or at least potentially, into more aggressive lesions. Alterations in genes ATF7IP and MSH3, related to heterochromatin formation and mismatch repair, could be important in corticotroph tumorigenesis. The main drawback of our study is the limited sample size. We are currently increasing the number of samples to corroborate our findings and to be able to perform a more comprehensive complementary phylogenetic analysis of our data. Finally, further research is needed to uncover the roles of these variants in corticotroph tumorigenesis. 5. Materials and Methods 5.1. Patients and Tumor Tissue Samples Ten pituitary tissues were collected: one ACTH-CA, one CCA, three SCA, and five ACTH-secreting PA causing CD, including the tumor of a patient who developed Nelson syndrome after bilateral adrenalectomy. All tumors included in the study were sporadic and were collected from patients diagnosed, treated and followed at the Endocrinology Service and the Neurosurgical department of Hospital de Especialidades, Centro Médico Nacional Siglo XXI of the Instituto Mexicano del Seguro Social, Hospital General de Mexico “Dr. Eduardo Liceaga” and Instituto Nacional de Neurologia y Neurocirugia “Manuel Velazquez”. All participating patients were recruited with signed informed consent and ethical approval from the Comisión Nacional de Ética e Investigación Científica of the Instituto Mexicano del Seguro Social, in accordance with the Helsinki declaration. CD was diagnosed according to our standard protocol. Briefly, the presence of hypercortisolism was documented based on two screening tests, namely a 24 h urinary free-cortisol level above 130 µg and the lack of suppression of morning (7:00–8:00) cortisol after administration of 1 mg dexamethasone the night before (23:00) to less than 1.8 µg/dL, followed by a normal or elevated plasma ACTH to ascertain ACTH-dependence. Finally, an overnight, high-dose (8 mg) dexamethasone test, considered indicative of a pituitary source, and a cortisol suppression > 69%, provided that a pituitary adenoma was clearly present on magnetic resonance imaging (MRI) of the sellar region. In none of the 10 patients included in the study was inferior petrosal venous sampling necessary to confirm the pituitary origin of the ACTH excess. Invasiveness was defined by the presence of tumor within the cavernous sinuses (CS). DNA was extracted from paraffin-embedded tumor tissues using the QIAamp DNA FFPE tissue kit. From frozen tumors, DNA was obtained using the Proteinase K-ammonium acetate protocol. 5.2. Construction and Sequencing of Whole Exome Libraries Exome libraries were prepared according to the Agilent SureSelect XT HS Human All exon v7 instructions. Briefly, 200 ng of DNA was enzymatically fragmented with Agilent SureSelect Enzymatic Fragmentation Kit. Fragmented DNA was end-repaired and dA-tail was added at DNA ends; then, molecular barcode adaptors were added, followed by AMPure XP bead purification. The adaptor-ligated library was amplified by PCR and purified by AMPure XP beads. DNA libraries were hybridized with targeting exon probes and purified with streptavidin-coated magnetic beads. The retrieved libraries were amplified by PCR and purified by AMPure XP beads and pooled for sequencing in NextSeq 500 using Illumina flow cell High Output 300 cycles chemistry. All quality controls of the libraries were carried out using Screen tape assays and quantified by Qubit fluorometer. Quality parameters included a DNA integrity number above 8 and a 100X sequencing depth aimed with at least 85% of coverage. 5.3. Bioinformatics Analysis The fastq files were subjected to quality control using FastQC v0.11.9, the adapters were removed using Cutadapt v3.4, the alignment was carried out with Burrows–Wheeler Alignment Tool v0.7.17 with the -M option to ensure compatibility with Picard and GRCh38 as a reference genome. The marking of duplicates as well as the sorting was carried out with Picard v2.26.4 with the AddOrReplaceReadGroups programs with the option SORT_ORDER = coordinate and MarkDuplicates, respectively. Variant calling was carried out using Genomic Analysis Toolkit (GATK) v4.2.2.0 following the Best Practices guide (available at https://gatk.broadinstitute.org/) [46] and with the parameters used by Genomic Data Commons (GDC), available at https://docs.gdc.cancer.gov/ [47]. The GATK tools used were CollectSequencingArtifactMetrics, GetPileupSummaries, CalculateContamination and Mutect2. Mutect2 was run with the latest filtering recommendations, including a Panel of Normal and a Germline Reference from the GATK database. Filtering was performed with the CalculateContamination, LearnReadOrientationModel and FilterMutectCalls tools with the default parameters. For the calculation of CNV GISTIC v2.0.23 was used with the parameters used by GDC. Catalog of Somatic Mutation in Cancer (COSMIC) was used to uncover pathogenic variants. For the analysis of variants and CNV, the maftool v2.10.0 and ComplexHeatmap 2.10.0 packages were used. All analyses were carried out on the GNU/Linux operating system under Ubuntu v20.01.3 or using the R v4.0.2 language in Rstudio v2021.09.0+351. A second bioinformatics pipeline was also used, SureCall software (Agilent) with the default parameters used for SNV variant calling. The variants found by both algorithms were taken as reliable SNV. Data were deposited in Sequence Read Archive hosted by National Center for Biotechnology Information under accession number PRJNA806516. Phylogenetic tree inference (PTI) was run by means of the default parameters using matrices for each sample. These matrices contain an identifier for each variant, mutant read counts, counts of reference reads and the gene associated with the variant. The only PTI parameter was Allele Frequency of Mutation and was used to improve the speed of the algorithm. Briefly, PTI uses an iterative process on the variants shared between the samples. First, it builds the base of the tree using the variants shared by all the samples; second, it eliminates these variants and establishes a split node; and third, it eliminates the variants of the sample that produced the division (split). PTI iteratively performs these three steps for all division possibilities. Each tree is given a score based on an aggregated variant count, and the tree with the highest score is chosen as the optimal tree. 5.4. Sanger Sequencing forConfirmation of Exome Findings Exome variant findings in exome sequencing were validated by Sanger sequencing using BigDye Terminator v3.1 Cycle Sequencing kit (ThermoFischer) in a 3500 Genetic Analyzer. Primers used for USP8 [48], TP53 [49], EGFR [50], AURKA [51], CDKN1A [52,53] and HSD3B1 sequencing have been previously reported. 5.5. Hormone and Transcription Factor Immunohistochemistry Paraffin-embedded, formalin-fixed tissue blocks were stained with hematoxylin–eosin and reviewed by a pathologist. Tumors were represented with a 2-fold redundancy. Sections (3 μm) were cut and placed onto coated slides. Immunostaining was performed by means of the HiDef detection HRP polymer system (Cell Marque, CA, USA), using specific antibodies against each pituitary hormone (TSH, GH, PRL, FSH, LH and ACTH) and the lineage-specific transcription factors TBX19, POU1F1 and NR5A1, as previously described [54]. Two independent observers performed assessment of hormones and transcription factors expression at different times. 5.6. Statistical Analysis Two-tailed Fisher exact tests and Student’s t tests were used to evaluate the relationship between the identified gene variants and clinicopathological features. A p value of <0.05 was considered statistically significant. Statistical software consisted of SPSS v28.0.1 Supplementary Materials The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/ijms23094861/s1. Author Contributions D.M.-R., K.T.-P. and M.M. conceived, designed and coordinated the project, performed experiments, analyzed, discussed data and prepared the manuscript. S.A.-E., G.S.-R., E.P.-M., S.V.-P., R.S., L.B.-A., C.G.-T., J.G.-C. and J.T.A.-S. performed DNA purification, library preparation, sequencing experiments, bioinformatics analysis and wrote the manuscript. A.-L.E.-d.-l.-M., I.R.-S., E.G.-A., L.A.P.-O., G.G., S.M.-J., L.C.-M., B.L.-F. and A.B.-L. provided biological samples and detailed patient information. All authors have read and agreed to the published version of the manuscript. Funding This work was partially supported by grants 289499 from Fondos Sectoriales Consejo Nacional de Ciencia y Tecnologia, Mexico, and R-2015-785-015 from Instituto Mexicano del Seguro Social (MM). Institutional Review Board Statement Protocol approved by the Comisión Nacional de Ética e Investigación Científica of the Instituto Mexicano del Seguro Social, in accordance with the Helsinki declaration (R-2019-785-052). Informed Consent Statement Informed consent was obtained from all subjects involved in the study. Data Availability Statement Data were deposited in Sequence Read Archive hosted by National Center for Biotechnology Information under accession number PRJNA806516. Acknowledgments Sergio Andonegui-Elguera is a doctoral student from Programa de Doctorado en Ciencias Biomédicas, Universidad Nacional Autónoma de México (UNAM) and received fellowship 921084 from CONACYT. 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  6. Compared with placebo, levoketoconazole improved cortisol control and serum cholesterol levels for adults with endogenous Cushing’s syndrome, according to results from the LOGICS study presented here. Safety and efficacy of levoketoconazole (Recorlev, Xeris Biopharma) for treatment of Cushing’s syndrome were established in the pivotal phase 3, open-label SONICS study. The phase 3, double-blind LOGICS study sought to demonstrate the drug specificity of levoketoconazole in normalizing mean urinary free cortisol (mUFC) level. “Treatment with levoketoconazole benefited patients with Cushing’s syndrome of different etiologies and a wide range in UFC elevations at baseline by frequent normalization of UFC,” Ilan Shimon, MD, professor at the Sackler Faculty of Medicine at Tel Aviv University and associate dean of the Faculty of Medicine at Rabin Medical Center and director of the Institute of Endocrinology in Israel, told Healio. “This is a valuable Cushing’s study as it includes a placebo-controlled randomized withdrawal phase.” LOGICS participants were drawn from a cohort of 79 adults with Cushing’s syndrome with a baseline mUFC at least 1.5 times the upper limit of normal who participated in a single-arm, open-label titration and maintenance phase of approximately 14 to 19 weeks. Researchers randomly assigned 39 of those participants plus five from SONICS who had normalized mUFC levels on stable doses of levoketoconazole for at least 4 weeks to continue to receive the medication (n = 22) or to receive placebo with withdrawal of the medication (n = 22) for 8 weeks. At the end of the withdrawal period, all participants received levoketoconazole for 8 more weeks. Primary endpoint was proportion of participants who lost mUFC normalization during the randomized withdrawal period, and secondary endpoints included proportion with normalized mUFC and changes in total and LDL cholesterol at the end of the restoration period. During the withdrawal period, 95.5% of participants receiving placebo vs. 40.9% of those receiving levoketoconazole experienced loss of mUFC response, for a treatment difference of –54.5% (95% CI, –75.7 to –27.4; P = .0002). At the end of the withdrawal period, 4.5% of participants receiving placebo vs. 50% of those receiving levoketoconazole maintained normalized mUFC, for a treatment difference of 45.5% (95% CI, 19.2-67.9; P = .0015). Among participants who had received placebo and lost mUFC response, 60% regained normalized mUFC at the end of the restoration period. During the withdrawal period, participants in the placebo group had increases of 0.9 mmol/L in total cholesterol and 0.6 mmol/L in LDL cholesterol vs. decreases of 0.04 mmol/L (P = .0004) and 0.006 mmol/L (P = .0056), respectively, for the levoketoconazole group. The increases seen in the placebo group were reversed when participants restarted the medication. The most common adverse events with levoketoconazole were nausea (29%) and hypokalemia (26%). Prespecified adverse events of special interest were liver-related (10.7%), QT interval prolongation (10.7%) and adrenal insufficiency (9.5%). “This study has led to the FDA decision to approve levoketoconazole for the treatment of Cushing’s syndrome after surgical failure or if surgery is not possible,” Shimon said. From https://www.healio.com/news/endocrinology/20220512/logics-levoketoconazole-improves-cortisol-control-in-endogenous-cushings-syndrome
  7. Osilodrostat is associated with rapid normalization of mean urinary free cortisol (mUFC) excretion in patients with Cushing disease and has a favorable safety profile, according to the results of a study published in the Journal of Clinical Endocrinology & Metabolism. The phase 3 LINC-4 study (ClinicalTrials.gov Identifier: NCT02697734) evaluated the safety and efficacy of osilodrostat, a potent, orally available 11β­-hydroxylase inhibitor, compared with placebo in patients with Cushing disease. The trial, which was conducted at 40 centers in 14 countries, included a 12-week, randomized, double-blind, placebo-controlled period that was followed by a 36-week, open-label osilodrostat treatment period with an optional extension. Eligible patients were aged 18 to 75 years with a confirmed diagnosis of persistent or recurrent Cushing disease after pituitary surgery and/or irradiation or de novo disease, as well as an mUFC level greater than 1.3 times the upper limit of normal (ULN). The patients were randomly assigned 2:1 to osilodrostat 2 mg twice daily or matching placebo, stratified by prior pituitary irradiation. The primary endpoint was the proportion of patients who achieved mUFC ≤ULN at week 12. The key secondary endpoint was the proportion of patients who achieved mUFC ≤ULN at week 36. A total of 73 patients (median age, 39.0 years; 83.6% women) were randomly assigned to either osilodrostat (n=48) or placebo (n=25) and received at least 1 study drug dose from November 2016 to March 2019. The participants had a median (interquartile range [IQR]) time since diagnosis of Cushing disease of 67.4 (26.4-93.8) months. The median treatment duration in the randomized, placebo-controlled period was 12.0 weeks in both the osilodrostat group (IQR, 2.0-13.0 weeks) and the placebo group (IQR, 11.7-13.7 weeks). The proportion of patients who achieved mUFC ≤ULN (≤138 nmol/24 h) at week 12 was significantly increased in those who received osilodrostat (n=37, 77.1%) vs those who received placebo (n=2, 8.0%), with an estimated odds ratio of 43.4 (95% CI, 7.1-343.2) in favor of osilodrostat (P <.0001). A total of 59 patients (80.8%; 95% CI, 69.9-89.1) also achieved the key secondary endpoint of mUFC ≤ULN at week 36, after 24 weeks of open-label osilodrostat. The most frequently occurring adverse events in the placebo-controlled period in the osilodrostat and placebo groups, respectively, were decreased appetite (37.5% vs 16.0%), arthralgia (35.4% vs 8.0%), nausea (31.3% vs 12.0%), and fatigue (25.0% vs 16.0%). A potential study limitation is that although osilodrostat exposure was greater than 1 year among the participants, some adverse effects may take longer to be observed. “This randomized, placebo-controlled trial demonstrates that osilodrostat is a highly effective treatment for Cushing disease, normalizing UFC excretion in 77% of patients after 12 weeks’ treatment,” stated the investigators. “Cortisol reductions were maintained throughout 48 weeks of treatment and were accompanied by improvements in clinical signs of hypercortisolism and quality of life. The safety profile was favorable.” Disclosure: This study was funded by Novartis Pharma AG. Some of the study authors declared affiliations with biotech, pharmaceutical, and/or device companies. Please see the original reference for a full list of disclosures. Reference Gadelha M, Bex M, Feelders RA, et al. Randomized trial of osilodrostat for the treatment of Cushing’s disease. J Clin Endocrinol Metab. Published online March 23, 2022. doi:10.1210/clinem/dgac178 From https://www.endocrinologyadvisor.com/home/topics/general-endocrinology/osilodrostat-effective-for-cushing-disease/
  8. Abstract Cushing’s syndrome (CS) secondary to ectopic adrenocorticotrophic hormone (ACTH)-producing prostate cancer is rare with less than 50 cases reported. The diagnosis can be challenging due to atypical and variable clinical presentations of this uncommon source of ectopic ACTH secretion. We report a case of Cushing’s syndrome secondary to prostate adenocarcinoma who presented with symptoms of severe hypercortisolism with recurrent hypokalaemia, limb oedema, limb weakness, and sepsis. He presented with severe hypokalaemia and metabolic alkalosis (potassium 2.5 mmol/L and bicarbonate 36 mmol/L), with elevated 8 am cortisol 1229 nmol/L. ACTH-dependent Cushing’s syndrome was diagnosed with inappropriately normal ACTH 57.4 ng/L, significantly elevated 24-hour urine free cortisol and unsuppressed cortisol after 1 mg low-dose, 2-day low-dose, and 8 mg high-dose dexamethasone suppression tests. 68Ga-DOTANOC PET/CT showed an increase in DOTANOC avidity in the prostate gland, and his prostate biopsy specimen was stained positive for ACTH and markers for neuroendocrine differentiation. He was started on ketoconazole, which was switched to IV octreotide in view of liver dysfunction from hepatic metastases. He eventually succumbed to the disease after 3 months of his diagnosis. It is imperative to recognize prostate carcinoma as a source of ectopic ACTH secretion as it is associated with poor clinical outcomes, and the diagnosis can be missed due to atypical clinical presentations. 1. Introduction Ectopic secretion of adrenocorticotropic hormone (ACTH) is responsible for approximately 10–20% of all causes of Cushing syndrome [1]. The classic sources of ectopic ACTH secretion include bronchial carcinoid tumours, small cell lung carcinoma, thymoma, medullary thyroid carcinoma (MTC), gastroenteropancreatic neuroendocrine tumours (NET), and phaeochromocytomas [2]. Ectopic adrenocorticotropic syndrome (EAS) is diagnostically challenging due to its variable clinical manifestations; however, prompt recognition and treatment is critical. Ectopic ACTH production from prostate carcinoma is rare, and there are less than 50 cases published to date. Here, we report a case of ectopic Cushing’s syndrome secondary to prostate adenocarcinoma who did not present with the typical physical features of Cushing’s syndrome, but instead with features of severe hypercortisolism such as hypokalaemia, oedema, and sepsis. 2. Case Presentation A 61-year-old male presented to our institution with recurrent hypokalaemia, lower limb weakness, and oedema. He had a history of recently diagnosed metastatic prostate adenocarcinoma, for which he was started on leuprolide and finasteride. Other medical history includes poorly controlled diabetes mellitus and hypertension of 1-year duration. He presented with hypokalaemia of 2.7 mmol/L associated with bilateral lower limb oedema and weakness, initially attributed to the intake of complementary medicine, which resolved with potassium supplementation and cessation of the complementary medicine. One month later, he was readmitted for refractory hypokalaemia of 2.5 mmol/L and progression of the lower limb weakness and oedema. On examination, his blood pressure (BP) was 121/78 mmHg, and body mass index (BMI) was 24 kg/m2. He had no Cushingoid features of rounded and plethoric facies, supraclavicular or dorsocervical fat pad, ecchymoses, and no purple striae on the abdominal examination. He had mild bilateral lower limb proximal weakness and oedema. His initial laboratory findings of severe hypokalaemia with metabolic alkalosis (potassium 2.5 mmol/L and bicarbonate 36 mmol/L), raised 24-hour urine potassium (86 mmol/L), suppressed plasma renin activity and aldosterone, central hypothyroidism, and elevated morning serum cortisol (1229 nmol/L) (Table 1) raised the suspicion for endogenous hypercortisolism. Furthermore, hormonal evaluations confirmed ACTH-dependent Cushing’s syndrome with inappropriately normal ACTH (56 ng/L) and failure of cortisol suppression after 1 mg low-dose, 2-day low-dose, and 8 mg high-dose dexamethasone suppression tests (Table 2). His 24-hour urine free cortisol (UFC) was significantly elevated at 20475 (59–413) nmol/day. Table 1 Investigations done during his 2nd admission. Table 2 Diagnostic workup for hypercortisolism. To identify the source of excessive cortisol secretion, magnetic resonance imaging (MRI) of the pituitary fossa and computed tomography (CT) of the thorax, abdomen, and pelvis were performed. Pituitary MRI was unremarkable, and CT scan showed the known prostate lesion with extensive liver, lymph nodes, and bone metastases (Figure 1). To confirm that the prostate cancer was the source of ectopic ACTH production, gallium-68 labelled somatostatin receptor positron emission tomography (PET)/CT (68Ga-DOTANOC) was done, which showed an increased DOTANOC avidity in the inferior aspect of the prostate gland (Figure 2). Immunohistochemical staining of his prostate biopsy specimen was requested, and it stained positive for ACTH and markers of neuroendocrine differentiation (synaptophysin and CD 56) (Figures 3 and 4), establishing the diagnosis of EAS secondary to prostate cancer. Figure 1 CT thorax abdomen and pelvis showing prostate cancer (blue arrow) with liver metastases (red arrow). Figure 2 Ga68-DOTANOC PET/CT demonstrating increased DOTANOC avidity seen in the inferior aspect of the right side of the prostate gland (red arrow). Figure 3 Hematoxylin and eosin staining showing acinar adenocarcinoma of the prostate featuring enlarged, pleomorphic cells infiltrating as solid nests and cords with poorly differentiated glands (Gleason score 5 + 4 = 9). Figure 4 Positive ACTH immunohistochemical staining of prostate tumour (within the circle). The patient was started on potassium chloride 3.6 g 3 times daily and spironolactone 25 mg once daily with normalisation of serum potassium. His BP was controlled with the addition of lisinopril and terazosin to spironolactone and ketoconazole, and his blood glucose was well controlled with metformin and sitagliptin. To manage the hypercortisolism, he was treated with ketoconazole 400 mg twice daily with an initial improvement of serum cortisol from 2048 nmol/L to 849 nmol/L (Figure 5). Systemic platinum and etoposide-based chemotherapy was recommended for the treatment of his prostate cancer after a multidisciplinary discussion, but it was delayed due to severe bacterial and viral infection. With the development of liver dysfunction, ketoconazole was switched to intravenous octreotide 100 mcg three times daily as metyrapone was not readily available in our country. However, the efficacy was suboptimal with marginal reduction of serum cortisol from 3580 nmol/L to 3329 nmol/L (Figure 5). The patient continued to deteriorate and was deemed to be medically unfit for chemotherapy or bilateral adrenalectomy. He was referred to palliative care services, and he eventually demised due to cancer progression within 3 months of his diagnosis. Figure 5 The trend in cortisol levels on pharmacological therapy. 