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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. 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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. Recordati Rare Diseases, a US biopharma that forms part of the wider Italian group, has presented multiple positive data sets on Isturisa (osilodrostat) at the annual ENDO 2022 meeting in Atlanta, Georgia. Isturisa is a cortisol synthesis inhibitor indicated for the treatment of adult patients with Cushing’s disease for whom pituitary surgery is not an option or has not been curative. Among the data presented, the Phase III LINC 4 study demonstrated that Isturisa maintained normal mean urinary free cortisol long-term in patients with Cushing’s disease while the Phase III LINC 3 study found adrenal hormone levels changed during early treatment with the drug while stabilizing during long-term treatment. The ILLUSTRATE study also showed patients treated with a prolonged titration interval tended to have greater persistence with therapy. Mohamed Ladha, president and general manager for North America, Recordati Rare Diseases, said: “The data from these studies reinforces the efficacy and safety of Isturisa as a treatment for patients with Cushing’s disease. “We are pleased to share these data with the endocrine community and are excited to provide patients with a much-needed step forward in the management of this rare, debilitating, and potentially life-threatening condition.” Cushing’s disease is a rare, serious illness caused by a pituitary tumor that leads to overproduction of cortisol by the adrenal glands. Excess cortisol can contribute to an increased risk of morbidity and mortality. Treatment for the condition seeks to lower cortisol levels to a normal range. Isturisa, which was approved by the US Food and Drug Administration in March 2020, works by inhibiting 11-beta-hydroxylase, an enzyme responsible for the final step of cortisol biosynthesis in the adrenal gland. From https://www.thepharmaletter.com/article/results-reinforce-efficacy-of-recordati-s-isturisa-in-cushing-s-disease
  3. MaryO'Note: I found this article very simplistic. What do you think? This article is for informational purposes only and is not a substitute for professional medical advice, diagnosis or treatment. Contact a qualified medical professional before engaging in any physical activity, or making any changes to your diet, medication or lifestyle. Imagine the heart-pounding rush of adrenaline you’d get while bungee jumping or zip lining — that’s what Angela Yawn felt all the time before receiving her diagnosis. In a span of six years, the 49-year-old gained 52 kg (115 lbs) and suffered from joint swelling, headaches, skin redness and a racing heart. “I would put my hand on my chest because it made me feel like that’s what I needed to do to hold my heart in,” Yawn, who lives in Griffin, U.S., told Today. “I noticed it during the day, but at night when I was trying to lie down and sleep, it was worse because I could do nothing but hear it beat, feel it thump.” Yawn recalled being the most frustrated with the weight gain, as she’d put on 1 kg (2 lbs) a day while only eating 600 calories. “I was going crazy,” she said. After dozens of doctors couldn’t piece together her seemingly unrelated symptoms, Yawn sought out the help of an endocrinologist in February 2021. Blood tests and an MRI confirmed that Yawn had a tumour in her pituitary gland — a small, pea-sized organ at the base of the brain — that caused the gland to release excess adrenocorticotropic hormones. As a result, she became inundated with cortisol, a steroid the body releases in response to danger or stress. This combination of factors led to her diagnosis — Cushing’s disease. Read on to learn more about Cushing’s disease, signs and symptoms as well as how it can be prevented. What is Cushing’s disease? “Cushing’s disease is a rare but serious condition that is caused by a pituitary tumour," a specialist from the University of California, Los Angeles (UCLA) pituitary team tells Yahoo Canada. "The gland releases excessive adrenocorticotropic hormones and cortisol into the blood over a long period of time. It’s a hormonal disorder that is sometimes called hypercortisolism, and you will need to see an endocrinologist or someone who specializes in hormonal-related diseases to confirm your diagnosis and to help you receive proper care.” Cushing’s disease is not the same as Cushing’s syndrome, which refers to elevated levels of cortisol in the blood and is much more common than Cushing’s disease. Unlike the disease, Cushing’s syndrome can be caused by taking medications that have cortisol such as prednisone, asthma inhalers and joint steroid injections. Who is at risk for Cushing’s disease? Cushing’s disease is incredibly rare, resulting in only 10 to 15 new cases per million people in the United States each year, according to UCLA Health. “It’s most commonly found in people between the ages of 20 and 50, and affects about three times more women than men,” the UCLA source, who asked not to be named, says. “However, you might be more at risk if you have high blood pressure, if you’re overweight or if you have type 2 diabetes.” What are the signs and symptoms of Cushing’s disease? Although each person may have a unique combination of symptoms, patients typically experience changes to their physical appearance, according to Mayo Clinic. “It’s very common to see rapid weight gain, red cheeks and bruising of the skin,” the UCLA source says. “I’ve also seen patients with generalized fatigue, depression, high blood pressure, a rapid heartbeat and loss of vision.” “The symptoms can seem random or unrelated, which is why it can be so hard to diagnose,” they add. To establish if you have the disease, your doctor will conduct a physical exam and ask you about your symptoms and medical history. Generally, the first step in diagnosing Cushing's disease is determining the state of excess cortisol in the blood. Afterwards, an MRI will determine if a pituitary tumour is visible. If you have symptoms of Cushing’s disease, you should make an appointment to see a doctor or endocrinologist. How is Cushing’s disease treated? In the last decade, treatment options have changed thanks to several breakthroughs in pituitary science. “Surgery to remove the tumour is normally the first treatment option. It’s minimally invasive, has a fairly high success rate and it’s the only long-term cure for Cushing’s disease at the moment,” explains the UCLA source. If surgery isn’t an option or doesn’t solve the problem, medication and radiation therapy are other ways to treat the disease. “No matter the stage of the disease at the time of diagnosis, treating it requires an experienced specialist or team of doctors familiar with pituitary tumours,” the UCLA source adds. How can I prevent Cushing’s disease? “There’s no tried and true method of preventing the condition,” the source explains. “But if you’re at risk or if you think you have the disease, I always recommend having a doctor monitor your cortisol levels on a regular basis.” The UCLA source also recommends implementing healthy lifestyle changes that can help prevent high blood pressure. Examples include reducing stress, getting adequate sleep, exercising regularly and eating a healthy diet that's rich in fruits, vegetables and whole grains. Adapted from https://ca.news.yahoo.com/what-is-cushings-disease-experts-warn-rare-serious-condition-120015725.html
  4. 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. Neuropsychiatric findings in Cushing syndrome and exogenous glucocorticoid administration. Endocrinol Metab Clin N Am 42: 477-488, 2013. 3. Chen YF, Li YF, Chen X, Sun QF. Neuropsychiatric disorders and cognitive dysfunction in patients with Cushing's disease. CMJ 26: 3156-3160, 2013. 4. Priego T, Martín AI, González-Hedström D, Granado M, López-Calderón A. Role of hormones in sarcopenia. Vitam Horm 115: 535-570, 2021. 5. Grossman R, Mukherjee D, Chaichana KL, et al. Complications and death among elderly patients undergoing pituitary tumour surgery. Clin Endocrinol 73: 361-368, 2010. 6. Tiemensma J, Kokshoom NE, Biermasz NR, et al. Subtle cognitive impairments in patients with long-term cure of Cushing's disease. J Clin Endocrinol Metab 95: 2699-2714, 2010. 7. Brunetti A, Fulham MJ, Aloj L, et al. Decreased brain glucose utilization in patients with Cushing's disease. J Nucl Med 39: 786-790, 1998. 8. Liu S, Wang Y, Xu K, et al. Brain glucose metabolism is associated with hormone level in Cushing's disease: A voxel-based study using FDG-PET. Neuroimage Clin 12: 415-419, 2016. 9. Cheng H, Gao L, Hou B, et al. Reversibility of The cerebral blood flow in Patients with Cushing's Disease after Surgery Treatment. Metabolism 104: 154050, 2020. 10. Forget H, Lacroix A, Somma M, Cohen H. Cognitive decline in patients with Cushing's syndrome. J Int Neuropsychol Soc 6: 20-29, 2000. 11. Kim KJ, Filosa JA. Advanced in vitro approach to study neurovascular coupling mechanisms in the brain microcirculation. J Physiol 590: 1757-1770, 2012. 12. McEwen BS, Bowles NP, Gray JD, et al. Mechanisms of stress in the brain. Nat Neurosci 18: 1353-1363, 2015. 13. Rajkowska G, Miguel-Hidalgo JJ. Gliogenesis and glial pathology in depression. CNS Neurol Disord Drug Targets 6: 219-233, 2007. 14. Iuchi T, Akaike M, Mitsui T, et al. Glucocorticoid excess induces superoxide production in vascular endothelial cells and elicits vascular endothelial dysfunction. Circ Res 92: 81-87, 2003. 15. Cheng H, Gao L, Hou B, et al. Reversibility of the cerebral blood flow in patients with Cushing's disease after surgery treatment. Metabolism 104: 154050, 2020. 16. Frimodt-Møller KE, Møllegaard Jepsen JR, Feldt-Rasmussen U, et al. Hippocampal volume, cognitive functions, depression, anxiety, and quality of life in patients with Cushing syndrome. J Clin Endocrinol Metab 104: 4563-4577, 2019. 17. Siegel S, Kirstein CF, Grzywotz A, et al. Neuropsychological functioning in patients with cushing's disease and Cushing's syndrome. Exp Clin Endocrinol Diabetes 129: 194-202, 2021. 18. Chow Y, Masiak J, Mikołajewska E, et al. Limbic brain structures and burnout-A systematic review. Adv Med Sci 63: 192-198, 2018. 19. Starkman MN, Gebarski SS, Berent S, Schteingart DE. Hippocampal formation volume, memory dysfunction, and cortisol levels in patients with Cushing's syndrome. Biol Psychiatry 32: 756-765, 1992. 20. Goldberg AL, Tischler M, Demartina G, Griffin G. Hormonal regulation of protein degradation and synthesis in skeletal muscle. Fed Proc 39: 31-36, 1980. 21. Miller BS, Ignatoski KM, Daignault S, et al. A quantitative tool to assess degree of sarcopenia objectively in patients with hypercortisolism. Surgery 150: 1178-1185, 2011. 22. Delivanis D, Iñiguez-Ariza N, Zeb M, et al. Impact of hypercortisolism on skeletal muscle mass and adipose tissue mass in patients with adrenal adenomas. Clin Endocrinol 88: 209-216, 2018. 23. Kim JH, Kwak MK, Ahn SH, et al. Alteration in skeletal muscle mass in women with subclinical hypercortisolism. Endocrine 61: 134-143, 2018. 24. Gonzalez Rodriguez E, Marques-Vidal P, Aubry-Rozier B, et al. Diurnal salivary cortisol in sarcopenic postmenopausal women: the OsteoLaus Cohort. Calcif Tissue Int 109: 499-509, 2021. 25. Pivonello R, Fleseriu M, Newell-Price J, 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 Endocrinol 8: 748-761, 2020. 26. Lau D, Rutledge C, Aghi MK. Cushing's disease: current medical therapies and molecular insights guiding future therapies. Neurosurg Focus 38: E11, 2015. 27. Villar-Taibo R, Díaz-Ortega C, Sifontes-Dubo M, et al. Pituitary surgery in elderly patients: a safe and effective procedure. Endocrine 2: 814-822, 2021. From https://www.jstage.jst.go.jp/article/internalmedicine/advpub/0/advpub_8326-21/_article
  5. Abstract Cushing syndrome is a rare disease that rarely presents as acute psychosis. In this case, the patient presented with acute psychosis and agitation as the first manifestations of the disease which led to the admission of the patient to a psychiatry hospital for one month, as it was difficult to restrain her sufficiently for performing appropriate diagnostic tests due to disturbing behavior. She responded well to treatment with olanzapine and lorazepam to treat the patient’s agitation, and successfully complete her evaluation. Thereafter, she was diagnosed with a pituitary tumor and underwent pituitary lesion resection via a microscopic transsphenoidal as needed. Two months after surgery, her cortisol levels returned to baseline, and she became calmer and decreased the tensity of her psychosis; however, it was only five months after surgery that her psychotic symptoms and disturbed behavior ceased. Introduction Cushing syndrome is comprised of a group of symptoms induced by prolonged exposure to high blood cortisol levels [1]. It is a rare disease, occurring in approximately 2.4 per million individuals per year [2]. Psychiatric and cognitive manifestations of Cushing syndrome occur in 70%-85% of patients, with irritability, emotional lability, and depression occurring most commonly. Rarer symptoms include mania, panic attacks, anxiety, suicidal ideation, and acute psychosis [3-5]. In this article, we describe a patient with Cushing syndrome who developed psychosis with agitation as the first manifestation of Cushing syndrome. The patient was difficult to manage since her agitation and refusal to undergo evaluation prevented her from receiving outpatient care. Case Presentation A 22-year-old woman with a three-month history of an increase in appetite, binge eating, and weight gain. After two weeks of her initial symptoms, she started to have grandiose and persecutory delusions, auditory hallucinations, decreased need for sleep, agitation, irritability, and aggression for which she went to a private psychiatry clinic and was given 10 mg olanzapine oral at night. After a month of starting oral olanzapine, she was not improving and was admitted to the psychiatry ward for evaluation. During her admission period, she started to have cognitive symptoms including worsened memory, attention, and orientation. After one month of admission with no improvement on medication, she was noted to have moon face and high blood pressure, and her laboratory investigation showed mild hypokalemia, high cortisol level, and adrenocorticotropic hormone (ACTH), elevated liver enzymes, and mild hypertriglyceridemia. A magnetic resonance imaging (MRI) scan of the brain revealed a 6 × 2-mm hyperintense lesion in the anterior pituitary on a T2-weighted image; therefore, she was transferred to our hospital for further work up and management as we have the endocrine facility. She had no past psychiatric history or family history of psychiatric illnesses, nor a history of substance abuse. She also had no past medical history and was not on any medication prior to this presentation. The patient was admitted to the endocrine department to evaluate the possibility of Cushing syndrome. Her blood pressure (150/98), heart rate (128 BPM), and respiratory rate (30 BPM) were elevated. She was treated with losartan, amlodipine, and spironolactone. Basic labs were done (Table 1). Therefore, insulin therapy was initiated. The evaluation of the patient’s condition was difficult as she was aggressive and uncooperative due to a lack of insight. Her primary team planned for sedation with anesthesia to facilitate a clinical evaluation; however, no intensive care unit bed was available. Lab test Patient result Reference values cortisol levels 1549 nmol/L 140 to 690 nmol/L ACTH (Adrenocorticotropic Hormone) 54 pg/mL 10 to 50 pg/mL ALT (Alanine transaminase) 305 U/L 7 to 56 U/L AST (Aspartate aminotransferase) 112 U/L 8 to 33 U/L Alkaline phosphatase 141 IU/L 44 to 147 IU/L Hemoglobin A1c 7.3% 5.7% to 6.4% Table 1: Lab results for the patient when she first came to our hospital Psychiatry was consulted to manage agitation. We started her on 5 mg olanzapine oral twice daily, and 2 mg lorazepam three times daily intravenous when oral was not possible. Maximum dosage of 5 mg olanzapine and 2 mg lorazepam every four hours were administered as required to manage agitation. Her ECG showed a QTC of 464. One-to-one nurse observation was initiated to detect risky behaviors. The patient slept well and became calmer and more cooperative throughout evaluations when receiving medication. One-to-one nurse observation was discontinued after five days, and lorazepam administration was reduced to two times daily. She remained easily provoked with grandiose and persecutory delusions, auditory hallucinations, and confusion. As the patient calmed, the primary team continued clinical evaluations. A contrast-enhanced MRI showed a focal non-deforming and hypo-enhancing lesion, measuring 7 mm (AP) x 6 mm (TV) x 6 mm (CC), in the anterior pituitary (Figures 1, 2). A minimal leftward deviated pituitary stalk with normal thickness was also identified. An 8 mg dexamethasone suppression test revealed cortisol levels had decreased from 1,500 to 900 nmol/L. The 24-hour cortisol level was not determined, as the patient was easily provoked. Inferior petrosal sinus sampling was performed under general anesthesia. These results are consistent with central Cushing disease. Figure 1: Coronal T1-weighted MRI of the pituitary gland with contrast showed a hypoenhancing nodular lesion at the midline of the anterior pituitary, with mild eccentric to the right Figure 2: Brain MRI sagittal view showing focal anterior pituitary hypoenhancing lesion at the midline and eccentric to the right Treatment with 250 mg metyrapone twice daily was initiated and the patient was scheduled for pituitary lesion resection via a microscopic transsphenoidal approach by neurosurgery. Her blood tests began normalizing post-surgery except for low cortisol (Table 2), and her vital signs were within normal range. Medications regulating blood pressure and glucose levels were decreased to monotherapy and discontinued thereafter. And 40 and 20 mg doses of hydrocortisone administered in the morning and night, respectively, were tapered to 5 mg twice daily over a period of two months after the surgery, and cortisol levels were regulated reaching 167 nmol/L. Agitation and irritability, grandiose and persecutory delusion and auditory hallucination tensity were reduced, with intact cognitive and memory function. Therefore, medication dosages were gradually reduced, starting with lorazepam. Lab Test Patient result Reference values cortisol levels 68 nmol/L 140 to 690 nmol/L ACTH (Adrenocorticotropic Hormone) 25 pg/ml 10 to 50 pg/mL ALT (Alanine transaminase) 17.2 U/L 7 to 56 U/L AST (Aspartate aminotransferase) 19.2 U/L 8 to 33 U/L Alkaline phosphatase 121 IU/L 44 to 147 IU/L TSH (Thyroid Stimulating Hormone) 1.8 mIU/L 0.5 to 5.0 mIU/L Table 2: Lab results after the surgery. Before discharge, the patient’s psychotropic medications were withheld by the primary team for two days due to oversedation. Upon discharge, due to the side effects of olanzapine, the patient was switched to oral risperidone 1 mg at night, with 0.5 mg oral clonazepam twice daily as needed for agitation and psychosis. Throughout follow-up, the patient experienced ongoing psychosis with disturbed behavior even though she is using received clonazepam twice daily. Therefore, her dosage of risperidone was increased to 2 mg orally at night, and oral clonazepam (0.5 to 1 mg) was administered three times daily as needed to manage agitation. After three months of discharge (five months from surgical intervention), her levels of agitation and irritability decreased, delusions and auditory hallucinations ceased, and she returned to baseline, and clonazepam was discontinued and risperidone dosage was tapered to 0.5 mg with observation and follow up in the clinic, and no symptom relapse was observed. The complete discontinuation of her medications is planned next visit while monitoring the patient for signs of relapse. Discussion Cushing syndrome may initially present as psychosis, which may be misdiagnosis as a primary psychotic disorder, delaying the proper diagnosis and management. Our patient presented to a psychiatry hospital before being referred to us because she resisted psychosis treatment, the resistance to treatment of primary illness due to psychiatric manifestation is not uncommon, as Fujii et al. [6] reported the management of a patient who resisted schizophrenia treatment for 10 years before being diagnosed with Cushing syndrome. Agitation with psychosis is likely the main obstacle for properly evaluating, diagnosing, and treating patients with Cushing syndrome. In our patient, we aimed to reduce her agitation to facilitate clinical evaluation. The organic cause of psychosis often responds poorly to antipsychotic medication and exhibits a challenge in managing agitation which necessitate the utilization of highly sedating medications, to facilitate further clinical evaluation. Shah et al. [7] reported similar difficulty treating a patient with agitation despite prescribing lorazepam and 1 mg haloperidol twice daily, agitation was poorly controlled. In our case, the patient responds to a high dose of Olanzapine with lorazepam in a better way than the case report that was managed with haloperidol with lorazepam. Psychiatric symptoms secondary to medical conditions usually occur transiently and they resolve after treatment of the primary cause, however, the duration for complete resolution of symptoms is unknown. In our case, the patient gradually improved for three months prior to achieving remission, whereas a patient reported by Wu et al. [8] went into complete remission one-month post-cortisol level correction. Conclusions Cushing syndrome, like many other endocrine diseases, can present as treatment-resistant psychiatric symptoms, which may be missed and treated as a primary psychiatric illness due to the lack of proper assessment and management. In this study, we tried to correlate the psychiatric symptoms with Cushing syndrome, the challenges we faced, and the response to the treatment. Our case report gives an insight into possible rare secondary causes of psychosis and advice a thorough evaluation of patients. References Your bibliography. (2021). Accessed: March 27, 2021: https://www.ncbi.nlm.nih.gov/books/NBK470218/. Etxabe J, Vazquez JA: Morbidity and mortality in Cushing's disease: an epidemiological approach. Clin Endocrinol (Oxf). 1994, 40:479-84. 10.1111/j.1365-2265.1994.tb02486.x Starkman MN, Schteingart DE: Neuropsychiatric manifestations of patients with Cushing’s syndrome. Relationship to cortisol and adrenocorticotropic hormone levels. Arch Intern Med. 1981, 215:9. 10.1001/archinte.1981.00340020077021 Dorn LD, Burgess ES, Dubbert B, et al.: Psychopathology in patients with endogenous Cushing's syndrome: 'atypical' or melancholic features. Clin Endocrinol (Oxf). 1995, 43:433-42. 10.1111/j.1365-2265.1995.tb02614.x Sharma ST, Nieman LK, Feelders RA: Cushing's syndrome: epidemiology and developments in disease management. Clin Epidemiol. 2015, 7:281-93. 10.2147/CLEP.S44336 Fujii Y, Mizoguchi Y, Masuoka J, et al.: Cushing’s syndrome and psychosis: a case report and literature review. Prim Care Companion CNS Disord. 2018, 20:18br02279. 10.4088/PCC.18br02279 Shah K, Mann I, Reddy K, John G: A case of severe psychosis due to Cushing’s syndrome secondary to primary bilateral Macronodular adrenal hyperplasia. Cureus. 2019, 11:e6162. 10.7759/cureus.6162 Wu Y, Chen J, Ma Y, Chen Z: Case report of Cushing’s syndrome with an acute psychotic presentation. Shanghai Arch Psychiatry. 2016, 28:169-72. 10.11919/j.issn.1002-0829.215126 From https://www.cureus.com/articles/98986-cushings-syndrome-with-acute-psychosis-a-case-report
