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I'm not sure I like this! STORY: Could artificial intelligence be used to make brain surgery safer? At this university in London, trainee surgeon Danyal Khan is taking part in a mock operation during which he's assisted by a real-time video feed, as is typical in brain surgery. But what's new here is that the footage is being analyzed by AI to help Khan better understand what he's seeing. The AI system, which is under development at University College London (UCL), highlights sensitive or critical structures in the brain. Neurosurgeon Hani Marcus believes it has the potential to make brain surgery safer and more effective: "So I'm very bullish that in the medium to long term, the A.I. will be helping lots of surgeons do lots of operations better than they otherwise can." Marcus says the AI system analyzed video of more than 200 pituitary gland tumor operations, and gained around 10 years-worth of experience in a fraction of the time. That knowledge means the AI can now not only help navigate to the correct area of the brain, but also know what should be happening at any stage of the procedure, making it a valuable training aid. "So, what we're really trying to do is apply AI or artificial intelligence to support surgeons doing brain tumor surgery at the base of the brain. And what this practically entails is us training the AI with hundreds of videos, telling it, if you like, what structures are what and then at some point over that period, the AI becomes really good itself at recognising things, and able to support other surgeons who're perhaps less experienced in advising them what to do next." Assistant Professor of Robotics and A.I. Sophia Bano explains how that might look in a real operation: "There can be scenarios where clinicians, unintentionally, are very close to a very critical structure such as the optic nerve. This can have, any damage or a slight more pressure on the optic nerve, can have long term complications on the patient side. So, this whole tool will alert the surgeon during the procedure if there is any risk of potential complication so they can recalibrate themselves during the procedure." Khan, who was also involved in developing the software that is now helping him learn, says the A.I. system has been a valuable tool in his training. It could also provide him with step-by-step guidance during a procedure, similar to having a senior surgeon standing over your shoulder. "I think as I progress as a surgeon, there might be stages where I wonder, you know, have I done enough of a particular part of the procedure and should I move on? And actually having that sort of assistant in the background as a reassurance to look at and say, 'well, yeah, actually, at this stage, out of the hundreds of videos of experts that this algorithm has watched, the experts would probably start moving on to the next phase'. It's a useful double check." According to UCL, the system could be ready to be used in operating theaters within two years. From https://news.yahoo.com/ai-could-help-brain-surgery-080156167.html

In a recent study published in Hypertension Research, scientists examine the endocrine causes of hypertension (HTN) and investigate the efficacy of treatments to alleviate HTN. What is HTN? About 30% of the global population is affected by HTN. HTN is a modifiable cardiovascular (CV) risk factor that is associated with a significant number of deaths worldwide. There are two types of HTN known as primary and secondary HTN. As compared to primary HTN, secondary HTN causes greater morbidity and mortality. The most common endocrine causes of HTN include primary aldosteronism (PA), paragangliomas and pheochromocytomas (PGL), Cushing’s syndrome (CS), and acromegaly. Other causes include congenital adrenal hyperplasia, mineralocorticoid excess, cortisol resistance, Liddle syndrome, Gordon syndrome, and thyroid and parathyroid dysfunction. What is PA? PA is the most common endocrine cause of hypertension, which is associated with excessive aldosterone secretion by the adrenal gland and low renin secretion. It is difficult to estimate the true prevalence of PA due to the complexity of its diagnosis. Typically, the plasma aldosterone-to-renin ratio (ARR) is measured to diagnose PA. The diagnosis of PA can also be confirmed using other diagnostic tools like chemiluminescent enzyme immunoassays (CLEIAs) and radio immune assay (RIA). Continuous aldosterone secretion is associated with organ damage due to chronic activation of the mineralocorticoid (MR) receptor in many organs, including fibroblasts and cardiomyocytes. An elevated level of aldosterone causes diastolic dysfunction, endothelial dysfunction, left ventricular hypertrophy, and arterial stiffness. Increased aldosterone secretion also leads to obstructive sleep apnea and increases the risk of osteoporosis. This is why individuals with PA are at a higher risk of cardiovascular events (CVDs), including heart failure, myocardial infarction, coronary artery disease, and atrial fibrillation. PA is treated by focusing on normalizing potassium and optimizing HTN and aldosterone secretion. Unilateral adrenalectomy is a surgical procedure proposed to treat PA. Young patients who are willing to stop medication are recommended surgical treatment. The most common pharmaceutical treatment for PA includes mineralocorticoid receptor antagonists such as spironolactone and eplerenone. Pheochromocytomas and paragangliomas PGL are tumors that develop at the thoracic-abdominal-pelvic sympathetic ganglia, which are present along the spine, as well as in the parasympathetic ganglia located at the base of the skull. The incidence rate of PGL is about 0.6 for every 100,000 individuals each year. PGL tumors synthesize excessive catecholamines (CTN), which induce HTN. Some of the common symptoms linked to HTN associated with PGL are palpitations, sweating, and headache. PGL can be diagnosed by determining metanephrines (MN) levels, which are degraded products of CTN. Bio-imaging tools also play an important role in confirming the diagnosis of PGL. Excessive secretion of CTN increases the risk of CVDs, including Takotsubo adrenergic heart disease, ventricular or supraventricular rhythm disorders, hypertrophic obstructive or ischaemic cardiomyopathy, myocarditis, and hemorrhagic stroke. Excessive CTN secretion also causes left ventricular systolic and diastolic dysfunction. Typically, PGL treatment is associated with surgical procedures. Two weeks before the surgery, patients are treated with alpha-blockers. For these patients, beta-blockers are not used as the first line of treatment without prior use of alpha-adrenergic receptors. Patients with high CTN secretion are treated with metyrosine, as this can inhibit tyrosine hydroxylase. Hydroxylase converts tyrosine into dihydroxyphenylalanine, which is related to CTN synthesis. What is CS? CS, which arises due to persistent exposure to glucocorticoids, is a rare disease with an incidence rate of one in five million individuals each year. The most common symptoms of CS include weight gain, purple stretch marks, muscle weakness, acne, and hirsutism. A high cortisol level causes cardiovascular complications such as HTN, hypercholesterolemia, and diabetes. CS is diagnosed based on the presence of two or more biomarkers that can be identified through pathological tests, such as salivary nocturnal cortisol, 24-hour urinary-free cortisol, and dexamethasone suppression tests. CS is treated through surgical procedures based on the detected lesions. Patients with severe CS are treated with steroidogenic inhibitors, such as metyrapone, ketoconazole, osilodrostat, and mitotane. Pituitary radiotherapy and bilateral adrenalectomy are performed when other treatments are not effective. Acromegaly Acromegaly arises due to chronic exposure to growth hormone (GH), leading to excessive insulin-like growth factor 1 (IGF1) synthesis. This condition has a relatively higher incidence rate of 3.8 million person-years. Clinical symptoms of acromegaly include thickened lips, widened nose, a rectangular face, prominent cheekbones, soft tissue overgrowth, or skeletal deformities. Prolonged exposure to GH leads to increased water and sodium retention, insulin resistance, reduced glucose uptake, and increased systemic vascular resistance. These conditions increase the risk of HTN and diabetes in patients with acromegaly. Acromegalic patients are also at a higher risk of cancer, particularly those affecting the thyroid and colon. Acromegaly is diagnosed using the IGF1 assay, which determines IGF1 levels in serum. After confirming the presence of high IGF1 levels, a GH suppression test must be performed to confirm the diagnosis. Bioimaging is also conducted to locate adenoma. Acromegaly is commonly treated through surgical procedures. Patients who refuse this line of treatment are treated with somatostatin receptor ligands, growth hormone receptor antagonists, dopaminergic agonists, or radiotherapy. Journal reference: De Freminville, J., Amar, L., & Azizi, M. (2023) Endocrine causes of hypertension: Literature review and practical approach. Hypertension Research; 1-14. doi:10.1038/s41440-023-01461-1 From https://www.news-medical.net/news/20231015/Hormones-and-high-blood-pressure-Study-reveals-endocrine-culprits-and-targeted-treatments.aspx

Niall Cavanagh, now aged 48, bravely fought and beat a brain tumour diagnosis when he was a teenager. He shared his experience with The Kerryman in an interview conducted ahead of the seventeenth annual International Brain Tumour Awareness Week. This week-long event, running from October 28th to November 4th, aims to raise awareness about brain tumours and support those affected by them. Niall’s journey was not an easy one. Leading up to his diagnosis in 1992, he experienced symptoms such as excessive thirst, urination, severe headaches, vomiting, and stunted growth. It was when he went for an eye examination for double vision that the examiner noticed something seriously wrong with his retinas. Further tests revealed a germinoma brain tumour pressing on the pituitary gland. To relieve the pressure caused by the tumour, Niall underwent an emergency ventriculoperitoneal shunt procedure. This involved inserting a tube from his brain to his abdomen to drain the excess cerebrospinal fluid. He also underwent extensive radiotherapy to shrink the tumour and prevent its spread. The tumour affected Niall’s pituitary gland, resulting in a condition known as hypopituitarism. This condition causes a deficiency in various hormones, including growth hormone and anti-diuretic hormone. Niall experienced adverse effects on his physical and mental health due to the tumour and subsequent treatments. Despite the challenges, Niall gained a clearer perspective on life. He learned to appreciate what is truly important and developed compassion through his own struggles with depression and anxiety. He emphasized that each person’s experience with a brain tumour is unique, and it’s essential to show support and understanding to others facing similar battles. Niall’s health has gradually improved over the years, although he still faces challenges due to a weakened immune system. However, he remains resilient and has pursued higher education, obtaining two degrees in IT and a Masters in information systems. He currently works part-time in an administrative role with the Renewable Energy Centre in Killarney. Throughout his journey, Niall received invaluable support from his family and various organizations, including the Cork Brain Tumour Support Group (now Brain Tumour Ireland), the Pituitary Foundation, and Headway in Tralee. Niall’s story serves as an inspiration and a reminder of the importance of raising awareness and providing support to those affected by brain tumours. International Brain Tumour Awareness Week aims to continue spreading awareness and fostering understanding of this life-changing condition. Sources: – The Kerryman From https://www.expresshealthcaremgmt.com/news2/kerry-man-reflects-on-beating-brain-tumour-diagnosis-as-a-teenager-you-have-to-sink-or-swim/156637/