3. Discussion Ectopic ACTH secretion is an uncommon cause of Cushing’s syndrome accounting for approximately 9–18% of the patients with Cushing’s syndrome [3]. Clinical presentation is highly variable depending on the aggressiveness of the underlying malignancy, but patients typically present with symptoms of severe hypercortisolism such as hypokalaemiaa, oedema, and proximal weakness which were the presenting complaints of our patient [4]. The classical symptoms of Cushing’s syndrome are frequently absent due to the rapid clinic onset resulting in diagnostic delay [5]. Prompt diagnosis and localisation of the source of ectopic ACTH secretion are crucial due to the urgent need for treatment initiation. The usual sources include small cell lung carcinoma, bronchial carcinoid, medullary thyroid carcinoma, thymic carcinoid, and pheochromocytoma. CT of the thorax, abdomen, and pelvis should be the first-line imaging modality, and its sensitivity varies with the type of tumour ranging from 77% to 85% [6]. Functional imaging such as 18-fluorodeoxyglucose-PET and gallium-68 labelled somatostatin receptor PET/CT can be useful in localising the source of occult EAS, determining the neuroendocrine nature of the tumour or staging the underlying malignancy [3, 6]. As prostate cancer is an unusual cause of EAS, we proceeded with 68Ga-DOTANOC PET/CT in our patient to localise the source of ectopic ACTH production. The goals of management in EAS include treating the hormonal excess and the underlying neoplasm as well as managing the complications secondary to hypercortisolism [3]. Prompt management of the cortisol excess is paramount as complications such as hyperglycaemia, hypertension, hypokalaemia, pulmonary embolism, sepsis, and psychosis can develop especially when UFC is more than 5 times the upper limit of normal [3]. Ideally, surgical resection is the first-line management, but this may not be feasible in metastatic, advanced, or occult diseases. Pharmacological agents are frequently required with steroidogenesis inhibitors such as ketoconazole and metyrapone, which reduce cortisol production effectively and rapidly [3, 6], the main drawback of ketoconazole being its hepatic toxicity. The efficacy of ketoconazole is reported to be 44%, metyrapone 50–75%, and ketoconazole-metyrapone combination therapy 73% [3, 7]. Mitotane, typically used in adrenocortical carcinoma, is effective in controlling cortisol excess but has a slow onset of action [3, 8]. Etomidate infusion can be used for short-term rapid control of severe symptomatic hypercortisolism and can serve as a bridge to definitive therapy [9]. Mifepristone, a glucocorticoid receptor antagonist, is indicated mainly in difficult to control hyperglycaemia secondary to hypercortisolism [8]. Somatostatin analogue has been proposed as a possible pharmacological therapy due to the expression of somatostatin receptors by ACTH secreting tumours [8, 10]. Bilateral adrenalectomy should be considered in patients with severe symptomatic hypercortisolism and life-threatening complications who cannot be optimally managed with medical therapies, especially in patients with occult EAS or metastatic disease [3, 8]. Bilateral adrenalectomy results in immediate improvement in cortisol levels and symptoms secondary to hypercortisolism [11]. However, surgical complications, morbidity, and mortality are high in patients with uncontrolled hypercortisolism [8], and our patient was deemed by his oncologist and surgeon to have too high a risk for bilateral adrenalectomy. For the treatment of prostate carcinoma, platinum and etoposide-based chemotherapies have been used, but their efficacy is limited with a median survival of 7.5 months [4, 12]. The side effects of chemotherapy can be severe with an enhanced risk of infection due to both cortisol and chemotherapy-mediated immunosuppression. Prompt control of hypercortisolism prior to chemotherapy and surgical procedure is strongly suggested to attenuate life-threatening complications such as infection, thrombosis, and bleeding with chemotherapy or surgery as well as to improve prognosis [3, 13]. There are rare reports of ectopic ACTH secretion from prostate carcinoma. These tumours were predominantly of small cell or mixed cell type, and pure adenocarcinoma with neuroendocrine differentiation are less common [4, 5]. There is a strong correlation between the prognosis and the types of malignancy in patients with EAS, and patients with prostate carcinoma have a poor prognosis [4]. These patients had metastatic disease at presentation, and the median survival was weeks to months despite medical treatment, chemotherapy, and even bilateral adrenalectomy [4], as seen with our patient who passed away within 3 months of his diagnosis. In conclusion, adenocarcinoma of the prostate is a rare cause of EAS. The diagnosis and management are complex and challenging requiring specialised expertise with multidisciplinary involvement. The presentation can be atypical, and it is imperative to suspect and recognise prostate carcinoma as a source of ectopic ACTH secretion. Prompt initiation of treatment is important, as it is a rapidly progressive and aggressive disease associated with intense hypercortisolism resulting in high rates of mortality and morbidity. Data Availability The data used to support the findings of this study are included within the article. Conflicts of Interest The authors declare that there are no conflicts of interest. 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  9. More than three-quarters of adults with Cushing’s disease assigned osilodrostat had a normalized mean urinary free cortisol level at 12 weeks and maintained a normal level at 36 weeks, according to data from the LINC 4 phase 3 trial. In findings published in The Journal of Clinical Endocrinology & Metabolism, 77% of adults with Cushing’s disease randomly assigned to osilodrostat (Isturisa, Recordati) had mean urinary free cortisol (UFC) levels reduced to below the upper limit of normal at 12 weeks compared with 8% of adults assigned to placebo. Most adults with Cushing's disease taking 2 mg twice daily osilodrostat had normalized mean UFC levels at 12 weeks compared with placebo. Data were derived from Gadelha M, et al. J Clin Endocrinol Metab. 2022;doi:10.1210/clinem/dgac178. “Osilodrostat is a highly effective treatment for Cushing’s disease, normalizing urinary free cortisol excretion in 77% of patients after 12 weeks’ treatment,” Mônica Gadelha, MD, professor of endocrinology at The Federal University of Rio de Janeiro, and colleagues wrote. “Cortisol reductions were maintained throughout 48 weeks of treatment and were accompanied by improvements in clinical signs of hypercortisolism and quality of life.” Gadelha and colleagues enrolled 73 adults aged 18 to 75 years with Cushing’s disease from 40 centers in 14 countries into the LINC 4 phase 3 trial. Participants were randomly assigned to 2 mg osilodrostat twice daily (n = 48) or placebo (n = 25) for 12 weeks. Urinary samples were collected at weeks 2, 5 and 8 to measure mean UFC, and dosage was adjusted based on efficacy and tolerability. After 12 weeks, participants from both groups received osilodrostat in a 36-week open-label treatment period. All participants restarted the open-label portion of the trial at 2 mg osilodrostat unless they were on a lower dose at week 12. Dose adjustments in the open-label phase were made using the same guidelines in the randomized, double-blind, placebo-controlled trial. The primary endpoint was the efficacy of osilodrostat at achieving a mean UFC below the upper limit of normal of 138 nmol per 24 hours at 12 weeks vs. placebo; the key secondary endpoint was the percentage of participants achieving a normal mean UFC at 36 weeks. At 12 weeks, the percentage of adults with a normalized mean UFC level was higher in the osilodrostat group compared with placebo (77.1% vs. 8%; P < .0001). At 36 weeks, 80.8% of all participants had a normal mean UFC level. The overall response rate was 79.5% at 48 weeks. Median time to first controlled mean UFC response was 35 days for those randomly assigned to osilodrostat as well as those randomly assigned to placebo who crossed over to osilodrostat for the open-label phase. At 48 weeks, 84% of participants were receiving 10 mg or less of osilodrostat per day, including 56% receiving 4 mg or less daily. At 12 weeks, the osilodrostat group had several cardiovascular and metabolic-related improvements, including systolic and diastolic blood pressure, HbA1c, HDL cholesterol, body weight and waist circumference. No changes were observed in the placebo group. “The improvements in cardiovascular and metabolic parameters were sustained throughout osilodrostat treatment and have the potential to alleviate the burden of comorbidities in many patients with Cushing’s disease,” the researchers wrote. At 12 weeks, 52.5% of those receiving osilodrostat had a reduction in supraclavicular fat pad and 50% had a reduction in dorsal fat pad. At least 25% of participants also had improvements in facial redness, striae, proximal muscle atrophy and central obesity. Improvements were sustained through week 48. During the placebo-controlled trial, grade 3 and 4 adverse events occurred for about 20% of participants in both groups. For the entire study, 38.4% of adults reported grade 3 and 4 adverse events, with the most common being hypertension. Eight participants discontinued the study due to adverse events. From https://www.healio.com/news/endocrinology/20220408/osilodrostat-normalizes-urinary-free-cortisol-in-most-adults-with-cushings-disease
  10. Highlights • Cushing syndrome (CS) is a rare disorder with a variety of underlying etiologies. • CS is expected to affect 0.2 to 5 people per million per year. • Adrenal-dependent CS is an uncommon variant of CS. • This study reports a rare occurrence of pituitary and adrenal adenoma with CS. Abstract Introduction Cushing syndrome is a rare disorder with a variety of underlying etiologies, that can be exogenous or endogenous (adrenocorticotropic hormone (ACTH)-dependent or ACTH-independent). The current study aims to report a case of ACTH-independent Cushing syndrome with adrenal adenoma and nonfunctioning pituitary adenoma. Case report A 37–year–old female presented with amenorrhea for the last year, associated with weight gain. She had a moon face, buffalo hump, and central obesity. A 24-hour urine collection for cortisol was performed, revealing elevated cortisol. Cortisol level was non-suppressed after administering dexamethasone. MRI of the pituitary revealed a pituitary microadenoma, and the CT scan of the abdomen with adrenal protocol revealed a left adrenal adenoma. Discussion Early diagnosis may be postponed due to the variety of clinical presentations and the referral of patients to different subspecialists based on their dominant symptoms (gynecological, dermatological, cardiovascular, psychiatric); it is, therefore, critical to consider the entire clinical presentation for correct diagnosis. Conclusion Due to the diversity in the presentation of CS, an accurate clinical, physical and endocrine examination is always recommended. Keywords Cushing syndrome Cushing's disease Adrenal adenoma Pituitary adenoma Urine free cortisol 1. Introduction Cushing syndrome (CS) is a collection of clinical manifestations caused by an excess of glucocorticoids [1]. CS is a rare disorder with a variety of underlying etiologies that can be exogenous due to continuous corticosteroid therapy for any underlying inflammatory illness or endogenous due to either adrenocorticotropic hormone (ACTH)-dependent or ACTH-independent [2], [3]. Cushing syndrome is expected to affect 0.2 to 5 people per million per year. Around 10% of such cases involve children [4], [5]. ACTH-dependent glucocorticoid excess owing to pituitary adenoma accounts for the majority (60–70%) of endogenous CS, with primary adrenal causes accounting for only 20–30% and ectopic ACTH-secreting tumors accounting for the remaining 5–10% [6]. Adrenal-dependent CS is an uncommon variant of CS caused mostly by benign (90%) or malignant (8%) adrenal tumors or, less frequently, bilateral micronodular (1%) or macronodular (1%) adrenal hyperplasia [7]. The current study aims to report a case of ACTH-independent Cushing syndrome with adrenal adenoma and nonfunctioning pituitary adenoma. The report has been arranged in line with SCARE guidelines and includes a brief literature review [8]. 2. Case report 2.1. Patient's information A 37–year–old female presented with amenorrhea for the last year, associated with weight gain. She denied having polyuria, polydipsia, headaches, visual changes, dizziness, dryness of the skin, cold intolerance, or constipation. She had no history of chronic disease and denied using steroids. She visited an internist, a general surgeon, and a gynecologist and was treated for hypothyroidism. She was put on Thyroxin 100 μg daily, and oral contraceptive pills were given for her menstrual problems. Last time, the patient was referred to an endocrinology clinic, and they reviewed the clinical and physical examinations. 2.2. Clinical examination She had a moon face, buffalo hump, central obesity, pink striae over her abdomen, and proximal weakness of the upper limbs. After reviewing the history and clinical examination, CS was suspected. 2.3. Diagnostic assessment Because the thyroid function test revealed low thyroid-stimulating hormone (TSH), free T3, and freeT4, the patient was sent for a magnetic resonance imaging (MRI) of the pituitary, which revealed a pituitary microadenoma (7 ∗ 6 ∗ 5) mm (Fig. 1). Since the patient was taking thyroxin and oral contraceptive pills, the investigations were postponed for another six weeks due to the contraceptive pills' influence on the results of the hormonal assessment for CS. After six weeks of no medication, a 24-hour urinary free cortisol (UFC) was performed three times, revealing elevated cortisol levels (1238, 1100, and 1248) nmol (normal range, 100–400) nmol. A dexamethasone suppression test was done (after administering dexamethasone tab 1 mg at 11 p.m., serum cortisol was measured at 9 a.m.). The morning serum cortisol level was 620 nmol (non-suppressed), which normally should be less than 50 nmol. The ACTH level was below 1 pg/mL. Download : Download high-res image (103KB) Download : Download full-size image Fig. 1. Contrast enhanced T1W weighted MRI (coronal section) showing small 7 mm hypo-enhanced microadenoma (yellow arrow) in right side of pituitary gland with mild superior bulge. Based on these findings, ACTH independent CS was suspected. The computerized tomography (CT) scan of the abdomen with adrenal protocol revealed a left adrenal adenoma (33 mm × 25 mm) without features of malignancy (Fig. 2). Download : Download high-res image (168KB) Download : Download full-size image Fig. 2. Computed tomography scan of the abdomen with IV contrast, coronal section, showing 33 mm × 25 mm lobulated enhanced left adrenal tumor (yellow arrow), showing absolute washout on dynamic adrenal CT protocol, consistent with adrenal adenoma. 2.4. Therapeutic intervention The patient was referred to the urologist clinic for left adrenalectomy after preparation for surgery and perioperative hormonal management. She underwent laparoscopic adrenalectomy and remained in the hospital for two days. The histopathology results supported the diagnosis of adrenal adenoma. 2.5. Follow-up She was released home after two days on oral hydrocortisone 20 mg in the morning and 10 mg in the afternoon. After one month of follow-up, serum cortisol was 36 nmol, with the resolution of some features such as weight reduction (3 kg) and skin color (pink striae became white). 3. Discussion Cushing's syndrome is a serious and well-known medical condition that results from persistent exposure of the body to excessive glucocorticoids, either from endogenous or, most frequently, exogenous sources [9]. The average age of diagnosis is 41.4 years, with a female-to-male ratio of 3:1 [10]. ACTH-dependent CS accounts for almost 80% of endogenous CS, while ACTH-independent CS accounts for nearly 20% [10]. This potentially fatal condition is accompanied by several comorbidities, including hypertension, diabetes, coagulopathy, cardiovascular disease, infections, and fractures [11]. Exogenous CS, also known as iatrogenic CS, is more prevalent than endogenous CS and is caused by the injection of supraphysiologic glucocorticoid dosages [12]. ACTH-independent CS is induced by uncontrolled cortisol release from an adrenal gland lesion, most often an adenoma, adrenocortical cancer, or, in rare cases, ACTH-independent macronodular adrenal hyperplasia or primary pigmented nodular adrenal disease [13]. The majority of data suggests that early diagnosis is critical for reducing morbidity and mortality. Detection is based on clinical suspicion initially, followed by biochemical confirmation [14]. The clinical manifestation of CS varies depending on the severity and duration of glucocorticoid excess [14]. Some individuals may manifest varying symptoms and signs because of a rhythmic change in cortisol secretion, resulting in cyclical CS [15]. The classical symptoms of CS include weight gain, hirsutism, striae, plethora, hypertension, ecchymosis, lethargy, monthly irregularities, diminished libido, and proximal myopathy [16]. Neurobehavioral presentations include anxiety, sadness, mood swings, and memory loss [17]. Less commonly presented features include headaches, acne, edema, abdominal pain, backache, recurrent infection, female baldness, dorsal fat pad, frank diabetes, electrocardiographic abnormalities suggestive of cardiac hypertrophy, osteoporotic fractures, and cardiovascular disease from accelerated atherosclerosis [10]. The current case presented with amenorrhea, weight gain, moon face, buffalo hump, and skin discoloration of the abdomen. Similar to the current case, early diagnosis may be postponed due to the variety of clinical presentations and the referral of patients to different subspecialists based on their dominant symptoms (gynecological, dermatological, cardiovascular, psychiatric); it is, therefore, critical to consider the entire clinical presentation for correct diagnosis [18]. Weight gain may be less apparent in children, but there is frequently an arrest in growth with a fall in height percentile and a delay in puberty [19]. The diagnosis and confirmation of the etiology can be difficult and time-consuming, requiring a variety of laboratory testing and imaging studies [20]. According to endocrine society guidelines, the initial assessment of CS must include one or more of the three following tests: 24-hour UFC measurement; evaluation of the diurnal variation of cortisol secretion by assessing the midnight serum or salivary cortisol level; and a low-dose dexamethasone suppression test, typically the 1 mg overnight test [21]. Although UFC has sufficient sensitivity and specificity, it does not function well in milder cases of Cushing's syndrome [22]. In CS patients, the typical circadian rhythm of cortisol secretion is disrupted, and a high late-night cortisol serum level is the earliest and most sensitive diagnostic indicator of the condition [23]. In the current case, the UFC was elevated, and cortisol was unsuppressed after administration of dexamethasone. All patients with CS should have a high-resolution pituitary MRI with a gadolinium-based contrast agent to prove the existence or absence of a pituitary lesion and to identify the source of ACTH between pituitary adenomas and ectopic lesions [24]. Adrenal CT scan is the imaging modality of choice for preoperatively localizing and subtyping adrenocortical lesions in ACTH-independent Cushing's syndrome [9]. MRI of the pituitary gland of the current case showed a microadenoma and a CT scan of the adrenals showed left adrenal adenoma. Surgical resection of the origin of the ACTH or glucocorticoid excess (pituitary adenoma, nonpituitary tumor-secreting ACTH, or adrenal tumor) is still the first-line treatment of all forms of CS because it leaves normal adjacent structures and results in prompt remission and inevitable recovery of regular adrenal function [12], [25]. Laparoscopic (retroperitoneal or transperitoneal) adrenalectomy has become the gold standard technique for adrenal adenomas since it is associated with fewer postoperative morbidity, hospitalization, and expense when compared to open adrenalectomy [17]. In refractory cases, or when a patient is not a good candidate for surgery, cortisol-lowering medication may be employed [26]. The current case underwent left adrenalectomy. Symptoms of CS, such as central obesity, muscular wasting or weakness, acne, hirsutism, and purple striae generally improve first and may subside gradually over a few months or even a year; nevertheless, these symptoms may remain in 10–30% of patients [27]. Glucocorticoid replacement is essential after adrenal-sparing curative surgery until the pituitary-adrenal function returns, which might take up to two years, especially if adrenal adenomas have been resected [25]. Chronic glucocorticoid excess causes lots of new co-morbidities, lowering the quality of life and increasing mortality. The most common causes of mortality in CS are cardiovascular disease and infections [28]. After one month of follow-up, serum cortisol was 36 nmol, and several features, such as weight loss (3 kg) and skin color, were resolved (pink striae became white). In conclusion, the coexistence of adrenal adenoma and pituitary adenoma with CS is a rare possibility. Due to the diversity in the presentation of CS, an accurate clinical, physical and endocrine examination is always recommended. Laparoscopic adrenalectomy is the gold standard for treating adrenal adenoma. Consent Written informed consent was obtained from the patient's family for publication of this case report and accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal on request. Provenance and peer review Not commissioned, externally peer-reviewed. Ethical approval Approval is not necessary for case report (till 3 cases in single report) in our locality. The family gave consent for the publication of the report. Funding None. Guarantor Fahmi Hussein Kakamad, Fahmi.hussein@univsul.edu.iq. Research registration number Not applicable. CRediT authorship contribution statement Abdulwahid M. Salh: major contribution of the idea, literature review, final approval of the manuscript. Rawa Bapir: Surgeon performing the operation, final approval of the manuscript. Fahmi H. Kakamad: Writing the manuscript, literature review, final approval of the manuscript. Soran H. Tahir, Fattah H. Fattah, Aras Gh. Mahmood, Rawezh Q. Salih, Shaho F. Ahmed: literature review, final approval of the manuscript. Declaration of competing interest None to be declared. References [1] S.M. Ahmed, S.F. Ahmed, S. Othman, B.A. Abdulla, S.H. Mohammed, A.M. Salih, et al. 