  6. 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
  7. Crinetics Pharmaceuticals, Inc. (Nasdaq: CRNX) today announced positive results from the multiple-ascending dose (MAD) portion of a first-in-human Phase 1 clinical study of CRN04894, the company's first-in-class, investigational, oral, nonpeptide adrenocorticotropic hormone (ACTH) antagonist that is being developed for the treatment of Cushing’s disease, congenital adrenal hyperplasia (CAH) and other conditions of excess ACTH. Following administration of CRN04894, results showed serum cortisol below normal levels and a marked reduction in 24-hour urine free cortisol excretion in the presence of sustained, disease-like ACTH concentrations. “The design of our Phase 1 healthy volunteer study allowed us to demonstrate CRN04894’s potent pharmacologic activity in the presence of ACTH levels that were in similar range to those seen in CAH and Cushing’s disease patients,” said Alan Krasner, M.D., Crinetics’ chief medical officer. “The observation of dose-dependent reductions in serum cortisol levels to below the normal range even in the presence of high ACTH indicates that CRN04894 was effective in blocking the key receptor responsible for regulating cortisol secretion. We believe this is an important finding that may be predictive of CRN04894’s efficacy in patients.” ACTH is the key regulator of the hypothalamic-pituitary adrenal (HPA) axis controlling adrenal activation. It is regulated by cortisol via a negative feedback loop that acts to inhibit ACTH secretion. This feedback loop is dysregulated in diseases of excess ACTH. In Cushing’s disease, a benign pituitary tumor drives excess ACTH secretion even in the presence of excess cortisol. While in CAH, an enzyme deficiency results in excess androgen synthesis without normal cortisol synthesis, allowing unchecked ACTH production and requiring lifelong glucocorticoid use. In both diseases, excess ACTH drives over-stimulation of the adrenal gland and leads to a host of symptoms including infertility, adrenal rest tumors, and metabolic complications in CAH and, in Cushing’s disease, symptoms include hypertension, central obesity, neuropsychiatric disorders and metabolic complications. To our knowledge, no other ACTH antagonists are currently in clinical development for diseases of ACTH excess such as Cushing’s disease or CAH. The 49 healthy adults evaluated in the multiple ascending dose portion of the Phase 1 study were administered 40, 60 or 80 mg doses of CRN04894, or placebo, daily for 10 days. After 10 days of dosing was complete, evaluable participants were administered an ACTH challenge to stimulate adrenal activation to disease relevant levels. Safety and pharmacokinetic data were consistent with expectations from the single-ascending dose cohorts in the Phase 1 study. There were no discontinuations due to treatment-related adverse events and no serious adverse events reported. Glucocorticoid deficiency was the most common treatment-related adverse event in the MAD cohorts. This was an expected extension of pharmacology given the mechanism of action of CRN04894. CRN04894 showed consistent oral bioavailability in the MAD cohorts with a half-life of approximately 24 hours, which is anticipated to support once-daily dosing. Participants in the MAD cohorts who were administered once nightly CRN04894 experienced a dose-dependent suppression of adrenal function as measured by suppression of serum cortisol production of 17%, 29% and 37% on average from baseline over 24 hours for the 40, 60 or 80 mg dosing groups respectively, (despite requirement for glucocorticoid supplementation in some of these subjects to prevent clinical adrenal insufficiency), compared to an average 2% increase in serum cortisol for individuals receiving placebo. The strong, dose-dependent suppression of serum and urine free cortisol was achieved despite ACTH levels in subjects in the 60 and 80 mg cohorts similar to those typically seen in patients with CAH and Cushing’s disease. Even when an additional exogenous ACTH challenge was administered on top of the already increased ACTH levels, cortisol levels remained below the normal range in subjects receiving CRN04894, indicating clinically significant suppression of adrenal activity. “Due to its central position in HPA axis, ACTH is the obvious target for inhibiting excessive stimulation of the adrenal in diseases of ACTH excess. Even though the field of endocrinology has known about its clinical significance for more than 100 years, we are not aware of any other ACTH antagonist that has entered clinical development. This is an important milestone for endocrinology and for our company.” said Scott Struthers, Ph.D., founder and chief executive officer of Crinetics. “We are very excited to initiate patient studies in Cushing’s disease and CAH with CRN04894, which will be our third home-grown NCE to demonstrate pharmacologic proof-of-concept and enter patient trials.” Crinetics plans to present additional details of safety, efficacy, and biomarker results from the CRN04894 Phase 1 study at an endocrinology-focused medical meeting in 2022. Data Review Conference Call Crinetics will hold a conference call and live audio webcast today, May 25, 2022, at 8:00 a.m. Eastern Time to discuss results from the MAD cohorts of the Phase 1 study of CRN04894. To participate, please dial 1-877-407-0789 (domestic) or 1-201-689-8562 (international) and refer to conference ID 13730000. To access the webcast, click here. Following the live event, a replay will be available on the Events page of the Company’s website. About the CRN04894 Phase 1 Study Crinetics has completed enrollment of the 88 healthy volunteers in this double-blind, randomized, placebo-controlled Phase 1 study. Participants were divided into multiple cohorts in the single ascending dose (n=39) and multiple ascending dose (n=49) portions of the study. In both the SAD and MAD portions of the study, safety and pharmacokinetics were assessed. In addition, pharmacodynamic responses were evaluated before and after challenges with injected synthetic ACTH to assess pharmacologic effects resulting from exposure to CRN04894. From https://www.streetinsider.com/Corporate+News/Crinetics+Pharmaceuticals+(CRNX)+Reports+Positive+Top-line+Results+Including+Strong+Adrenal+Suppression+from+CRN04894+Phase+1+Study/20126484.html
  8. Published: May 15, 2022 (see history) DOI: 10.7759/cureus.25015 Cite this article as: Iturregui J, Shi G (May 15, 2022) Recurrent Metatarsal Fractures in a Patient With Cushing Disease: A Case Report. Cureus 14(5): e25015. doi:10.7759/cureus.25015 Abstract Cushing syndrome (CS) can result from excess exposure to exogenous or endogenous glucocorticoids. The most common endogenous cause of CS is an adrenocorticotropic hormone (ACTH)-secreting pituitary adenoma, known as Cushing disease (CD). Patients typically present with characteristics including truncal obesity, moon facies, facial plethora, proximal muscle weakness, easy bruising, and striae. Insufficiency fractures of the metatarsals are a rare presentation for CS. A 39-year-old premenopausal woman presented to the orthopedic outpatient clinic with recurrent metatarsal fractures and no history of trauma. A metabolic bone disease was suspected, and after further evaluation by endocrinology services, the CD was diagnosed. Surgical resection was performed, and pathology confirmed the presence of a pituitary adenoma. Multiple, recurrent, non-traumatic metatarsal fractures can be the initial presentation of CD in a premenopausal woman. Introduction Cushing syndrome (CS) is a rare clinical and metabolic disorder caused by excessive exposure to glucocorticoids. In the United States, an estimated 10 to 15 people per million population are affected by CS each year, while studies in Europe report an incidence of 0.7 to 2.4 per million people affected annually [1,2]. Furthermore, CS more commonly affects women [2]. Common characteristics of CS include truncal obesity, moon facies, proximal muscle weakness, fatigue, facial plethora, supraclavicular fullness, peripheral edema, weight gain, striae, easy bruising, acne, hirsutism, amenorrhea, dorsocervical "buffalo" hump, depression, hypertension, impaired glucose tolerance, and osteoporosis [1,3,4]. The most common cause of CS is exogenous glucocorticoid therapy. Meanwhile, endogenous cortisol hypersecretion commonly results from either an adrenocorticotropic hormone (ACTH)-secreting pituitary adenoma or a cortisol-secreting adrenal tumor. When CS is caused by a pituitary adenoma, this is referred to as Cushing disease (CD). CD is the most common endogenous cause of CS, accounting for 80-85% of cases [1,5]. Whether a patient’s CS is caused by exogenous or endogenous sources, excessive exposure to steroids can have deleterious effects on the bones, resulting in secondary osteoporosis. The decrease in bone mass and microarchitectural changes increase the risk of fragility fractures, with reported rates as high as 30-67% [6]. The most commonly reported fracture site in CS patients is the vertebrae; however, other reported fracture sites include the ribs, sternum, wrist, elbow, shoulder, pelvis, hip, femoral condyles, tibia, fibula, calcaneus, metatarsals, and phalanges [4,6-16]. There are reports of metatarsal fractures occurring in patients diagnosed with endogenous CS [3,6,7,16-19]. However, to the best of our knowledge, there are no reports of multiple, recurrent, bilateral metatarsal fractures as the initial presentation in a pre-menopausal woman with CD. Here, we present a case of a premenopausal woman with recurrent metatarsal stress fractures who was diagnosed with CD after further evaluation. Case Presentation A 39-year-old premenopausal woman was evaluated by her primary care physician due to right foot pain after feeling a pop while walking. She reported swelling and some bruising along the lateral aspect of her foot. Her exercise regimen consisted of walking twice a week for 30 minutes at each session. She did not report any traumatic injuries to her foot. Imaging revealed a fifth metatarsal fracture (Figure 1). The patient was placed in a cast walker boot and referred to orthopedics for further evaluation. Orthopedic management included no weight bearing on her right foot and continuing using the cast walker boot or a postop shoe, with reevaluation in four weeks. Figure 1: Oblique radiograph of the right foot demonstrating a mildly displaced transverse fracture of the proximal fifth metatarsal (arrow). At the time of evaluation, the patient was 161.5 cm tall, weighed 101 kg, and had a BMI of 38.86 kg/m2. Her medical history included hypertension, hyperglycemia, hyperlipidemia, hypothyroidism, obesity, anxiety, obstructive sleep apnea, and colon polyps. The patient reported a history of metatarsal fractures in her left foot in 2008, which healed slowly and without surgical intervention. She also underwent bunion and bunionette surgery on her left foot. Her medications included alprazolam, levothyroxine, lisinopril, bimatoprost, ergocalciferol, meloxicam, and ondansetron. She was a former smoker (2007-2010), a daily wine drinker, and had an active job working as a nurse. Her family history included lung cancer and alcohol abuse in her father; hypertension, hypothyroidism, and alcohol abuse in her mother; and osteoporosis and end-stage renal disease secondary to polycystic kidney disease in her sister. At the three-month follow-up visit, the fracture line remained clearly visible, and minimal callus had formed at the fracture site. Surgical fixation was recommended and performed four months after the fracture occurred. Six months after her right foot's fifth metatarsal fracture, she developed new-onset swelling and tenderness over the middle metatarsals dorsally in her right foot with no history of trauma. Radiographs demonstrated new second and third metatarsal neck fractures (Figure 2). Conservative management with a postop shoe for six weeks and re-evaluation was recommended. In the interim between her initial right foot fifth metatarsal fracture and the new right foot second and third metatarsal fractures, the patient was diagnosed with diabetes mellitus type II, treated with a plant-based diet, hospitalized for urolithiasis, and diagnosed with depression. She was started on bupropion. Figure 2: Anteroposterior radiograph of bilateral feet demonstrating second and third metatarsal neck fractures of the right foot (arrows). Due to the recurrent metatarsal stress fractures with no associated trauma, the patient was referred to endocrinology for workup of metabolic bone disease. Her physical exam revealed no abnormalities, and her overall workup was negative. Bone mineral density results demonstrated osteopenia in the lumbar spine (T-score: -1.8) and left femoral neck (T-score: -1.0), and normal bone density in the left total hip (T-score: -0.80). Six months following her right foot's second and third metatarsal fractures, the patient developed right great toe and second toe swelling and bruising. Two months later, after trying supportive tennis shoes and reducing weightbearing on her right foot, she did not notice any improvement and sought orthopedic care. Radiographs revealed a new subacute fracture of the right second proximal phalanx (Figure 3). A magnetic resonance imaging (MRI) scan was ordered, which revealed a first metatarsal shaft stress fracture as well (Figure 4). She underwent conservative management with a Cam walker boot and was referred to endocrinology for re-evaluation for suspected endocrinopathy. Figure 3: AP radiograph of bilateral feet demonstrating a subacute fracture of the second proximal phalanx of the right foot (arrow). Figure 4: T1-weighted sagittal MRI of the right foot demonstrating a first metatarsal shaft stress fracture (arrow). At her endocrinology visit, a physical exam revealed some facial hair, frontal hair loss, and a significant dorsocervical and anterior cervical fat pad. A Cushingoid face shape, facial redness, acne, oligomenorrhea, incremental weight gain over the last decade, centripetal adiposity, easy bruising, and lower leg swelling were also reported. Bone mineral density results reported spine and hip Z-scores within the expected range for age, indicating no osteoporosis. Since she had features of hypercortisolism, labs to evaluate for Cushing syndrome were ordered. The 11:00 pm salivary cortisol levels were elevated to 173 ng/dL and 168 ng/dL in two samples. The 1 mg dexamethasone suppression test failed to suppress her cortisol levels, with an elevated cortisol value of 29 mcg/dL. The 24-hour urine-free cortisol level was elevated at 135 mcg/24 hours. These lab results confirmed a diagnosis of Cushing syndrome. Her ACTH was elevated at 86 pg/mL, which indicated an ACTH-dependent CS. Pituitary MRI demonstrated a 1.1 cm × 1.5 cm × 1.1 cm pituitary lesion, representing a pituitary macroadenoma (Figure 5). The patient underwent endoscopic endonasal transsphenoidal pituitary tumor resection with the goal of treating her Cushing disease and preventing further fragility fractures. Pathology evaluation confirmed a pituitary adenoma. Figure 5: T1-weighted coronal MRI of the pituitary demonstrating a 1.1 cm × 1.5 cm × 1.1 cm cystic sellar mass which represents a pituitary macroadenoma (arrow). Discussion This is a case of a 39-year-old woman who presented with recurrent metatarsal fractures with no history of trauma, raising suspicion of a metabolic bone disease. The patient also developed centripetal weight gain, glucose intolerance, kidney stones, depression/anxiety, and Cushingoid features. A laboratory workup performed by endocrinology services confirmed a diagnosis of ACTH-dependent CS. An MRI revealed a pituitary lesion which represented a pituitary macroadenoma, for which surgical resection was performed. Pathology confirmed a pituitary adenoma. The association of multiple, non-traumatic metatarsal fractures occurring in premenopausal women with endogenous CS has been reported in the literature [3,7,19]. However, to the best of our knowledge, this is the first report presenting a premenopausal woman with multiple, recurrent metatarsal fractures as the initial manifestation of CD. Several mechanisms play a role in glucocorticoid-induced bone loss, which is more prominent in trabecular bone compared to cortical bone [3,4,6,8]. Normally, trabecular bone has a greater bone turnover rate than cortical bone. In the presence of excess glucocorticoids, trabecular bone has greater sensitivity to glucocorticoids and undergoes slower bone turnover. The most significant effects of excess glucocorticoids on bones are decreased osteoblast function and quantity, which explain the reduced trabecular bone turnover rate [4,10]. The proposed mechanisms for this are glucocorticoid-induced inhibition of osteoblast proliferation and genesis, as well as induction of osteoblast and osteocyte apoptosis [4,10,11]. Furthermore, glucocorticoids decrease bone protein synthesis (e.g., osteocalcin), type I collagen formation, and alkaline phosphatase activity [4]. Additional effects include greater bone resorption, inhibition of intestinal calcium absorption, inhibition of renal calcium reabsorption, and decreased secretion of gonadal steroids and growth hormones [8]. Glucocorticoids also induce protein catabolism, which can result in muscle weakness, decreased bone stimulation from weakened muscle contraction, and further bone loss and debility [4]. Multiple fragility fractures in the foot with no history of trauma or overuse are uncommon. When evaluating a patient with this presentation, secondary causes for these fractures need to be investigated. Differential diagnoses include osteoporosis, Charcot foot, multiple myeloma, celiac disease, avascular necrosis, and endocrine disorders such as hyperthyroidism, primary hyperparathyroidism, or CS, among others [3,6,7]. There is a high rate of fragility fractures due to secondary osteoporosis in CS patients, with the vertebrae being most commonly affected [6]. LiYeung and Lui [7] and Albon et al. [19] each reported a case of a pre-menopausal woman who initially presented with multiple metatarsal fractures secondary to an adrenal adenoma causing CS. In each case, the patient’s densitometry indicated osteoporosis. However, in our case and the case reported by Molnar et al. [3] of a pre-menopausal woman with multiple fractures due to CD (recurrent fractures were not reported), the bone densitometries performed did not indicate osteoporosis. The patients reported by LiYeung and Lui [7], Albon et al. [19], and Molnar et al. [3] did not demonstrate marked clinical characteristics of CS. In comparison to our patient, she did have multiple Cushingoid features upon her second evaluation by endocrinology. Furthermore, in all our cases, the patients were first evaluated for metatarsal fractures as the initial presentation, which resulted in a diagnosis of endogenous CS after further evaluation. Finally, early recognition and treatment of CS are important, as there is an increased risk of morbidity and mortality as the condition progresses [20]. In addition, the treatment of CS can reverse the bone loss that occurs with excess glucocorticoid exposure [4,10]. This case also highlights the importance of collaboration between physicians in the different branches of medicine. Conclusions Excess glucocorticoid exposure can have deleterious effects on the bones, increasing the risk for secondary osteoporosis and fragility fractures. There needs to be an index of suspicion for metabolic bone disease, including endogenous CS caused by CD, as the underlying etiology of multiple, recurrent, atraumatic metatarsal fractures in pre-menopausal women. Early diagnosis and management of CD can lower the risk of morbidity and mortality as well as reverse bone loss. References Guaraldi F, Salvatori R: Cushing syndrome: maybe not so uncommon of an endocrine disease. J Am Board Fam Med. 2012, 25:199-208. 10.3122/jabfm.2012.02.110227 Valassi E, Santos A, Yaneva M, et al.: The European Registry on Cushing's syndrome: 2-year experience. Baseline demographic and clinical characteristics. Eur J Endocrinol. 2011, 165:383-92. 10.1530/EJE-11-0272 Molnar V, Zekan P, Dušek T, Ivković A: Multiple metatarsal fractures: the first manifestation of Cushing’s disease—a case report. J Am Podiatr Med Assoc. 2021, 111:10.7547/19-024 Han JY, Lee J, Kim GE, et al.: A case of cushing syndrome diagnosed by recurrent pathologic fractures in a young woman. J Bone Metab. 2012, 19:153-8. 10.11005/jbm.2012.19.2.153 Barahona MJ, Sucunza N, Resmini E, et al.: Deleterious effects of glucocorticoid replacement on bone in women after long-term remission of Cushing's syndrome. J Bone Miner Res. 2009, 24:1841-6. 10.1359/jbmr.090505 Papadakis G, Uebelhart B, Goumaz M, Zawadynski S, Rizzoli R: An unusual case of hypercortisolism with multiple weight-bearing bone fractures. Clin Cases Miner Bone Metab. 2014, 10:213-7. LiYeung LL, Lui TH: Bilateral adrenal adenoma presented as multiple metatarsal and phalangeal fractures. J Orthop Case Rep. 2015, 5:77-8. 10.13107/jocr.2250-0685.353 Trementino L, Appolloni G, Ceccoli L, Marcelli G, Concettoni C, Boscaro M, Arnaldi G: Bone complications in patients with Cushing's syndrome: looking for clinical, biochemical, and genetic determinants. Osteoporos Int. 2014, 25:913-21. 10.1007/s00198-013-2520-5 Abdel-Kader N, Cardiel MH, Navarro Compan V, Piedra Priego J, González A: Cushing's disease as a cause of severe osteoporosis: a clinical challenge. Reumatol Clin. 2012, 8:278-9. 10.1016/j.reuma.2011.11.004 Lee HJ, Je JH, Seo JH, Na YJ, Yoo HJ: Multiple spontaneous rib fractures in patient with Cushing’s syndrome. J Bone Metab. 2014, 21:277-82. 10.11005/jbm.2014.21.4.277 Poonuru S, Findling JW, Shaker JL: Lower extremity insufficiency fractures: an underappreciated manifestation of endogenous Cushing's syndrome. Osteoporos Int. 2016, 27:3645-9. 10.1007/s00198-016-3712-6 Belaya ZE, Hans D, Rozhinskaya LY, et al.: The risk factors for fractures and trabecular bone-score value in patients with endogenous Cushing's syndrome. Arch Osteoporos. 2015, 10:44. 10.1007/s11657-015-0244-1 Tajika T, Shinozaki T, Watanabe H, Yangawa T, Takagishi K: Case report of a Cushing's syndrome patient with multiple pathologic fractures during pregnancy. J Orthop Sci. 2002, 7:498-500. 10.1007/s007760200087 Baron E, Sheinfeld M, Migdal EA, Hardoff R: Multiple pathologic fractures mimicking bone metastases in a patient with Cushing's syndrome. Clin Nucl Med. 1996, 21:506-8. 10.1097/00003072-199606000-00027 Bosch S, Bogaerts S: Pituitary adenoma presenting with bilateral calcaneal stress fracture: a case report. JOSPT Cases. 2021, 1:109-111. Kostoglou-Athanassiou I, Spiliotis G, Athanassiou L, Myriokefalitakis I: Cushing’s syndrome in a patient with systemic lupus erythematosus. Endocrine Abstracts. 2018, 56:106. 10.1530/endoabs.56.P106 Kaur K, Findling JW: Cushing’s disease. A Case-Based Guide to Clinical Endocrinology. Davies TF (ed): Humana Press, Totowa; 2008. 27-33. 10.1007/978-1-60327-103-5_3 Ontell FK, Shelton DK: Multiple stress fractures. An unusual presentation of Cushing's disease. West J Med. 1995, 162:364-6. Albon L, Rippin J, Franklyn J: “My feet are killing me!” An unusual presentation of Cushing’s syndrome. Endocrine Abstracts. 2003, 5:26. 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 From https://www.cureus.com/articles/91295-recurrent-metatarsal-fractures-in-a-patient-with-cushing-disease-a-case-report