Abstract Cushing’s disease (CD) is an extremely rare diagnosis in children. In this report, we present the case of an almost 16-year-old, short and thin boy with CD, the first symptoms of which were spinal pain and vertebral fractures as a result of osteoporosis. In light of his growth retardation and short stature, the boy underwent diagnostics, which excluded growth hormone (GH) deficiency, hypothyroidism and celiac disease. Finally, based on cortisol profile results, dexamethasone suppression tests and bilateral sampling during catheterization of the inferior petrosal sinuses, CD was diagnosed. Keywords: Cushing’s disease; obesity; growth retardation; osteoporosis 1. Introduction Cushing’s disease (CD) is an extremely rare diagnosis in children; however, if it occurs, it is more likely to present in older children [1,2]. It is a type of ACTH-dependent Cushing’s syndrome (CS), in which the pituitary gland is the source of ACTH secretion. The highest incidence of CD occurs in children aged 12.3–14.1 years [3]. The incidence of CD during this developmental age is approximately 5% of that seen in adults (with an annual incidence of 0.89–1 per million pediatric patients) [1,2,4]. The rarest form of ACTH-dependent CS in children is ectopic Cushing’s syndrome (ECS), associated with ectopic production of ACTH or CRH, most commonly by neuroendocrine tumors such as bronchial carcinoids, gastrointestinal tumors, medullary thyroid carcinoma, or pheochromocytomas [2,4,5]. Children with ECS constitute 1% of patients with CS in the developmental age [2]. An even rarer disease is ACTH-independent Cushing’s syndrome—associated with adrenal lesions (adenoma, carcinoma, bilateral macronodular adrenal hyperplasia (BMAH), or primary pigmented nodular adrenocortical disease (PPNAD)) [2]. Regarding CD, ACTH is secreted in an overwhelming majority of cases by pituitary corticotropic microadenomas and—less commonly—by macroadenomas, the latter occurring in only 10% of adult CD cases and even more rarely in children (2%) [1,3]. Long-term hypercortisolemia can also lead to bone-mineralization disorders, including osteoporosis, especially in the bones of the central skeleton [4,6,7]. In children, the most common features of CD are rapid weight gain (93–98%), growth retardation (63–100%) and/or facial changes (63–100%) [4]. Mood disturbances, muscle weakness, osteopenia, and headaches are less frequent symptoms. Limited data are available about bone mineral density (BMD) in children with CD. Lonser et al. [8] observed fractures in 7% of patients with CD that were studied. Chronic glucocorticoid excess associated with CD has negative effects on bone turnover, leading to bone-mineralization disorders in both adults and children. Multiple factors contribute to decreased bone mineral density in CD, including the direct effect of glucocorticoids on osteoclasts and osteoblasts, both impairing bone formation and enhancing bone resorption. Glucocorticoids also act to decrease gastrointestinal calcium absorption and renal calcium reabsorption. Bone loss occurs more frequently in Cushing’s syndrome caused by adrenal tumors in CD [6,7]. An additional factor involved in bone-mineralization disorders, particularly in adult patients with CD, may be hypogonadotropic hypogonadism. Reproductive and sexual dysfunctions are highly prevalent in CS, with higher frequency observed in patients with pituitary-related CS, compared to those with adrenal-related CS. Hypogonadism is identified in as much as 50–75% of men with CS and menstrual irregularities are present in 43–80% of women diagnosed with this condition. During active disease, there is a significant reduction in plasma testosterone and gonadotropin levels in men [7,9]. These testosterone levels typically normalize during remission of the disease. Pivonello et al. [7] suggest that the lack of testosterone normalization three months after CS treatment indicates the need for administration of testosterone to protect the patient’s bone mass. In children, cortisol excess can also suppress gonadotropin, TSH and growth hormone secretion, contributing to the absence of pubertal characteristics or inhibiting its progression in patients who have already entered puberty [1]. So far, to our knowledge, there have been no reports on children where bone-mineralization disorders (without weight gain and hirsutism) are the first sign of CD. 2. Case Presentation We present the case of an almost 16-year-old boy with short stature who, in May 2021, was referred to the Osteoporosis Outpatient Clinic of the Polish Mother’s Memorial Hospital—Research Institute (PMMH-RI) in Lodz, Poland, due to severe back pain. Low bone mass was diagnosed via dual-energy X-ray absorptiometry (DXA). Initially, it seemed that the occurrence of those symptoms might be related to steroid therapy, because in November 2020 (just after SARS-CoV-2 infection) the child had developed severe abdominal pain, accompanied by an increase in the activity of liver enzymes, and after excluding an infectious cause, autoimmune hepatitis was diagnosed. Deflazacort (Calcort) therapy was prescribed in gradually reduced doses, with the initial dose being 24 mg in the morning and 18 mg in the afternoon. This therapy was discontinued on 1 October 2021. As early as on the fifth day of glucocorticosteroid treatment, pain presented in the lumbar spine region, increasing with movement. Initially, the pain was intermittent, then it became constant. No painkillers were needed. On 7 May 2021, on the basis of DXA, low bone mass was diagnosed (Z-score Spine: −4.2, Z-score TBLH: −1.9). In June 2021 (while still undergoing treatment with steroids) the boy was admitted to the Department of Endocrinology and Metabolic Diseases PMMH-RI for further diagnostics (Table 1). Table 1. The medical