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  11. This is one of the suggestions from the Cushing’s Awareness Challenge post: Our “Official mascot” is the zebra. Our mascot In med school, student doctors are told “When you hear hoofbeats, think horses, not zebras“. According to Wikipedia: “Zebra is a medical slang term for a surprising diagnosis. Although rare diseases are, in general, surprising when they are encountered, other diseases can be surprising in a particular person and time, and so “zebra” is the broader concept. The term derives from the aphorism ‘When you hear hoofbeats behind you, don’t expect to see a zebra’, which was coined in a slightly modified form in the late 1940s by Dr. Theodore Woodward, a former professor at the University of Maryland School of Medicine in Baltimore. Since horses are the most commonly encountered hoofed animal and zebras are very rare, logically you could confidently guess that the animal making the hoofbeats is probably a horse. A zebra cup my DH bought me 🙂 By 1960, the aphorism was widely known in medical circles.” Why? Because those of us who DO have a rare disorder know from personal experience what it feels like to be dismissed by a doctor or in many cases, multiple doctors. Many physicians have completely lost the ability to even imagine that zebras may exist! Cushing’s is too rare – you couldn’t possible have that. Well… rare means some people get it. Why couldn’t it be me? Although one of my signature images has a zebra, many have rainbows or butterflies in them so I guess that I consider those my own personal mascots. I posted this in 2010 in 40 Days of Thankfulness: Days Twenty-Two through Thirty Butterflies are something else again. I like them because I would like to think that my life has evolved like a butterfly’s, from something ugly and unattractive to something a big easier on the eye. My Cushie self was the caterpillar, post-op is more butterfly-ish, if not in looks, in good deeds. From July, 2008
  12. Researchers published the study covered in this summary on Research Square as a preprint that has not yet been peer reviewed. Key Takeaways Among women who underwent pituitary surgery to treat Cushing disease subsequent pregnancy had no apparent effect on Cushing disease recurrence, in a single-center review of 113 women treated over a 30-year period. Why This Matters No single factor predicts the recurrence of Cushing disease during long-term follow-up of patients who have undergone pituitary surgery. This is the first study to assess the effect of pregnancy on Cushing disease recurrence in a group of reproductive-age women who initially showed post-surgical remission. Study Design Retrospective study of 355 patients with confirmed Cushing disease who were admitted to a single tertiary hospital in Brazil between 1990 and 2020. All patients had transsphenoidal surgery, with a minimum follow-up of 6 months and median follow-up of 83 months. Remission occurred in 246 of these patients. The current analysis focused on 113 of the patients who achieved remission, were women, were 45 years old or younger at time of surgery (median 32 years old), and had information available on their obstetric history. Ninety-one of these women (81%) did not become pregnant after their surgery, and 22 (19%) became pregnant after surgery. Key Results Among the 113 women in the main analysis 43 (38%) had a Cushing disease recurrence, a median of 48 months after their pituitary surgery. Following surgery, 11 women in each of the two subgroups (recurrence, no recurrence) became pregnant. Although the subgroup with recurrence had a higher incidence of pregnancy (11/43; 26%) compared with those with no recurrence (11/70; 16%) Kaplan-Meier analysis showed that survival free of Cushing disease recurrence was similar and not significantly different in the women with a postsurgical pregnancy and those who did not become pregnant (P=.531). The review also showed that, of the women who became pregnant, several obstetrical measures were similar between patients who had a recurrence and those who remained in remission, including number of pregnancies per patient, maternal weight gain, type of delivery (normal or cesarean), delivery time (term or premature), neonatal weight, and neonatal size. The review also showed roughly similar rates of maternal and fetal complications in these two subgroups of women who became pregnant. Limitations The study was retrospective and included a relatively small number of patients. The authors collected information on obstetric history for some patients by telephone or email contacts. Disclosures The study received no commercial funding. None of the authors had disclosures. This is a summary of a preprint research study , " Pregnancy After Pituitary Surgery Does Not Influence the Recurrence of Cushing's Disease, " written by researchers at the Sao Paulo (Brazil) University Faculty of Medicine on Research Square provided to you by Medscape. This study has not yet been peer reviewed. The full text of the study can be found on researchsquare.com.
  13. The study covered in this summary was published on Research Square as a preprint and has not yet been peer reviewed. Key Takeaways A study of 78 patients who underwent elective transsphenoidal adenomectomy to remove a pituitary tumor or other lesions within the pituitary fossa at a single center in the UK suggests that postoperative plasma levels of copeptin — a surrogate marker for levels of arginine vasopressin (antidiuretic hormone) — can rule out development of central (neurogenic) diabetes insipidus caused by a deficiency of arginine vasopressin following pituitary surgery. The researchers suggest using as a cutoff a copeptin level of >3.4 pmol/L at postoperative day 1 to rule out diabetes insipidus. Such a cutoff yields the following: A high sensitivity of 91% for ruling out diabetes insipidus. A negative predictive value of 97%. Only 1 of 38 patients with a copeptin value >3.4 pmol/L at day 1 postoperatively developed diabetes insipidus. A low specificity of 55%, meaning that copeptin level is not useful for diagnosing diabetes insipidus Why This Matters An estimated 1% to 67% of patients who undergo pituitary gland surgery develop diabetes insipidus, often soon after surgery, although it is often transient. Diagnosing diabetes insipidus in such patients requires a combination of clinical assessment, the monitoring of fluid balance, and determining plasma and urine sodium and osmolality. Currently, clinical laboratories in the UK do not have assays for arginine vasopressin, which has a short half-life in vivo and is unstable ex vivo, even when frozen, and is affected by delayed or incomplete separation from platelets. Copeptin, an arginine vasopressin precursor, is much more stable and measurable by commercial immunoassays. The findings suggest that patients who have just undergone pituitary gland surgery and are otherwise healthy and meet the copeptin cutoff for ruling out diabetes insipidus could be discharged 24 hours after surgery and that there is no need for additional clinical and biochemical monitoring. This change would ease demands on the healthcare system. Study Design The study reviewed 78 patients who underwent elective transsphenoidal adenomectomy to remove a pituitary tumor from November 2017 to June 2020 at the John Radcliffe Hospital in Oxford, United Kingdom. Patients remained in hospital for a minimum of 48 hours after their surgery. Clinicians collected blood and urine specimens preoperatively and at day 1, day 2, day 8, and week 6 post surgery. The patients were restricted to 2 L of fluid a day postoperatively to prevent masking of biochemical abnormalities through compensatory drinking. Diabetes insipidus was suspected when patients' urine output was >200 mL/h for 3 consecutive hours or >3 L/d plus high plasma sodium (>145 mmol/L) and plasma osmolality (> 295 mosmol/kg) plus inappropriately low urine osmolality. Definitive diagnosis of diabetes insipidus was based on clinical assessment, urine and plasma biochemistry, and need for treatment with desmopressin (1-deamino-8-D-arginine vasopressin). Key Results The median age of the patients was 55, and 53% were men; 92% of the lesions were macroadenomas; the most common histologic type was gonadotroph tumor (47%). Among the 78 patients, 11 (14%) were diagnosed with diabetes insipidus postoperatively and required treatment with desmopressin; of these, seven patients (9%) continued taking desmopressin after 6 weeks (permanent diabetes insipidus), but the other four did not need to take desmopressin for more than a week. Patients who developed diabetes insipidus were younger than other patients (mean age, 46 vs 56), and 8 of the 11 patients who developed diabetes insipidus (73%) were women. Preoperative copeptin levels were similar in the two groups. At day 1, day 8, and 6 weeks postoperatively, copeptin levels were significantly lower in the diabetes insipidus group; there were no significant differences at day 2, largely because of an outlier result. An area under the receiver operating characteristic curve (AUC; C-statistic) analysis showed that on day 1 after surgery, copeptin levels could account for 74.22% of the incident cases of diabetes insipidus (AUC, 0.7422). On postop day 8, the AUC for copeptin was 0.8015, and after 6 weeks, the AUC associated with copeptin was 0.7321. Limitations Blood samples for copeptin tests from patients who underwent pituitary surgery were collected at specified times and were frozen for later analysis; performing the test in real time might alter patient management. The study may have missed peak copeptin levels by not determining copeptin levels sooner after pituitary gland surgery, inasmuch as other researchers have reported better predictive values for diagnosing diabetes insipidus from samples taken 1 hour after extubation or <12 hours after surgery. Disclosures The study did not receive commercial funding. The authors report no relevant financial relationships. This is a summary of a preprint research study, "Post-Operative Copeptin Analysis Predicts Which Patients Do Not Develop Diabetes Insipidus After Pituitary Surgery," by researchers from John Radcliffe Hospital, Oxford University Hospitals NHS Foundation Trust, in the United Kingdom. Preprints from Research Square are provided to you by Medscape. This study has not yet been peer reviewed. The full text of the study can be found on researchsquare.com. Read the article here: https://www.medscape.com/viewarticle/970357#vp_1
  14. Abstract Summary The pandemic caused by severe acute respiratory syndrome coronavirus 2 is of an unprecedented magnitude and has made it challenging to properly treat patients with urgent or rare endocrine disorders. Little is known about the risk of coronavirus disease 2019 (COVID-19) in patients with rare endocrine malignancies, such as pituitary carcinoma. We describe the case of a 43-year-old patient with adrenocorticotrophic hormone-secreting pituitary carcinoma who developed a severe COVID-19 infection. He had stabilized Cushing’s disease after multiple lines of treatment and was currently receiving maintenance immunotherapy with nivolumab (240 mg every 2 weeks) and steroidogenesis inhibition with ketoconazole (800 mg daily). On admission, he was urgently intubated for respiratory exhaustion. Supplementation of corticosteroid requirements consisted of high-dose dexamethasone, in analogy with the RECOVERY trial, followed by the reintroduction of ketoconazole under the coverage of a hydrocortisone stress regimen, which was continued at a dose depending on the current level of stress. He had a prolonged and complicated stay at the intensive care unit but was eventually discharged and able to continue his rehabilitation. The case points out that multiple risk factors for severe COVID-19 are present in patients with Cushing’s syndrome. ‘Block-replacement’ therapy with suppression of endogenous steroidogenesis and supplementation of corticosteroid requirements might be preferred in this patient population. Learning points Comorbidities for severe coronavirus disease 2019 (COVID-19) are frequently present in patients with Cushing’s syndrome. ‘Block-replacement’ with suppression of endogenous steroidogenesis and supplementation of corticosteroid requirements might be preferred to reduce the need for biochemical monitoring and avoid adrenal insufficiency. The optimal corticosteroid dose/choice for COVID-19 is unclear, especially in patients with endogenous glucocorticoid excess. First-line surgery vs initial disease control with steroidogenesis inhibitors for Cushing’s disease should be discussed depending on the current healthcare situation. Keywords: Adult; Male; Other; Belgium; Pituitary; Adrenal; Neuroendocrinology; Oncology; Insight into disease pathogenesis or mechanism of therapy; February; 2022 Background The pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has had a significant impact on the health care systems to date. The clinical presentation of coronavirus disease 2019 (COVID-19) is diverse, ranging from asymptomatic illness to respiratory failure requiring admission to the intensive care unit (ICU). Risk factors for severe course include old age, male gender, comorbidities such as arterial hypertension, diabetes mellitus, chronic lung-, heart-, liver- and kidney disease, malignancy, immunodeficiency and pregnancy (1). Little is known about the risk of COVID-19 in patients with rare endocrine malignancies, such as pituitary carcinoma. Case presentation This case concerns a 43-year-old man with adrenocorticotrophic hormone (ACTH)-secreting pituitary carcinoma (with cerebellar and cervical drop metastases) with a severe COVID-19 infection. He had previously received multiple treatment modalities including surgery, radiotherapy, ketoconazole, pasireotide, cabergoline, bilateral (subtotal) adrenalectomy and temozolomide chemotherapy as described elsewhere (2). His most recent therapy was a combination of immune checkpoint inhibitors consisting of ipilimumab (3 mg/kg) and nivolumab (1 mg/kg) (anti-CTLA-4 and anti-PD-1, respectively) every 3 weeks for four cycles, after which maintenance therapy with nivolumab (240 mg) every 2 weeks was continued. Residual endogenous cortisol production was inhibited with ketoconazole 800 mg daily. He had stabilized disease with a decrease in plasma ACTH, urinary free cortisol and stable radiological findings (2). Surgical resection of the left adrenal remnant was planned but was not carried out due to the development of a COVID-19 infection. In March 2021, he consulted our emergency department for severe respiratory complaints. He had been suffering from upper respiratory tract symptoms for one week, with progressive dyspnoea in the last three days. He tested positive for SARS-CoV-2 the day before admission. On examination, his O2 saturation was 72%, with tachypnoea (40/min) and bilateral pulmonary crepitations. His temperature was 37.2°C, blood pressure 124/86 mmHg and pulse rate 112 bpm. High-flow oxygen therapy was initiated but yielded insufficient improvement (O2 saturation of 89% and tachypnoea 35/min). He was urgently intubated for respiratory exhaustion. Investigation Initial investigations showed type 1 respiratory insufficiency with PaO2 of 52.5 mmHg (normal 75–90), PaCO2 of 33.0 mmHg (normal 36–44), pH of 7.47 (normal 7.35–7.45) and a P/F ratio of 65.7 (normal >300). His inflammatory parameters were elevated with C-reactive protein level of 275.7 mg/L (normal <5·0) and white blood cell count of 7.1 × 10⁹ per L with 72.3% neutrophils. His most recent morning plasma ACTH-cortisol level (measured using the Elecsys electrochemiluminescence immunoassays on a Cobas 8000 immunoanalyzer [Roche Diagnostics]) before his admission was 213 ng/L (normal 7.2–63) and 195 µg/L (normal 62–180) respectively, while a repeat measurement 3 weeks after his admission demonstrated increased cortisol levels of 547 µg/L (possibly iatrogenic due to treatment with high-dose hydrocortisone) and a decreased ACTH of 130 ng/L. Treatment On admission, he was started on high-dose dexamethasone therapy for 10 days together with broad-spectrum antibiotics for positive sputum cultures containing Serratia, methicillin-susceptible Staphylococcus aureus and Haemophilus influenzae. Thromboprophylaxis with an intermediate dose of low molecular weight heparin (tinzaparin 14 000 units daily for a body weight of 119 kg) was initiated. A ‘block-replacement’ regimen was adopted with the continuation of ketoconazole (restarted on day 11) in view of his endocrine treatment and the supplementation of hydrocortisone at a dose depending on the current level of stress. The consecutive daily dose of hydrocortisone and ketoconazole is shown in Fig. 1. View Full Size Figure 1 ‘Block-replacement’ therapy with ketoconazole and hydrocortisone/dexamethasone. Dexamethasone 10 mg daily was initially started as COVID-19 treatment, followed by hydrocortisone at a dose consistent with current levels of stress. Ketoconazole was restarted on day 11 and titrated to a dose of 800 mg daily to suppress endogenous glucocorticoid production. Citation: Endocrinology, Diabetes & Metabolism Case Reports 2022, 1; 10.1530/EDM-21-0182 Download Figure Download figure as PowerPoint slide Outcome and follow-up He developed multiple organ involvement, including metabolic acidosis, acute renal failure requiring continuous venovenous hemofiltration, acute coronary syndrome type 2, septic thrombophlebitis of the right jugular vein, and critical illness polyneuropathy. He was readmitted twice to the ICU, for ventilator-associated pneumonia and central line-associated bloodstream infection respectively. He eventually recovered and was discharged from the hospital to continue his rehabilitation. Discussion We describe the case of a patient with severe COVID-19 infection with active Cushing’s disease due to pituitary carcinoma, who was treated with high-dose dexamethasone followed by ‘block-replacement’ therapy with hydrocortisone in combination with off-label use of ketoconazole as a steroidogenesis inhibitor. His hospitalization was prolonged by multiple readmissions to the ICU for infectious causes. Our case illustrates the presence of multiple comorbidities for a severe and complicated course of COVID-19 in a patient with active Cushing’s disease. Dexamethasone was initially chosen as the preferred corticosteroid therapy, in analogy with the RECOVERY trial, in which dexamethasone at a dose of 6mg once daily (oral or i.v.) resulted in lower 28-day mortality in hospitalized patients with COVID-19 requiring oxygen therapy or invasive mechanical ventilation (3). However, the optimal dose/choice of corticosteroid therapy is unclear, especially in a patient population with pre-existing hypercortisolaemia. A similar survival benefit for hydrocortisone compared to dexamethasone has yet to be convincingly demonstrated. This may be explained by differences in anti-inflammatory activity but could also be due to the fact that recent studies with hydrocortisone were stopped early and were underpowered (4, 5). Multiple risk factors for a complicated course of COVID-19 are present in patients with Cushing’s syndrome and might increase morbidity and mortality (6, 7). These include a history of obesity, arterial hypertension and impaired glucose metabolism. Prevention and treatment of these pre-existing comorbidities are essential. Patients with Cushing’s syndrome also have an increased thromboembolic risk, which is further accentuated by the development of severe COVID-19 infection (6, 7). Thromboprophylaxis with low molecular weight heparin is associated with lower