  9. — More than half of patients saw physical manifestations fully resolve by week 72 by Kristen Monaco, Staff Writer, MedPage Today May 16, 2022 SAN DIEGO -- Osilodrostat (Isturisa) improved many physical features associated with Cushing's disease, according to additional findings from the phase III LINC-3 study. Among 137 adults with Cushing's disease, a 39.5% improvement in central obesity scores was observed from baseline to week 72 with osilodrostat, reported Alberto Pedroncelli, MD, PhD, of Recordati AG in Basel, Switzerland. Not only was central obesity the most common physical manifestation associated with hypercortisolism among these Cushing's disease patients, but it was also more frequently rated as severe at baseline, Pedroncelli explained during the American Association of Clinical Endocrinology (AACE) annual meeting. Osilodrostat treatment also led to a 34.9% improvement in proximal muscle atrophy at week 72, along with a 34.4% improvement in hirsutism scores. By week 72, nearly all physical manifestations of hypercortisolism saw significant improvement -- marked by more than 50% of patients scoring these physical traits as nonexistent: Dorsal fat pat: 50.6% Central obesity: 30.6% Supraclavicular fat pad: 51.8% Facial rubor: 64.7% Hirsutism in women: 53.1% Proximal muscle atrophy: 61.2% Striae: 63.5% Ecchymoses: 87.1% Most of these physical manifestation improvements were notable soon after treatment initiation with osilodrostat, Pedroncelli pointed out. When stratified according to testosterone levels, hirsutism scores remained either stable or improved in the majority of patients who had normal or above normal testosterone levels. More women with normal testosterone levels over time experienced improvements in hirsutism versus those with levels above the upper limit of normal, who mostly remained stable. Osilodrostat is an oral agent that was first FDA approved in March 2020 for adults with Cushing's disease who either cannot undergo pituitary gland surgery or have undergone the surgery but still have the disease. Available in 1 mg, 5 mg, and 10 mg film-coated tablets, the drug acts as a potent oral 11-beta-hydroxylase inhibitor -- the enzyme involved in the last step of cortisol synthesis. Osilodrostat is taken orally twice daily, once in the morning and once in the evening. Approval was based upon findings from the LINC-3 and LINC-4 trials, which found osilodrostat was able to normalize cortisol levels in 53% of patients, based on mean 24-hour urinary free cortisol (UFC) concentrations. During an initial 10-week randomization phase, 86% of patients maintained their complete cortisol response if they remained on osilodrostat versus only 29% of those who were switched to placebo. As expected, 77.4% of the 137 adults included in the trial were women. The median participant age was 40 and about 47 months had passed since their initial diagnosis. A total of 87.6% underwent previous pituitary surgery and 16.1% underwent previous pituitary irradiation. At baseline, median and mean 24-hour UFC levels were 3.5 nmol and 7.3 nmol, respectively, based on two or three urine samples. Participants had an average body weight of 176.4 lb, body mass index (BMI) of 30, and 41 in waist circumference at baseline. Throughout the trial, all measures dropped, reaching the nadir at week 72: body weight of 165 lb, BMI of 27, and 37.8 in waist circumference. The most common side effects reported with the agent include adrenal insufficiency, fatigue, nausea, headache, and edema. Kristen Monaco is a staff writer, focusing on endocrinology, psychiatry, and nephrology news. Based out of the New York City office, she’s worked at the company since 2015. Disclosures The study was supported by Recordati AG. Pedroncelli reported employment with Recordati. Primary Source American Association of Clinical Endocrinology Source Reference: Pedroncelli AM, et al "Osilodrostat therapy improves physical features associated with hypercortisolism in patients with Cushing's disease: findings from the phase III LINC 3 study" AACE 2022. From https://www.medpagetoday.com/meetingcoverage/aace/98745
  10. 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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  11. A worldwide, observational study of adults and adolescents with growth hormone deficiency (GHD) found long-term GH replacement was safe. These findings were published in the Journal of Clinical Endocrinology & Metabolism. Data for this long-term follow-up study were sourced from the KIMS Pfizer International Metabolic Database cohort. Patients (N=15,809) with confirmed GHD were prescribed GH by their primary care physician. Adverse events were evaluated at up to 18 years (mean, 5.3 years). The median age of study participants was 44.8 (range, 5.6-91.2) years, 50.5% were girls or women, 94.4% were White, 57.6% were true-naive to treatment at baseline, 59.7% had pituitary or hypothalamic tumor, 21.6% had idiopathic or congenital GHD, and 67.8% had at least 2 pituitary deficiencies. Patients were administered a mean GH dosage of 0.30±0.30 mg/d. At year 15, patients (n=593) had a 1.7-kg/m2 increase in body mass index (BMI), a 4.3-kg increase in weight, a 0.4-cm decrease in height, a 6.2-cm increase in waist circumference, a 0.03 increase in waist to hip ratio, a 6.3-mm Hg increase in systolic blood pressure, a 1.0-mm Hg increase in diastolic blood pressure, and a 0.5-bpm decrease in heart rate. Approximately one-half of the patients (51.2%) experienced at least 1 adverse event, but few patients (18.8%) reported treatment-related adverse events. The most common all-cause adverse events included arthralgia (4.6%), peripheral edema (3.9%), headache (3.6%), influenza (2.8%), depression (2.8%), and recurrence of pituitary tumor (2.7%). The most common treatment-related adverse events were peripheral edema (3.1%) and arthralgia (2.6%). The rate of all-cause (P =.0141) and related (P =.0313) adverse events was significantly related with age at enrollment, with older patients (aged ³45 years) having higher rates than younger patients. The rate of all-cause and related adverse events was higher among patients with pituitary or hypothalamic tumor, adult-onset GHD, and insulin-like growth factor 1 standard deviation score greater than 0; those who had prior pituitary radiation treatment; and those who took a GH dosage of no more than 0.30 mg/d (all P £..014). A total of 1934 patients discontinued treatment, and 869 patients reduced their dose due to adverse events. Study discontinuation was highest among patients with idiopathic or congenital GHD (45.0%). At least 1 serious adverse event occurred among 4.3% of patients. The most common serious events included recurrence of pituitary tumor (n=154; 1.0%) and death (n=21; 0.1%). The highest mortality rate was observed among patients who enrolled at 45 years of age and older (4.7%). In total, 418 patients who had no history of cancer at baseline were diagnosed with cancer after starting GH treatment, which equated to a standardized incidence ratio of 0.92 (95% CI, 0.83-1.01). This study was limited as data were collected during routine clinical practice and no predefined windows or reporting were set. This study found that GH replacement therapy was safe at up to an 18-year follow-up among adolescents and adults. Disclosure: Multiple authors declared affiliations with industry. Please refer to the original article for a full list of disclosures. Reference Johannsson G, Touraine P, Feldt-Rasmussen U, et al. Long-term safety of growth hormone in adults with growth hormone deficiency: overview of 15,809 GH-treated patients. J Clin Endocrinol Metab. Published online April 3, 2022. doi:10.1210/clinem/dgac199 From https://www.endocrinologyadvisor.com/home/topics/general-endocrinology/safety-of-long-term-growth-hormone-treatment-assessed/
  12. Abstract Context Arginine-vasopressin and CRH act synergistically to stimulate secretion of ACTH. There is evidence that glucocorticoids act via negative feedback to suppress arginine-vasopressin secretion. Objective Our hypothesis was that a postoperative increase in plasma copeptin may serve as a marker of remission of Cushing disease (CD). Design Plasma copeptin was obtained in patients with CD before and daily on postoperative days 1 through 8 after transsphenoidal surgery. Peak postoperative copeptin levels and Δcopeptin values were compared among those in remission vs no remission. Results Forty-four patients (64% female, aged 7-55 years) were included, and 19 developed neither diabetes insipidus (DI) or syndrome of inappropriate anti-diuresis (SIADH). Thirty-three had follow-up at least 3 months postoperatively. There was no difference in peak postoperative copeptin in remission (6.1 pmol/L [4.3-12.1]) vs no remission (7.3 pmol/L [5.4-8.4], P = 0.88). Excluding those who developed DI or SIADH, there was no difference in peak postoperative copeptin in remission (10.2 pmol/L [6.9-21.0]) vs no remission (5.4 pmol/L [4.6-7.3], P = 0.20). However, a higher peak postoperative copeptin level was found in those in remission (14.6 pmol/L [±10.9] vs 5.8 (±1.4), P = 0.03]) with parametric testing. There was no difference in the Δcopeptin by remission status. Conclusions A difference in peak postoperative plasma copeptin as an early marker to predict remission of CD was not consistently present, although the data point to the need for a larger sample size to further evaluate this. However, the utility of this test may be limited to those who develop neither DI nor SIADH postoperatively. Cushing disease, copeptin, cortisol, remission Issue Section: Clinical Research Article Arginine vasopressin (AVP) and CRH act synergistically as the primary stimuli for secretion of ACTH, leading to release of cortisol [1, 2]. The role of AVP in the hypothalamic-pituitary-adrenal (HPA) axis is via release from the parvocellular neurons of the paraventricular nuclei (and possibly also from the magnocellular neurons of the paraventricular and supraoptic nuclei), the secretion of which is stimulated by stress [3-6]. AVP release results in both independent stimulation of ACTH release and potentiation of the effects of CRH [3, 7-9]. Additionally, there is evidence that glucocorticoids act by way of negative feedback to suppress AVP secretion [10, 11-20]. Further, parvocellular neurons of the hypothalamic paraventricular nuclei have been shown to increase AVP production and neurosecretory granule size after adrenalectomy, and inappropriately elevated plasma AVP has been reported in the setting of adrenal insufficiency with normalization of plasma AVP after glucocorticoid administration [21-24]. This relationship of AVP and its effect on the HPA axis has been used in the diagnostic evaluation of Cushing syndrome (CS) [14] and evaluation of remission after transsphenoidal surgery (TSS) in Cushing disease (CD) by administration of desmopressin [25]. Copeptin makes up the C-terminal portion of the AVP precursor pre-pro-AVP. Copeptin is released from the posterior pituitary in stoichiometric amounts with AVP, and because of its longer half-life in circulation, it is a stable surrogate marker of AVP secretion [26-28]. Plasma copeptin has been studied in various conditions of the anterior pituitary. In a study by Lewandowski et al, plasma copeptin was measured after administration of CRH in assessment of HPA-axis function in patients with a variety of pituitary diseases. An increase in plasma copeptin was observed only in healthy subjects but not in those with pituitary disease who had an appropriately stimulated serum cortisol, and the authors concluded that copeptin may be a sensitive marker to reveal subtle alterations in the regulation of pituitary function [7]. Although in this study and others, plasma copeptin was assessed after pituitary surgery, it has not, to the best of our knowledge, been studied as a marker of remission of CD before and after pituitary surgery [7, 29]. In this study, plasma copeptin levels were assessed as a surrogate of AVP secretion before and after TSS for treatment of CD. Because there is evidence that glucocorticoids exert negative feedback on AVP, we hypothesized that there would be a greater postoperative increase in plasma copeptin in those with CD in remission after TSS resulting from resolution of hypercortisolemia and resultant hypocortisolemia compared with those not in remission with persistent hypercortisolemia and continued negative feedback. In other words, we hypothesized that an increase in copeptin could be an early marker of remission of CD after TSS. We aimed to complete this assessment by comparison of the peak postoperative copeptin and change in copeptin from preoperative to peak postoperative copeptin for those in remission vs not in remission postoperatively. Subjects and Methods Subjects Adult and pediatric patients with CD who presented at the Eunice Kennedy Shriver National Institute of Child Health and Human Development under protocol 97-CH-0076 and underwent TSS between March 2016 and July 2019 were included in the study. Exclusion criteria included a prior TSS within 6 weeks of the preoperative plasma copeptin sample or a preoperative diagnosis of diabetes insipidus, renal disease, or cardiac failure. Written informed consent was provided by patients aged 18 years and older and by legal guardians for patients aged < 18 years to participate in this study. Written informed assent was provided by patients aged 7 years to < 18 years. The 97-CH-0076 study (Investigation of Pituitary Tumors and Related Hypothalamic Disorders) has been approved by the Eunice Kennedy Shriver National Institute of Child Health and Human Development institutional review board. Clinical and Biochemical Data Clinical data were extracted from electronic medical records. Age, sex, body weight, body mass index (BMI), pubertal stage (in pediatric patients only), and history of prior TSS were obtained preoperatively during the admission for TSS. Clinical data obtained postoperatively included TSS date, histology, development of central diabetes insipidus (DI) or (SIADH), time from TSS to most recent follow-up, and clinical remission status at postoperative follow-up. Preoperatively, serum sodium, 24-hour urinary free cortisol (UFC), UFC times the upper limit of normal (UFC × ULN), midnight (MN) serum cortisol, MN plasma ACTH, and 8 AM plasma ACTH were collected. Postoperatively, serum sodium, serum and urine osmolality, urine specific gravity, serum cortisol, and plasma ACTH were collected. For serum cortisol values < 1 mcg/dL, a value of 0.5 mcg/dL was assigned for the analyses; for plasma ACTH levels < 5 pg/mL, a value of 2.5 pg/mL was assigned. Additionally, plasma copeptin levels were obtained preoperatively and on postoperative days (PODs) 1 through 8 after TSS at 8:00 AM. Peak postoperative copeptin was the highest plasma copeptin on PODs 1 through 8. The delta copeptin (Δcopeptin) was determined by subtracting the preoperative copeptin from the peak postoperative copeptin; hence, a positive change indicated a postoperative increase in plasma copeptin. Plasma copeptin was measured using an automated immunofluorescent sandwich assay on the BRAHMS Kryptor Compact PLUS Copeptin-proAVP. The limit of detection for the assay was 1.58 pmol/L, 5.7% intra-assay coefficient of variation, and 11.2% inter-assay coefficient of variation, with a lower limit of analytical measurement of 2.8 pmol/L. For those with multiple preoperative plasma copeptin values within days before surgery, an average of preoperative copeptin levels was used for analyses. Diagnosis of CD was based on guidelines published by the Endocrine Society and as previously described for the adult and pediatric populations [30, 31]; diagnosis was further confirmed by either histologic identification of an ACTH-secreting pituitary adenoma in the resected tumor specimen, decrease in cortisol and ACTH levels postoperatively, and/or clinical remission after TSS at follow-up evaluation. All patients were treated with TSS at the National Institutes of Health Clinical Center by the same neurosurgeon. Remission after surgical therapy was based on serum cortisol of < 5 μg/dL during the immediate postoperative period, improvement of clinical signs and symptoms of cortisol excess at postoperative follow up, nonelevated 24-hour UFC at postoperative follow-up, nonelevated midnight serum cortisol at postoperative follow up when available, and continued requirement for glucocorticoid replacement at 3 to 6 months’ postoperative follow-up. Diagnosis of SIADH was based on development of hyponatremia (serum sodium < 135 mmol/L) and oliguria (urine output < 0.5 mL/kg/h). Diagnosis of DI was determined by development of hypernatremia (serum sodium > 145 mmol/L), dilute polyuria (urine output > 4 mL/kg/h), elevated serum osmolality, and low urine osmolality. Statistical Analyses Results are presented as median (interquartile range [IQR], calculated as 25th percentile-75th percentile) or mean ± SD, as appropriate, and frequency (percentage). Where appropriate, we compared results using parametric or nonparametric testing; however, the median (IQR) and the mean ± SD were both reported to allow for comparisons with the appropriate testing noted. Subgroup analyses were completed comparing those who developed water balance disorders included patients who developed DI only (but not SIADH), those who developed SIADH only (but not DI), and those with no water balance disorder; hence, for these subgroup analyses, those who developed both DI and SIADH postoperatively (n = 4) were excluded. Preoperative copeptin, peak postoperative copeptin, and Δcopeptin were compared between those with and without remission at follow-up, using either t test or Wilcoxon rank-sum test, depending on the distribution of data. These were done in all patients combined, as well as within each subgroup. The same tests were used for comparing other continuous variables (eg, age, BMI SD score [SDS], cortisol excess measures) between those with and without remission. Categorical data (eg, sex, Tanner stage) were analyzed using the Fisher exact test. Comparisons of copeptin levels among the subgroups (DI, SIADH, neither) were carried out using mixed models and the Kruskal-Wallis test, as appropriate. Post hoc pairwise comparisons were adjusted for multiplicity using the Bonferroni correction, and as applicable, only corrected P values are reported. Mixed models for repeated measures also analyzed copeptin, serum sodium, and cortisol data for PODs 1 through 8. In addition, maximum likelihood estimation (GENMOD) procedures analyzed the effects of copeptin and serum sodium on the remission at follow-up. Correlation analyses were done with Spearman ρ. All analyses were tested for the potential confounding effects of age, sex, BMI SDS, and pubertal status, and were adjusted accordingly. For plasma copeptin reported as < 2.8 pmol/L, a value of 1.4 pmol/L (midpoint of 0 and 2.8 pmol/L) was used; sensitivity analyses repeated all relevant comparisons using the threshold limit of 2.8 pmol/L instead of 1.4 pmol/L. Odds ratios (OR) and 95% CIs, other magnitudes of the effect, data variability, and 2-sided P values provided the statistical evidence for the conclusions. Statistical analyses were performed in SAS version 9.4 software (SAS Institute, Inc, Cary, NC). Results Patient Characteristics Forty-four adult and pediatric patients, aged 7 to 55 years (77.2% were < 18 years old), with CD were included in the study. The cohort included 28 female patients (64%), and the median BMI SDS was 2.2 (1.1-2.5). Thirty-four percent (15/44) had prior pituitary surgery (none within the prior 6 weeks). Seventy-five percent (33/44) had postoperative follow-up evaluations available, with median follow-up of 13.5 months (11.3-16.0). Of those 33 patients, 85% were determined to be in remission at follow-up. Comparing those in remission vs no remission, there was no difference in age, sex, BMI SDS, pubertal status (in pediatric ages only), preoperative measures of cortisol excess (UFC × ULN, PM serum cortisol, MN plasma ACTH, AM plasma ACTH), duration of follow-up, or development of DI or SIADH. There was a lower postoperative serum cortisol nadir in those in remission at follow-up compared with those not in remission at follow-up, as expected, because a postoperative serum cortisol < 5 μg/dL was included in defining remission status. Postoperatively, 8/44 (18%) developed DI, 13/44 (30%) developed SIADH, 4/44 (9%) developed both DI and SIADH, and 19/44 (43%) developed no water balance disorder (Table 1). There were no differences by remission status when assessing these subgroups (ie, DI, SIADH, and no water balance disorder) separately. Table 1. Demographic and clinical characteristics of subjects All subjects, n = 44 All subjects by remission status, n = 33 All subjects by remission status, excluding those with DI or SIADH, n = 13 Remission, n = 28 No remission, n = 5 P Remission, n = 10 No remission, n = 3 P Age, median (range), y 14.5 (7-55) 17.4 ± 10.7 14.5 (12.5-17.5) 15.6 ± 13.2 11.0 (9.0-12.0) 0.11 13.7 ± 3.1 14.0 (13.0-15.0) 19.7 ± 16.8 11.0 (9.0-39.0) 0.60a Sex  Female 28 (64%) 22 (78.6%) 3 (60.0%) 0.57 9 (90.0%) 2 (66.7%) 0.42 BMI SDS 2.2 (1.1-2.5) 1.7 ± 1.0 2.0 (0.9-2.5) 2.2 ± 0.4 2.2 (2.1-2.3) 0.70 1.7 ± 1.1 2.0 (0.7-2.5) 2.0 ± 0.4 2.1 (1.5-2.3) 0.65a Pubertal status Female (n = 19) (n = 15) (n = 2) 0.51 (n = 8) (n = 1) 0.44   Tanner 1-2 6 4 (26.7%) 1 (50.0%) 3 (37.5%) 1 (25.0%)   Tanner 3-5 13 11 (73.3%) 1 (50.0%) 5 (62.5%) 0 Male (n = 14) (n = 5) (n = 2) (n = 1) (n = 1) --- Testicular volume < 12, mL 10 4 (80.0%) 2 (10.00%) 1 (100.0%) 1 (100.0%) Testicular volume ≥ 12, mL 4 1 (20.0%) 0 1.0 0 0 Preoperative UFC ULN 3.3 (1.2-6.1) 4.9 ± 6.1 2.6 (1.0-7.6) 3.2 ± 1.3 3.7 (3.0-3.9) 0.70 7.2 ± 8.4 3.9 (1.8-9.1) 3.8 ± 0.7 3.9 (3.0-4.4) 0.93 Preoperative PM cortisol 11.9 (9.2-14.8) 13.3 ± 4.7 12.2 (9.2-16.8) 10.8 ± 2.1 11.5 (9.0-11.6) 0.30 13.3 ± 6.0 11.2 (8.4-16.5) 11.1 ± 2.6 11.6 (8.3-13.6) 0.57a Preoperative MN ACTH 43.4 (29.3-51.6) 44.2 ± 25.5 46.1 (27.6-50.5) 40.9 ± 15.3 11.5 (9.0-11.6) 0.74 36.6 ± 16.6 37.4 (29.1-48.8) 34.0 ± 9.4 39.3 (23.1-39.5) 0.67 Preoperative AM ACTH 44.6 (31.4-60.5) 46.9 ± 28.9 44.0 (29.8-56.2) 48.6 ± 28.8 58.7 (21.7-60.5) 0.84 35.2 ± 16.2 40.3 (28.0-44.0) 45.4 ± 24.6 58.7 (17.0-60.5) 0.41a Postoperative cortisol nadir 0.5 (0.5-0.5) 0.7 ± 0.7 0.5 (0.5-0.5) 7.8 ± 6.6 5.2 (2.2-12.3) <0.001 0.6 ± 0.3 0.5 (0.5-0.5) 8.1 ± 7.9 5.2 (2.1-17.0) 0.003 Duration of follow-up 13.5 (11.3-16.0) 15.3 ± 7.9 14.0 (12.0-16.5) 14.0 ± 13.0 11.0 (6.0-14.0) 0.30 18.6 ± 11.2 15.5 (12.0-27.0) 16.7 ± 17.2 11.0 (3.0-36.0) 0.82a DI only 8 (18%) 7/8 (87.5%) 1/8 (12.5%) 0.91 --- --- --- SIADH only 13 (30%) 8/9 (88.9%) 1/9 (11.1%) Neither DI/SIADH 19 (43%) 10/13 (76.9%) 3/13 (23.1%) Both DI and SIADH 4 (9%) 3/3 (100%) 0/3 Demographic and clinical characteristics of all subjects (n = 44) with Cushing disease. Data are also presented by remission status for all subjects with postoperative follow-up (n = 33) and by remission status after excluding those who developed DI or SIADH postoperatively with postoperative follow-up (n = 13). Both median (IQR) and mean ± SD reported to allow for comparisons, with P value provided using appropriate testing depending on distribution of data sets. Data are mean ± SD, median (25th-75th IQR), or frequency (percentage) are reported, except for age, which is presented as median (range). Abbreviations: AM, 7:30-8 PM; BMI, body mass index; DI, diabetes insipidus; IQR, interquartile range; MN, midnight; N/A, not applicable; SDS, SD score; SIADH, syndrome of inappropriate antidiuresis; UFC, urinary free cortisol; ULN, upper limit of normal. p-values below the threshold of 0.05 are in bold. aP value indicates comparison using parametric testing, as appropriate for normally distributed data. Open in new tab Preoperative copeptin levels were higher in males (7.0 pmol/L [5.1-9.6]) than in females (4.0 pmol/L [1.4-5.8], P = 0.004) (Fig. 1). Age was inversely correlated with preoperative copeptin (rs = -0.35, P = 0.030) and BMI SDS was positively correlated with preoperative copeptin (rs = 0.54, P < 0.001) (Fig. 2). Figure 1. Open in new tabDownload slide Preoperative plasma copeptin and sex. Preoperative plasma copeptin in all patients, comparing by sex. A higher preoperative plasma copeptin was found in males (7.0 pmol/L [5.1-9.6]) than in females (4.0 pmol/L [1.4-5.8], P = 0.004). Horizontal lines = median. Whiskers = 25th and 75th interquartile ranges. Figure 2. Open in new tabDownload slide Preoperative plasma copeptin and BMI SDS. Association of preoperative plasma copeptin and BMI SDS in all patients. A BMI SDS was positively associated with a preoperative plasma copeptin (rs = 0.54, P < 0.001). Shaded area = 95% confidence interval. Copeptin Before and After Transsphenoidal Surgery for CD Among the 33 patients with postoperative follow-up, there was no difference in peak postoperative copeptin for patients in remission vs those not in remission (6.1 pmol/L [4.3-12.1] vs 7.3 pmol/L [5.4-8.4], P = 0.88). There was also no difference in the Δcopeptin for those in remission vs not in remission (2.3 pmol/L [-0.5 to 8.2] vs 0.1 pmol/L [-0.1 to 2.2], P = 0.46) (Fig. 3). Including all subjects, the mean preoperative copeptin was 5.6 pmol/L (±3.4). For patients with follow-up, there was no difference in preoperative copeptin for those in remission (4.8 pmol/L [±2.9]) vs no remission (6.0 pmol/L [±2.0], P = 0.47). POD 1 plasma copeptin ranged from < 2.8 to 11.3 pmol/L. Figure 3. Open in new tabDownload slide (A) Peak postoperative plasma copeptin in all patients, comparing those in remission with no remission (6.1 pmol/L [4.3-12.1] vs 7.3 pmol/L [5.4-8.4], P = 0.88). (B) ΔCopeptin (preoperative plasma copeptin subtracted from postoperative peak plasma copeptin) in all patients, comparing those in remission with no remission (2.3 pmol/L [-0.5 to 8.2] vs 0.1 pmol/L [-0.1 to 2.2], P = 0.46). Horizontal lines = median. Whiskers = 25th and 75th interquartile ranges. When those who developed DI or SIADH were excluded, there was no difference in peak postoperative copeptin in those in remission vs no remission (10.2 pmol/L [6.9-21.0] vs 5.4 pmol/L [4.6-7.3], P = 0.20). However, because the distribution of the peak postoperative copeptins was borderline normally distributed, parametric testing was also completed for this analysis, which showed a higher peak postoperative copeptin in remission (14.6 pmol/L [±10.9]) vs no remission (5.8 [±1.4], P = 0.03). There was no difference in the Δcopeptin for those in remission vs not in remission (5.1 pmol/L [0.3-19.5] vs 1.1 pmol/L [-0.1 to 2.2], P = 0.39) (Fig. 4). Preoperative copeptin was not different for those in remission (4.7 pmol/L [±2.4]) vs no remission (4.9 pmol/L [±20.3], P = 0.91). There was no association between serum cortisol and plasma copeptin over time postoperatively (Fig. 5). Figure 4. Open in new tabDownload slide (A) Peak postoperative plasma copeptin excluding those who developed DI or SIADH, comparing those in remission with no remission (10.2 pmol/L [6.9-21.0] vs 5.4 pmol/L [4.6-7.3], P = 0.20). (B) ΔCopeptin (preoperative plasma copeptin subtracted from postoperative peak plasma copeptin) excluding those who developed DI or SIADH, comparing those in remission with no remission (5.1 pmol/L [0.3-19.5] vs 1.1 pmol/L [-0.1 to 2.2], P = 0.39). Horizontal lines = median. Whiskers = 25th and 75th interquartile ranges. Figure 5. Open in new tabDownload slide Plasma copeptin and serum cortisol vs postoperative day for patients who did not develop DI or SIADH. Plasma copeptin (indicated by closed circle) and serum cortisol (indicated by “x”). Results shown as (median, 95% CI). All analyses here were repeated adjusting for serum sodium, and there were no differences by remission status for preoperative, peak postoperative, or Δcopeptin for all subjects or after excluding those who developed a water balance disorder (data not shown). Copeptin and Water Balance Disorders As expected, peak postoperative copeptin appeared to be different among patients who developed DI, SIADH, and those without any fluid balance disorder (P = 0.029), whereas patients with DI had lower median peak postoperative copeptin (4.4 pmol/L [2.4-6.9]) than those who developed no fluid abnormality (10.0 pmol/L [5.4-16.5], P = 0.04), the statistical difference was not present after correction for multiple comparisons (P = 0.13). Peak postoperative copeptin of patients with SIADH was 9.4 pmol/L (6.5-10.4) and did not differ from patients with DI (P = 0.32) or those with no fluid abnormality (P = 1.0). There was a difference in Δcopeptin levels among these subgroups (overall P = 0.043), which appeared to be driven by the lower Δcopeptin in those who developed DI (-1.2 pmol/L [-2.6 to 0.1]) vs in those with neither DI or SIADH (3.1 pmol/L [0-9.6], P = 0.05). However, this pairwise comparison did not reach statistical significance, even before correction for multiple comparisons (P = 0.16) (Fig. 6). Preoperative copeptin levels were also not different among the subgroups (P = 0.54). Figure 6. Open in new tabDownload slide (A) Peak postoperative plasma copeptin, comparing those who developed DI, SIADH, or neither (P = 0.029 for comparison of all 3 groups). (B) ∆ Copeptin (preoperative plasma copeptin subtracted from postoperative peak plasma copeptin), comparing those who developed DI, SIADH, or neither (P = 0.043 for comparison of all 3 groups). Horizontal lines = median. Whiskers = 25th and 75th interquartile ranges. Top brackets = pairwise comparisons. P values presented are after Bonferroni correction for multiple comparisons. Association of Sodium and Copeptin Longitudinal data, adjusting for subgroups (ie, DI, SIADH, neither), were analyzed. As expected, there was a group difference (P = 0.003) in serum sodium over time (all DI was missing preoperative serum sodium), with the difference being driven by DI vs SIADH (P = 0.007), and SIADH vs neither (P = 0.012). There was no group difference in plasma copeptin over POD by water balance status (P = 0.16) over time (Fig. 7). There was also no effect by remission status at 3 to 6 months for either serum sodium or plasma copeptin. Figure 7. Open in new tabDownload slide (A) Serum sodium and (B) plasma copeptin by POD and water balance status longitudinal data, adjusting for subgroups (ie, DI, SIADH, neither). Data points at point 0 on the x-axis indicate preoperative values. As expected, there was a group difference (P = 0.003) in serum sodium over time (all with DI were missing preoperative serum sodium), with the difference being driven by DI vs SIADH (P = 0.007), and SIADH vs neither (P = 0.012). There was no group difference in plasma copeptin over POD by water balance status (P = 0.16) over time. Higher serum sodium levels from PODs 1 through 8 itself decreased the odds of remission (OR, 0.56; 95% CI, 0.42-0.73; P < 0.001) in all CD patients. Copeptin levels from these repeated measures adjusting for serum sodium did not correlate with remission status at 3 to 6 months’ follow-up (P = 0.38). There were no differences in preoperative, peak postoperative, or delta sodium levels by remission vs no remission in all patients and in those with no water balance disorders. Discussion AVP and CRH act synergistically to stimulate the secretion of ACTH and ultimately cortisol [1, 2], and there is evidence that glucocorticoids act by way of negative feedback to suppress AVP secretion [10, 11-20]. Therefore, we hypothesized that a greater postoperative increase in plasma copeptin in those with CD in remission after TSS because of resolution of hypercortisolemia and resultant hypocortisolemia, compared with those not in remission with persistent hypercortisolemia and continued negative feedback, would be observed. Although a clear difference in peak postoperative and Δcopeptin was not observed in this study, a higher peak postoperative copeptin was found in those in remission after excluding those who developed DI/SIADH when analyzing this comparison with parametric testing, and it is possible that we did not have the power to detect a difference by nonparametric testing, given our small sample size. Therefore, postoperative plasma copeptin may be a useful early marker to predict remission of CD after TSS. The utility of this test may be limited to those who do not develop water balance disorders postoperatively. If a true increase in copeptin occurs for those in remission after treatment of CD, it is possible that this could be due to the removal of negative feedback from cortisol excess on pre-pro-AVP secretion, as hypothesized in this study. However, it is also possible that other factors may contribute to an increase in copeptin postoperatively, including from the stress response of surgery and postoperative hypocortisolism and resultant stimulation of pre-pro-AVP secretion from these physical stressors and/or from unrecognized SIADH. It was anticipated that more severe hypercortisolism to be negatively correlated with preoperative plasma copeptin because of greater negative feedback on AVP. However, no association was found between preoperative plasma copeptin and markers of severity of hypercortisolism (MN cortisol, AM ACTH, UFC × ULN) in this study. Similarly, we would expect that the preoperative plasma copeptin would be lower compared with healthy individuals. However, comparisons of healthy individuals may be difficult because the fluid and osmolality status at the time of the sample could influence the plasma copeptin, and depending on those factors, copeptin could be appropriately low. A healthy control group with whom to compare the preoperative values was not available for this study, and the thirsted state was not standardized for the preoperative copeptin measurements. Future studies could be considered to determine if preoperative plasma copeptin is lower in patients with CD, or other forms of CS, compared with healthy subjects, with all subjects thirsted for an equivalent period. Further, if preoperative plasma copeptin is found to be lower in thirsted subjects with CS than a thirsted healthy control group, the plasma copeptin could potentially be a diagnostic test to lend support for or against the diagnosis of endogenous CS. In the comparisons of those who developed DI, SIADH, or neither, no difference was found in the Δcopeptin. Peak copeptin was lower in DI compared with those without DI or SIADH (but not different from SIADH). Again, it is possible that there is a lower peak postoperative copeptin and change in copeptin in those with DI, but we may not have had the power to detect this in all of our analyses. These comparisons of copeptin among those with or without water balance disorders postoperatively are somewhat consistent with a prior study showing postoperative copeptin as a good predictor of development of DI, in which a plasma copeptin < 2.5 pmol/L measured on POD 0 accurately identified those who developed DI, and plasma copeptin > 30 pmol/L ruled out the development of DI postoperatively [29]. In the current study, 3 of 6 subjects with DI had a POD 1 plasma copeptin < 2.5 pmol/L, and none had a POD 1 plasma copeptin > 30 pmol/L. However, the study by Winzeler et al found that copeptin measured on POD 0 (within 12 hours after surgery) had the greatest predictive value, and POD 0 plasma copeptin was not available in our study. Further, we used the preoperative, peak, and delta plasma copeptin for analyses, so the early low copeptin levels may not have been captured in our data and analyses. Additionally, this study revealed that increasing levels of serum sodium have lower odds of remission. Those who have an ACTH-producing adenoma that is not identified by magnetic resonance imaging and visual inspection intraoperatively have lower rates of remission and are more likely to have greater manipulation of the pituitary gland intraoperatively [32-36], and the latter may result in greater damage to the pituitary stalk or posterior pituitary, increasing the risk for development of DI and resultant hypernatremia. A higher preoperative copeptin was associated with male sex and increasing BMI SDS. Increasing preoperative copeptin was also found in pubertal boys compared with pubertal girls, with no difference in copeptin between prepubertal boys and girls. It is particularly interesting to note that these associations were only in the preoperative plasma copeptin levels, but not the postoperative peak copeptin or Δcopeptin. Because the association of higher plasma in adult males and pubertal males in comparison to adult females and pubertal females, respectively, have been reported by others [26, 37-40], it raises the question of a change in the association of sex and BMI with plasma copeptin in the postoperative state. An effect of BMI or sex was not found by remission status, so it does not seem that the postoperative hypocortisolemic state for those in remission could explain this loss of association. However, this study may not have been powered to detect this. Strengths of this study include the prospective nature of the study. Further, this is the first study assessing the utility of copeptin to predict remission after treatment of CD. Limitations of this study include the small sample size because of the rarity of the condition, difficulty in clinically diagnosing DI and SIADH, potential effect of post-TSS fluid balance disorders (particularly for those who may have developed transient partial DI or transient SIADH), lack of long-term follow-up, lack of any postoperative follow-up in 11 of the 44 total subjects, as well the observational nature of the study. Further, it is possible that pubertal status, sex, and BMI may have affected copeptin levels, which may have not been consistently detected because of lack of power. Lack of data on the timing of hydrocortisone replacement is an additional limitation of this study because postoperative glucocorticoid replacement could affect AVP secretion via negative feedback. Additional studies are needed to assess to further assess the role of vasopressin and measurement of copeptin in patients before and after treatment of CD. A clear difference in peak postoperative plasma copeptin as an early marker to predict remission of CD after TSS was not found. Further studies with larger sample sizes are needed to further evaluate postoperative plasma copeptin as an early marker to predict remission of CD, though the utility of this test may be limited to those who do not develop water balance disorders postoperatively. Future studies comparing copeptin levels before and after treatment of adrenal CS would be of particular interest because this would minimize the risk of postoperative DI or SIADH which also influence copeptin levels. Additionally, comparison of thirsted preoperative plasma copeptin in those with endogenous CS and thirsted plasma copeptin in healthy controls could potentially provide evidence of whether or not preoperative plasma copeptin is lower in patients with CD, or other forms of CS, compared with healthy subjects. Further, if this is found to be true, it could potentially be a diagnostic test to lend support for or against endogenous CS. Abbreviations AVP arginine vasopressin BMI body mass index CD Cushing disease CS Cushing syndrome DI diabetes insipidus HPA hypothalamic-pituitary-adrenal IQR interquartile range MN midnight OR odds ratio POD postoperative day SDS SD score SIADH syndrome of inappropriate antidiuresis TSS transsphenoidal surgery UFC urinary free cortisol ULN upper limit of normal Acknowledgments The authors thank the patients and their families for participating in this study. Funding This work was supported by the Intramural Research Program, Eunice Kennedy Shriver National Institute of Child Health & Human Development (NICHD), National Institutes of Health. Disclosures C.A.S. holds patents on technologies involving PRKAR1A, PDE11A, GPR101, and related genes, and his laboratory has received research funding support by Pfizer Inc. for investigations unrelated to this project. C.A.S. is associated with the following pharmaceutical companies: ELPEN, Inc., H. Lunbeck A/S, and Sync. Inc. Clinical Trial Information ClinicalTrials.gov registration no. NCT00001595 (registered November 4, 1999). Data Availability Some or all datasets generated during and/or analyzed during the current study are not publicly available but are available from the corresponding author on reasonable request. Published by Oxford University Press on behalf of the Endocrine Society 2022. This work is written by (a) US Government employee(s) and is in the public domain in the US. From https://academic.oup.com/jes/article/6/6/bvac053/6564309?login=false
  13. 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/
  14. She experienced extreme weight gain, thin skin and a racing heart. It took years to finally solve the medical mystery. Angela Yawn went to a dozen doctors before finally getting a diagnosis for her life-disrupting symptoms.Courtesy Angela Yawn April 27, 2022, 10:52 AM EDT / Source: TODAY By A. Pawlowski When a swarm of seemingly unrelated symptoms disrupted Angela Yawn’s life, she thought she was going crazy. She gained weight — 115 pounds over six years — even as she tried to eat less. Her skin tore easily and bruises would stay on her body for months. Her face would suddenly turn blood red and hot to the touch as if she had a severe sunburn. She suffered from joint swelling and headaches. She felt tired, anxious and depressed. Her hair was falling out. Then, there was the racing heart. “I would put my hand on my chest because it made me feel like that’s what I needed to do to hold my heart in,” Yawn, 49, who lives in Griffin, Georgia, told TODAY. “I noticed it during the day, but at night when I was trying to lie down and sleep, it was worse because I could do nothing but hear it beat, feel it thump." Yawn, seen here before the symptoms began, had no problems with weight before.Courtesy Angela Yawn Yawn was especially frustrated by the weight gain. Even when she ate just 600 calories a day — consuming mostly lettuce leaves — she was still gaining about 2 pounds a day, she recalled. A doctor told her to exercise more. Yawn gained 115 pounds over six years. "When the weight really started to pile on, I stayed away from cameras as I felt horrible about myself and looking back at this picture is still very embarrassing for me but I wanted (people) to see what this disease has the potential to do if not diagnosed," she said.Courtesy Angela Yawn In all, Yawn went to a dozen doctors and was treated for high blood pressure and congestive heart failure, but nothing helped. As a last resort, she sought out an endocrinologist in February of 2021 and broke down in her office. “That was the last hope I had of just not lying down and dying because at that point, that’s what I wanted to do,” Yawn said. “I thought the problem was me. I thought that I’m making up these issues, that maybe I’m bipolar. I was going crazy.” What is Cushing disease? When the endocrinologist suddenly started listing all of her symptoms without being prompted, Yawn stopped crying. Blood tests and an MRI finally confirmed the doctor’s suspicion: Yawn had a tumor in her pituitary gland — a pea-size organ at the base of the brain — that was causing the gland to release too much adrenocorticotropic hormone. That, in turn, flooded her body with cortisol, a steroid hormone that’s normally released in response to stress or danger. The resulting condition is called Cushing disease. Imagine the adrenaline rush you’d get while jumping out of an airplane and skydiving — that’s what Yawn felt all the time, with harmful side-effects. Yawn was making six times the cortisol she needed, said Dr. Nelson Oyesiku, chair of neurosurgery at UNC Health in Chapel Hill, North Carolina, who removed her tumor last fall. “That’s a trailer load of cortisol. Day in, day out, morning, noon and night, whether you need it or not, your body just keeps making this excess cortisol. It can wreak havoc in the body physiology and metabolism,” Oyesiku told TODAY. The steroid regulates blood pressure and heart rate, which is why Yawn's skin was flushed and her heart was racing, he noted. It can regulate how fat is burned and deposited in the body, which is why Yawn was gaining weight. Other effects of the steroid's overproduction include fatigue, thin skin with easy bruising, mental changes and high blood sugar. Cushing disease is rare, affecting about five people per million each year, so most doctors will spend their careers without ever coming across a case, Oyesiku said. That’s why patients often go years without being diagnosed: When they complain of blood sugar problems or a racing heart, they’ll be treated for much more common issues like diabetes or high blood pressure. Pituitary gland is hard to reach Removing Yawn’s tumor in September of 2021 would require careful maneuvering. If you think of the head as a ball, the pituitary gland sits right at the center, between the ears, between the eyes and about 4 inches behind the nose, Oyesiku said. It’s called the “master gland” because it regulates other glands in the body that make hormones, he noted. The location of the pituitary gland makes it heard to reach.janulla / Getty Images It’s a very difficult spot to reach. To get to it, Oyesiku made an incision deep inside Yawn’s nose in a small cavity called the sphenoid sinus. Using a long, thin tube that carried a light and a camera, he reached the tiny tumor — about the size of a rice grain — and removed it using special instruments. The surgery took four hours. The potential risk is high: The area is surrounded by vessels that carry blood to the brain, and it’s right underneath optic nerves necessary for a person to see. If things go wrong, patients can become blind, brain dead, or die. Recovery from surgery Today, Yawn is slowly returning to normal. She has lost 41 pounds and continues to lose weight. Her hair is no longer falling out. But patients sometimes require months or even a few years to adjust to normal cortisol levels. “It takes some time to unwind the effects of chronic exposure to steroids, so your body has to adapt to the new world order as the effects of the steroids recede,” Oyesiku said. "My life was on hold for five years... I'm trying not to be too impatient," Yawn said.Courtesy Angela Yawn Yawn’s body was so used to that higher cortisol level that she’s had to rely on steroid supplements to feel normal after the surgery. It’s like an addict going through withdrawal, she noted. The next step is finishing another cycle of supplements and then slowly tapering off them so that her body figures out how to function without the steroid overload. “I am definitely moving in the right direction,” she said. "I hope that I’ll get back to that woman I used to be — in mind, body and spirit." From https://www.today.com/health/health/cushing-disease-pituitary-gland-tumor