history and the course of diagnostics and treatment. The patient was a second child, born at 40 weeks of gestational age, weighing 4150 g, measuring 56 cm, and achieving a 10-point Apgar score. During infancy, he received vitamin D supplementation in accordance with Polish recommendations at that time. However, after his first year of life, the supplementation was not taken regularly. The boy received vaccinations according to the standard immunization schedule. There was no significant family medical history. During the physical examination, apart from the presence of short stature, no other notable abnormalities were detected. The skin was clear, without pathological lesions; no features of hyperandrogenism were observed. The boy’s body weight was 47.4 kg (3rd–10th centile); his height, 162 cm (<3rd centile); and height SDS, −2.36; while his BMI was 18.06 kg/m2 (10th–25th centile). Pubarche was assessed as stage 4 according to the Tanner scale; the volume of the testes was 10–12 mL each. After available anthropometric measurements from the patient’s medical history were plotted on the growth chart for sex and chronological age, it became evident that the boy experienced growth retardation from the age of 11 (Figure 1). Figure 1. Growth chart for boys. The red line represents growth retardation from the age of 11. Apart from slightly increased calcium excretion in the 24 h urine collection (Calcium: 9.52 mmol/24 h), there were no significant abnormalities in the laboratory tests assessing calcium–phosphate metabolism (Calcium: 2.41 mmol/L, Phosphorus: 1.3 mmol/L). Serum parathormone (PTH) and vitamin D concentrations remained normal (PTH: 22.9 pg/mL, 25(OH)D: 46.7 ng/mL). Due to the described pain complaints, a thoracolumbar spine X-ray was performed. A decrease in the height of the Th5-Th9 vertebrae and central lowering of the upper border plate of the L4 and L5 were observed (Figure 2). Figure 2. A thoracolumbar spine X-ray with multilevel vertebral fractures. Decrease in the height of the Th5–Th9 vertebrae and central lowering of the upper border plate of the L4 and L5 were found. Magnetic resonance imaging (MRI) of the spine confirmed multilevel vertebral fractures, which, together with the presence of low bone mass on DXA examination, allowed a diagnosis (according to ISCD guidelines) of osteoporosis to be made. Treatment included calcium supplements and cholecalciferol. The parents did not consent to treatment with bisphosphonates (sodium pamidronate), which is an off-label treatment. In light of the patient’s short stature and growth retardation, an endocrinological assessment was conducted. The possibility of growth hormone (GH) deficiency and hypothyroidism as underlying causes for the growth retardation was ruled out. Gonadotropin and androgen levels were adequate for the pubertal stage (FSH—8.3 IU/L, LH—4.7 IU/L, testosterone—4.750 ng/mL, DHEA-S—230.30 µg/dL (normal range: 70.2–492), 17-OH-progesterone—0.78 ng/mL). The bone age was assessed to be 15 years. Alongside continued steroid therapy for autoimmune hepatitis, profiles of cortisol and ACTH secretion were performed. Due to the patient’s elevated cortisol levels during night hours (cortisol 24:00—10.7 µg/dL), an overnight dexamethasone suppression test (DST) and low-dose dexamethasone suppression test (LDDST) were performed. After administering 1 mg dexamethasone (23:00), his morning cortisol level (8:00) still remained elevated (cortisol—3.4 µg/dL). However, after administering 0.5 mg dexamethasone every 6 h for the next 2 days, cortisol levels (8:00) normalized (cortisol—1.0 µg/dL). An MRI of the pituitary gland showed only a poorly demarcated area in the anterior part of the glandular lobe, measuring approximately 2.0 × 3.5 × 5.0 mm on T2W images (Figure 3). A follow-up MRI examination was recommended, which was performed during the child’s next hospitalization in January 2022. The previously described area was still very faint. Figure 3. MRI examination image depicting the poorly demarcated area in the anterior part of the glandular lobe. The arrows point to a structure suspected of being an adenoma. In October 2021, the administration of deflazocort as a treatment was discontinued. During hospitalization in January 2022, the diurnal pattern of ACTH and cortisol secretion was re-evaluated, yet no consistent diurnal rhythm was observed; cortisol levels remained elevated at night. For this reason, overnight DST and then LDDST were carried out again (Figure 4), in which no suppression of cortisol concentrations was obtained. Only after a high-dose DST (HDDST), in which a high 1.5 mg of dexamethasone was administered every 6 h (125 µg/kg/24 h), was cortisol secretion suppressed. Figure 4. Laboratory findings indicating the diagnosis of ACTH-dependent Cushing’s syndrome. Based on the above results, CD was suspected as the cause of osteoporosis and growth retardation. In February 2022, a CRH test was performed upon the patient, which revealed a four-fold increase in ACTH levels and a two-fold increase in serum cortisol levels (Table 2). Table 2. The results of human CRH (hCRH) stimulation test and bilateral inferior petrosal sinus sampling (BIPSS). The CRH stimulation test was administered in the morning using human synthetic CRH (Ferring) at a dose of 1 μg/kg of body weight. During the test, cortisol and ACTH levels were measured in serum at the following time points: −15, 0, 15, 30, 60, and 90 min (see Table 2). As part of the diagnostic process, urinary free cortisol excretion was also measured over two consecutive days. Only on the first day was there a slight elevation in urinary free cortisol concentration, measuring 183.60 μg/24 h (normal range: 4.3–176). The measurement performed on the second day showed a normal urinary free cortisol concentration of 145.60 μg/24 h (normal range: 4.3–176). On 2 March 2022, the patient underwent a bilateral inferior petrosal sinus sampling (BIPSS). Human CRH stimulation was also used during the procedure. The presence of ACTH-dependent hypercortisolemia of pituitary origin was confirmed. The outcome of the CRH stimulation during the BIPSS is presented in Table 2. The boy qualified for transsphenoidal surgery (TSS) of the pituitary adenoma and was successfully operated on (8 March 2022). Postoperative histopathological examination revealed features of a corticotroph-rich pituitary adenoma. 