mortality in COVID-19 patients with high sepsis‐induced coagulopathy score or high D-dimer levels (8) and is presently widely used in the treatment of severe COVID-19 disease (9). Subsequently, this treatment is indicated in hospitalized COVID-19 patients with Cushing’s syndrome. It is unclear whether therapeutic anticoagulation dosing could provide additional benefits (6, 7). An algorithm based on the International Society on Thrombosis and Hemostasis-Disseminated Intravascular Coagulation score was proposed to evaluate the ideal anticoagulation therapy in severe/critical COVID-19 patients, with an indication for therapeutic low molecular weight heparin dose at a score ≥5 (9). Furthermore, the chronic cortisol excess induces suppression of the innate and adaptive immune response. Patients with Cushing’s syndrome, especially when severe and active, should be considered immunocompromised and have increased susceptibility for viral and other (hospital-acquired) infections. Prophylaxis for Pneumocystis jirovecii with trimethoprim/sulfamethoxazole should therefore be considered (6, 7). Additionally, there is a particular link between the pathophysiology of COVID-19 and Cushing’s syndrome. The SARS-CoV-2 virus (as well as other coronaviruses) enter human cells by binding the ACE2 receptor. The transmembrane serine protease 2 (TMPRSS2), expressed by endothelial cells, is additionally required for the priming of the spike-protein of SARS-CoV-2, leading to viral entry. TMPRSS2 was studied in prostate cancer and found to be regulated by androgen signalling. Consequently, the androgen excess frequently associated with Cushing’s syndrome might be an additional risk factor for contracting COVID-19 via higher TMPRSS2 expression (10), especially in women, in whom the effect of excess androgen would be more noticeable compared to male patients with Cushing’s syndrome. Treating Cushing’s syndrome with a ‘block-replacement’ approach, with suppression of endogenous steroidogenesis and supplementation of corticosteroid requirements, is an approach that should be considered, especially in severe or cyclic disease. The use of this method might decrease the need for monitoring and reduce the occurrence of adrenal insufficiency (7). Our patient was on treatment with ketoconazole, which was interrupted at initial presentation and then restarted under the coverage of a hydrocortisone stress regimen. Ketoconazole was chosen because of its availability. Advantages of ketoconazole over metyrapone include its antifungal activity with the potential for prevention of invasive pulmonary fungal infections, as well as its antiandrogen action (especially in female patients) and subsequent inhibition of TMPRSS2 expression (10). Regular monitoring of the liver function (every month for the first 3 months, at therapy initiation or dose increase) is necessary. Caution is needed due to its inhibition of multiple cytochrome P450 enzymes (including CYP3A4) and subsequently greater risk of drug-drug interactions vs metyrapone (7, 10). Another disadvantage of ketoconazole is the need for oral administration. In our patient, ketoconazole was delivered through a nasogastric tube. i.v. etomidate is an alternative in case of an unavailable enteral route. Finally, as a general point, the first-line treatment of a patient with a novel diagnosis of Cushing’s disease is transsphenoidal surgery. Recent endocrine recommendations pointed out the possibility of initial disease control with steroidogenesis inhibitors in patients without an indication for urgent intervention during a high prevalence of COVID-19 (7). This would allow the optimalization of metabolic parameters; emphasizing that the short-to mid-term prognosis is related to the cortisol excess and not its cause. Surgery could then be postponed until the health situation allows for safe elective surgery (7). This decision depends of course on the evolution of COVID-19 and the healthcare system in each country and should be closely monitored by policymakers and physicians. Declaration of interest The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported. Funding This work did not receive any specific grant from any funding agency in the public, commercial, or not-for-profit sector. Patient consent Written informed consent for publication of their clinical details and/or clinical images was obtained from the patient. Author contribution statement J M K de Filette is an endocrinologist-in-training and was the main author. All authors were involved in the clinical care of the patient. All authors contributed to the reviewing and editing process and approved the final version of the manuscript. References 1↑ Gao Y-D, Ding M, Dong X, Zhang J-J, Kursat Azkur A, Azkur D, Gan H, Sun Y-L, Fu W, Li W, et al.Risk factors for severe and critically ill COVID-19 patients: a review. Allergy 2021 76 428–455.(https://doi.org/10.1111/all.14657) Search Google Scholar Export Citation 2↑ Sol B, de Filette JMK, Awada G, Raeymaeckers S, Aspeslagh S, Andreescu CE, Neyns B, Velkeniers B. Immune checkpoint inhibitor therapy for ACTH-secreting pituitary carcinoma: a new emerging treatment? European Journal of Endocrinology 2021 184 K1–K5. (https://doi.org/10.1530/EJE-20-0151) Search Google Scholar Export Citation 3↑ The RECOVERY Collaborative Group. Dexamethasone in hospitalized patients with Covid-19. New England Journal of Medicine 2021 3 84 693–704.(https://doi.org/10.1056/nejmoa2021436) Search Google Scholar Export Citation 4↑ Angus DC, Derde L, Al-Beidh F, Annane D, Arabi Y, Beane A, van Bentum-Puijk W, Berry L, Bhimani Z & Bonten M et al.Effect of hydrocortisone on mortality and organ support in patients with severe COVID-19: the REMAP-CAP COVID-19 corticosteroid domain randomized clinical trial. JAMA 2020 324 1317–1329. (https://doi.org/10.1001/jama.2020.17022) Search Google Scholar Export Citation 5↑ Dequin PF, Heming N, Meziani F, Plantefève G, Voiriot G, Badié J, François B, Aubron C, Ricard JD & Ehrmann S et al.Effect of hydrocortisone on 21-day mortality or respiratory support among critically ill patients with COVID-19: a randomized clinical trial. JAMA 2020 324 1298–1306. (https://doi.org/10.1001/jama.2020.16761) Search Google Scholar Export Citation 6↑ Pivonello R, Ferrigno R, Isidori AM, Biller BMK, Grossman AB, Colao A. COVID-19 and Cushing’s syndrome: recommendations for a special population with endogenous glucocorticoid excess. Lancet: Diabetes and Endocrinology 2020 8 654–656. (https://doi.org/10.1016/S2213-8587(2030215-1) Search Google Scholar Export Citation 7↑ Newell-Price J, Nieman LK, Reincke M, Tabarin A. ENDOCRINOLOGY IN THE TIME OF COVID-19: Management of Cushing’s syndrome. European Journal of Endocrinology 2020 183 G1–G7. (https://doi.org/10.1530/EJE-20-0352) Search Google Scholar Export Citation 8↑ Tang N, Bai H, Chen X, Gong J, Li D, Sun Z. Anticoagulant treatment is associated with decreased mortality in severe coronavirus disease 2019 patients with coagulopathy. Journal of Thrombosis and Haemostasis 2020 18 1094–1099. (https://doi.org/10.1111/jth.14817) Search Google Scholar Export Citation 9↑ Carfora V, Spiniello G, Ricciolino R, Di Mauro M, Migliaccio MG, Mottola FF, Verde N, Coppola N & Vanvitelli COVID-19 Group. Anticoagulant treatment in COVID-19: a narrative review. Journal of Thrombosis and Thrombolysis 2021 51 642–648. (https://doi.org/10.1007/s11239-020-02242-0) Search Google Scholar Export Citation 10↑ Barbot M, Ceccato F, Scaroni C. Consideration on TMPRSS2 and the risk of COVID-19 infection in Cushing’s syndrome. Endocrine 2020 69 235–236. (https://doi.org/10.1007/s12020-020-02390-6) Search Google Scholar Export Citation From https://edm.bioscientifica.com/view/journals/edm/2022/1/EDM21-0182.xml
  15. https://doi.org/10.1002/ccr3.5337 Abstract A 50-year-old woman with adrenal Cushing's syndrome and chronic hepatitis C developed an acute exacerbation of chronic hepatitis C before adrenectomy. After administration of glecaprevir/pibrentasvir was started, her transaminase levels normalized promptly and a rapid virological response also was achieved. Laparoscopic left adrenectomy was then performed safely. 1 INTRODUCTION Reports of reactivation of hepatitis C virus (HCV) and acute exacerbation of chronic hepatitis C associated with immunosuppressive therapy and cancer drug therapy are rarer than for hepatitis B virus (HBV) but have been made occasionally. In HBV infection, viral reactivation and acute hepatitis caused by an excess of endogenous cortisol due to Cushing's syndrome have been reported, but no acute exacerbation of chronic hepatitis C has been reported so far. Here, we report a case of acute exacerbation of chronic hepatitis C during the course of adrenal Cushing's syndrome. 2 CASE REPORT A woman in her 50s underwent a CT scan at a nearby hospital to investigate treatment-resistant hypertension and was found to have a left adrenal mass. Her blood tests showed low ACTH and HCV antibody positivity, and she was referred to our hospital because she was suspected of having Cushing's syndrome and chronic hepatitis C. There is nothing special to note about her medical or family history. She had never smoked and drank very little. Her physical findings on admission were 164.5 cm tall, 92.6 kg in weight, and a BMI of 34.2 kg/m2. Her blood pressure was 179 / 73 mmHg, pulse 64 /min (rhythmic), body temperature 36.8°C, and respiratory rate 12 /min. She had findings of central obesity, moon face, buffalo hump, and red skin stretch marks. Her blood test findings (Table 1) showed an increase in ALT, HCV antibody positivity, and an HCV RNA concentration of 4.1 log IU/mL. The virus was genotype 2. Cortisol was within the reference range, but ACTH was as low, less than 1.5 pg/mL. Her bedtime cortisol level was 7.07 μg/dL, which was above her reference of 5 μg/dL, suggesting the loss of diurnal variation in cortisol secretion. Testing showed the amount of cortisol by 24-hour urine collection was 62.1 μg/day, and this level of cortisol secretion was maintained. In an overnight low-dose dexamethasone suppression test, cortisol after loading was 6.61 μg/dL, which exceeded 5 μg/dL, suggesting that cortisol was autonomously secreted. Her contrast-enhanced CT scan (Figure 1) revealed a tumor with a major axis of about 30 mm in her left adrenal gland. MRI scans showed mild hyperintensity in the “in phase” (Figure 2A) and decreased signal in the “out of phase” (Figure 2B), suggesting her adrenal mass was an adenoma. Based on the above test results, she was diagnosed with chronic hepatitis C and adrenal Cushing's syndrome. She agreed to receive treatment with direct acting antiviral agents (DAAs) after resection of the left adrenal tumor. However, two months later, she had liver dysfunction with AST 116 U/L and ALT 213 U/L (Figure 3). HBV DNA was undetectable at the time of liver injury, but the HCV RNA concentration increased to 6.4 logIU/mL. Therefore, an acute exacerbation of chronic hepatitis C was suspected, and a percutaneous liver biopsy was performed. The biopsy revealed an inflammatory cell infiltration, mostly composed of lymphocytes and plasma cells and mainly in the portal vein area (Figure 4). Fibrosis and interface hepatitis were also observed, and spotty necrosis was evident in the hepatic lobule. No clear fat deposits were found in the hepatocytes, ruling out NASH or NAFLD. According to the New Inuyama classification, hepatitis equivalent to A2-3/F1-2 was considered. Because HBV DNA was not detected, no new drug was used, and no cause of liver damage, such as biliary atresia, was found; the patient was diagnosed with liver damage due to reactivation of HCV, with acute exacerbation of chronic hepatitis C. The treatment policy was changed, in order to treat hepatitis C before the left adrenal resection, and administration of glecaprevir/pibrentasvir was started. A blood test two weeks after the start of treatment confirmed normalization of AST and ALT, and a rapid virological response was achieved (Figure 3). Subsequently, HCV RNA remained negative, no liver damage was observed, and laparoscopic left adrenectomy was safely performed nine months after the initial diagnosis. The pathological findings were adrenal adenoma, and no atrophy was observed in the attached normal adrenal cortical gland. After the operation, hypertension improved and weight loss was obtained (92.6 kg (BMI: 34.2 kg/m2) before the operation, but 77.0 kg (BMI: 28.5 kg/m2) one year after the operation). ACTH increased, and the adrenal Cushing's syndrome was considered to have been cured. Regarding HCV infection, the sustained virological response has been maintained to date, more than 2 years after the completion of DAA therapy, and the follow-up continues. TABLE 1. Laboratory data on admission Hematology Chemistry WBC 6100 /μL TP 8.2 g/dL DHEA-S 48 /μL RBC 526 x 104 /μL Alb 3.4 g/dL PRA 0.7 ng/mL/h Hb 15.8 g/dL T-Bil 0.3 mg/dL ALD 189 pg/mL Ht 49.1 % AST 33 U/L PLT 25.5 x 104 /μL ALT 46 U/L Serological tests LDH 201 U/L CRP <0.10 mg/dL ALP 292 U/L HBsAg (-) γ-GTP 77 U/L anti-HBs (-) Coagulation BUN 13 mg/dL anti-HBc (+) PT 126.1 % Cr 0.63 mg/dL HBeAg (-) APTT 27.5 sec HbA1c 6.2 % anti-HBe (+) Cortisol 7.46 μg/dL anti-HCV (+) ACTH <1.5 pg/mL FBS 82 mg/dL Genetic tests Na 138 mmol/L HBV DNA Undetectable Cl 105 mmol/L HCV RNA 4.1 LogIU/Ml K 3.6 mmol/L HCV genotype 2 Ca 9.0 mg/dL Abbreviations: Hematology: WBC, white blood cells; RBC, red blood cells; Hb, hemoglobin; Ht, hematocrit; PLT, platelets. Coagulation: PT, prothrombin time; APTT, activated partial thromboplastin time. Chemistry: TP, total protein; Alb, albumin; T-Bil, total bilirubin; AST, aspartate transaminase; ALT, alanine aminotransferase; LDH, lactate dehydrogenase; ALP, alkaline phosphatase; γGTP, γ-glutamyl transpeptidase; BUN, blood urea nitrogen; Cr, creatinine; HbA1c, Hemoglobin A1c; FBS, fasting blood sugar; Na, sodium; Cl, chlorine; K, potassium; Ca, calcium; DHEA-S, dehydroepiandrosterone sulfate; PRA, plasma renin activity; ALD, aldosterone. Serological tests: CRP, C-reactive protein; HBsAg, hepatitis B surface antigen; anti-HBs, hepatitis B surface antibody; anti-HBc, hepatitis B core antibody; HBeAg, hepatitis B e antigen; anti-HBe, hepatitis B e antibody; anti-HCV, hepatitis C virus antibody. Genetic tests: HBV DNA, hepatitis B virus deoxyribonucleic acid; HCV RNA, hepatitis C virus ribonucleic acid. FIGURE 1 Open in figure viewerPowerPoint Contrast-enhanced CT examination. Contrast-enhanced CT examination revealed a tumor (arrow) with a major axis of about 30 mm in the left adrenal gland FIGURE 2 Open in figure viewerPowerPoint MRI image of the adrenal lesion. MRI showed mild hyperintensity in the "in phase" (A) and decreased signal in the "out of phase" (B), suggesting adrenocortical adenoma (arrow) FIGURE 3 Open in figure viewerPowerPoint Changes in serum transaminase and HCV RNA levels. All showed rapid improvement by administration of direct acting antivirals. ALT: alanine aminotransferase, AST: aspartate transaminase, HCV RNA: hepatitis C virus ribonucleic acid FIGURE 4 Open in figure viewerPowerPoint Pathological findings of tissues obtained by percutaneous liver biopsy. Infiltration of inflammatory cells, which was mostly composed of lymphocytes and plasma cells and a small number of neutrophils, was observed mainly in the portal vein area. This was accompanied by fibrous enlargement and interface hepatitis. Although the arrangement of hepatocytes was maintained in the hepatic lobule, spotty necrosis was observed in some parts. No clear fat deposits were found in the hepatocytes, and NASH or NAFLD was a negative finding. According to the New Inuyama classification, hepatitis equivalent to A2-3/F1-2 was considered (a; ×100, b; ×200, scale bar = 500 µm) 3 DISCUSSION Reactivation of HBV can cause serious liver damage. Therefore, it is recommended to check the HBV infection status before starting anticancer chemotherapy or immunotherapy and to continue monitoring for the presence or absence of reactivation thereafter.1, 2 On the other hand, there are fewer reports of the reactivation of HCV, and many aspects of the pathophysiology of HCV reactivation remain unclear. In this case, it is possible that chronic hepatitis C was acutely exacerbated due to endogenous cortisol secretion in Cushing's syndrome. Although the definition of HCV reactivation has not been defined, several studies3-5 have defined an increase of HCVRNA of 1.0 log IU/ml or more as HCV reactivation. In addition, the definition of acute exacerbation of chronic hepatitis C is that ALT increases to more than three times the upper limit of the reference range.3, 4, 6 Mahale et al. reported a retrospective study in which acute exacerbation of chronic hepatitis C due to cancer medication was seen in 11% of 308 patients.3 Torres et al. also reported that, in a prospective study of 100 patients with cancer medication, HCV reactivation was found in 23%.4 Given these reports, HCV reactivation potentially could occur quite frequently. However, Torres et al. reported that only 10% of all patients had acute exacerbations, none of which led to liver failure.4 Such data suggest that HCV reactivation may often be overlooked in actual cases without aggravation. Thus, the frequency of aggravation due to hepatitis virus reactivation is thought to be lower for HCV than for HBV. However, there are some reports of deaths from acute exacerbation of chronic hepatitis C.7-10 In addition, if severe hepatitis develops following viral reactivation, mortality rates have been reported to be similar for HBV and HCV.8, 11 Thus, reactivation of HCV is considered to be a pathological condition that requires caution, similar to HBV. Torres et al. reported that administration of rituximab or corticosteroids is a significant independent risk factor.4 In addition, there are reports of acute exacerbation of chronic hepatitis C due to corticosteroids administered as antiemetics and as immunosuppressive therapy.12-14 Therefore, excess cortisol can reactivate not only HBV but also HCV. The mechanism by which HCV is reactivated with cortisol is assumed to be decreased cell-mediated immunity due to rapid apoptosis of circulating T cells caused by glucocorticoids,4 enhancement of HCV infectivity by upregulation of viral receptor expression on the hepatocyte surface,15 and enhanced viral replication.16 In addition, there is a report that genotype 2 is more common in cases with acute exacerbation of chronic hepatitis C,4, 13 which is consistent with this case. Regarding HBV reactivation due to Cushing's syndrome, three cases of acute exacerbation of chronic hepatitis B have been reported.17-19 It is believed that Cushing's syndrome caused a decrease in cell-mediated immunity and humoral immunity due to an endogenous excess of cortisol, resulting in an acute exacerbation of chronic hepatitis B.13 As described above, because an excess of cortisol can cause reactivation of HCV, it is considered that a decrease in immunocompetence due to Cushing's syndrome, which is an excess of endogenous cortisol, can also cause reactivation of HCV and acute exacerbation of chronic hepatitis. However, as far as we can determine, no cases of Cushing's syndrome causing HCV reactivation or acute exacerbation of chronic hepatitis C have been reported and similar cases may be latent. Among the reports of acute exacerbation of hepatitis B due to adrenal Cushing's syndrome, there is a case in which the liver damage and viral load were improved only by adrenalectomy.17 Therefore, it is also possible that hepatitis C was improved by adrenal resection in this case. However, general anesthesia associated with adrenalectomy and the use of various drugs used for postoperative physical management should be avoided, if possible, in situations where some severe liver damage is present. In addition, reactivation of immunity due to rapid depletion of glucocorticoid, following resection of an adrenal tumor, may lead to exacerbation of liver damage. In this case, the amount of HCV and hepatic transaminase levels were improved rapidly by glecaprevir/pibrentasvir treatment, and the operation could be performed safely. If Cushing's syndrome is complicated by an acute exacerbation of hepatitis C, clinicians should consider including treatment strategies such as in this case. Summarizing the above, when liver damage appears in HCV-infected patients with Cushing's syndrome, it will be necessary to distinguish the acute exacerbation and reactivation of chronic hepatitis C. Treatment with DAAs may then be considered to be effective for reactivation of HCV and acute exacerbation of chronic hepatitis. 4 CONCLUSION We report a case of chronic hepatitis C with acute exacerbation during the course of Cushing's syndrome. At the time of cancer drug therapy and in the state of endogenous and extrinsic corticosteroid excess, it is necessary to pay attention not only to acute exacerbation of chronic hepatitis B but also to hepatitis C. ACKNOWLEDGEMENTS All authors would like to thank the patient and his family for allowing this case study. CONFLICT OF INTEREST The authors have no conflict of interests. AUTHOR CONTRIBUTIONS TO and KM were collected and analyzed the data and wrote and edited the manuscript. KH, ST, HO, KT, KM, and JK were involved in the patient's care and provided advice on the preparation of this case report. ETHICAL APPROVAL This study complied with the standards of the Declaration of Helsinki and the current ethical guidelines. CONSENT Written informed consent was obtained from the patient to publish this report in accordance with the journal's patient consent policy. From https://onlinelibrary.wiley.com/doi/10.1002/ccr3.5337