  15. https://doi.org/10.1016/j.aace.2022.04.003Get rights and content Under a Creative Commons license Open access Highlights • We describe a rare case of a patient with a sparsely granulated corticotroph pituitary macroadenoma with pituitary apoplexy who underwent transsphenoidal resection resulting in remission of hypercortisolism. • Corticotroph adenomas are divided into densely granulated, sparsely granulated and Crooke’s cell tumors. • macroadenomas account for 7-23% of patients with pituitary corticotroph adenomas • Sparsely granulated corticotroph tumors are associated with longer duration of Cushing disease prior to diagnosis, larger tumor size at diagnosis, decreased immediate remission rate, increased proliferative marker Ki-67 and increased recovery time of hypothalamic-pituitary-adrenal axis after surgery. • Granulation pattern is an important clinicopathological distinction impacting the behavior and treatment outcomes of pituitary corticotroph adenomas Abstract Background /Objective: Pituitary corticotroph macroadenomas, which account for 7% to 23% of corticotroph adenomas, rarely present with apoplexy. The objective of this report is to describe a patient with a sparsely granulated corticotroph tumor (SGCT) presenting with apoplexy and remission of hypercortisolism. Case Report A 33-year-old male presented via ambulance with sudden onset of severe headache and nausea/vomiting. Physical exam revealed bitemporal hemianopsia, diplopia from right-sided third cranial nerve palsy, abdominal striae, facial plethora, dorsal and supraclavicular fat pad. Magnetic resonance imaging (MRI) demonstrated a 3.2 cm mass arising from the sella turcica with hemorrhage compressing the optic chiasm, extension into the sphenoid sinus and cavernous sinus. Initial investigations revealed plasma cortisol of 64.08 mcg/dL (Reference Range (RR), 2.36 – 17.05). He underwent emergent transsphenoidal surgery. Pathology was diagnostic of SGCT. Post-operatively, cortisol was <1.8ug/dL (RR, 2.4 – 17), adrenocorticotropic hormone (ACTH) 36 pg/mL (RR, 0 – 81), thyroid stimulating hormone (TSH) 0.07 uIU/mL (RR, 0.36 - 3.74), free thyroxine 1 ng/dL (RR, 0.8 – 1.5), luteinizing hormone (LH) <1 mIU/mL (RR, 1 – 12), follicle stimulating hormone (FSH) 1 mIU/mL (RR, 1 – 12) and testosterone 28.8 ng/dL (RR, 219.2 – 905.6) with ongoing requirement for hydrocortisone, levothyroxine, testosterone replacement and continued follow-up. Discussion Corticotroph adenomas are divided into densely granulated, sparsely granulated and Crooke’s cell tumors. Sparsely granulated pattern is associated with larger tumor size and decreased remission rate after surgery. Conclusion This report illustrates a rare case of hypercortisolism remission due to apoplexy of a SGCT with subsequent central adrenal insufficiency, hypothyroidism and hypogonadism. Keywords pituitary apoplexy pituitary macroadenoma pituitary tumor sparsely granulated corticotroph tumor Cushing disease Introduction The incidence of Cushing Disease (CD) is estimated to be between 0.12 to 0.24 cases per 100,00 persons per year1,2. Of these, 7-23% are macroadenomas (>1 cm)3, 4, 5. Pituitary apoplexy is a potentially life-threatening endocrine and neurosurgical emergency which occurs due to infarction or hemorrhage in the pituitary gland. Apoplexy occurs most commonly in non-functioning macroadenomas with an estimated prevalence of 6.2 cases per 100,000 persons and incidence of 0.17 cases per 100,00 persons per year6. Corticotroph macroadenoma presenting with apoplexy is uncommon with only a handful of reports in the literature7. We present a case of a sparsely granulated corticotroph (SGCT) which presented with apoplexy leading to remission of hypercortisolism and subsequent central adrenal insufficiency. Case Presentation A 33-year-old male who was otherwise healthy and not on any medications presented to a community hospital with sudden and severe headache accompanied by hypotension, nausea, vomiting, bitemporal hemianopsia and diplopia. Computed Tomography (CT) scan of the brain demonstrated a hyperattenuating 2.0 cm x 2.8 cm x 1.5 cm mass at the sella turcica with extension into the right cavernous sinus and encasement of the right internal carotid arteries (Figure 1A). He was transferred to a tertiary care center for neurosurgical management with endocrinology consultation post-operatively. Download : Download high-res image (404KB) Download : Download full-size image Figure 1. hyperattenuating 2.0 cm x 2.8 cm x 1.5 cm mass at the sella turcica on unenhanced CT (A); MRI demonstrated a 1.9 cm x 3.2 cm x 2.4 cm heterogeneous mass on T1 (B) and T2-weighted imaging (C) showing small hyperintense areas in solid part of the sella mass with flattening of the optic chiasm, remodeling/dehiscence of the floor of the sella and extending into the right cavernous sinus with at least partial encasement of the ICA In retrospect, he reported a 3-year history of ongoing symptoms of hypercortisolism including increased central obesity, dorsal and supraclavicular fat pad, facial plethora, abdominal purple striae, easy bruising, fatigue, decreased libido and erectile dysfunction. Notably, at the time of presentation he did not have a history of diabetes, hypertension, osteoporosis, fragility fractures or proximal muscle weakness. He fathered 2 children previously. His physical examination was significant for Cushingoid facies, facial plethora, dorsal and supraclavicular fat pads and central obesity with significant axillary and abdominal wide purple striae (Figure 2). Neurological examination revealed bitemporal hemianopsia, right third cranial nerve palsy with ptosis and impaired extraocular movement. The fourth and sixth cranial nerves were intact as was the rest of his neurological exam. These findings were corroborated by Ophthalmology. Download : Download high-res image (477KB) Download : Download full-size image Figure 2. Representative images illustrating facial plethora (A); abdominal striae (B, C); supraclavicular fat pad (D); dorsal fat pad (E) Initial laboratory data at time of presentation to the hospital included elevated plasma cortisol of 64.08ug/dL (RR, 2.36 – 17.05), ACTH was not drawn at the time of presentation, normal TSH 0.89 mIU/L (RR, 0.36 – 3.74), free thyroxine 0.91ng/dL (RR, 0.76 – 1.46), evidence of central hypogonadism with low total testosterone 28.8 ng/dL (RR, 219.2 – 905.6) and inappropriately normal luteinizing hormone (LH) 1mIU/mL (RR, 1 – 12) and follicle stimulating hormone (FSH) 3mIU/mL (RR, 1 – 12), low prolactin <1 ng/mL (RR, 3 – 20), and normal insulin growth factor – 1 (IGF–1) 179ng/mL (RR, 82 – 242). A pituitary gland dedicated MRI was performed to further characterize the mass, which re-demonstrated a 1.9 cm x 3.2 cm x 2.4 cm heterogenous mass at the sella turcica extending superiorly and flattening the optic chiasm, remodeling of the floor of the sella and bulging into the sphenoid sinus and extending laterally into the cavernous sinus with encasement of the right internal carotid artery (ICA). As per the radiologist’s diagnostic impression, this appearance was most in keeping with a pituitary macroadenoma with apoplexy (Figure 1B – C). The patient underwent urgent TSS and decompression with no acute complications. Pathological examination of the pituitary adenoma showed features characteristic of sparsely granulated corticotroph pituitary neuroendocrine tumor (adenoma)8, with regional hemorrhage and tumor necrosis (apoplexy). The viable tumor exhibited a solid growth pattern (Figure 3A), t-box transcription factor (T-pit) nuclear immunolabeling (Figure 3B), diffuse cytoplasmic CAM5.2 (low molecular weight cytokeratin) immunolabeling (Figure 3C), and regional weak to moderate intense granular cytoplasmic ACTH immuno-staining (Figure 3D). The tumor was immuno-negative for: pituitary-specific positive transcription factor 1 (Pit-1) and steroidogenic factor 1 (SF-1) transcription factors, growth hormone, prolactin, TSH, FSH, LH, estrogen receptor-alpha, and alpha-subunit. Crooke hyalinization was not identified in an adjacent compressed fragment of non-adenomatous anterior pituitary tissue. Ki-67 immunolabeling showed a 1.5% proliferative index (11 of 726 nuclei). Download : Download high-res image (2MB) Download : Download full-size image Figure 3. Hematoxylin phloxine saffron staining showing adenoma with solid growth pattern (A); immunohistochemical staining showing T-pit reactivity of tumor nuclei (B); diffuse cytoplasmic staining for cytokeratin CAM5.2 (C); and regional moderately intense granular cytoplasmic staining for ACTH (D). Scale bar = 20 μm Post-operatively, he developed transient central diabetes insipidus requiring desmopressin but resolved on discharge. His postoperative cortisol was undetectable, ACTH 36 pg/mL (RR, 0 - 81), TSH 0.07 mIU/mL (RR, 0.36 - 3.74), free thyroxine 1 ng/dL (RR, 0.8 - 1.5), LH <1mIU/mL (RR, 1 - 12), FSH 1 mIU/mL (RR, 1 - 12) and testosterone 28.8 ng/dL (RR, 219.2 - 905.6) (Table 1 and Figure 4). One month later, he reported 15 pounds of weight loss and a 5-inch decrease in waist circumference. He also noted a reduction in the dorsal and supraclavicular fat pads, facial plethora, and Cushingoid facies as well as fading of the abdominal stretch marks. His visual field defects and right third cranial nerve palsy resolved on follow up with ophthalmology post-operatively. Repeat MRI six months post-operatively showed minor residual soft tissue along the floor of the sella. He is being followed by Neurosurgery, Ophthalmology, and Endocrinology for monitoring of disease recurrence, visual defects, and management of hypopituitarism. Table 1. Pre- and post-operative hormonal panel POD -1 POD 0 POD1 POD2 POD3 POD16 6 -9 months Comments Cortisol(2.4 – 17 ug/dL) 64↓ 32↓ 11↓ <1.8↓ <1.8↓ 1.8↓ HC started POD3 post bloodwork ACTH(0 – 81 pg/mL) 41↓ 36↓ 28↓ 13↓ TSH(0.36 - 3.74 uIU/mL) 0.89 0.43 0.12↓ 0.07↓ 0.05↓ 0.73 Thyroxine, free(0.8 – 1.5 ng/dL) 0.9 0.9 1.1 1 2.1↑ 1 Levothyroxine started POD4 LH(1 – 12 miU/mL) 1↓ <1↓ 1↓ 3 FSH(1 – 12 mIU/mL) 3↓ 1↓ 1↓ 3 Testosterone(219.2 – 905.6 ng/dL) 28.8↓ <20↓ 175.9↓ Testosterone replacement started as outpatient Testosterone, free(160 - 699 pmol/L) <5.8↓ 137↓ IGF-1(82 – 242 ng/mL) 179 79 GH(fasting < 6 mIU/L) 4.5 <0.3 Prolactin(3 – 20 ng/mL) <1↓ <1↓ POD, postoperative day; HC, hydrocortisone; ACTH, adrenocorticotropic hormone; TSH, thyroid stimulating hormone; LH, luteinizing Hormone; FSH, follicle stimulating hormone; IGF-1, insulin like growth factor - 1; GH, growth hormone Download : Download high-res image (259KB) Download : Download full-size image Figure 4. Trend of select pituitary hormonal panel with key clinical events denoted by black arrows. Discussion Microadenomas account for the majority of corticotroph tumors, but 7% – 23% of patients are diagnosed with a macroadenoma3, 4, 5. It is even rarer for a corticotroph macroadenoma to present with apoplexy with only a handful of case reports or series in the literature7. Due to its rarity, appropriate biochemical workup on presentation, such as including an ACTH with the blood work, may be omitted especially if the patient is going for emergent surgery. In this case, the undetectable prolactin can reflect loss of anterior pituitary function and also suggest a functioning corticotroph adenoma due to the inhibitory effect of long term serum glucocorticoids on prolactin secretion9. After undergoing TSS, the patient developed central adrenal insufficiency, hypothyroidism and hypogonadism requiring hormone replacement. Presumably, the development of adrenal insufficiency demonstrated the remission of hypercortisolism as a result of apoplexy and/or TSS. The ACTH remains detectable likely representing residual tumor that was not obliterated by apoplexy nor excised by TSS given it location near the carotid artery and cavernous sinus. The presence of adrenal insufficiency in the setting of detectable ACTH is not contradictory as the physiological hypothalamic-pituitary-adrenal axis has been suppressed by the long-term pathological production of ACTH. IGF-1 and prolactin also failed to recover post-operatively. In CD where the production of IGF-1 and prolactin are attenuated by elevated cortisol, it would then be expected that IGF-1 and prolactin recover after hypercortisolism remission. However, the absence of this observation in our case is likely a sequalae of the apoplexy and extensive surgery leading to pituitary hypofunction. We also want to highlight features of the pre-operative radiographical findings which can provide valuable insight into the subsequent histology. Previous literature has shown that, on T2-weight MRI, silent corticotroph adenomas are strongly correlated with characteristic a multimicrocystic appearance while nonfunctional gonadotroph macroadenomas are not correlated with this MRI finding10. The multimicrocystic appearance is described as small hyperintense areas with hyperintense striae in the solid part of the tumor (Figure 1C)10. This is an useful predictive tool for silent corticotroph adenomas with a sensitivity of 76%, specificity of 95% and a likelihood ratio of 15.310. The ability to distinguish between silent corticotroph macroadenoma and other macroadenomas is important for assessing rate of remission and recurrence risk. In 2017, the WHO published updated classification for pituitary tumors. In this new classification, corticotroph adenomas are further divided into densely granulated, sparsely granulated and Crooke’s cell tumors11. DGCT are intensely Periodic Acid Schiff (PAS) stain positive and exhibit strong diffuse pattern of ACTH immunoreactivity, whereas SGCT exhibit faintly positive PAS alongside weak focal ACTH immunoreactivity4,12. Crooke’s cell tumors are characterized by Crooke’s hyaline changes in more than 50% of the tumor cells4. In the literature, SGCT account for an estimated 19-29% of corticotroph adenomas13, 14, 15. The clinicopathological relevance of granulation pattern in corticotroph tumors was unclear until recently. In multiple studies examining granulation pattern and tumor size, SGCT were statistically larger13,15,16. Hence, we suspect that many of the previously labelled silent corticotroph macroadenomas in the literature were SGCT. The traditional teaching of CD has been “small tumor, big Cushing and big tumor, small Cushing” which reflects the inverse relationship between tumor size and symptomatology17. This observation appears to hold true as Doğanşen et al. found a trend towards longer duration of CD in SGCT of 34 months compared to 26 months in DGCT based on patient history13,17. It has been postulated that the underlying mechanism of the inverse relationship between tumor size and symptomatology is impaired processing of proopiomelanocortin resulting in less effective secretion of ACTH in corticotroph macroadenomas3. Doğanşen et al. also found that the recurrence rate was doubled for SGCT, while Witek et al. showed that SGCT were less likely to achieve remission postoperatively13,16. Similar to other cases of SGCT, the diagnosis was only arrived retrospective after pathological confirmation10. Interestingly, the characteristic Crooke’s hyaline change of surrounding non-adenomatous pituitary tissue was not observed as one would expect in a state of prolonged glucocorticoid excess in this case. Although classically described, the absence of this finding does not rule out CD. As evident in a recent retrospective study where 10 out of 144 patients with CD did not have Crooke’s hyaline change18. In patients without Crooke’s hyaline change, the authors found a lower remission rate of 44.4% compared to 73.5% in patients with Crooke’s hyaline change. Together with the detectable post-operative ACTH, sparsely granulated pattern and absence of Crooke’s hyaline change in surrounding pituitary tissue, the risk of recurrence is increased. These risk factors emphasize the importance of close monitoring to ensure early detection of recurrence. Declaration of Interests ☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. ☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Conclusion We present a case of a sparsely granulated corticotroph macroadenoma presenting with apoplexy leading to remission of hypercortisolism and development of central adrenal insufficiency, hypothyroidism and hypogonadism requiring hormone replacement. References 1 J. Lindholm, S. Juul, J.O. Jørgensen, et al. Incidence and late prognosis of cushing's syndrome: a population-based study J Clin Endocrinol Metab, 86 (1) (2001), pp. 117-123 View Record in ScopusGoogle Scholar 2 J. Etxabe, J.A. Vazquez Morbidity and mortality in Cushing's disease: an epidemiological approach Clin Endocrinol (Oxf), 40 (4) (1994), pp. 479-484 View PDF CrossRefView Record in ScopusGoogle Scholar 3 L. Katznelson, J.S. Bogan, J.R. Trob, et al. Biochemical assessment of Cushing's disease in patients with corticotroph macroadenomas J Clin Endocrinol Metab, 83 (5) (1998), pp. 1619-1623 View Record in ScopusGoogle Scholar 4 L.V. Syro, F. Rotondo, M.D. Cusimano, et al. Current status on histological classification in Cushing's disease Pituitary, 18 (2) (2015), pp. 217-224 View PDF CrossRefView Record in ScopusGoogle Scholar 5 Y.S. Woo, A.M. Isidori, W.Z. Wat, et al. Clinical and biochemical characteristics of adrenocorticotropin-secreting macroadenomas J Clin Endocrinol Metab, 90 (8) (2005), pp. 4963-4969 View PDF CrossRefView Record in ScopusGoogle Scholar 6 C. Briet, S. Salenave, J.F. Bonneville, E.R. Laws, P. Chanson Pituitary Apoplexy Endocr Rev, 36 (6) (2015), pp. 622-645 View PDF CrossRefView Record in ScopusGoogle Scholar 7 K. Siwakoti, S.B. Omay, S.E. Inzucchi SPONTANEOUS RESOLUTION OF PRIMARY HYPERCORTISOLISM OF CUSHING DISEASE AFTER PITUITARY HEMORRHAGE AACE Clin Case Rep, 6 (1) (2020), pp. e23-e29 ArticleDownload PDFCrossRefView Record in ScopusGoogle Scholar 8 S.L. Asa, O. Mete What's new in pituitary pathology? Histopathology, 72 (1) (2018), pp. 133-141 View PDF CrossRefView Record in ScopusGoogle Scholar 9 M.E. Freeman, B. Kanyicska, A. Lerant, G. Nagy Prolactin: Structure, Function, and Regulation of Secretion Physiological Reviews, 80 (4) (2000), pp. 1523-1631 View PDF CrossRefView Record in ScopusGoogle Scholar 10 L. Cazabat, M. Dupuy, A. Boulin, et al. Silent, but not unseen: multimicrocystic aspect on T2-weighted MRI in silent corticotroph adenomas Clin Endocrinol (Oxf), 81 (4) (2014), pp. 566-572 View PDF CrossRefView Record in ScopusGoogle Scholar 11 M.B.S. Lopes The 2017 World Health Organization classification of tumors of the pituitary gland: a summary Acta Neuropathol, 134 (4) (2017), pp. 521-535 View PDF CrossRefView Record in ScopusGoogle Scholar 12 W. Saeger, J. Honegger, M. Theodoropoulou, et al. Clinical Impact of the Current WHO Classification of Pituitary Adenomas Endocr Pathol, 27 (2) (2016), pp. 104-114 View PDF CrossRefView Record in ScopusGoogle Scholar 13 S. Doğanşen, B. Bilgiç, G.Y. Yalin, S. Tanrikulu, S. Yarman Clinical Significance of Granulation Pattern in Corticotroph Pituitary Adenomas Turk Patoloji Derg, 35 (1) (2019), pp. 9-14 Google Scholar 14 O. Mete, A. Cintosun, I. Pressman, S.L. Asa Epidemiology and biomarker profile of pituitary adenohypophysial tumors Mod Pathol, 31 (6) (2018), pp. 900-909 View PDF CrossRefView Record in ScopusGoogle Scholar 15 B. Rak, M. Maksymowicz, M. Pękul, G. Zieliński Clinical, Biological, Radiological Pathological and Immediate Post-Operative Remission of Sparsely and Densely Granulated Corticotroph Pituitary Tumors: A Retrospective Study of a Cohort of 277 Patients With Cushing's Disease Front Endocrinol (Lausanne), 12 (2021) 672178 Google Scholar 16 P. Witek, G. Zieliński, K. Szamotulska, M. Maksymowicz, G. Kamiński Clinicopathological predictive factors in the early remission of corticotroph pituitary macroadenomas in a tertiary referral centre Eur J Endocrinol, 174 (4) (2016), pp. 539-549 View PDF CrossRefView Record in ScopusGoogle Scholar 17 A.M. McNicol Tumors of the pituitary gland. S. L. Asa. AFIP atlas of tumor pathology, third series The Journal of Pathology, 188 (1) (1999), pp. 115-116 View Record in ScopusGoogle Scholar 18 A. Akirov, V. Larouche, I. Shimon, et al. Significance of Crooke's Hyaline Change in Nontumorous Corticotrophs of Patients With Cushing Disease Front Endocrinol (Lausanne), 12 (2021) 620005 Google Scholar From https://www.sciencedirect.com/science/article/pii/S2376060522000268#!