3. Discussion Osteoporosis, like CD, is extremely rare in the developmental age population. Bone-mineralization disorders among children may be primary (e.g., osteogenesis imperfecta), or secondary to other diseases or their treatment (e.g., with glucocorticosteroids). This case report presents a boy with osteoporosis, the cause of which was originally attributed to the treatment of autoimmune hepatitis with glucocorticosteroids. Steroid therapy is the most common cause of bone-mineralization disorders in children. However, osteoporosis is a late complication of steroid treatment. Briot et al. [10] demonstrated that the risk of fractures increases as early as 3 months after initiating steroid therapy. An additional factor increasing the risk of fractures is the dose of glucocorticosteroids used, corresponding to 2.5–5 mg of prednisolone per day [10]. In the case of the present patient, the appearance of spinal pain and thus vertebral fractures could not have been related to the deflazacort treatment started 5 days earlier. The bone-mineralization disorder must therefore have occurred much earlier. For this reason, the authors considered it necessary to search for other endocrine causes of osteoporosis development, including hypogonadism, growth hormone deficiency or Cushing’s syndrome/disease. The serum vitamin D concentration can also influence bone mineral density. Every patient with mineralization disorders, especially with osteoporosis, requires a thorough assessment of calcium–phosphate metabolism [11]. Until the initiation of steroid therapy in March 2021, the patient did not undergo regular vitamin D supplementation. At the start of deflazacort treatment, his serum 25(OH)D concentration was 12.4 ng/mL. Consequently, additional cholecalciferol supplementation at a dose of 3000 IU/day was introduced. In a subsequent measurement conducted in June 2021, the concentration was within the reference range [25(OH)D: 46.7 ng/mL]. Considering the lack of regular supplementation before March 2021, it can be assumed that in October 2020, when the boy experienced SARS-CoV-2 infection, his serum vitamin D concentration was likely decreased as well, which could have had a further negative impact on the patient’s bone mineralization. Scientific reports indicate that adequate vitamin D levels reduce the risk of viral infections, including SARS-CoV-2 [12]. Di Filippo et al. [13] demonstrated that vitamin D deficiency observed in 68.2% of SARS-CoV-2-infected individuals correlated with a more severe course of the infection. In our patient, the course of COVID-19 was asymptomatic, and the diagnosis was established based on positive IgM antibody titers against SARS-CoV-2. The vitamin deficiency was most likely associated with irregular supplementation and lack of exposure to UV radiation (due to lockdown measures in Poland at that time). A reduced serum 25(OH)D concentration could have contributed to worsened bone mineral density and increased susceptibility to SARS-CoV-2 infection; however, it is the chronic hypercortisolism characteristic of CD that most likely led to the development of osteoporosis with accompanying fractures. Another factor necessitating further diagnostic investigation into CD was the patient’s growth retardation observed since the age of 11. Both the pubertal state of the boy, and his gonadotropin and testosterone serum levels, allowed us to exclude hypogonadism. Maximum spontaneous nocturnal secretion of the growth hormone was 31.84 ng/mL. The diagnosis of CD was established on the basis of elevated cortisol levels at night and the lack of cortisol suppression in the test after administering dexamethasone. Final confirmation of the diagnosis was obtained in a post-CRH stimulation test. In pediatric cases, the absence of typical diurnal variation in serum corticosolemia, especially the nocturnal decline, and the inability to suppress cortisol secretion at midnight, are highly sensitive indicators of hypercortisolemia [6,8]. Consequently, in our patient, osteoporosis was a complication of diagnosed CD. The patient in question was not obese, which is the predominant symptom of CD. This symptom, according to Ferrigno et al. [1], is present in 92–98% of examined children diagnosed with CD. Storr et al. [14] showed that facial changes and facial swelling were observed in 100% of subjects with CD, whereas Lonser et al. [8] observed this in only 63% of children with CD. In our patient, no changes in facial appearance were observed. Other symptoms typical of CS, such as hirsutism, acne, or bruises, were not noticed either. These symptoms were observed in all children with CD studied by Wędrychowicz et al. [3]. Non-specific symptoms of this condition may include mood changes, depression and emotional vacillation [1,8]. However, our patient’s parents did not observe any changes in the boy’s behavior. The indication for initiating the whole diagnostic process was (in addition to osteoporosis) growth retardation. Ferrigno et al. [1] point out that chronic hypercortisolemia most often leads to growth disorders accompanied by excessive weight gain. This is an early, highly sensitive and characteristic sign of CD. Short stature is not always observed and occurs in one in two children diagnosed with CD. The patient we present was short (height—162 cm (<3rd centile hSDS: −2.36)); growth retardation was observed from the age of 11 years. The occurrence of vertebral fractures and the accompanying pain as the initial symptoms of hypercortisolism, the absence of obesity, and the confirmation of CD, an exceedingly rare condition in