  16. Join our Rare Disease Day virtual panel discussion as BioNews columnists from a variety of our rare communities participate in a lively conversation with fellow patient advocate Liza Bernstein. This window into often overlooked aspects of life with a rare disease will provide a variety of patient perspectives. Topics will include awareness and advocacy, equity, mental health, empowerment, and more. We invite everyone to join us for this signature event and look forward to your participation in the Q and A! Panelists: Paris Dancy, Columnist, Cushing’s Disease News Michelle Gonzaba, Columnist, Myasthenia Gravis News Claire Richmond, Columnist, Porphyria News Sherry Toh, Columnist, SMA News Today Hosted by Liza Bernstein, Patient Advocate & Sr. Director Patient & Community Engagement Time Feb 28, 2022 02:00 PM in Central Time (US and Canada) Register at https://us06web.zoom.us/webinar/register/WN_dylme0wBRCyH8TfQ7B6x-w
  17. Abstract Summary Here, we describe a case of a patient presenting with adrenocorticotrophic hormone-independent Cushing’s syndrome in a context of primary bilateral macronodular adrenocortical hyperplasia. While initial levels of cortisol were not very high, we could not manage to control hypercortisolism with ketoconazole monotherapy, and could not increase the dose due to side effects. The same result was observed with another steroidogenesis inhibitor, osilodrostat. The patient was finally successfully treated with a well-tolerated synergitic combination of ketoconazole and osilodrostat. We believe this case provides timely and original insights to physicians, who should be aware that this strategy could be considered for any patients with uncontrolled hypercortisolism and delayed or unsuccessful surgery, especially in the context of the COVID-19 pandemic. Learning points Ketoconazole–osilodrostat combination therapy appears to be a safe, efficient and well-tolerated strategy to supress cortisol levels in Cushing syndrome. Ketoconazole and osilodrostat appear to act in a synergistic manner. This strategy could be considered for any patient with uncontrolled hypercortisolism and delayed or unsuccessful surgery, especially in the context of the COVID-19 pandemic. Considering the current cost of newly-released drugs, such a strategy could lower the financial costs for patients and/or society. Keywords: Adult; Male; White; France; Adrenal; Adrenal; Novel treatment; December; 2021 Background Untreated or inadequately treated Cushing’s syndrome (CS) is a morbid condition leading to numerous complications. The latter ultimately results in an increased mortality that is mainly due to cardiovascular events and infections. The goal of the treatment with steroidogenesis inhibitors is normalization of cortisol production allowing the improvement of comorbidities (1). Most studies dealing with currently available steroidogenesis inhibitors used as monotherapy reported an overall antisecretory efficacy of roughly 50% in CS. Steroidogenesis inhibitors can be combined to better control hypercortisolism. To the best of our knowledge, we report here for the first time a patient treated with a ketoconazole–osilodrostat combination therapy. Case presentation Here, we report the case of Mr D.M., 53-years old, diagnosed with adrenocorticotrophic hormone (ACTH)-independent CS 6 months earlier. At diagnosis, he presented with resistant hypertension, hypokalemia, diabetes mellitus, easy bruising, purple abdominal striae and major oedema of the lower limbs. Investigations A biological assessment was performed, and the serum cortisol levels are depicted in Table 1. ACTH levels were suppressed (mean levels 1 pg/mL). Mean late-night salivary cortisol showed a four-fold increase (Table 2), and mean 24 h-urinary cortisol showed a two-fold increase. Serum cortisol was 1000 nmol/L at 08:00 h after 1 mg dexamethasone dose at 23:00 h. The rest of the adrenal hormonal workup was within normal ranges (aldosterone: 275 pmol/L and renin: 15 mIU/L). An adrenal CT was performed (Fig. 1) and exhibited a 70-mm left adrenal mass (spontaneous density: 5 HU and relative washout: 65%) and a 45-mm right adrenal mass (spontaneous density: −2 HU and relative washout: 75%). The case was discussed in a multidisciplinary team meeting, which advised to perform 18F-FDG PET-CT and 123I-Iodocholesterol scintigraphy before considering surgery. A genetic screening was performed, testing for ARMC5 and PRKAR1A pathogenic variants. View Full Size Figure 1 Adrenal CT depicting the bilateral macronodular adrenocortical hyperplasia. Citation: Endocrinology, Diabetes & Metabolism Case Reports 2021, 1; 10.1530/EDM-21-0071 Download Figure Download figure as PowerPoint slide Table 1 Serum cortisol levels at diagnosis (A), using ketoconazole monotherapy (B), using osilodrostat monotherapy (C) and using osilodrostat–ketoconazole combination therapy (D). Serum cortisol (nmol/L) 08:00 h 24:00 h 16:00 h 20:00 h 12:00 h 16:00 h A. At diagnosis 660 615 716 566 541 561 B. Ketoconazole monotherapy 741 545 502 224 242 508 C. Osilodrostat monotherapy 658 637 588 672 486 692 D. Osilodrostat–ketoconazole combination 436 172 154 103 135 274 Table 2 Salivary cortisol levels at diagnosis (A), using ketoconazole monotherapy (B), using osilodrostat monotherapy (C) and using osilodrostat-ketoconazole combination therapy (D). Salivary cortisol (nmol/L) 23:00 h 12:00 h 13:00 h Mean A. At diagnosis 47 62 38 49 B. Ketoconazole monotherapy 20 15 21 18 C. Osilodrostat monotherapy 85 90 56 77 D. Osilodrostat–ketoconazole combination 10 14 9 11 Treatment As this condition occurred during the COVID-19 pandemic, it was decided to first initiate steroidogenesis inhibitors to lower the patient’s cortisol levels. Initially, ketoconazole was initiated and uptitrated up to 1000 mg per day based on close serum cortisol monitoring, with a three-fold increase of liver enzymes and poor control of cortisol levels (Table 1). In the absence of biological efficacy, ketoconazole was replaced by osilodrostat, which was gradually increased up to 30 mg per day (10 mg at 08:00 h and 20 mg at 20:00 h) without reaching normal cortisol levels (Table 1) and with slightly increased blood pressure levels. Considering the lack of efficacy of anticortisolic drugs used as monotherapy, we combined osilodrostat (30 mg per day) to ketoconazole (600 mg per day), that is, at the last maximal tolerated dose as monotherapy of each drug. Outcome This combination of steroidogenesis inhibitors achieved a good control in cortisol levels, mimicking a physiological circadian rhythm (Table 1D). The patient did not exhibit any side effect and the control of cortisol levels resulted in a rapid improvement of hypertension, kalemia, diabetes control and disappearance of lower limbs oedema. The patient underwent a 18F-FDG PET-CT that did not exhibit any increased uptake in both adrenal masses and a 123I-Iodocholesterol scintigraphy exhibiting a highly increased uptake in both adrenal masses, predominating in the left adrenal mass (70 mm). Unilateral adrenalectomy of the larger mass was then performed, and as the immediate post-operative serum cortisol level was 50 nmol/L, hydrocortisone was administered at a dose of 30 mg per day, with a stepwise decrease to 10 mg per day over 3 months. Pathological examination exhibited macronodular adrenal hyperplasia with a 70-mm adreno cortical adenoma (WEISS score: 1 and Ki67: 1%). The genetic screening exhibited a c.1908del p.(Phe637Leufs*6) variant of ARMC5 (pathogenic), located in exon 5. The patient has no offspring and is no longer in contact with the rest of his family. Discussion The goal of the treatment with steroidogenesis inhibitors is normalization of cortisol production allowing the improvement of comorbidities (1). Most studies dealing with currently available steroidogenesis inhibitors used as monotherapy reported an overall antisecretory efficacy of roughly 50% in CS. This rate of efficacy was probably underestimated in retrospective studies due to the lack of adequate uptitration of the dose; For example, the median dose reported in the French retrospective study on ketoconazole was only 800 mg/day, while 50% of the patients were uncontrolled at the last follow-up (2). Steroidogenesis inhibitors can be combined to better control hypercortisolism. Up to now, such combinations, mainly ketoconazole and metyrapone, were mainly reported in patients with severe CS (median urinary-free Cortisol (UFC) 30- to 40-fold upper-limit norm (ULN)) and life-threatening comorbidities (3, 4). Normal UFC was reported in up to 86% of these patients treated with high doses of ketoconazole and metyrapone. Expected side effects (such as increased liver enzymes for ketoconazole or worsened hypertension and hypokalemia for metyrapone) were reported in the majority of the patients. The fear of these side effects probably explains the lack of uptitration in previous reports. Combination of steroidogenesis inhibitors has previously been described by Daniel et al. in the largest study reported on the use of metyrapone in CS; 29 patients were treated with metyrapone and ketoconazole or mitotane, including 22 in whom the second drug was added to metyrapone monotherapy because of partial efficacy or adverse effects. The final median metyrapone dose in patients controlled with combination therapy was 1500 mg per day (5). Combination of adrenal steroidogenesis inhibitors should not be reserved to patients with severe hypercortisolism. In the case shown here, the association was highly effective in terms of secretion, using lower doses than those applied as a single treatment, but without the side effects previously observed with higher doses of each treatment used as a monotherapy. To our knowledge, the association of ketoconazole and osilodrostat had never been reported. Ketoconazole blocks several enzymes of the adrenal steroidogenesis such as CYP11A1, CYP17, CYP11B2 (aldosterone synthase) and CYP11B1 (11-hydroxylase), leading to decreased cortisol and occasionally testosterone concentrations. Though liver enzymes increase is not dose-dependent, it usually happens at doses exceeding 400–600 mg per day (2). Osilodrostat blocks CYP11B1 and CYP11B2; a combination should thus allow for a complete blockade of these enzymes that are necessary for cortisol secretion. Short-term side effects such as hypokalemia and hypertension are similar to those observed with metyrapone, due to increased levels of the precursor deoxycorticosterone, correlated with the dose of osilodrostat (6). As for our patient, the occurrence of side effects should not lead to immediately switch to another drug, but rather to decrease the dose and add another cortisol-lowering drug. Moreover, considering the current cost of newly-released drugs such a strategy could lower financial costs for patients and/or society. Another point to take into account is the current COVID-19 pandemic, for which, as recently detailed in experts’ opinion (7), the main aim is to reach eucortisolism, whatever the way. Indeed patients presenting with CS usually also present with comorbidities such as obesity, hypertension, diabetes mellitus and immunodeficiency (8). Surgery, which represents the gold standard strategy in the management of CS (1, 9), might be delayed to reduce the hospital-associated risk of COVID-19, with post-surgical immunodepression and thromboembolic risks (7). Because immunosuppression and thromboembolic diathesis are common CS features (9, 10), during the COVID-19 pandemic, the use of steroidogenesis inhibitors appears of great interest. In these patients, combing steroidogenesis inhibitors at intermediate doses might allow for a rapid control of hypercortisolism without risks of major side effects if a single uptitrated treatment is not sufficient. Obviously, the management of associated comorbidities would also be crucial in this situation (11). To conclude, we report for the first time a case of CS, in the context of primary bilateral macronodular adrenocortical hyperplasia successfully treated with a well-tolerated combination of ketoconazole and osilodrostat. While initial levels of cortisol were not very high, we could not manage to control hypercortisolism with ketoconazole monotherapy, and could not increase the dose due to side effects. The same result was observed with another steroidogenesis inhibitor, osilodrostat. This strategy could be considered for any patient with uncontrolled hypercortisolism and delayed or unsuccessful surgery, especially in the context of the COVID-19 pandemic. Declaration of interest F C and T B received research grants from Recordati Rare Disease and HRA Pharma Rare Diseases. Frederic Castinetti is on the editorial board of Endocrinology, Diabetes and Metabolism case reports. Frederic Castinetti was not involved in the review or editorial process for this paper, on which he is listed as an author. Funding This work did not receive any specific grant from any funding agency in the public, commercial or not-for-profit sector. Patient consent Informed written consent has been obtained from the patient for publication of the case report. Author contribution statement V A was the patient’s physician involved in the clinical care and collected the data. T B and F C supervised the management of the patient. F C proposed the original idea of this case report. V A drafted the manuscript. F C critically reviewed the manuscript. T B revised the manuscript into its final version. References 1↑ Nieman LK, Biller BMK, Findling JW, Murad MH, Newell-Price J, Savage MO, Tabarin A & Endocrine Society. Treatment of Cushing’s syndrome: an Endocrine Society clinical practice guideline. Journal of Clinical Endocrinology and Metabolism 2015 100 2807–2831. (https://doi.org/10.1210/jc.2015-1818) Search Google Scholar Export Citation 2↑ Castinetti F, Guignat L, Giraud P, Muller M, Kamenicky P, Drui D, Caron P, Luca F, Donadille B & Vantyghem MC et al.Ketoconazole in Cushing’s disease: is it worth a try? Journal of Clinical Endocrinology and Metabolism 2014 99 1623–1630. (https://doi.org/10.1210/jc.2013-3628) Search Google Scholar Export Citation 3↑ Corcuff JB, Young J, Masquefa-Giraud P, Chanson P, Baudin E, Tabarin A. Rapid control of severe neoplastic hypercortisolism with metyrapone and ketoconazole. European Journal of Endocrinology 2015 172 473–481. (https://doi.org/10.1530/EJE-14-0913) Search Google Scholar Export Citation 4↑ Kamenický P, Droumaguet C, Salenave S, Blanchard A, Jublanc C, Gautier JF, Brailly-Tabard S, Leboulleux S, Schlumberger M & Baudin E et al.Mitotane, metyrapone, and ketoconazole combination therapy as an alternative to rescue adrenalectomy for severe ACTH-dependent Cushing’s syndrome. Journal of Clinical Endocrinology and Metabolism 2011 96 2796–2804. (https://doi.org/10.1210/jc.2011-0536) Search Google Scholar Export Citation 5↑ Daniel E, Aylwin S, Mustafa O, Ball S, Munir A, Boelaert K, Chortis V, Cuthbertson DJ, Daousi C & Rajeev SP et al.Effectiveness of metyrapone in treating Cushing’s syndrome: a retrospective multicenter study in 195 patients. Journal of Clinical Endocrinology and Metabolism 2015 100 4146–4154. (https://doi.org/10.1210/jc.2015-2616) Search Google Scholar Export Citation 6↑ Pivonello R, Fleseriu M, Newell-Price J, Bertagna X, Findling J, Shimatsu A, Gu F, Auchus R, Leelawattana R & Lee EJ et al.Efficacy and safety of osilodrostat in patients with Cushing’s disease (LINC 3): a multicentre phase III study with a double-blind, randomised withdrawal phase. Lancet: Diabetes and Endocrinology 2020 8 748–761. (https://doi.org/10.1016/S2213-8587(2030240-0) Search Google Scholar Export Citation 7↑ Newell-Price J, Nieman LK, Reincke M, Tabarin A. ENDOCRINOLOGY IN THE TIME OF COVID-19: Management of Cushing’s syndrome. European Journal of Endocrinology 2020 183 G1–G7. (https://doi.org/10.1530/EJE-20-0352) Search Google Scholar Export Citation 8↑ Kakodkar P, Kaka N, Baig MN. A comprehensive literature review on the clinical presentation, and management of the pandemic coronavirus disease 2019 (COVID-19). Cureus 2020 12 e7560. (https://doi.org/10.7759/cureus.7560) Search Google Scholar Export Citation 9↑ Pivonello R, De M, Cozzolino A, Colao A. The treatment of Cushing’s disease. Endocrine Reviews 2015 36 385–486. (https://doi.org/10.1210/er.2013-1048) Search Google Scholar Export Citation 10↑ Hasenmajer V, Sbardella E, Sciarra F, Minnetti M, Isidori AM, Venneri MA. The immune system in Cushing’s syndrome. Trends in Endocrinology and Metabolism 2020 31 655–669. (https://doi.org/10.1016/j.tem.2020.04.004) Search Google Scholar Export Citation 11↑ Pivonello R, Ferrigno R, Isidori AM, Biller BMK, Grossman AB, Colao A. COVID-19 and Cushing’s syndrome: recommendations for a special population with endogenous glucocorticoid excess. Lancet: Diabetes and Endocrinology 2020 8 654–656. (https://doi.org/10.1016/S2213-8587(2030215-1) Search Google Scholar Export Citation From https://edm.bioscientifica.com/view/journals/edm/2021/1/EDM21-0071.xml?body=fullHtml-9967
  18. Cushing’s disease is a progressive pituitary disorder in which there is an excess of cortisol in the body. While the disease can be treated surgically, this option is not possible for all patients. This is one of the approved medications that assist in controlling cortisol levels in people with Cushing’s disease. Recorlev Recorlev was approved by the FDA in December 2021 to treat those Cushing’s patients for whom surgery is not a choice or has failed to lower cortisol levels. The medication is an oral cortisol synthesis inhibitor that prevents the adrenal glands — sitting atop the kidneys — from producing too much cortisol, thereby easing Cushing’s symptoms. Recorlev (levoketoconazole) is a treatment that Strongbridge Biopharma — now acquired by Xeris Pharmaceuticals — developed for endogenous Cushing’s syndrome. Endogenous Cushing’s is a form of the disease in which symptoms occur because the body produces too much cortisol. Abnormally high cortisol levels in Cushing’s syndrome may be primarily due to a tumor in the brain’s pituitary gland — a type of the condition called Cushing’s disease. The first treatment option is surgery to remove those tumors. However, in some patients, this procedure is not an option or is ineffective at lowering cortisol levels. Recorlev was approved by the U.S. Food and Drug Administration (FDA) in December 2021 to treat those Cushing’s patients for whom surgery is not a choice or has failed to lower cortisol levels. How does Recorlev works? Cortisol plays several important roles in the body, including regulating salt and sugar levels, blood pressure, inflammation, breathing, and metabolism. Too much cortisol, however, throws the body off balance, causing a wide range of symptoms, such as obesity, high blood sugar levels, bone problems, and fatigue. Recorlev is an oral cortisol synthesis inhibitor that prevents the adrenal glands — sitting atop the kidneys — from producing too much cortisol, thereby easing Cushing’s symptoms. Recorlev in clinical trials Recorlev’s approval was mainly supported by data from two Phase 3 clinical trials: one called SONICS (NCT01838551) and the other LOGICS (NCT03277690). SONICS was a multicenter, open-label, three-part trial that evaluated the safety and effectiveness of Recorlev in 94 patients with endogenous Cushing’s syndrome who were not candidates for radiation therapy or surgery, and whose cortisol levels in the urine were at least 1.5 times higher than normal. Top-line data from SONICS revealed that nearly a third of patients saw their urinary cortisol levels drop to a normal range after six months of maintenance treatment with Recorlev, without requiring any dose increments in that period of time. A subgroup analysis of the study also showed Recorlev helped control cortisol and blood sugar levels in patients with both Cushing’s and diabetes. The study also showed that Recorlev was able to lessen symptoms, ease depression, and improve patients’ quality of life. LOGICS was a double-blind, randomized, withdrawal and rescue study that assessed the safety, efficacy, and pharmacological properties of Recorlev in patients with endogenous Cushing’s syndrome who had previously participated in SONICS, or who had never been treated with Recorlev. After a period of taking Recorlev, some participants were switched to a placebo while others remained on the medication. This design allowed researchers to assess the effects of treatment withdrawal. According to patients who stopped using Recorlev and moved to a placebo saw their urine cortisol levels rise in response to the lack of treatment, compared with those who remained on Recorlev. Additional data from the study also showed that patients who switched to a placebo lost Recorlev’s cholesterol-lowering benefits. Safety data from an ongoing open-label Phase 3 extension study called OPTICS (NCT03621280) also supported Recorlev’s approval. This trial, which is expected to conclude in June 2023, is designed to assess the long-term effects of Recorlev in patients who completed one or both previous studies, for up to three years. Other details Recorlev’s starting dose is 150 mg twice daily and should be taken orally with or without food. The maximum recommended dose is 1,200 mg per day, given as 600 mg twice daily. The most common side effects associated with Recorlev include nausea, vomiting, increased blood pressure, abnormally low blood potassium levels, fatigue, headache, abdominal pain, and unusual bleeding. Liver enzymes should be monitored before and during the treatment since this therapy can cause hepatotoxicity, or liver damage, in some individuals. For this reason, it is contraindicated in people with liver diseases such as cirrhosis. Recorlev should be immediately stopped if signs of hepatotoxicity are observed. Recorlev also can influence heartbeat. As such, patients with certain heart conditions should be closely monitored before and during treatment. Hypocortisolism, or lower-than-normal levels of cortisol, also may occur during treatment with Recorlev. For this reason, patients should have their cortisol levels closely monitored, and lessen or interrupt treatment if necessary. Recorlev interacts with medicines on which certain liver enzymes act, such as CYP3A4. Treatment also is an inhibitor of P-gp, OCT2, and MATE1, which are transporters of certain medicines. The use of Recorlev with these medicines may increase the risk of adverse reactions.