  16. 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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  17. Sponsor: Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD) Information provided by (Responsible Party): National Institutes of Health Clinical Center (CC) ( Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD) Brief Summary: Background: The pituitary gland produces hormones. A tumor in this gland can cause it to produce too much of the hormone cortisol. Too much cortisol in the body causes Cushing disease. This disease causes many problems. Some of these problems might persist after the disease is cured. Objective: To find out the long-term effects of exposure to high levels of cortisol during childhood and adolescence. Eligibility: People ages 10-42years who were diagnosed with Cushing disease before age 21 and are now cured and have normal or low cortisol levels People related to someone with Cushing disease Design: Participants will be screened with a medical history. Participants will complete an online survey. This will include questions about their or their child s physical and mental health. All participants will be seen at 5 -year intervals after cure of Cushing disease (5yr, 10yr, 15yr, 20yr (last visit)) Participants who have a relative with Cushing disease will have a medical history and blood tests or cheek swabs. Participants who have the disease will have: Physical exam Blood tests Cheek swab DXA scan: A machine will x-ray the participant s body to measure bone mineral content. For participants who are still growing, a hand x-ray Participants with the disease may also have: Hormone stimulation test: Participants will get a hormone or another substance that will be measured. Serial hormone sampling: Participants blood will be measured several times through a thin plastic tube in an arm vein. Urine tests: Participants urine may be collected over 24 hours. MRI: Participants may have a dye injected into a vein. They will lie on a table that slides into a machine. The machine will take pictures of the body. Read more https://clinicaltrials.gov/ct2/show/NCT03831958#eligibility
  18. The LINC 4 study demonstrated superiority of Isturisa® (osilodrostat) over placebo in achieving cortisol normalisation during the 12-week, double-blind, randomised phase (77% vs 8%, P<0.0001). Isturisa provided rapid and sustained control of cortisol secretion in the majority of patients throughout the 48-week core phase of the study. PUTEAUX, France, March 29, 2022--(BUSINESS WIRE)--Recordati Rare Diseases announce today the publication of positive results from the Phase III LINC 4 study of Isturisa in The Journal of Clinical Endocrinology & Metabolism.1 These data reinforce Isturisa as an effective and well-tolerated oral therapy for patients with Cushing’s disease. Isturisa is indicated in the EU for the treatment of adult patients with endogenous Cushing’s syndrome,2 a rare and debilitating condition of hypercortisolism that is most commonly caused by a pituitary adenoma (Cushing’s disease).3 The LINC 4 study augments the efficacy and safety data for Isturisa in patients with Cushing’s disease, confirming the results from the Phase III LINC 3 study. This study in 73 adults is the first Phase III study of a medical treatment in patients with Cushing’s disease to include an upfront, randomised, double-blind, placebo-controlled period during which 48 patients received Isturisa and 25 received placebo for the first 12 weeks, followed by an open-label period during which all patients received Isturisa until week 48; thereafter, patients could enter an optional extension phase. Key findings published in the manuscript entitled ‘Randomised trial of osilodrostat for the treatment of Cushing’s disease’ include:1 LINC 4 met the primary endpoint: Isturisa was significantly superior to placebo at normalising mUFC at the end of a 12-week randomised, double-blind period (77% vs 8%; P<0.0001). Effects of Isturisa were rapid. Over one-quarter of patients randomised to Isturisa achieved normal mUFC as early week 2 and 58% achieved control by week 5. The key secondary endpoint was also met, with 81% of all patients in the study having normal mUFC at week 36. Improvements in cardiovascular and metabolic parameters of Cushing’s disease, including blood pressure and blood glucose metabolism, were seen by week 12 and were maintained throughout the study. Physical features of hypercortisolism improved during Isturisa treatment, including fat pads, facial rubor, striae, and muscle wasting. Improvements were observed by week 12, with continued improvement throughout the study to week 48. Patient-reported QoL scores (CushingQoL and Beck Depression Inventory) also improved during Isturisa treatment. Isturisa was well tolerated in the majority of patients, with no unexpected adverse events (AEs). The most common AEs overall were decreased appetite, arthralgia, fatigue and nausea. "These results show convincingly that osilodrostat is an effective treatment for Cushing’s disease," said Peter J. Snyder MD, Professor of Medicine at the University of Pennsylvania. "Osilodrostat rapidly lowered cortisol excretion to normal in most patients with Cushing’s disease and maintained normal levels throughout the core phase of the study. Importantly, this normalisation was accompanied by improvements in cardiovascular and metabolic parameters, which increase morbidity and mortality in Cushing’s disease." "These compelling data build on the positive Phase III LINC 3 study, published in The Lancet Diabetes & Endocrinology in 2020,4 demonstrating that Isturisa enables most patients with Cushing’s disease to gain rapid control of their cortisol levels, which in turn provides relief from a host of undesirable symptoms," said Alberto Pedroncelli, Clinical Development & Medical Affairs Lead, Global Endocrinology, Recordati AG. "Recordati Rare Diseases is committed to improving the lives of patients with this rare, debilitating and life-threatening condition. I would like to thank everyone who has contributed to LINC 4 and the LINC clinical programme." "I had Cushing's disease for 8 years without being diagnosed," said Thérèse Fournier from L'association "Surrénales". "I was trapped in a vicious circle of missed diagnoses and worsening physical and psychological symptoms that became life-threatening. I lost everything – my job, my house, my partner, my friends – I was isolated. When I finally received my diagnosis, I was relieved because I knew the truth. Since my surgery, I have been learning to live again, enjoying the moments that make a life. I am still on the path to remission, but I feel deeply happy, even if I carry this journey that nobody can understand." About Cushing’s syndrome Cushing’s syndrome is a rare disorder caused by chronic exposure to excess levels of cortisol from either an exogenous (eg medication) or an endogenous source.5 Cushing’s disease is the most common cause of endogenous Cushing’s syndrome and arises as a result of excess secretion of adrenocorticotropic hormone from a pituitary adenoma, a tumour of the pituitary gland.5,6 There is often a delay in diagnosing Cushing’s syndrome, which consequently leads to a delay in treating patients.7 Patients who are exposed to excess levels of cortisol for a prolonged period have increased comorbidities associated with the cardiovascular and metabolic systems, which consequently reduce QoL and increase the risk of mortality.3,6 To alleviate the clinical signs associated with excess cortisol exposure, the primary treatment goal in Cushing’s syndrome is to reduce cortisol levels to normal.8 About LINC 4 LINC 4 is a multicentre, randomised, double-blind, 48-week study with an initial 12-week placebo-controlled period to evaluate the safety and efficacy of Isturisa® in patients with Cushing’s disease. The LINC 4 study enrolled patients with persistent or recurrent Cushing’s disease or those with de novo disease who were ineligible for surgery; 73 randomised patients were treated with Isturisa® (n=48) or placebo (n=25).1 The primary endpoint of the study is the proportion of randomised patients with a complete response (mUFC ≤ULN) at the end of the placebo-controlled period (week 12). The key secondary endpoint is the proportion of patients with an mUFC ≤ULN at week 36.1,9 About Isturisa® Isturisa® is an oral inhibitor of 11β-hydroxylase (CYP11B1), which catalyses the final step of cortisol synthesis in the adrenal glands.2 Isturisa® is available as 1 mg, 5 mg and 10 mg film-coated tablets.2 Isturisa® is approved for the treatment of adult patients with endogenous Cushing’s syndrome in the EU and is now available in France, Germany, Greece and Austria.2 Isturisa® was granted marketing authorisation by the European Commission on 9 January 2020. For detailed recommendations on the appropriate use of this product, please consult the summary of product characteristics.2 References 1. Gadelha M, Bex M, Feelders RA et al. Randomised trial of osilodrostat for the treatment of Cushing's disease. J Clin Endocrinol Metab 2022; dgac178, https://doi.org/10.1210/clinem/dgac178. 2. Isturisa® summary of product characteristics. May 2020. 3. Ferriere A, Tabarin A. Cushing's syndrome: Treatment and new therapeutic approaches. Best Pract Res Clin Endocrinol Metab 2020;34:101381. 4. Pivonello R, Fleseriu M, Newell-Price J 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 Endocrinol 2020;8:748-61. 5. Lacroix A, Feelders RA, Stratakis CA et al. Cushing's syndrome. Lancet 2015;386:913-27. 6. Pivonello R, Isidori AM, De Martino MC et al. Complications of Cushing's syndrome: state of the art. Lancet Diabetes Endocrinol 2016;4:611-29. 7. Rubinstein G, Osswald A, Hoster E et al. Time to diagnosis in Cushing's syndrome: A meta-analysis based on 5367 patients. J Clin Endocrinol Metab 2020;105:dgz136. 8. Nieman LK, Biller BM, Findling JW et al. Treatment of Cushing's syndrome: An Endocrine Society clinical practice guideline. J Clin Endocrinol Metab 2015;100:2807-31. 9. ClinicalTrials.gov. NCT02697734; available at https://clinicaltrials.gov/ct2/show/NCT02697734 (accessed March 2021). Recordati Rare Diseases, the company’s EMEA headquarters are located in Puteaux, France, with global headquarter offices in Milan, Italy. For a full list of products, please click here: www.recordatirarediseases.com/products. Recordati, established in 1926, is an international pharmaceutical group, listed on the Italian Stock Exchange (Reuters RECI.MI, Bloomberg REC IM, ISIN IT 0003828271), with a total staff of more than 4,300, dedicated to the research, development, manufacturing and marketing of pharmaceuticals. Headquartered in Milan, Italy, Recordati has operations in Europe, Russia and the other C.I.S. countries, Ukraine, Turkey, North Africa, the United States of America, Canada, Mexico, some South American countries, Japan and Australia. An efficient field force of medical representatives promotes a wide range of innovative pharmaceuticals, both proprietary and under license, in several therapeutic areas including a specialized business dedicated to treatments for rare diseases. Recordati is a partner of choice for new product licenses for its territories. Recordati is committed to the research and development of new specialties with a focus on treatments for rare diseases. Consolidated revenue for 2021 was € 1,580.1 million, operating income was € 490.2 million and net income was € 386.0 million. For further information: Recordati website: www.recordatirarediseases.com This document contains forward-looking statements relating to future events and future operating, economic and financial results of the Recordati group. By their nature, forward-looking statements involve risk and uncertainty because they depend on the occurrence of future events and circumstances. Actual results may therefore differ materially from those forecast as a result of a variety of reasons, most of which are beyond the Recordati group’s control. The information on the pharmaceutical specialties and other products of the Recordati group contained in this document is intended solely as information on the Recordati group’s activities and therefore, as such, it is not intended as medical scientific indication or recommendation, nor as advertising. View source version on businesswire.com: https://www.businesswire.com/news/home/20220325005169/en/ Contacts Celine Plisson, MD Medical Affairs Director Telephone: +33(0)147739463 Email: PLISSON.C@recordati.com Related Quotes Symbol Last Price Change % Change REC Emles Real Estate Credit ETF 22.89 +0.13 +0.57% TRENDING 1. Oil Climbs After Two-Day Drop as Investors Assess Ukraine Talks 2. Stocks Fall, Oil Rises as Russia Concerns Return: Markets Wrap 3. Truckmaker MAN to shorten hours of up to 11,000 workers on Ukraine crisis 4. UPDATE 1-Sri Lanka suffers long power cuts as currency shortage makes fuel scarce 5. German inflation rises more than expected in March From https://finance.yahoo.com/news/recordati-rare-diseases-announce-publication-070000542.html?guccounter=1&guce_referrer=aHR0cHM6Ly93d3cuZ29vZ2xlLmNvbS8&guce_referrer_sig=AQAAABds3nKexRHBGxK9BEM1W93vciZ-QM8hw9-QOcCELZxkbW8U7OfcYw-GBd_tvRaS9mnjvRvdhI1sQaBD2jkR6yvqhUZsHw6f7CfO78LaGvdTRWhjaIy7b5IcPvPpOCGzR0Ex1_8t1TFNqxMkSr7OmIxfDflflXrh4cHI8Ze3okeU
  19. An analysis of nationwide data from Sweden provides an overview of the increased risk of death associated with Cushing's disease was present even after biochemical remission. New data from an analysis of patient data over nearly 30 years suggests the increased risk of mortality associated with Cushing’s disease persists even after treatment. A 4:1 matched analysis comparing data from 371 patients with Cushing’s disease with 1484 matched controls, indicated risk of mortality was 5-fold greater among those not in remission compared to matched controls, but even those in remission at the last follow-up were at a 50% greater risk of mortality compared to controls. “To our knowledge, this is the first study that investigated mortality in an unselected cohort of patients treated for Cushing’s disease and followed up in comparison to mortality in matched controls. The mortality rate was more than doubled in patients with Cushing’s disease, and not being in remission was a strong predictor of premature death,” wrote investigators. With a lack of consensus surrounding the impact of biochemical remission on life expectancy in patients with Cushing’s disease, a team of investigators from multiple institutions in Sweden designed their study with the intent of assessing this association with mortality in a time-to-event analysis of an unselected nationwide Cushing’s disease cohort. Using the Swedish Pituitary Registry, investigators identified 371 patients with Cushing’s disease for inclusion in their analysis. The Swedish Pituitary Register is a nationwide registry that collected data on the majority of Swedish patients with Cushing’s disease. For the current study, investigators included all patients with Cushing’s disease from the register diagnosed between May 1991-September 2018 and followed these patients until the date of death, date of emigration, or December 26, 2018. From the register, investigators obtained data related to date of diagnosis, age, sex, treatment, and biochemical remission status evaluations. The median age at diagnosis was 44 (IQR, 32-56) years and the median follow-up was 10.6 (IQR, 5.7-18) years. The remissions rates for the study cohort were 80%, 92%, 96%, 91%, and 97% at the 1-, 5-, 10-, 15- and 20-year follow-ups, respectively. These patients were matched in a 4:1 based on age, sex, and residential area at the diagnosis data, yielding a cohort of 1484 matched controls. Upon analysis, the overall risk of mortality was greater among those with Cushing’s disease compared to the matched controls (HR, 2.1 [95% CI, 1.5-2.8]). Investigators pointed out increased risk was observed among patients in remission at the last follow-up (n=303; HR, 1.5 [95% CI, 1.02-2.2]), those in remission after a single pituitary surgery (n=177; HR, 1.7 [95% CI, 1.03-2.8]), and those not in remission (n=31; HR, 5.6 [95% CI, 2.7-11.6]). Additionally, results indicated cardiovascular disease and infections were the most overrepresented cases of death, accounting for 32 and 12 of the 66 total instances of mortality. “The findings of the present study confirm and complement previous findings of increased overall mortality in Cushing’s disease patients, having a more than doubled HR for death compared to matched controls. Most importantly, an increased HR persisted among patients who had been successfully treated and reached a Cushing’s disease biochemical cure,” investigators added. This study, “Increased mortality persists after treatment of Cushing’s disease: A matched nationwide cohort study,” was published in the Journal of the Endocrine Society. From https://www.endocrinologynetwork.com/view/medicaid-expansion-under-aca-may-have-reduced-rate-of-major-diabetes-related-amputations
  20. 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.