the pediatric population, collectively underscore the uniqueness of our patient’s disease presentation. A case involving a child with such an atypical course of ACTH-dependent CS has not been described before. Han et al. [15] reported a case of a 28-year-old lean woman (BMI: 19 kg/m²) with ACTH-independent CS due to a left adrenal adenoma, where, similarly to our patient, the initial manifestation of hypercortisolism was compression fractures of the thoracic vertebrae. The authors emphasize that vertebral fractures may affect 30–50% of patients with Cushing’s syndrome, with a higher frequency observed in patients with ACTH-independent CS compared to those in whom hypercortisolism results from the presence of pituitary adenoma [15]. The lack of obesity in a patient with hypercortisolism could be attributed to malnutrition, which accompanies the growth process in ECS. Hence, a crucial aspect was the differential diagnosis between CD and ECS. To this end, we performed a stimulation test using hCRH. We considered cut-off points for diagnosing CD to be a 35% increase in ACTH concentration at 15 and/or 30 min, and at least a 20% increase in cortisol concentration at 30 and 45 min [16,17]. In the case of ECS, a significant rise in CRH and cortisol concentrations is not observed. Recently published reports emphasize the need to explore new cut-off points to enhance the sensitivity and specificity of this test. Detomas et al. [5] indicate that an increase in ACTH ≥ 31% and cortisol ≥ 12% in the 30th minute of CRH tests allows for a highly sensitive and specific differentiation between CD and ECS. The authors highlight that measuring these hormones at the 60 min stage of the test does not provide diagnostic benefits. Notably, the study employed ovine CRH, which exhibits stronger and more prolonged stimulatory effects compared to the hCRH available in Europe that was used to diagnose our patient [5]. Conversely, Elenius et al. [16] suggest that optimal values for distinguishing between CD and ECS in the CRH stimulation test involve an increase in ACTH and/or cortisol levels of more than 40% during the test. In our patient, an over four-fold increase in ACTH levels and a more than two-fold increase in cortisol levels were observed at the 30 min mark of the test, thus independently and definitively excluding ECS regardless of the adopted cut-off points. Our patient’s case also demonstrates that MRI is not a perfect method of visualizing an ACTH-secreting pituitary adenoma. In the first MRI examination performed upon our patient, a poorly demarcated area (2.0 × 3.5 × 5.0 mm) was described in the anterior part of the glandular lobe; in the examination performed 6 months later, this area maintained poor visibility, while laboratory results at the time clearly indicated an ACTH-dependent form of CS. It was only the bilateral inferior petrosal sinus sampling (BIPSS) that allowed a clear diagnosis. Data from the literature indicate that microadenomas smaller than 3–4 mm are visible on MRI in only half of cases. In two large studies including children, pituitary adenomas were found on MRI in 63% and 55% of cases [18]. Among the patients with CD studied by Wędrychowicz et al. [3], pituitary adenomas were described on MRI in all of them, but in two patients (50%) this was only achieved upon follow-up. In the standard procedure, in the absence of a pituitary lesion in the MRI examination, it is recommended that a BIPSS be performed. In the case of our patient, this examination was necessary to make a definitive diagnosis. When analyzing the results of the BIPSS with hCRH stimulation, we employed the classical cut-offs for the ACTH IPS:P (Inferior Petrosal Sinus: Peripheral) ratio (i.e., ≥2 at baseline and ≥3 after hCRH stimulation) [1]. This allowed the confirmation of CD and determination of the pituitary adenoma’s localization, followed by the procedure for its surgical removal. The optimal cut-off values for the IPS:P ratio remain controversial. There are ongoing efforts to establish new, more precise cut-off points. Detomas et al. [19] demonstrated that an IPS:P ratio ≥ 2.1 during desmopressin stimulation in the BIPSS most accurately differentiates CD from ECS. Conversely, Chen et al. [20] showed that the optimal pre-desmopressin stimulation IPS:P ratio cut-off is 1.4, and post-stimulation it is 2.8. Both studies suggest the utilization of lower cut-off values for the IPS:P ratio than those traditionally adopted. Chen et al. [20] also advocate for avoiding stimulation during BIPSS. In most cases, the IPS:P ratio before stimulation is sufficient for diagnosing CD. According to the authors, desmopressin stimulation should be reserved for patients with ambiguous MRI findings or with a pituitary adenoma with diameter less than 6 mm. However, considering that the concentration of ACTH in the right inferior petrosal sinus in our patient was over 4 times higher than in the peripheral vessel and nearly 14 times higher after hCRH stimulation, regardless of the applied criteria, CD could be unequivocally diagnosed in our patient, and the lateralization of the microadenoma could be determined with certainty. The rarity of CD, and the diagnostic difficulties stemming from its oligosymptomatic or atypical course, encourage description in the form of case reports. Eviz et al. [21] delineate the occurrence of cerebral cortical atrophy in two children with ECS. Additionally, other researchers have underscored the potential for thyroid disorders to manifest alongside hypercorticosolemia [22]. Although obesity typically stands out as a primary symptom of CD, Pomahacova et al. [23] reported a case involving two children with CD who maintained normal body weight, mirroring our patient’s situation. The symptoms that prompted diagnostic investigation in these instances included weakness, sleep disturbances and growth retardation. Interestingly, growth retardation, along with facial changes, was observed in all examined children with CD [23]. Nonetheless, to the best of our knowledge, we have yet to encounter a case report resembling ours. Therefore, it remains crucial to share our experiences. 