  19. Neuroendocrine pulmonary tumors in people with Cushing syndrome (CS) are associated with increased nodal metastasis, higher recurrence, and lower disease-free survival compared with quiescent bronchopulmonary tumors, according to results from an observational case series published in JAMA Network Open. Researchers said their study shows these tumors are not biologically aggressive and underlying carcinoid biology may not be as important as symptomatic hormonal physiology. Patients (n=68) with CS who underwent curative-intent pulmonary surgery at the National Cancer Institute (NCI) between 1982 and 2020 were retrospectively reviewed for clinical outcomes on the basis of tumor etiology. Outcomes were compared among groups of patients with adrenocorticotropic hormone-secreting carcinoid tumors who were treated at the National Institutes of Health in 2021 (n=68), Hôpital Européen Georges-Pompidou in 2011 (n=14), the Mayo Clinic in 2005 (n=23), and Massachusetts General Hospital in 1997 (n=7). Patients who underwent surgery at the NCI were aged median 41 years (range, 17-80 years), 42.6% were men, 81.8% were White, and mean follow-up after surgery was 16 months (range, 0.1-341 months). Most patients had T status 1a (55.9%). The pathological stages were IA1 (37.3%), IA2 (23.7%), IA3 (1.7%), IIB (16.9%), IIIA (20.3%), or unknown (13.2%). The patients with typical carcinoid tumors (83.8%) underwent lobectomy (70.2%), wedge (22.8%), segmentectomy (5.3%), and pneumonectomy (1.7%) surgical approaches. Patients with atypical carcinoid tumors (16.2%) underwent lobectomy (72.7%) and wedge (27.3%) approaches. Stratified by surgical approach, lobectomy recipients were younger (P =.01) and more had node-positive atypical carcinoid tumors (P =.01). After surgery, morbidity occurred among 19.1% of patients; overall mortality was 1.5%. Disease-free survival at 5 years following surgery was 73.4% (95% CI, 48.7%-87.6%) and 55.1% (95% CI, 26.3%-76.5%) at 10 years. Disease-free survival was 75.4% (95% CI, 49.2%-89.3%) at 5 years and 50.2% (95% CI, 18.3%-75.7%) at 10 years for typical carcinoid tumors and remained stable at 75.0% among those with atypical carcinoid tumors. Median follow-up after surgery was 16 months (range, 0.1-341 months). At the time of last follow-up, 76.4% of the patient population was alive and tumor free. The overall incidence of persistence/recurrence was 16.2%. Recurrent disease occurred in 7 patients and persistent disease in 4 patients. Only one of this group had an atypical carcinoid tumor. Mean time to recurrence in patients with recurrent disease was 76 months with a median of 55 months. The adrenocorticotropic hormone-secreting carcinoid cohort from multiple institutions was aged median 39 years, 46.4% were men, 72.3% underwent lobectomy or pneumonectomy, 18.7% had morbidity, and 0.9% mortality. The majority of these groups had typical carcinoid tumors (83.9%) with a mean size of 1.1 cm (range, 0.1-10 cm) and 39.4% had lymph node positivity. Recurrence occurred among 12.6% of patients and persistence among 5.4% of patients. Among the recurrence cohort, 85.7% had typical carcinoid tumors. Time to recurrence was >6 years. Disease-free survival was 73% at five years and 55% at 10 years. This study was limited by the small group sizes, however, due to the rarity of this cancer it was not possible to include more individuals. “Ectopic adrenocorticotropic hormone secreting carcinoid tumors with Cushing syndrome appear to be associated with increased metastasis to lymph nodes, higher recurrence (mostly local), and lower overall disease-free survival at 5 and 10 years than quiescent bronchial carcinoid tumors, irrespective of histologic subtype,” the researchers wrote. “Nevertheless, we contend these tumors are not biologically aggressive since these patients have distinct, prolonged survival and delayed time to recurrence.” The researchers also noted that “the current staging system applied to these tumors raises questions about prognostic accuracy. Extrapolation may suggest that the underlying carcinoid biology may not be as important as the symptomatic hormonal physiology.” They suggested future studies may test “whether a lung-sparing surgical approach coupled with routine lymphadenectomy is an optimal intervention in this scenario when normal endocrine functioning is restored and CS sequelae resolve.” Reference Seastedt KP, Alyateem GA, Pittala K, et al. Characterization of outcomes by surgical management of lung neuroendocrine tumors associated with Cushing syndrome. JAMA Netw Open. 2021;4(9):e2124739. doi:10.1001/jamanetworkopen.2021.24739 From https://www.endocrinologyadvisor.com/home/topics/general-endocrinology/cushing-syndrome-and-lungs-and-neuoendocrine-tumors/
  20. Ahead of its New Year's Day decision deadline at the FDA, Xeris Biopharma has snagged an approval for Recorlev, a drug formerly known as levoketoconazole. Based on results from phase 3 studies called SONICS and LOGICS, the FDA approved the drug for adults with Cushing’s syndrome. Xeris picked up Recorlev earlier this year in its acquisition of rare disease biotech Strongbridge Biopharma. It's planning to launch in the first quarter of 2022. Recorlev's approval covers the treatment of endogenous hypercortisolemia in adults with Cushing’s syndrome who aren't eligible for surgery or haven't responded to surgery. Endogenous Cushing's disease is caused by a benign tumor in the pituitary gland that prompts the body to produce elevated levels of cortisol, which over time triggers a range of devastating physical and emotional symptoms for patients. In the SONICS study, the drug significantly cut and normalized mean urinary free cortisol concentrations without a dose increase, according to the company. The LOGICS trial confirmed the drug's efficacy and safety, Xeris says. Cushion's is a potentially fatal endocrine disease, and patients often experience years of symptoms before an accurate diagnosis, the company says. After a diagnosis, they're presented with limited effective treatment options. Following the approval, the company's "experienced endocrinology-focused commercial organization can begin rapidly working to help address the needs of Cushing’s syndrome patients in the U.S. who are treated with prescription therapy,” Xeris CEO Paul R. Edick said in a statement. Aside from its forthcoming Recorlev launch, Xeris markets Gvoke for severe hypoglycemia and Keveyis for primary periodic paralysis. Back in October, the company partnered up with Merck to help reformulate some of the New Jersey pharma giant's monoclonal antibody drugs. From https://www.fiercepharma.com/pharma/xeris-biopharma-scores-fda-approval-for-endogenous-cushing-s-syndrome-drug-recorlev
  21. This article was originally published here J Endocr Soc. 2021 Nov 24;6(1):bvab176. doi: 10.1210/jendso/bvab176. eCollection 2022 Jan 1. ABSTRACT CONTEXT: Acromegaly (ACM) and Cushing’s disease (CD) are caused by functioning pituitary adenomas secreting growth hormone and ACTH respectively. OBJECTIVE: To determine the impact of race on presentation and postoperative outcomes in adults with ACM and CD, which has not yet been evaluated. METHODS: This is a retrospective study of consecutive patients operated at a large-volume pituitary center. We evaluated (1) racial distribution of patients residing in the metropolitan area (Metro, N = 124) vs 2010 US census data, and(2) presentation and postoperative outcomes in Black vs White for patients from the entire catchment area (N = 241). RESULTS: For Metro area (32.4% Black population), Black patients represented 16.75% ACM (P = .006) and 29.2% CD (P = .56). Among the total 112 patients with ACM, presentations with headaches or incidentaloma were more common in Black patients (76.9% vs 31% White, P = .01). Black patients had a higher prevalence of diabetes (54% vs 16% White, P = .005), significantly lower interferon insulin-like growth factor (IGF)-1 deviation from normal (P = .03) and borderline lower median growth hormone levels (P = .09). Mean tumor diameter and proportion of tumors with cavernous sinus invasion were similar. Three-month biochemical remission (46% Black, 55% White, P = .76) and long-term IGF-1 control by multimodality therapy (92.3% Black, 80.5% White, P = .45) were similar. Among the total 129 patients with CD, Black patients had more hypopituitarism (69% vs 45% White, P = .04) and macroadenomas (33% vs 15% White, P = .05). At 3 months, remission rate was borderline higher in White (92% vs 78% Black, P = 0.08), which was attributed to macroadenomas by logistic regression. CONCLUSION: We identified disparities regarding racial distribution, and clinical and biochemical characteristics in ACM, suggesting late or missed diagnosis in Black patients. Large nationwide studies are necessary to confirm our findings. PMID:34934883 | PMC:PMC8677529 | DOI:10.1210/jendso/bvab176 From https://www.docwirenews.com/abstracts/journal-abstracts/racial-disparities-in-acromegaly-and-cushings-disease-a-referral-center-study-in-241-patients/
  22. 6. Cushing syndrome This disorder occurs when your body makes too much of the hormone cortisol over a long period of time, according to the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK). Although cortisol is notorious for driving up your stress, this hormone has other tasks on its docket, including regulating the way you metabolize food, the Mayo Clinic says. So, when you produce too much of it, it can interfere with your metabolism and cause you to gain weight, Peter LePort, M.D., a bariatric surgeon and medical director of MemorialCare Surgical Weight Loss Center at Orange Coast Medical Center in Fountain Valley, California, tells SELF. Beyond weight gain, symptoms of Cushing syndrome include deposits of fat-based tissue at the midsection, upper back, face, and between the shoulders, stretch marks due to rapid weight gain, thinning skin prone to bruising, increased body hair, irregular or missing periods, and more, according to the Mayo Clinic. Check out the other 12 at https://www.self.com/story/conditions-weight-gain-loss
  23. A retrospective analysis of data from more than 170 patients with Cushing syndrome and hyperglycemia provides insight into the effects of curative treatment on hyperglycemia among these patients. Irina Bancos, MD An analysis of retrospective data from a 20-year period details the impact of curative treatment on hyperglycemia among patients with Cushing syndrome. Led by a team of investigators from the Mayo Clinic in Rochester, MN, the study examined a cohort of 174 adult patients with Cushing Syndrome and determined 2-in-3 patients with hyperglycemia experienced resolution or improvement of hyperglycemia after a curative procedure. “This is the first study to analyze the quantitative changes based on the time from the curative surgery, to assess the changes in the intensity of hyperglycemia therapy and identify predictors for hyperglycemia improvement,” wrote investigators. A team led by Irina Bancos, MD, endocrinologist at the Mayo Clinic Rochester, designed the current study with an interest in examining the impact of curative procedures on hyperglycemia and its management in patients with Cushing syndrome from electronic medical record data of patients treated at a referral center from 2000-2019. The primary purpose of the study was to assess the impact of curative procedures on extent of hyperglycemia and the secondary aim was to investigators how baseline factors might influence improvement of hyperglycemia at follow-up. For inclusion in the analysis, patients needed to be at least 18 years of age, diagnosed with Cushing syndrome, and have hyperglycemia treated with a curative procedure from January 1, 2000-November 1, 2019. For the purpose of analysis, Cushing syndrome was diagnosed based on clinical evaluation by an endocrinologist and diagnosed according to the most recent guidelines. Hyperglycemia was defined according to American Diabetes Association guidelines. The primary outcome of interest for the study was the resolution of hyperglycemia following resolution of Cushing syndrome. For the purpose of analysis, resolution was defined as absence of hyperglycemia without the need for antihyperglycemic therapy. Secondary outcomes of interest included changes in HbA1c, and the intensity of hyperglycemia management. Overall, 174 patients were identified for inclusion in the study. This cohort had a median age of diagnosis of 51 (range, 16-82) years and 73% (n=127) were women. When assessing subtype of Cushing syndrome, the most common form was pituitary Cushing syndrome (60.9%), followed by ectopic (14.4%), and adrenal (24.7%). The median baseline HbA1c was 6.9% (range, 4.9-13.1), 24% of patients were not on any therapy for hyperglycemia, 52% were on oral medications, and 37% were on insulin (mean daily units, 58; range, 10-360). When assessing differences between subtypes, results indicated those with pituitary Cushing syndrome were younger at the time of surgery (P=.0009), and included more women (P=.0023), and reported a longer duration of symptoms prior to diagnosis. Investigators noted patients with pituitary Cushing syndrome also had the highest clinical severity score (P <.0001), but patients with ectopic Cushing syndrome had the highest biochemical severity score (P <.0001). Following Cushing syndrome remission and at the end of follow-up, which occurred at a median of 10.5 months, 21% of patients demonstrated resolution of hyperglycemia, 47% demonstrated improvement, and 32% had no change or worsening hyperglycemia. When assessing secondary end points, results indicate HbA1c decreased by 0.84% (P <.0001) and daily insulin dose decreased by a mean of 30 units (P <.0001). Further analysis indicated hypercortisolism severity score (severe vs moderate/mild: OR, 2.4; 95% CI, 1.1-4.9) and Cushing syndrome subtype (nonadrenal vs adrenal: OR, 2.9; 95% CI, 1.3-6.4) were associated with hyperglycemia improvement, but not type of hyperglycemia (diabetes vs prediabetes: OR, 2,1; 95% CI, 0.9-4.9) at the end of follow-up. “We demonstrated that almost 70% of patients with CS demonstrate either resolution or improvement in hyperglycemia following CS remission. As a group, patients demonstrate a decrease in HbA1c, and can be treated with less insulin and fewer non-insulin agents. Patients with more severe hyperglycemia, ACTH-dependent CS, and more severe CS are more likely to improve after surgery,” added investigators. This study, “The impact of curative treatment on hyperglycemia in patients with Cushing syndrome,” was published in The Journal of the Endocrine Society. From https://www.endocrinologynetwork.com/view/obesity-overweight-responsible-for-1-in-5-future-thyroid-cancers-in-australia
  24. https://doi.org/10.1016/j.amsu.2021.102978Get rights and content Under a Creative Commons license open access Highlights • Cushing syndrome is an abnormality resulting from high level of blood glucocorticoids. • Iatrogenic Cushing syndrome due to the overuse of topical corticosteroids is rarely reported. • This report presents a case of topical corticosteroid induced iatrogenic Cushing syndrome in an infant. Abstract Introduction Cushing syndrome (CS) is an endocrinological abnormality that results from a high level of glucocorticoids in the blood. Iatrogenic CS due to the overuse of topical corticosteroids is rarely reported. The current study aims to present a rare case of topical corticosteroid induced iatrogenic CS in an infant. Case presentation A 4-month-old female infant presented with an insidious onset of face puffiness that progressed over a 2-month period. The mother reported to have used a cream containing Betamethasone corticosteroid 5–8 times a day for a duration of 3 months to treat diaper dermatitis. Laboratory findings revealed low levels of adrenocorticotrophic hormone (ACTH) and serum. Abdominal ultrasound showed normal adrenal glands. The topical corticosteroid was halted and physiologic topical hydrocortisone doses were administered. Clinical discussion Infants are more likely to acquire topical corticosteroid induced iatrogenic CS due to their thin and absorptive skin, higher body surface area, and the high prevalence of conditions that necessitates the use of these medications. Most iatrogenic CS cases following topical steroid application have been reported in infants with diaper dermatitis that are most commonly treated with Clobetasol and Bethamethasone. Conclusion Infants are susceptible to develop CS due to topical corticosteroid overuse. Hence, physicians need to consider this in infantile CS cases, and take appropriate measures to avoid their occurrence. Previous article in issue Next article in issue Keywords Cushing syndrome Infant Iatrogenic Topical corticosteroid 1. Introduction Cushing syndrome (CS) is a reversible endocrinological abnormality that results from high level of cortisol or other glucocorticoids in the blood [1]. It can be caused by either endogenous factors such as excess steroid production and secretion due to adrenal or pituitary tumors, or exogenously through prolonged use of corticosteroid medications resulting in iatrogenic CS [2]. Iatrogenic CS due to the overuse of oral or parenteral corticosteroids is common, however, while topical corticosteroids are one of the most widely prescribed medications by dermatologists, they are less frequently reported to cause iatrogenic CS [3,4]. Even though CS is very rare in the pediatric population with an annual incidence of only 5 cases per million, children of the pediatric age have a higher risk of developing iatrogenic CS, which is likely due to the high prevalence of conditions that necessitates the use of topical corticosteroids and the thinness of their skin that can more easily absorb the steroid [5,6]. The aim of the current study is to present a rare case of topical corticosteroid induced iatrogenic CS in an infant. SCARE guidelines are considered in writing this report [7]. 2. Case presentation 2.1. Patient information A 4-month-old female infant presented with an insidious onset of puffiness of the face; the swelling progressed over a period of 2 months without any other associated symptoms. The infant's prenatal, developmental, and family history were insignificant, and she was born full term to consanguineous parents via caesarian delivery. After delivery she did not require neonatal intense care unit (NICU) and was discharged in good health. She has been given both bottle and breastfeeding every one to two hrs, and she has received all the required vaccinations at their proper times. The mother reported to have used a topical corticosteroid cream (Optizol-B cream; a combination of Clotrimazole and Betamethasone) for a period of 3 months with a dose of 5–8 times a day to treat diaper dermatitis of the infant. 2.2. Clinical findings The infant's physical examination revealed facial puffiness (Moon face) with no body edema, and cutaneous examination showed the diaper rash without any other cutaneous manifestations. The infant was vitally stable with no dysmorphic features and no skeletal deformities. Her growth parameters were within normal limits, and her systemic examination was unremarkable. 