  21. Although Dr. Friedman is at the forefront of Cushing’s Disease, he was not invited to be part of the Pituitary Society Consensus Guidelines on Cushing’s Disease published in Lancet Diabetes and Endocrinology in 2021, many of his ideas on Cushing’s Disease that he has been advocating for years were included in the recent guidelines. In this informative webinar, Dr. Friedman will discuss The use of imaging for the diagnosis of Cushing’s Disease The need for multiple testing to diagnose episodic Cushing’s Disease The importance of UFC and salivary cortisol testing The use of medication trial prior to surgery The use of ketoconazole for the medication trial and longer-term treatment Dr. Friedman will also discuss new Cushing’s medications. Sunday • April 3 • 6 PM PST Via Zoom Click here to join the meeting orhttps://us02web.zoom.us/j/4209687343?pwd=amw4UzJLRDhBRXk1cS9ITU02V1pEQT09OR+16699006833,,4209687343#,,,,*111116#Slides will be available on the day of the talk here. You can also click to read the consensus guidelines There will be plenty of time for questions using the chat button. For more information, email us at mail@goodhormonehealth.com
  22. Although Dr. Friedman is at the forefront of Cushing’s Disease, he was not invited to be part of the Pituitary Society Consensus Guidelines on Cushing’s Disease published in Lancet Diabetes and Endocrinology in 2021, many of his ideas on Cushing’s Disease that he has been advocating for years were included in the recent guidelines. In this informative webinar, Dr. Friedman will discuss The use of imaging for the diagnosis of Cushing’s Disease The need for multiple testing to diagnose episodic Cushing’s Disease The importance of UFC and salivary cortisol testing The use of medication trial prior to surgery The use of ketoconazole for the medication trial and longer-term treatment Dr. Friedman will also discuss new Cushing’s medications. Sunday • April 3 • 6 PM PST Via Zoom Click here to join the meeting orhttps://us02web.zoom.us/j/4209687343?pwd=amw4UzJLRDhBRXk1cS9ITU02V1pEQT09OR+16699006833,,4209687343#,,,,*111116#Slides will be available on the day of the talk here. You can also click to read the consensus guidelines There will be plenty of time for questions using the chat button. For more information, email us at mail@goodhormonehealth.com
  23. 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
  24. Abstract Corticotroph pituitary adenomas commonly cause Cushing’s disease (CD), but some of them are clinically silent. The reason why they do not cause endocrinological symptoms remains unclear. We used data from small RNA sequencing in adenomas causing CD (n = 28) and silent ones (n = 20) to explore the role of miRNA in hormone secretion and clinical status of the tumors. By comparing miRNA profiles, we identified 19 miRNAs differentially expressed in clinically functioning and silent corticotroph adenomas. The analysis of their putative target genes indicates a role of miRNAs in regulation of the corticosteroid receptors expression. Adenomas causing CD have higher expression of hsa-miR-124-3p and hsa-miR-135-5p and lower expression of their target genes NR3C1 and NR3C2. The role of hsa-miR-124-3p in the regulation of NR3C1 was further validated in vitro using AtT-20/D16v-F2 cells. The cells transfected with miR-124-3p mimics showed lower levels of glucocorticoid receptor expression than control cells while the interaction between miR-124-3p and NR3C1 3′ UTR was confirmed using luciferase reporter assay. The results indicate a relatively small difference in miRNA expression between clinically functioning and silent corticotroph pituitary adenomas. High expression of hsa-miR-124-3p in adenomas causing CD plays a role in the regulation of glucocorticoid receptor level and probably in reducing the effect of negative feedback mediated by corticosteroids. Keywords: neuroendocrine pituitary tumors; Cushing’s disease; silent corticotroph adenoma; miRNA; hsa-miR-124-3p; NR3C1; glucocorticoid receptor 1. Introduction Pituitary adenomas (also referred to as pituitary neuroendocrine tumors, PitNETs) represent about 10–20% of intracranial neoplasms in adults. They may originate from different kinds of secretory pituitary cells including corticotroph ACTH-secreting cells. Corticotroph adenomas commonly cause ACTH-dependent Cushing’s disease, but a significant proportion of these tumors are endocrinologically non-functioning and classified as subclinical/silent corticotroph adenomas (SCAs) [1]. CD-causing ACTH tumors are commonly small microadenomas with approximately 50% being smaller than 5 mm, which is challenging for MRI diagnostics [2]. In contrary, SCAs are commonly diagnosed due to neurological symptoms related to tumor mass at the stage of large macroadenomas. Frequently they show invasive growth and increased proliferation index [1]. According to current recommendations, SCAs are now referred to as “high-risk” pituitary adenomas which refers to their fast and invasive growth, high risk of recurrence and resistance to medical therapy [3,4]. They are recognized to be more aggressive than other clinically nonfunctioning pituitary tumors such as those of gonadotroph origin or null-cell adenomas [5]. The mechanism underlying the difference in secretory activity of CD-causing and subclinical tumors is unclear and only a few studies focused on this issue were published. The results indicated a role of the expression levels of particular genes/proteins involved in the regulation of POMC expression and pro-hormone conversion into ACTH as well as genes involved in pituitary differentiation [6,7,8,9,10,11,12,13]. However, it also appears that both active and silent corticotroph adenomas share a similar overall gene expression profile [14,15]. The aim of this study was to compare the profiles of microRNA (miRNA) expression in clinically functioning and silent corticotroph adenomas and to identify miRNAs that play a role in different ACTH secretory activity. 2. Results 2.1. Patients Characteristics The study included 28 patients with CD and 20 patients suffering from SCA. All patients with CD had clear clinical signs and symptoms of hypercortisolism verified according to biochemical criteria including elevated midnight cortisol levels and 24 h urinary free cortisol (UFC). Patients with SCA had no clinical or biochemical signs of hypercortisolism and showed normal levels of midnight cortisol and 24 h UFC. Patients with CD had significantly higher morning serum cortisol levels than patients with SCAs (p = 0.0002) while no significant difference was observed in the morning serum ACTH levels. No difference in cortisol/ACTH ratio was observed between CD and SCA patients. All the adenoma samples were ACTH-positive upon immunohistochemical staining against pituitary hormones (ACTH, GH, TSH, FSH, LH, α-subunit) and had characteristic ultrastructural features of corticotroph adenoma. Forty-one adenomas were positive only for ACTH, while seven ACTH-positive adenomas showed additional moderate/weak immunoreactivity for α-subunit. Increased proliferation assessed by Ki67 index ≥ 3% was observed in a similar proportion of CD and SCA patients, seven tumors causing CD and five SCAs. A higher proportion of sparsely vs. densely granulated adenomas was observed in SCAs than in CD-related adenomas, but the difference did not cross a significance threshold (p = 0.0787). No difference in the proportion of invasive/noninvasive adenomas was observed in clinically functioning and silent corticotroph adenomas. All SCAs were macroadenomas, while tumors causing CD included 17 macroadenomas and 11 microadenomas. No significant differences in preoperative clinical parameters, including 24 h UFC, morning serum ACTH level, morning and midnight serum cortisol level, cortisol/ACTH ratio, were observed between CD patients with micro- and macroadenomas. Irrespectively, a correlation between tumors size and ACTH level (Spearman R= 0.4678; p = 0.0121) and a negative correlation between cortisol/ACTH ratio (Spearman R= −0.4015; p = 0.0342) was observed in CD patients. No correlation was found between the remaining biochemical parameters and tumor size. Overall, the patients’ characteristics are presented in Table 1, while details including both the clinical and histopathological data are shown in Supplementary Table S1. Table 1. Summary of clinical features of patients with Cushing’s disease and silent corticotroph adenomas. 2.2. Identification of miRNAs Differentially Expressed in Corticotroph Adenomas Causing CD and Subclinical Cortiotroph Adenomas NGS data on miRNA expression of 48 corticotroph adenomas from previous investigation were used to compare miRNA expression levels between adenomas causing CD (n = 24) and subclinical corticotroph adenomas (n = 20). Sequencing of small RNA libraries produced approximately 2,497,367 reads per sample, which were mapped to the human genome (hg19) and used for quantification of expression levels of known miRNAs, according to miRBase 22 release. Sequencing reads were annotated to 1917 miRNAs. Measurements of 1902 mature miRNAs expression were included in the analysis, after filtering out the miRNAs with low expression. When miRNA profiles of adenomas causing CD and SCAs were compared, a total of 19 differentially expressed miRNAs were found that met the criteria of adjusted p-value < 0.05. This set included 16 miRNAs with higher expression in tumors causing CD: hsa-miR-129-2-3p, hsa-miR-129-5p, hsa-miR-124-3p, hsa-miR-132-5p, hsa-miR-129-1-3p, hsa-miR-135b-5p, hsa-miR-27a-3p, hsa-miR-10b-5p, hsa-miR-9-3p, hsa-miR-6506-3p, hsa-miR-6864-5p, hsa-let-7b-5p, hsa-miR-670-3p, hsa-miR-22-5p, hsa-miR-346 and hsa-miR-9-5p, Three miRNAs with lower expression in CD patients were found: hsa-miR-1909-3p, hsa-miR-4319 and hsa-miR-181b-3p. Details are presented in Table 2 and Figure 1A,B. Figure 1. MiRNA expression profiling in corticotroph adenomas. (A). Difference in miRNA expression between functioning and silent corticotroph adenomas. Volcano plot showing differentially expressed miRNAs. Significance and fold change thresholds are marked with dashed lines. (B). Heat map representing the expression of differentially expressed miRNAs and clustering the samples of adenomas causing Cushing’s disease (CD) and silent corticotroph adenomas (SCA). (C). The correlation between the expression levels of differentially expressed miRNAs and POMC expression or hormonal laboratory measurements in patients: morning plasma ACTH level, morning and midnight plasma cortisol levels and 24 h urinary free cortisol; * indicate p-value < 0.05; ** indicate p-value < 0.01; *** indicate p-value < 0.001 Table 2. The list of miRNAs differentially expressed in corticotroph pituitary adenomas causing CD and silent corticotroph adenomas. 2.3. The Correlation of miRNA Expression and Patients’ Clinical Data Since the clustering of the tumors based on the expression of differentially expressed miRNAs did not clearly separate functioning and silent adenomas, we determined whether the expression of the identified differentially expressed miRNAs is directly related to the results of patients’ laboratory tests as well as POMC expression, measured in tumor samples with qRT-PCR. For this purpose, Spearman’s correlation was applied to calculate a correlation matrix. We observed a significant positive correlation between 13 miRNAs out of 19 differentially expressed miRNAs and at least one of clinical laboratory parameters: serum ACTH, morning cortisol level, midnight cortisol level or 24 h UFC. For 11 miRNAs, with higher expression in patients with CD a positive correlation was observed, while a negative correlation was observed for 3 miRNAs that have lower expression in patients with CD. Four of the differentially expressed miRNAs, hsa-miR-9-3p, hsa-miR-9-5p, hsa-miR-27a-3p and hsa-miR-6506-3p, are correlated with POMC expression level in tumor tissue. The absolute value of correlation coefficient ranged between 0.31 and 0.55 which indicates a weak/moderate relationship. Details are presented in Figure 1C. 2.4. Funtional Enrichment Analysis of Differentially Expressed miRNAs To investigate the possible functional role of the identified miRNAs with different expression levels in CD tumors and SCAs, we used the information on experimentally validated miRNA targets gathered in the miRtarbase release 8.0 database. High confidence known miRNA targets that were validated with luciferase reporter assay, reported in miRtarbase, were included in the analysis. The enrichment of the genes reported as miRNA targets of our 19 miRNAs of interest was determined with gene set over-representation analysis (GSOA) based on Gene Ontology (GO) Molecular Function and GO Biological Processes. The list of all the genes reported in miRTarbase as validated with reporter gene assay was used as reference. As a result, we found 30 GO Molecular Function terms and 293 GO Biological Processes terms as significantly enriched with genes that are targets of the 19 differentially expressed miRNAs. Top 10 enriched terms were related mainly to steroid hormone activity, regulation of transcription and regulation of stem cell differentiation, as shown in Figure 2. Details are presented in Supplementary Table S2. We paid special attention to the terms that refer to steroid hormone action, i.e., steroid hormone receptor activity (GO:0003707), nuclear receptor activity (GO:0004879), ligand-activated transcription factor activity (GO:0098531), as well as steroid hormone-mediated signaling pathway (GO:0043401) and hormone-mediated signaling pathway (GO:0009755). Importantly, the miRNA target genes that were overrepresented in these terms included NR3C1 and NR3C2 that encode for adrenal hormones glucocorticoid receptor (GR) and mineralocorticoid receptor (MR), respectively. According to the miRtarbase 9.0 database, hsa-miR-124-3p is a negative regulator of NR3C1 gene [16] while both hsa-miR-124-3p and hsa-miR-135b-5p downregulate MR [17]. Figure 2. Gene set over-representation analysis of putative target genes of miRNAs differentially expressed in clinically functioning and silent corticotroph adenomas. Using the PubMed search, we found additional evidence strongly supporting the role of hsa-miR-124-3p in the regulation of NR3C1 [18,19,20,21] as well as the role of hsa-miR-135b-5p in downregulating NR3C2 [22,23]. 2.5. Comparison of the Expression of NR3C1 and NR3C2 in Corticotroph Adenomas Causing CD and Silent Adenomas We determined the expression levels of NR3C1 and NR3C2 in corticotroph adenomas with qRT-PCR. We observed a significantly lower expression of both genes in samples from CD patients (n = 24) as compared to SCAs (n = 24); fold change (FC) 0.49 p = 0.0166 and FC 0.37 p = 0.0132, for NR3C1 and NR3C2, respectively. However, the observed difference is rather slight and a notable dispersion of the results was observed (Figure 3). The differences in NR3C1 and NR3C2 expression correspond to the differences in hsa-miR-124-3p and hsa-miR-135b-5p levels. Patients with CD have higher levels of both miRNAs and lower levels of NR3C1 and NR3C2 mRNA (Figure 3). Unfortunately, we did not find a direct correlation between the expression levels of hsa-miR-124-3p and NR3C1 or hsa-miR-135b-5p and NR3C2. Figure 3. The expression levels of NR3C1 and NR3C2 measured with qRT-PCR as well as hsa-miR-124-3p and hsa-miR-135b-5p measured with small RNA sequencing in tumor samples from CD patients and silent corticotroph adenomas; * indicate p-value < 0.05 2.6. Investigtion of miRNA-Related Regulation of NR3C1 In Vitro Transfecting the cultured cells with miRNA mimics is the commonly used approach of in vitro validation of specific miRNA–mRNA interaction. We used mice corticotroph tumor AtT-20/D16v-F2 cells for in vitro experiment and initially verified whether these cells do express Nr3c1 and Nr3c2 genes using deposited RNAseq data from a previous experiment on AtT-20 cells (GSE132324; Gene Expression Omnibus) and qRT-PCR. This showed that the AtT-20/D16v-F2 have relatively high expression of Nr3c1 but do not express Nr3c2. Thus, we focused on the regulatory role of miR-124-3p on Nr3c1 expression. We used miRBase [24] and Targetscan [25] to determine whether miR-124-3p is evolutionarily conserved in humans and mice and whether it targets NR3C1 in both species. It confirmed that miR-124-3p is broadly conserved and it shares the same sequence of mature miRNA in humans and mice. Importantly, GR is among highly rated miR-124-3p predicted targets in both humans and mice and two highly conserved miR-124-3p binding motifs in 3′UTR of this gene were identified in these two species (Figure 4A). Figure 4. Role of mir-124-3p in regulation of glucocorticoid receptor gene. (A). Putative hsa-mir-124-3p target sites in 3′UTR of NR3C1. (B). Reduced expression of Nr3c1 gene expression and glucocorticoid receptor (GR) protein level in AtT-20/D16v-F2 cells treated with hsa-miR-124-3p mimics. (C). Results of luciferase reporter gene assay, showing the interaction between Nr3c1 3′UTR site 2 and mir-124-3p; * indicate p-value < 0.05; ns—not significant. When we transfected AtT-20/D16v-F2 cells with miR-124-3p miRNA mimic and unspecific negative control miRNA mimic, we observed a significant decrease in Nr3c1 expression in cells treated with miR-124-3p miRNA mimic (Figure 4B). It was significantly lower than in cells treated with unspecific miRNA mimic. This difference was also clearly visible at the protein level. The GR level was reduced in cells treated with miR-124-3p miRNA mimic as compared to control (Figure 4B). Two fragments of Nr3c1 3′UTR including each of putative miR-124-3p binding motifs were cloned in plasmid vector into 3′ region of the firefly luciferase gene. AtT-20/D16v-F2 cells were transfected with empty vector, vector with miR-124-3p binding site 1 and vector miR-124-3p binding site 2. Each of the three variants of the cells were cotransfected with miR-124-3p miRNA mimic or unspecific miRNA mimic that served as a negative control. Luminescence was developed 48 h after transfection and detected with microplate reader. As a result, we observed a significant decrease in luminescence in the cells with introduced plasmid with miR-124-3p binding site 2 treated with miR-124-3p mimic as compared to the cells transfected with the same plasmid construct but with control miRNA mimic. This observation confirms the interaction between miR-124-3p and 3′ UTR of Nr3c1 at putative binding site 2 (Figure 4C). The experiment did not confirm an interaction between miR-124-3p and 3′ UTR of Nr3c1 at binding site 1 since no significant difference of luminescence was found in cells transfected with plasmid vector harboring this binding motif treated with miR-124-3p mimic and the same cells treated with negative miRNA mimic (Figure 4C). 3. Discussion Based on the clinical manifestation and biochemical tests results, pituitary corticotroph adenomas can be divided into functioning adenomas causing Cushing’s disease and SCAs. These two subtypes of tumors also differ in terms of some characteristics in MRI [2,26] and pathological features [27]. In contrast to CD-causing adenomas which are commonly small microadenomas, SCAs are diagnosed as macroadenomas due to neurological symptoms related to tumor mass. They are characterized by invasive growth, high risk of recurrence and resistance to medical therapy and are therefore referred to as “high-risk” pituitary adenomas according to current classification [3,4]. In our study, the SCAs were larger than functioning counterparts, as expected. A clear prevalence of women is observed among CD patients according to literature data [28], while it is not observed in patients suffering from SCAs. Our SCA group contained near equal representation of women and men as in previous reports [29,30]; however, some studies indicated female prevalence in SCAs [31]. Comparing functioning and silent corticotroph adenomas, we did not observe difference in patients’ age as well as differences in invasive growth status, ratio of adenomas with increased proliferation index and proportions of sparsely and densely granulated adenomas that may suggest the lack of difference in the tumors’ “aggressiveness”. Importantly, limitations for generalization of our results should be noted. The number of patients included in the analysis is relatively low and the group is not representative of the general population, especially in the case of patients suffering from Cushing’s disease. Since the main goal of our study was a molecular profiling of tumor tissue, we intentionally preselected large adenomas, which allowed us to have enough tissue for DNA/RNA isolation and successful molecular procedures. In our investigation, we observed a negative correlation between cortisol/ACTH ratio and tumor volume in functioning corticotroph adenomas as described previously [32]. However, we did not observe any difference between micro- and macroadenomas causing CD as compared to SCAs (data not shown) as was found in previous studies [12]. The reason why some of corticotroph adenomas exhibit excessive hormone secretion and the others remain clinically silent is unclear and only few attempts have been made to determine the possible molecular mechanism underlying this difference in secretory activity. They were mainly focused on investigating the expression of the selected genes or proteins by comparing subclinical and functioning corticotroph adenomas. These studies indicated different expression levels of prohormone convertase 1/3 POMC, genes encoding somatostatin receptors, corticotropin releasing hormone receptor 1, vasopressin receptor (V1BR), corticosteroid 11-beta-dehydrogenase as well as NEUROD1 and TPIT [6,7,8,9,10,11,12,13]. However, whole transcriptome studies indicated that adenomas causing CD and subclinical corticotroph adenomas share a very common gene expression profile and a very low number of differentially expressed genes can be found by comparing transcriptome of silent and CD-causing ACTH tumors [14,15]. In our study, we determined the miRNA expression profile of 28 clinically functioning adenomas and 20 SCAs with next-generation sequencing of small RNA fraction. This allowed for the quantification of over 1900 miRNA annotated to current version of miRbase database and comparing their expression in two groups of tumor samples. We found a significant difference only in the expression levels of 19 miRNAs, that represent less than 1% of the miRNAs included in the analysis. This result resembles the observation from previous comparison of whole transcriptome profiles in functioning adenomas and SCAs where only 34 differentially expressed genes were found. Generally, both observations indicate a very common molecular profile of