4. Conclusions Cushing’s disease is an extremely rare diagnosis in children. In Poland, there is no statistical record of occurrences of this disease among children. Wędrychowicz et al. reported that in their single Polish center, between 2012 and 2018, they identified four cases of children aged 7–15 who were diagnosed with CD [3]. The case we present shows that obesity, commonly considered as a predominant symptom of CD, is not necessarily observed in patients with this diagnosis in the developmental age population. Among children, it is growth disturbance that may be the first manifestation. On the other hand, a late complication of CD may be osteoporosis, so whenever a child is diagnosed with a bone-mineralization disorder, the cause of its development should be sought. Diagnosis should be pursued until all potential causes of the described symptoms, including the rarest ones, are definitively ruled out—even if the clinical presentation, as in the case of our patient, initially does not point towards the final diagnosis. Thus far, no case of a child with CD exhibiting such subtle symptomatology has been described in the literature. The challenges in diagnosis we encountered primarily resulted from the atypical clinical outcome of CD in our patient—normal body weight, absence of hyperandrogenism, mood disturbances not apparent to caregivers and the patient’s immediate environment, as well as normal progression of puberty, did not immediately lead to the consideration of endocrinological causes of osteoporosis. The steroid therapy employed due to autoimmune hepatitis also complicated the diagnostic process. Only after discontinuing deflazacort treatment was it possible to definitively diagnose CD. Our patient required hydrocortisone replacement in gradually decreasing doses for a year following TSS. Considering that pituitary adenomas in children can be genetically predisposed (e.g., MEN 1 mutation, AIP mutation, USP8 mutation, and other rarer ones), genetic consultation was sought [1]. However, the conducted tests have thus far excluded the most common mutations in our patient. Due to the diagnosed osteoporosis, chronic supplementation with calcium and cholecalciferol was recommended, along with annual follow-up DXA scans. Studies indicate that patients in remission from CD experience a gradual improvement in bone mineral density [3]. While we can currently observe remission in our patient’s case, the advanced bone age of the child (indicating the completion of the growth process) left limited potential for significant improvement in final growth. The patient still requires regular endocrinological and neurosurgical follow-ups, hormonal assessments, and pituitary MRI examinations. Author Contributions Conceptualization—R.S. and A.Ł.; software—S.A.; formal analysis—R.S.; investigation—A.Ł. and G.Z.; data curation—A.Ł.; writing—original draft preparation—A.Ł., R.S. and S.A.; writing—review and editing—A.L.; visualization—S.A.; supervision—A.L. All authors have read and agreed to the published version of the manuscript. 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Abstract Background and Objectives Crooke cell adenomas (CCA) are a rare, aggressive subset of adrenocorticotrophin secreting pituitary corticotroph adenomas (sCTA) found in 5–10% of patients with Cushing’s disease. Multiple studies support worse outcomes in CCA but are limited by small sample size and single-institution databases. We compared outcomes in CCA and sCTA using a multicenter, international retrospective database of high-volume skull base centers. Methods Patients surgically treated for pituitary adenoma from January 2017 through December 2020 were included. Results 2826 patients from 12 international centers were compared (n=20 CCA and n=480 sCTA). No difference in baseline demographics, tumor characteristics or postoperative complications was seen. Microsurgical approaches (60% CCA vs. 62.3% sCTA) were most common. Gross total resection (GTR) was higher in CCA patients (100% vs. 83%, p=0.05). Among patients that had GTR according to intraoperative findings, fewer CCA patients had postoperative hormone normalization of pituitary function (50% vs. 77.8%, p<0.01) and remission of hypersecretion by 3-6 months (75% vs. 84.3%, p<0.01). This was present despite CCA having better local control rates (100% vs. 96%, p<0.01) and fewer patients with remnant on MRI (0% vs. 7.2%, p<0.01). A systematic literature review of 35 studies reporting on various treatment strategies reiterated the high rate of residual tumor, persistent hypercortisolism, and tumor-related mortality in CCA patients. Conclusion This modern, multicenter series of patients with CCA reflects their poor prognosis and reduced post-surgical hormonal normalization. Further work is necessary to better understand the pathophysiology of CCA to devise more targeted treatment approaches. References (0) Cited by (0) Previous presentations: none Previous publications: none Disclosures No relevant disclosures to report CREDIT statement Matthew Finlay: conceptualization, writing – review and editing Richard Drexler: conceptualization, writing – review and editing All: data curation, writing – review and editing Michael Karsy: conceptualization, data curation, methodology, writing – original draft, writing – review and editing, supervision Funding and Disclosures: none View full text From https://www.sciencedirect.com/science/article/abs/pii/S187887502301344X