2.3. Diagnostic approach Laboratory findings revealed low adrenocorticotropic hormone (ACTH) level in the blood measuring 5.9 p.m./l, a serum cortisol level of 24 nmol/l, and normal serum sodium and potassium levels of 144 mEq/l and 4.8 mmol/l, respectively. Abdominal ultrasonography (US) showed normal adrenal glands. 2.4. Therapeutic intervention The topical corticosteroid cream that contained Bethamethasone was halted and oral hydrocortisone was given (10 mg/m2) tapered over one month. The patient was given a card addressing Cushing syndrome to inform the health care providers in case of emergency situation or unexpected surgical intervention. 2.5. Follow-up and outcome The infant's facial puffiness was significantly improved after 7-month follow-up of the patient. 3. Discussion CS is an endocrinological disorder resulting from high glucocorticoid level in the blood, it is categorized into ACTH dependent (due to pituitary tumors or excess ACTH administration) or ACTH independent CS (due to adrenal neoplasms or excessive glucocorticoid intake) [8,9]. Under normal circumstances, ACTH is secreted by the pituitary gland which in turn stimulates the secretion of cortisol by the adrenal glands [10]. Prolonged exogenous corticosteroid administration can lead to a number of adverse effects based on potency and duration of the treatment, including the suppression of hypothalamic-pituitary-adrenal (HPA) axis and iatrogenic CS, severe infections, and failure to thrive [11]. While iatrogenic CS is frequent with prolonged administration of oral or parenteral corticosteroids, it is occurrence due to topical corticosteroids have rarely been reported [12]. Multiple factors can increase the probability of acquiring the condition, such as corticosteroid potency, amount and frequency of application, age, skin quality, presence of occlusion, and duration of application [4]. In general, infants are more likely to develop topical corticosteroid induced iatrogenic CS, this is due to their thin and absorptive skin, higher body surface area, underdeveloped skin barrier, and the high prevalence of conditions that necessitates the use of these medications [5,6]. Most iatrogenic CS cases following topical steroid application have been reported in infants with diaper dermatitis [8]. This was also the case in this study. This is likely because the diaper area provides occlusion, the perineal skin has intrinsically absorptive properties, the steroid causes local skin atrophy, and percutaneous absorption is even more increased as the result of skin inflammation [13]. The most frequently used corticosteroid for the treatment of diaper dermatitis is reported to be Clobetasol followed by Bethamethasone, with a mean application duration of 2.75 (1–17) months to induce cortisol and ACTH levels suppression [4]. Typical clinical manifestations of CS include facial puffiness (Moon face), generalized body edema and obesity, hirsutism, buffalo hump, hypertension, skin fragility, and purple striae [3,5]. The causative corticosteroid in the current case was Bethamethasone that only resulted in facial puffiness (Moon face) without generalized body edema. A specific and definitive diagnostic approach for iatrogenic CS is currently lacking [5]. However, prolonged exogenously administered glucocorticoids can suppress ACTH secretion which results in dismissing the need for proper endogenous production of cortisol [14]. Hence, almost all iatrogenic CS cases are associated with low ACTH and cortisol levels which can aid in the diagnosis of the condition [8]. Same findings were observed in this case. According to multiple studies, exogenous corticosteroid administration can often lead to HPA axis suppression alongside CS [15,16]. However, topical corticosteroid induced iatrogenic CS has been reported without HPA axis suppression [8]. The management of these cases start with the cessation of the causative corticosteroid medication and administration of physiologic topical hydrocortisone [5]. The same approach was followed in this study. In order to prevent the development of this condition in the first-place; clinicians should avoid prescribing high potency corticosteroids in the treatment of infantile dermatological disorders and instead choose low potency topical steroids, and also parents should be advised not to overuse these medications and only apply a thin layer to the affected area [6]. In conclusion, even though iatrogenic CS in infants is rare, overuse of topical corticosteroids can lead to their occurrence. Hence, physicians need to consider extensive steroid use as a causative agent of infantile CS. Appropriate measures need to be taken to avoid their occurrence by prescribing less potent steroids, limiting the use of high potent steroids, and informing parents about adverse effects of steroid overuse in infants. Source of funding None is found. Author statement Soran Mohammed Ahmed: physician managing the case, follow up the patient, and final approval of the manuscript. Shaho F. Ahmed, Snur Othman, Berwn A. Abdulla, Shvan M.Hussein, Abdulwahid M.Salih, and Fahmi H. Kakamad: literature review, writing the manuscript, final approval of the manuscript. Patient consent Written informed consent was obtained from the patient for publication of this case report and accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal on request. Provenance and peer review Not commissioned, externally peer-reviewed. Guarantor Fahmi Hussein Kakamad. 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  25. Endocrinology Research Centre, 117292 Moscow, Russia Author to whom correspondence should be addressed. Academic Editor: Spyridon N. Karras Nutrients 2021, 13(12), 4329; https://doi.org/10.3390/nu13124329 Received: 12 November 2021 / Revised: 26 November 2021 / Accepted: 27 November 2021 / Published: 30 November 2021 (This article belongs to the Special Issue Vitamin D in the New Decade: Facts, Controversies, and Future Perspectives for Daily Clinical Practice) Download PDF Browse Figures Citation Export Abstract In this study we aimed to assess vitamin D metabolism in patients with Cushing’s disease (CD) compared to healthy individuals in the setting of bolus cholecalciferol treatment. The study group included 30 adults with active CD and the control group included 30 apparently healthy adults with similar age, sex and BMI. All participants received a single dose (150,000 IU) of cholecalciferol aqueous solution orally. Laboratory assessments including serum vitamin D metabolites (25(OH)D3, 25(OH)D2, 1,25(OH)2D3, 3-epi-25(OH)D3 and 24,25(OH)2D3), free 25(OH)D, vitamin D-binding protein (DBP) and parathyroid hormone (PTH) as well as serum and urine biochemical parameters were performed before the intake and on Days 1, 3 and 7 after the administration. All data were analyzed with non-parametric statistics. Patients with CD had similar to healthy controls 25(OH)D3 levels (p > 0.05) and higher 25(OH)D3/24,25(OH)2D3 ratios (p < 0.05) throughout the study. They also had lower baseline free 25(OH)D levels (p < 0.05) despite similar DBP levels (p > 0.05) and lower albumin levels (p < 0.05); 24-h urinary free cortisol showed significant correlation with baseline 25(OH)D3/24,25(OH)2D3 ratio (r = 0.36, p < 0.05). The increase in 25(OH)D3 after cholecalciferol intake was similar in obese and non-obese states and lacked correlation with BMI (p > 0.05) among patients with CD, as opposed to the control group. Overall, patients with CD have a consistently higher 25(OH)D3/24,25(OH)2D3 ratio, which is indicative of a decrease in 24-hydroxylase activity. This altered activity of the principal vitamin D catabolism might influence the effectiveness of cholecalciferol treatment. The observed difference in baseline free 25(OH)D levels is not entirely clear and requires further study. Keywords: vitamin D; pituitary ACTH hypersecretion; cholecalciferol; vitamin D-binding protein 1. Introduction Cushing’s disease (CD) is one of the disorders associated with endogenous hypercortisolism and is caused by adrenocorticotropic hormone (ACTH) hyperproduction originating from pituitary adenoma [1]. Skeletal fragility is a frequent complication of endogenous hypercortisolism, and fragility fractures may be the presenting clinical feature of disease. The prevalence of osteoporosis in endogenous hypercortisolism as assessed by dual-energy X-ray absorptiometry (DXA) or incidence of fragility fractures has been reported to be up to 50%. Osteoporosis in CD patients has a complex multifactorial pathogenesis, characterized by a low bone turnover and severe suppression of bone formation [2]. Exogenous glucocorticoids are used in the treatment of a wide range of diseases and it is estimated that 1–2% of the population is receiving long-term glucocorticoid therapy. As a consequence, glucocorticoid-induced osteoporosis is the most common secondary cause of osteoporosis [3]. Native vitamin D (in particular D3, or cholecalciferol) and its active metabolites (such as alfacalcidol) are universally considered as the essential components of the osteoporosis management [4,5]. The search for the optimal treatment of bone complications during chronic exposure to glucocorticoid excess provoked the investigation of vitamin D metabolism in this state. Early studies on this topic were focused predominantly on the general vitamin D status (assessed as 25(OH)D level) and on the levels of the active vitamin D metabolite (1,25(OH)2D). These studies showed inconsistent results, reporting that the chronic excess of glucocorticoids decreased [6,7,8,9], increased [10,11,12] or did not change [13,14,15] the levels of 25(OH)D or 1,25(OH)2D. A likely reason for such inconsistency might have been the high heterogeneity of the studied groups. Some of these studies were performed in humans [6,7,9,10,11,12,13,15] and some in animal models [8,14], and only several of them included subjects with specifically endogenous hypercortisolism [10,12,14,15]. Only two studies assessed both the levels of the active (1,25(OH)2D) and the inactive (24,25(OH)2D) vitamin D metabolites in endogenous hypercortisolism. One of them lacked control group and reported low-normal 24,25(OH)2D levels in patients with Cushing’s syndrome [10]. The second study by Corbee et al. reported similar circulating concentrations of 25(OH)D, 1,25(OH)2D and 24,25(OH)2D in studied groups of dogs regardless of either the presence of CD or hypophysectomy status [14]. Several experimental studies were performed to evaluate the impact of glucocorticoid excess on the enzymes involved in vitamin D metabolism. In mouse kidney glucocorticoid treatment increased 24-hydroxylase expression [16] and 24-hydroxylase activity [17]. An increased expression of 24-hydroxylase was also shown in rat osteoblastic and pig renal cell cultures treated with 1,25(OH)2D [18]. Dhawan and Christakos showed that 1,25(OH)2D-induced transcription of 24-hydroxylase was glucocorticoid receptor-dependent [19]. However, some works showed conflicting results. In particular, the steroid and xenobiotic receptor (SXR) which is activated by glucocorticoids [20], repressed 24-hydroxylase expression in human liver and intestine in work by Zhou et al. [21]. Lower 24-hydroxylase expression was observed in the brain and myocardium of glucocorticoid-treated rats [22] as well as in human osteosarcoma cells and human osteoblasts [23]. Nevertheless, based on experimental data, it has been suggested that the acceleration of 25(OH)D catabolism in the presence of glucocorticoid excess may predispose to vitamin D deficiency. Yet, relatively recent meta-analysis of the studies assessing 25(OH)D levels in chronic glucocorticoid users showed that serum 25(OH)D levels in these patients were suboptimal and lower than in healthy controls, but similar to steroid-naive disease controls [24]. Glucocorticoids also affect calcium and phosphorus homeostasis. In particular, they were shown to reduce gastrointestinal absorption by antagonizing vitamin D action (reducing the expression of genes for proteins involved in calcium transport—epithelial Ca channel TRPV6 and calcium-binding protein calbindin-D9K) [25]. Glucocorticoids increased fractional calcium excretion due to mineralocorticoid receptor-mediated action on epithelial sodium channels [26]. Hypercalciuria is highly prevalent in people with CD [27]. These effects might result in a negative calcium balance, although plasma ionized calcium was normal in people and dogs with hypercortisolism compared to control subjects [12,28]. Glucocorticoids also reduced tubular phosphate reabsorption by inhibiting tubular expression of the sodium gradient-dependent phosphate transporter, and induced phosphaturia [29], which was accompanied by phosphate lowering in humans [12]. Overall, current data on vitamin D status in hypercortisolism are conflicting and need clarification. In particular, clinical data on the state of vitamin D metabolism in the state of glucocorticoids excess are quite scarce. Studies were very heterogeneous in design, some lacked a control group, and the absolute majority of the studies were performed before the introduction of vitamin D measurement standardization [30]. Nevertheless, determining the optimal vitamin D treatment regimen in these high-risk patients is fairly relevant. The aim of this study was to assess vitamin D metabolism in patients with CD compared to healthy individuals particularly in the setting of cholecalciferol treatment. 2. Materials and Methods 2.1. Study Population and Design The study group included 30 adult patients with CD admitted for inpatient treatment at a tertiary pituitary center. Diagnosis of CD was established in accordance with the federal guidelines [31]. All patients were confirmed to be positive for endogenous hypercortisolism in at least two of the following tests: 24-h urine free cortisol (UFC) greater than the normal range for the assay and/or serum cortisol > 50 nmol/L after the 1-mg overnight dexamethasone suppression test and/or late-night salivary cortisol greater than 9.4 nmol/L). All patients also had morning ACTH ≥ 10 pg/mL and pituitary adenoma ≥ 6 mm identified by magnetic resonance imaging (MRI) or a positive for CD bilateral inferior petrosal sinus sampling (BIPSS). MRI was performed using a GE Optima MR450w 1.5T with Gadolinium (Boston, MA, USA). BIPSS was performed according to the standard procedure described elsewhere [32,33]. The control group included 30 apparently healthy adult individuals recruited from the staff and the faculty of the facility. Inclusion criteria were age from 18 to 60 for both groups and the presence of the disease activity for the study group (defined as the presence of endogenous hypercortisolism at the time of participation in the study). Exclusion criteria for both groups were: vitamin D supplementation for 3 months prior to the study; severe obesity (body mass index (BMI) ≥ 35 kg/m2); pregnancy; the presence of granulomatous disease, malabsorption syndrome, liver failure; decreased GFR (less than 60 mL/min per 1.73 m2); severe hypercalcemia (total serum calcium > 3.0 mmol/L); allergic reactions to vitamin D medications; 25(OH)D level more than 60 ng/mL (determined by immunochemiluminescence analysis). All patients were recruited in the period from October 2019 to April 2021. The study protocol (ClinicalTrials.gov Identifier: NCT04844164) was approved by the Ethics Committee of Endocrinology Research Centre, Moscow, Russia on 10 April 2019 (abstract of record No. 6), all patients signed informed consent to participate in the study. All participants received standard therapeutic dose (150,000 IU) of an aqueous solution of cholecalciferol (Aquadetrim®, Medana Pharma S.A., Sieradz, Poland) orally as a single dose [34]. Blood and urine samples were obtained before the intake as well as on days 1, 3 and 7 after administration; time points of sample collection were determined based on the authors’ previous work evaluating changes in 25(OH)D levels after a therapeutic dose of cholecalciferol [35]. The assessment included serum biochemical parameters (total calcium, albumin, phosphorus, creatinine, magnesium), parathyroid hormone (PTH), vitamin D-binding protein (DBP), vitamin D metabolites (25(OH)D3, 25(OH)D2, 1,25(OH)2D3, 3-epi-25(OH)D3 and 24,25(OH)2D3), free 25(OH)D and urine biochemical parameters (calcium- and phosphorus-creatinine ratios in spot urine). 2.2. Socio–Demographic and Anthropometric Data Collection At the baseline visit, patients underwent a questionnaire aimed to assess their lifestyle: the presence of unhealthy habits, physical activity level, balanced diet (consumption of dairy products, meat, coffee, soft drinks), exposure to ultraviolet (UV) radiation (solarium and sunscreen usage, traveling south and the number of daytime walks in the sunny weather in the 3 months preceding study participation). Smoking status was classified as current smoker, former smoker and non-smoker; current and former smokers were collectively referred to as total smokers. A unit of alcohol was defined as a glass of wine, a bottle of beer or a shot of spirits, approximating 10–12 g ethanol. Serving of dairy products was defined as 100 g of cottage cheese, 200 mL of milk, 125 g of yogurt or 30 g of cheese. Patients’ weight was measured in light indoor clothing with a medical scale to the nearest 100 g, and their height with a wall-mounted stadiometer to the nearest centimeter. BMI was calculated as weight in kilograms divided by height in meters squared. 