corticotroph adenomas, regardless of the functional status. In our study, the expression levels of 13 out of 19 identified differentially expressed miRNAs were also correlated with peripheral ACTH/cortisol levels, further supporting the role of these miRNAs in secretory activity of corticotroph adenomas. The possible role of miRNA in subclinical nature of SCAs was addressed in only one previous study by García-Martínez A et al. [33]. The authors compared the expression of 5 miRNAs in 24 functioning and 23 silent adenomas and observed a difference in hsa-miR-200a and hsa-miR-103 levels [33]. Their results were not confirmed by our investigation since these two miRNAs were not found among differentially expressed miRNAs. In our data, very a similar expression level of hsa-miR-200a was observed in clinically functioning and silent adenomas. In turn, a slightly higher expression of hsa-miR-103a-3p was observed in SCAs as previously reported, but the difference did not cross the significance threshold level. We should note that different methods were used for these two studies and technical and analytical differences could result in this discrepancy. Since miRNAs play a role in gene regulation, their effect should be investigated in the context of the function of targeted genes. The interaction between miRNA and its target mRNA 3′UTR can be predicted with in silico tools. Unfortunately, prediction results can be very difficult to interpret since a huge number of predicted interactions can be found for some miRNAs. For example, when using the Targetescan (http://www.targetscan.org; accessed on 28 February 2022) prediction tool [25], over 4000 target genes were predicted for each hsa-miR-9-3p, hsa-miR-1909-3p, hsa-miR-22-5p and hsa-miR-181b-3p that we found as differentially expressed in CD and SCA. Therefore, to investigate a possible functional relevance of differentially expressed miRNAs we used a database of experimentally validated miRNA targets [34]. Gene set over-representation analysis of miRNA target genes indicated their enrichment in the pathways of steroid hormone nuclear receptors functioning. This result indicates that miRNAs that have different expression levels in CD and SCAs play a role in the regulation of expression of genes involved in steroid hormone signaling at hormone receptor level. It is especially interesting since this group of compounds includes adrenal hormones that play a role in the regulation of the hypothalamic–pituitary–adrenal (HPA) axis. The particular enriched miRNA target genes included NR3C1 and NR3C2 that encode for corticosteroid hormone receptors (GR and MR, respectively). Both receptors are located in the cytoplasm where they bind glucocorticoids. Upon ligand binding, they are translocated to nucleus where they form dimers on DNA at glucocorticoid response elements (GREs). Glucocorticoid and mineralocorticoid receptors directly regulate the expression of target genes and/or influence the expression indirectly through the interaction with other transcription factors [35]. Glucocorticoids play a role in the basic mechanism of negative feedback of HPA axis. They act on hypothalamus, where high cortisol levels reduce secretion of corticotropin-releasing hormone (CRH), thus they directly reduce stimulation of ACTH secretion by anterior pituitary lobe. Glucocorticoids also inhibit the activity of pituitary cells indirectly. Corticotroph cells express GRs and their activation results in the reduction of POMC expression and secretion of ACTH [36,37]. In pituitary corticotroph adenomas, NR3C1 point mutations and loss of heterozygosity in NR3C1 locus were identified [38]. These mutations seem to affect the secretory activity and result in tumor resistance to corticosteroids [39]. Reduced expression of corticosteroid receptors in corticotroph adenomas has been reported in patients with resistance to high doses of dexamethasone [40]. These data indicate a role of GR in secretory activity of clinically functioning corticotroph adenomas. The expression of corticosteroid genes was previously investigated in CD-causing tumors and SCAs and no significant differences were found. However, it is worth noting that a low number of SCA patients was included in these studies: n = 9 [13], n = 8 [11] and n = 2 [41]. According to previously published results, hsa-miR-124-3p is a negative regulator of NR3C1 [16,18,19,20,21]. This was observed in acute lymphoblastic leukemia [19], adipocytes [20] and human embryonic kidney cells [21], where the reduced expression of NR3C1 upon an increase in hsa-miR-124-3p as well as a direct interaction between this miRNA and 3′UTR of GR gene were observed. Some additional clinical observations also suggest the role of hsa-miR-124-3p in the regulation of the response to cortiosteroids in patients with acute-on-chronic liver failure [18] and lymphoblastic leukemia [19]. Hsa-miRNA-124 also mediates corticosteroid resistance in T-cells of sepsis patients through the downregulation of GR [42]. Our analysis of the expression level of NR3C1 in corticotroph adenomas showed that tumors causing CD have lower gene expression and accordingly they exhibit higher levels of hsa-miR-124-3p. Subsequently, the role of hsa-miR-124-3p in NR3C1 downregulation was confirmed in mice AtT-20/D16v-F2 corticotroph cells using miRNA mimics and reporter gene assay. Transfection of AtT-20/D16v-F2 cells with hsa-miR-124-3p mimics resulted in reduced NR3C1 mRNA expression and GR protein level. We also confirmed the interaction between hsa-miR-124-3p and one of two predicted binding motifs in 3′UTR of NR3C1 with luciferase reporter gene assay. Since sequences of hsa-miR-124-3p and target sequence in 3′UTR of NR3C1 mRNA are the same in mice and in humans, we believe that results showing the regulation of the GR-encoding gene in mice AtT-20/D16v-F2 cells are also relevant to humans. Together, the available data indicate that in pituitary corticotrophs, hsa-miR-124-3p downregulates the expression of the GR gene. Since this receptor mediates the response of pituitary cells to cortisol, the expression of hsa-miR-124-3p appears to be an important element in the regulation of secretory activity of corticotroph cells. Based on these results, we can hypothesize that in CD, a high level of hsa-miR-124-3p contributes to lowering of GR expression and in consequence it plays a role in lowering the effect of glucocorticoid feedback on the activity of corticotroph adenoma. Hsa-miR-124-3p and hsa-miR-135b-5p can downregulate the expression level of MR, as proven in model HeLa cells [17]. Expression of both miRNAs is higher in corticotroph adenomas causing CD which corresponds to the lower expression of the NR3C2 gene in these tumors as compared to SCAs. Since the role of the MR receptor expression in pituitary cells is poorly understood, the functional implication of this observation is much less clear than in the case of GR downregulation. MR and GR have similar amino acid sequences, especially in DNA-binding domain, but they differ in affinity to corticosteroids. MR is specific for both mineralocorticoids and glucocorticoids while GR is specific predominantly for glucocorticoids. MRs have much higher affinity for glucocorticoids than GRs and are activated at basal glucocorticoid conditions, while GR occupancy is increased when glucocorticoid levels rise during the circadian peak or stress. Due to these differences, these two receptors play slightly different roles, despite the fact that they share a number of target genes [43]. MR expression is considered more tissue-specific than GR and was reported to be the most prevalent in kidney and adipose tissue but also in the hippocampus and hypothalamus [44]. However, the available databases of human expression pattern such as the Genotype-Tissue Expression project (https://gtexportal.org; accessed on 10 December 2021) or Protein atlas (https://www.proteinatlas.org; accessed on 10 December 2021) indicate that MR is widely expressed in multiple human tissues and organs including the pituitary gland. Unfortunately, a role of MR receptor in pathogenesis of pituitary tumors remains unknown. AtT-20 cells, which are the only available cell line model of corticotroph adenoma, do not express MR receptor, thus the procedure of experimental validation of the role of miRNA in NR3C2 silencing is not applicable. With a lack of experimental data on the exact role of MR, we can only hypothesize that miRNA-mediated silencing of NR3C2 may have the similar effect on HPA axis feedback as silencing of NR3C1. It may enhance ACTH secretion by reducing the direct inhibitory effect of glucocorticoids on neoplastic pituitary corticotrophs. The difference in expression of hsa-miR-124-3p and hsa-miR-135b-5p between subclinical and CD-causing adenomas is not big, thus we suppose that high expression of these miRNAs is not the only cause of difference in ACTH secretion. Presumably this is one of the mechanisms in the regulation of corticotrophs’ secretory activity. The model of miRNA-based corticosteroid receptor regulation does not undermine the role of previously described differences in the expression of convertase 1/3, POMC, somatostatin receptors or corticotropin releasing hormone receptor 1 or genes involved in differentiation of pituitary cells [6,7,8,9,10,11,12,13]. When considering the complex nature of the regulation of ACTH secretion, it can be assumed that multiple mechanisms may be involved in the silent character of subclinical adenomas. The low number of identified differentially expressed miRNAs or genes in silent and clinically functioning adenomas probably results from the intertumoral molecular heterogeneity of SCAs. This is also in line with clinical evidence indicating that some silent corticotroph adenomas can transform into clinically functioning ones while the others remain silent [1]. The misregulation of GR expression or NR3C1 mutation may have important therapeutical implications in CD patients. Non-selective GR antagonist Mifepristone was officially approved for treatment in patients with Cushing’s syndrome [45] while another new GR inhibitor, Relacorilant (CORT125134), is under clinical investigation for its use in this group of patients [46]. The further studies will be required to assess the role of GR abnormalities in response to GR-targeting treatment in CD. In our study, we focused mainly on the role of hsa-miR-124-3p and hsa-miR-135b-5p in the regulation of corticosteroid receptors, but the role of other differentially expressed miRNAs can also be elucidated, based on the function of putative target genes. In the pathways enrichment analysis of the putative targets, molecular functions related to transcriptional regulation were found among the top processes. Interestingly, five miRNAs, i.e., hsa-miR-132-5p, hsa-miR-135b-5p, hsa-miR-27a-3p, hsa-miR-9-3p and hsa-miR-9-5p, were previously reported to downregulate the expression of FOXO1 transcription factor [47,48,49,50,51]. FOXO1 plays an important role in the differentiation of pituitary cells [52] and secretion of gonadotropic hormones [53,54] and prolactin [55]. The role of FOXO1 in pituitary corticotroph cells was not investigated but it was shown to regulate POMC expression in POMC hypothalamic neurons [56]. In POMC, neurons of arcuate nucleus FOXO1 directly suppresses POMC expression. A similar mechanism was also observed in prolactin pituitary adenomas where FOXO1 suppresses the promoter of PRL gene [55]. It is possible that high expression of hsa-miR-132-5p, hsa-miR-135b-5p, hsa-miR-27a-3p, hsa-miR-9-3p and hsa-miR-9-5p in pituitary corticotroph adenomas reduces the level of FOXO1 and eventually contributes to the upregulation of POMC expression. In our data from corticotroph adenomas, we observed the correlation between levels of hsa-miR-9-3p/hsa-miR-9-5 and POMC expression, which also supports this concept, but the exact role of miRNAs in possible FOXO1-related regulation of secretory activity of corticotroph cells requires further functional investigation. 4. Materials and Methods 4.1. Patients and Tissue Samples Pituitary tumor samples from 48 patients were collected during transsphenoidal surgery. Formalin-fixed and paraffin-embedded (FFPE) tissue samples, including 28 samples from patients with Cushing’s disease and 20 samples of SCA were used for the study. Diagnosis of hypercortisolism was based on standard hormonal criteria: increased UFC in three 24 h urine collections, disturbances of cortisol circadian rhythm, increased serum cortisol levels accompanied by increased or not suppressed plasma ACTH levels at 8.00 and a lack of suppression of serum cortisol levels to <1.8 µg/dL during an overnight dexamethasone suppression test (1 mg at midnight). The pituitary etiology of Cushing’s disease was confirmed based on the serum cortisol levels or UFC suppression < 50% with a high-dose dexamethasone suppression test (2 mg q.i.d. for 48 h) or a positive result of a corticotrophin-releasing hormone stimulation test (100 mg i.v.) and positive pituitary magnetic resonance imaging. ACTH levels were assessed using IRMA (ELSA-ACTH, CIS Bio International, Gif-sur-Yvette Cedex, France). The analytical sensitivity was 2 pg/mL (reference range: 10–60 pg/mL). Serum cortisol concentrations were determined by the Elecsys 2010 electrochemiluminescence immunoassay (Roche Diagnostics, Mannheim, Germany). Sensitivity of the assay was 0.02 μg/dL (reference range: 6.2–19.4 μg/dL). UFC was determined after extraction (liquid/liquid with dichloromethane) by electrochemiluminescence immunoassay (Elecsys 2010, Roche Diagnostics)—reference range: 4.3–176 μg/24 h. All the tumors underwent detailed histopathological diagnosis including immunohistochemical staining with antibodies against particular pituitary hormones (ACTH, GH, TSH, FSH, LH, α-subunit) and Ki67 as well as ultrastructural analysis with electron microscopy. The SCAs were characterized by the following clinicopathological criteria: positive immunohistochemical staining for ACTH, lack of signs and symptoms of hypercortisolism (Cushing’s syndrome), negative hormonal evaluation and non-compliance with diagnostic criteria of the CD. Macroadenoma was defined as an adenoma with at least one diameter exceeding 10 mm, and the tumor volume was assessed with the diChiro Nelson formula (height × length × width × π/6). Invasive growth of the tumors was evaluated using Knosp grading [57]. Adenomas with Knosp grades 0, 1 and 2 were considered non-invasive, while those with Knosp 3 and 4 were considered invasive. Forty-three patients had a clear history of not using any drugs that control the overproduction of the cortisol or ACTH (ketoconazole, mitotane, metyrapone, osilodrostat, mifepristone, pasireotide) before surgical treatment. The information on preoperative pharmacological treatment was not available for 5 patients. Tumor tissue content of each FFPE sample ranged between 80 and 100% (median 99%), as assessed with histopathological examination. Patients’ characteristics are presented in Table 1 and details on each patient’s data are available in Supplementary Table S1. The study was approved by the local Ethics Committee of Maria Sklodowska-Curie National Research Institute of Oncology in Warsaw, Poland. Each patient provided informed consent for the use of tissue samples for scientific purposes. Total RNA from FFPE samples was purified with RecoverAll™ Total Nucleic Acid Isolation Kit for FFPE tissue (Thermo Fisher Scientific, Waltham, MA, USA) and measured using NanoDrop 2000 (Thermo Fisher Scientific). RNA was stored at −70 °C. 4.2. Micro RNA Expression Profiling For comparing the miRNA expression profiles in CD-causing and clinically silent adenomas, NGS data from our previous investigation of miRNA expression in corticotroph adenomas were used. The dataset is available at Gene Expression Omnibus, accession no GSE166279. Sequencing of small RNA fraction was performed in 48 tumor samples (28 CD patients and 20 SCA patients) with ion semiconductor sequencing technology, as described previously [58]. Briefly, Ion Total RNA-Seq Kit v2 (Thermo Fisher Scientific) was used for sequencing library construction, Ion Xpress™ RNA-Seq Barcode Kit was used for hybridization and ligation of RNA adapters. RNA reverse transcription and subsequent cDNA purification and library size selection were performed using Nucleic Acid Binding Beads and verified using Bioanalyzer 2100 with High Sensitivity DNA Kit (Agilent, Santa Clara, CA, USA). Ion Chef instrument, with Ion PI™ Hi-Q™ Chef Kit (Thermo Fisher Scientific) and Ion Proton sequencer (Thermo Fisher Scientific) were used for library preparation and sequencing, respectively. BamToFastq package was applied for converting unmapped bam files into fastq files. miRDeep2 was applied for read mapping to known human miRNAs (according to miRBase release 22) and reads quantification. Data normalization and differential expression analysis were performed using DESeq2. Filtration for low-expression miRNAs was applied as described previously. FC of expression calculated as the ratio of the normalized read-count value in CD-causing and silent adenomas was used as a measure of expression difference. Adjusted p-value < 0.05 was used as significance threshold. MiRtarbase release 9.0 database [34] was used to identify known miRNA target genes. PANTHER (http://pantherdb.org; accessed on 10 December 2021) [59] was used for gene set over-representation analysis. 4.3. qRT-PCR gene Expression Analysis One microgram of RNA was subjected to reverse transcription with Transcriptor First Strand cDNA Synthesis Kit (Roche Diagnostics). qRT-PCR reaction was carried out in 384-well format using 7900HT Fast Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) and Power SYBR Green PCR Master Mix (Thermo Fisher Scientific) in a volume of 5 μL, containing 2.25 pmol of each primer. The samples were amplified in triplicates. GAPDH was used as reference gene. Delta Ct method was used to calculate the relative expression level. PCR primers’ sequences are presented in Supplementary Table S3. 4.4. Cell Line Culture and miRNA Mimic Transfection AtT-20/D16v-F2 cells were purchased from ATCC collection and cultured in DMEM medium supplemented with 10% FBS, as recommended. MiRCURY LNA miRNA Mimics including hsa-miR-124-3p mimic (YM00471256, Qiagen, Hilden, Germany) and negative control mimic (YM00479902-ADB, Qiagen) were used. AtT-20/D16v-F2 cells were seeded at 5 × 104 per well of a 24-well plate in culture medium and transfected with 50 nM miRNA with 1% (v/v) HiPerFect Transfection Reagent (Qiagen), according to the manufacturer’s instructions. The next day, the culture medium was changed. In total, 48 h after transfection the cells were harvested and subjected to isolation of total RNA with RNeasy Mini Kit (Qiagen). The expression of the putative hsa-miR-124-3p target gene was determined with qRT-PCR. 4.5. Luciferase Reporter Gene Assay Hsa-miR-124-3p target sites in 3′UTR of NR3C1 were determined with Targetscan [25]. Each of two predicted hsa-miR-124-3p target sites were cloned into pmirGLO Dual-Luciferase miRNA Target Expression Vector (Promega, Madison, WI, USA). AtT-20/D16v-F2 cells (2 × 104/well) were seeded onto a 96-well plate in 100 µL culture medium. The next day, the cells were transfected with 100 ng of each plasmid vector, independently using 0.25% (v/v) lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA) in 10 µL of DMEM. The cells were subsequently transfected with either hsa-miR-124-3p mimic (YM00471256, Qiagen) or negative control mimic (YM00479902-ADB, Qiagen) in a final concentration of 50 nM using HiPerfectReagent (Qiagen). Culture medium was changed on the next day. Luciferase activity was measured with One-Glo Luciferase Assay System (Promega) 48 h after transfection. 4.6. Western Blotting Cells were lysed in ice cold RIPA buffer, incubated for 30 min in 4 °C and centrifuged at 12,500× g rpm for 20 min at 4 °C. Samples were resolved using SDS-PAGE and electrotransferred to polyvinylidene fluoride membranes (PVDF) (Thermo Fisher). GR protein was detected with monoclonal anti-Glucocorticoid Receptor antibody (ab183127, Abcam, Cambridge, UK), and secondary anti-rabbit antibody conjugated to HRP (#7074, Cell Signaling, Beverly, MA, USA). Glyceraldehyde-3-Phosphate Dehydrogenase (#MAB374, Millipore, Bedford, MA, USA) detected with mouse HRP-conjugated antibody (#7076 Cell Signaling) served as control. Visualization was performed with SuperSignal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific) and CCD digital imaging system Alliance Mini HD4 (UVItec Limited, Cambridge, UK). 4.7. Statistical Analysis A two-sided Mann–Whitney U-test was used for analysis of continuous variables. The Spearman correlation method was used for correlation analysis. Significance threshold of α = 0.05 was adopted. Data were analyzed using GraphPad Prism 6.07 (GraphPad Software, La Jolla, CA, USA). Hierarchical clustering analysis was carried out with Cluster 3.0, and the results were visualized using TreeView 1.6 software (Stanford University School of Medicine, Stanford, CA, USA). Supplementary Materials The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/ijms23052867/s1. Author Contributions Conceptualization, M.M. and M.B.; Methodology, M.B. and B.J.M.; Software, J.B.; Formal analysis, P.K., B.J.M. and M.B.; Investigation, B.J.M., P.K., N.R., M.B. and M.P.; Resources, J.K., G.Z., A.S. and T.M.; Data curation, J.B., B.J.M. and M.B.; Writing—original draft preparation, M.B., P.K. and B.J.M.; Writing—review and editing, all the authors; Visualization, M.B. and B.J.M.; Supervision, M.M.; Project administration M.B.; Funding acquisition, M.M. All authors have read and agreed to the published version of the manuscript. Funding This research was funded by National Science Centre, Poland, grant number 2021/05/X/NZ5/01874. Institutional Review Board Statement The study was conducted in accordance with the Declaration of Helsinki, and approved by the local Ethics Committee of Maria Sklodowska-Curie Institute—Oncology Center in Warsaw, Poland; approval no. number 44/2018, date of approval 26 July 2018. Informed Consent Statement Informed consent was obtained from all subjects involved in the study. Data Availability Statement Data from next-generation sequencing of small RNA fraction of 48 corticotroph adenoma samples are available at Gene Expression Omnibus, accession no GSE166279. Conflicts of Interest The authors declare no conflict of interest. References Ben-Shlomo, A.; Cooper, O. Silent Corticotroph Adenomas. Pituitary 2018, 21, 183–193. 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  25. 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. 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