2.3. Laboratory Measurements Morning ACTH (reference range 7–66 pg/mL), serum cortisol after a low-dose dexamethasone suppression test (cutoff value for suppression, 50 nmol/L [36]), late-night salivary cortisol (reference range 0.5–9.4 nmol/L [37]) were assayed by electrochemiluminescence assay using a Cobas 6000 Module e601 (Roche, Rotkreuz, Switzerland). The 24-h UFC (reference range 60–413 nmol/24 h) was measured by an immunochemiluminescence assay (extraction with diethyl ether) on a Vitros ECiQ (Ortho Clinical Diagnostics, Raritan, NJ, USA). Total 25(OH)D levels (25(OH)D2 + 25(OH)D3; reference range 30–100 ng/mL) at the baseline visit were determined by the immunochemiluminescence analysis (Liaison, DiaSorin, Saluggia, Italy). PTH levels were evaluated by the electrochemiluminescence immunoassay (ELECSYS, Roche, Basel, Switzerland; reference range for this and subsequent laboratory parameters are given in the Results section for easier reading). Biochemical parameters of blood serum and urine were assessed by the ARCHITECT c8000 analyzer (Abbott, Chicago, IL, USA) using reagents from the same manufacturer according to the standard methods. Serum DBP and free 25(OH)D levels were measured by enzyme-linked immunosorbent assay (ELISA) using commercial kits. The assay used for free 25(OH)D levels assessment (DIAsource, ImmunoAssays S.A., Ottignies-Louvain-la-Neuve, Belgium) has <6.2% intra- and inter-assay coefficient of variation (CV) at levels 5.8–9.6 pg/mL. The assay used for DBP levels assessment (Assaypro, St Charles, MO, USA) has 6.2% average intra-assay CV and 9.9% average inter-assay CV. The levels of vitamin D metabolites (25(OH)D3, 25(OH)D2, 1,25(OH)2D3, 3-epi-25(OH)D3 and 24,25(OH)2D3) in serum were determined by ultra-high performance liquid chromatography in combination with tandem mass spectrometry (UPLC-MS/MS) using an in-house developed method, described earlier [38]. With this technique, the laboratory participates in DEQAS quality assurance program (lab code 2388) and the results fall within the target range for the analysis of 25(OH)D and 1,25(OH)2D metabolites in human serum (Supporting Information, Figures S1 and S2). All UPLC-MS/MS measurements were made after the first successful completion (5/5 samples within the target range) of the DEQAS distributions for both analytes simultaneously. Each batch contained control samples (analytes in blank serum) with both high and low analyte concentrations. The samples were barcoded and randomized prior to the measurements to eliminate analyst-related errors. Serum samples (3 aliquots) collected at each visit were either transferred directly to the laboratory for biochemical analyzes, total 25(OH)D and PTH measurement (1 aliquot) or were stored at −80 °C avoiding repeated freeze-thaw cycles for measurement of DBP, free 25(OH)D and vitamin D metabolites at a later date (2 aliquots). Albumin-adjusted serum calcium levels were calculated using the formula [39]: total plasma calcium (mmol/L) = measured total plasma calcium (mmol/L) + 0.02 × (40 − measured plasma albumin (g/L)). Baseline free 25(OH)D levels were also calculated using the formula introduced by Bikle et al. [40,41]. The affinity constant for 25(OH)D and albumin binding (Kalb) used for the calculation was equal 6 × 105 M−1, and affinity constant for 25(OH)D and DBP binding (KDBP) was equal 7 × 108 M−1. Free 25(OH)D=total 25(OH)D1+Kalb∗albumin+KDBP∗DBP 2.4. Statistical Analysis Statistical analysis was performed using Statistica version 13.0 (StatSoft, Tulsa, OK, USA). All data were analyzed with non-parametric statistics and expressed as median [interquartile range] unless otherwise specified. Mann-Whitney U-test and Fisher’s exact two-tailed test were used for comparisons between two groups. Friedman ANOVA was performed to evaluate changes in indices throughout the study and pairwise comparisons using Wilcoxon test with adjustment for multiple comparisons (Bonferroni) were also made if the Friedman ANOVA was significant. Spearman rank correlation method was used to obtain correlation coefficients among indices. A p-value of less than 0.05 was considered statistically significant. When adjusting for multiple comparisons, a p-value greater than the significance threshold, but less than 0.05 was considered as a trend towards statistical significance. 3. Results The groups were similar in terms of age, sex and BMI (p > 0.05). Both groups consisted predominantly of young and middle-aged women and the majority of patients were overweight or moderately obese (Table 1). Patients from the study group presented with lower screening levels of total 25(OH)D (p < 0.05). Table 1. General characteristics of the patients at the baseline visits. For detailed description of the data format please refer to the Section 2. The features of the underlying disease course in the study group are listed in Table 2. 15 patients (50%) had diabetes mellitus with an almost compensated state at the time of participation in the study, and 7 patients (23%) reported a history of low-energy fractures. Table 2. Characteristics of the patients with Cushing’s disease (CD) in terms of the underlying disease. The groups did not differ significantly in the reported smoking status, the level of daily physical activity, dietary habits and UV exposure (p > 0.05) and although there was a slight difference in alcohol consumption (p < 0.05), the absolute values were minor in both groups (Table 3). Table 3. Questionnaire results. 3.1. Baseline Laboratory Evaluation Detailed results of laboratory studies are presented in Table 4 and Table 5. Table 4. Changes in the levels of the biochemical parameters and parathyroid hormone (PTH) during the study. Table 5. Changes in the levels of free 25(OH)D, vitamin D-binding protein (DBP) and vitamin D metabolites during the study. Patients with CD had several alterations in biochemical parameters, in particular, lower baseline serum creatinine and albumin levels, while magnesium levels were higher than in the control group (p < 0.05). They also had higher levels of urine phosphorus-creatinine ratio (p < 0.05). The rest of the studied biochemical parameters did not show significant difference between the groups (p > 0.05). 3 patients (10%) from the study group and 5 patients (17%) from the control group had secondary hyperparathyroidism, one patient with CD (3%) was diagnosed with mild primary hyperparathyroidism. As for the assessment of vitamin D metabolism, unexpectedly the levels of 25(OH)D3 occurred to be equal in the groups (p > 0.05), with only two patients (7%) from the study group and one patient (3%) from the control group having sufficient vitamin D levels, according to the Endocrine Society and the Russian Association of Endocrinologists guidelines (≥30 ng/mL [34,42]). The levels of the active vitamin D metabolite—1,25(OH)2D3—were equal between the groups as well (p > 0.05), whereas the levels of 3-epi-25(OH)D3 and 24,25(OH)2D3 were lower in CD patients. Further calculation of 25(OH)D3/24,25(OH)2D3 and 25(OH)D3/1,25(OH)2D3 ratios corresponded to the observed levels of metabolites: 25(OH)D3/24,25(OH)2D3 ratio was higher in the study group (p < 0.05) assuming lower 24-hydroxylase activity and 25(OH)D3/1,25(OH)2D3 ratio was equal between the groups (p > 0.05). Levels of free 25(OH)D were lower in CD patients (p < 0.05) and the levels of DBP did not differ between the groups (p > 0.05). Although calculated free 25(OH)D showed prominent positive correlation with the measured free 25(OH)D in both groups (r = 0.63 in the study group, r = 0.87 in the control group, p < 0.05), the association appeared to be weaker in the study group. In the control group, DBP levels correlated with both measured and calculated 25(OH)D levels (r = −0.48, p < 0.05 and r = −0.69, p < 0.05 respectively), while in patients with CD there was no association with measured free 25(OH)D levels (r = 0.04, p > 0.05 and r = −0.50, p < 0.05 respectively). Correlation with 24-h UFC in CD patients was observed for serum albumin level (r = −0.37, p < 0.05) and urine calcium-creatinine ratio (r = 0.51, p < 0.05) among assessed biochemical parameters, and only with 25(OH)D3/24,25(OH)2D3 ratio among the parameters of vitamin D metabolism (r = 0.36, p < 0.05). 3.2. Laboratory Evaluation after the Intake of Cholecalciferol All patients from the study group and 28 patients (93%) from the control group completed the study. The observed baseline differences in biochemical parameters mostly preserved during the follow-up. In the study group there was an increase in serum phosphorus levels by Day 1 (p = 0.006) and a tendency to an increase in the urine phosphorus-creatinine ratio by Day 7 (p = 0.02). Patients from the control group showed a clinically insignificant increase in serum creatinine levels by Day 1 (p = 0.002) and a non-significant trend towards an increase in serum total and albumin-adjusted calcium (p = 0.01 for both measurements). No change in PTH levels was observed in patients with CD during the follow-up (p > 0.05), while in the control group there was a tendency for PTH to decrease by Day 3 (p = 0.02). There were no new cases of hypercalcemia in both groups during the follow-up. One patient from the study group and one patient from the control group had persistently increased urine calcium-creatinine ratio throughout the study. Four patients from the study group (13%) and none from the control group developed hypercalciuria during the follow-up, however these patients had no clinical manifestations during the observation period. By Day 7, 25 patients (83%) from the study group and 22 patients (79%) reached sufficient 25(OH)D3 levels (≥30 ng/mL). Levels of 25(OH)D3 continued to increase by Day 3 in both groups (p < 0.001), after which tended to decrease in the study group (p = 0.01) and remained stable in the control group (p = 0.65). The increase in 25(OH)D3 after cholecalciferol intake was equal between the groups (18.5 [15.9; 22.5] ng/mL in the study group vs. 16.6 [13.1; 19.8] ng/mL in the control group, p > 0.05). In the presence of obesity, Δ25(OH)D3 was higher in the CD patients than in the control group (18.3 [14.2; 23.0] vs. 12.1 [10.0; 13.1] ng/mL, p < 0.05), while in non-obese patients no difference was observed (p > 0.05). Obese and non-obese patients with CD had equal Δ25(OH)D3 (18.3 [14.2; 23.0] vs. 19.6 [16.0; 21.5] ng/mL, p > 0.05), while in the control group it was significantly lower in obese patients (12.1 [10.0; 13.1] vs. 18.3 [15.3; 21.4] ng/mL, p < 0.05). BMI showed significant correlation with Δ25(OH)D3 only in the control group (r = −0.47, p < 0.05), while in CD patients there was no such association (r = −0.06, p > 0.05) (Figure 1). Figure 1. Relationship between Δ25(OH)D3 and BMI in groups. 1,25(OH)2D3 levels increased in CD patients by Day 1 and were stable during the follow-up in the control group. The rest of the studied parameters of vitamin D metabolism changed in a similar way between groups: 3-epi-25(OH)D3 levels increased until the Day 3, after which they decreased by the Day 7; 24,25(OH)2D3 levels showed more graduate elevation throughout the follow-up. In both groups 25(OH)D3/24,25(OH)2D3 ratios increased by Day 1, after which they decreased by Day 7, and 25(OH)D3/1,25(OH)2D3 ratios increased by Day 1, after which they remained stable. DBP levels didn’t change and free 25(OH)D levels showed an increase in both groups during the follow-up. The levels of 25(OH)D2 did not exceed 0.5 ng/mL in all examined individuals throughout the study. Among assessed parameters of vitamin D metabolism, higher 25(OH)D3/24,25(OH)2D3 ratios in the study group was the only difference between the groups which remained significant throughout the observation period (p < 0.05) (Figure 2). Figure 2. Dynamic evaluation of 25(OH)D3/24,25(OH)2D3 ratios in groups. 4. Discussion The main goal of our study was to evaluate the 25(OH)D3 levels and its response to the therapeutic dose of cholecalciferol in patients with CD as compared to healthy individuals. We observed no difference in baseline 25(OH)D3 assessed by UPLC-MS/MS between groups. Similar to our data were obtained in most studies conducted specifically in the state of endogenous hypercortisolism in humans [12,15] and dogs [14]. The study by Kugai et al. lacked control group and reported plasma levels of 25(OH)D corresponding to the vitamin D deficiency in most of the examined patients [10], while in our study only 2/3 of the patients with CD had 25(OH)D levels below 20 ng/mL. As for exogenous hypercortisolism, the meta-analysis aimed to explore serum 25(OH)D levels in glucocorticoid users showed lower levels than in healthy controls, but similar to steroid-naive disease controls, thus causing concern regarding the influence of the disease status on 25(OH)D levels [24]. Somewhat surprisingly, we obtained significantly discordant results in the study group when screening total 25(OH)D by ELISA and when measuring baseline 25(OH)D3 by UPLC-MS/MS, since the initial difference between the groups revealed by ELISA data with lower total 25(OH)D levels in the study group was not replicated by UPLC-MS/MS. It should be noted that our ELISA method did not participate in an external quality control program at the time of the study unlike UPLC-MS/MS; furthermore, a lower analytical performance was previously described for this technique with tendency for low specificity and lower measurement results [45]. When assessing other parameters of vitamin D metabolism, the most significant finding was the higher 25(OH)D3/24,25(OH)2D3 ratio in CD patients, both initially and during the observation after the intake of the cholecalciferol loading dose, indicating consistently reduced activity of 24-hydroxylase, the main enzyme of vitamin D catabolism. Earlier clinical and experimental studies also suggested altered activity of enzymes of vitamin D metabolism in hypercortisolism. However, these studies were heterogeneous and aimed predominantly at studying the activity of 1α-hydroxylase [7,8,10,11,12,14], which was not altered in patients with CD as compared to healthy individuals in our study. In the setting of the short-term glucocorticoid administration, Lindgren et al. showed transient increase in 24,25(OH)2D3 levels in rats [8], while in the study of Hahn et al. there was no change in 24,25(OH)2D3 levels [11]. Dogs with CD had similar 24,25(OH)2D3 levels before and after hypophysectomy as well as compared to control dogs [14]. The only study of considerably similar design by Kugai et al. reported low-normal 24,25(OH)2D3 in patients with Cushing’s syndrome [10], which is consistent with our result, as well as some experimental works indicative of suppression on CYP24A1 expression by glucocorticoids in human osteoblasts [23], liver and intestine [21] and in rat brain and myocardium [22]. However, in the present work, the activity of 24-hydroxylase in patients with hypercortisolism was for the first time evaluated by calculating the 25(OH)D3/24,25(OH)2D3 ratio, which has recently emerged as a new tool for vitamin D status assessment [46,47]. Given the correlation of this parameter with laboratory marker of the underlying disease activity (24-h UFC), a direct effect of cortisol overproduction on 24-hydroxylase activity might be assumed. Interestingly, it seems that the decreased activity of 24-hydroxylase observed in CD influenced the effectiveness of cholecalciferol treatment, decreasing the negative effect of obesity, as patients with CD had similar increase in 25(OH)D3 in obese and non-obese state and lacked correlation between Δ25(OH)D3 and BMI, as opposed to the control group. Moreover, the increase in 25(OH)D3 in obese patients from the control group was lower not only than in non-obese controls, but also than in obese patients with CD. Another intriguing finding was lower levels of free 25(OH)D observed in patients with CD despite similar DBP levels and lower albumin levels, which, on the contrary, allows one to expect higher values of free 25(OH)D. Considering the weaker correlation between the measured and calculated free 25(OH)D in patients with CD, as well as the lack of correlation of the measured 25(OH)D with the main transport protein, an altered affinity of DBP might be suspected. One possible explanation is protein glycosylation as a consequence of diabetes mellitus, which was present in half of the patients [38,48,49]. After cholecalciferol intake, which was accompanied by an increase in free 25(OH)D, the differences between the groups were leveled; therefore, another suggested explanation might be competitive binding to the ligand. Since actin binds DBP with high affinity [50] and considering catabolic action of glucocorticoids on muscle tissue [51], actin is a presumable competing ligand candidate. Although this is mostly speculative, as far as the authors are aware, the present work was the first to assess free vitamin D in the glucocorticoid excess, so the described findings require verification of reproducibility and further evaluation. The obtained discrepancies in the biochemical parameters characterizing calcium and phosphorus metabolism were generally consistent with the data of early studies discussed in the introduction [12,25,26,27,28,29], except for similar to controls serum phosphorus levels and lower prevalence of hypercalciuria. An interesting observation was the complete absence of the PTH decrease in patients with CD after receiving a loading dose of cholecalciferol. The mechanism of this phenomenon is not entirely clear, we tend to agree with the earlier hypothesis that this may be an adaptation to chronic urinary calcium loss [52]. Our research is distinguished by a number of important strengths: a prospective design, substantial sample of patients with CD, accounting for social and behavioral factors affecting vitamin levels D, comprehensive spectrum of vitamin D metabolism parameters investigated and participation in an external quality control program for vitamin D metabolites measurement. Nevertheless, the study also had several limitations: the amount of dietary vitamin D and phosphorus, as well as possible differences in DBP affinity to vitamin D metabolites due to genetic isoforms of DBP [53] or other possible involved parameters (e.g., fibroblast growth factor-23) were not taken into account. A few patients from both groups received therapy with possible impact on vitamin D and calcium metabolism within 3 months preceding the participation in the study (spironolactone, diuretics, proton pump inhibitors, oral contraceptives, antifungal treatment, antidepressants, barbiturates, antiepileptic drugs). The groups had a trend for differences in sex and BMI (p = 0.07 for both parameters). Also, the study lacked a study group of patients with remission of CD to test the hypotheses put forward, however, this is a promising direction for further research. 5. Conclusions We report that patients with endogenous ACTH-dependent hypercortisolism of pituitary origin have a consistently higher 25(OH)D3/24,25(OH)2D3 ratio than healthy controls, which is indicative of a decrease in 24-hydroxylase activity. This altered activity of the principal vitamin D catabolism might influence the effectiveness of cholecalciferol treatment. There is also a lack of clarity regarding the lower levels of free 25(OH)D observed in patients with CD, which require further study. To test the proposed hypotheses and to develop specialized clinical guidelines for these patients, longer-term randomized clinical trials are needed. Supplementary Materials The following are available online at https://www.mdpi.com/article/10.3390/nu13124329/s1, Method validation against DEQAS, Figure S1: Comparison between DEQAS data for 25(OH)D scheme and our lab results, Figure S2: Comparison between DEQAS data for 1,25(OH)2D scheme and our lab results. Author Contributions Conceptualization, L.R., E.P., A.P. and A.Z.; methodology, V.B., Z.B., L.R. and G.M.; formal analysis, A.P.; investigation, A.P., V.B., E.P., L.D. and A.Z.; data curation, A.P. and V.B.; writing—original draft preparation, A.P.; writing—review and editing, V.B., E.P., A.Z., Z.B., L.R.; visualization, V.B.; supervision, L.D., L.R., G.M. and N.M.; project administration, L.R. and N.M.; funding acquisition, L.R. and N.M. All authors have read and agreed to the published version of the manuscript. Funding This research was funded by the Russian Science Foundation, grant number 19-15-00243. Institutional Review Board Statement This study was performed in line with the principles of the Declaration of Helsinki. Approval was granted by the Ethics Committee of Endocrinology Research Centre, Moscow, Russia on 10 April 2019 (abstract of record No. 6). Informed Consent Statement Written informed consent was obtained from all individual participants included in the study. 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