Context: Intensity-modulated radiotherapy (IMRT) is a modern precision radiotherapy technique for the treatment of the pituitary adenoma. Objective: Aim to investigate the efficacy and toxicity of IMRT in treating Cushing’s Disease (CD). Methods: 70 of 115 patients with CD treated with IMRT at our institute from April 2012 to August 2021 were included in the study. The radiation doses were usually 45-50 Gy in 25 fractions. After IMRT, endocrine evaluations were performed every 6 months and magnetic resonance imaging (MRI) annually. Endocrine remission was defined as suppression of 1 mg dexamethasone test (DST) or normal 24-hour urinary free cortisol level (24hUFC). The outcome of endocrine remission, endocrine recurrence, tumor control and complications were retrieved from medical record. Results: At a median follow-up time of 36.8 months, the endocrine remission rate at 1, 2, 3 and 5 years were 28.5%, 50.2%, 62.5% and 74.0%, respectively. The median time to remission was 24 months (95%CI: 14.0-34.0). Endocrine recurrence was found in 5 patients (13.5%) till the last follow-up. The recurrence-free rate at 1, 2, 3 and 5 years after endocrine remission was 98.2%, 93.9%, 88.7% and 88.7%, respectively. The tumor control rate was 98%. The overall incidence of new onset hypopituitarism was 22.9%, with hypothyroidism serving as the most common individual axis deficiency. Univariate analysis indicated that only higher Ki-67 index (P=0.044) was significant favorable factors for endocrine remission. Conclusion: IMRT was a highly effective second-line therapy with low side effect profile for CD patients. Endocrine remission, tumor control and recurrence rates were comparable to previous reports on FRT and SRS. Introduction Cushing’s disease (CD) is characterized by hypersecretion of adrenocorticotropic hormone (ACTH) from pituitary adenoma. As the state of hypercortisolemia considerably increases morbidity and mortality, normalizing cortisol levels is regarded as the major treatment goal in patients with CD (1). Transsphenoidal selective adenomectomy (TSS) is now established as the first-line treatment of CD. Despite the satisfactory remission rate that can be achieved with TSS (ranging from 59-97%), delayed recurrences have also been reported in up to 50% of patients (2). The Endocrine Society guidelines suggest a shared decision-making approach in patients who underwent a noncurative surgery or for whom surgery was not possible (3). Second-line therapeutic options include repeat transsphenoidal surgery, medical therapy, radiotherapy and bilateral adrenalectomy. Radiotherapy (RT) is generally used in patients who have failed TSS or have recurrent CD, as well as in progressively growing or invasive corticotroph tumors (3, 4). Both stereotactic radiosurgery(SRS)and fractionated radiotherapy (FRT) have been used in the treatment of CD. Conventional radiotherapy as one of the technique for FRT has been used with a long experience, but its benefits were hindered by high risk of toxicity, mainly attributed to the harm to healthy surrounding structures (4). Previous studies on conventional RT in treating CD showed high efficacy (tumor control rate of 92-100% and hormonal control rate of 46-89%), but RT-induced hypopituitarism (30-58%) and recurrence (16-21%) were also commonly reported (1, 4–7). Modern precise radiotherapy, especially intensity-modulated radiotherapy (IMRT), can spare the surrounding normal structure better by a more conformal and precise dose distribution (8). However, a large cohort study on long-term efficacy and toxicity of IMRT for CD is still lacking. Therefore, in the current study, we aim to analyze the efficacy and toxicity of intensity-modulated radiotherapy (IMRT) in treating CD. We also investigated the predictors of endocrine remission in aid of further management. Methods Patient We collected 115 cases of Cushing’s disease treated at our center from April 2012 to August 2021. Patients were excluded under the following conditions: (1) follow-up time less than 3 months, (2) lacking evaluation of serum cortisol (F), adrenocorticotropic hormone (ACTH) or 24-hour urinary free cortisol (24hUFC) before or after RT, (3) underwent uni or bilateral adrenalectomy, (4) having received RT at other institutes before admitted to our center. At last, a total of 70 cases were included in this study. Radiotherapy parameters RT was administrated by a linear accelerator (6 MV X-ray). Intensity-modulated radiation therapy was applied for all patients. Including fix-filde IMRT (FF-IMRT), volumetric modulated arc therapy (VMAT) or Tomotherapy. We immobilized the patient with an individualized thermoplastic head mask and then conducted a computed tomography (CT) simulation scan at 2- to 3-mm intervals. The target volume and organs at risks (OARs) were delined with a contrast enhanced T1-weighted image (T1WI) magnetic resonance imaging (MRI) fusing with planning CT. The gross tumor volume (GTV) was defined with the lesion visible on MRI or CT. The clinical target volume (CTV) included microscopic disease, especially when the tumor invaded cavernous sinus and surrounding bones. The planning target volume (PTV) was defined as CTV plus a margin of 2- to 3-mm in three dimensions. The prescription dose was defined at 100% isodoseline to cover at least 95% PTV. The maximum dose was limited to less than 54 Gy for the brain stem and optic pathway structures. Radiotherapy was performed once a day and five fractions a week during five to six weeks. The total dose was 45-60 Gy, delivered in 25-30 fractions, with most patients (78.6%) receiving 45-50 Gy in 25 fractions. The fractionated dose was 1.8-2.0 Gy. Data collection and clinical evaluation Baseline characteristics were collected at the last outpatient visit before RT, including demographic characteristics, biochemical data, tumor characteristics and details of previous treatments. After RT, endocrine evaluations were performed every 6 months. Endocrine remission was considered when 1 mg dexamethasone suppression test (DST)<1.8 mg/dl. If 1mg DST results were lacking, then 24hUFC within the normal range was used as a remission criterion. Patients who regained elevated hormone levels after achieving remission were considered to have endocrine recurrence. For patients receiving medications that could interfere with the metabolism of cortisol, hormonal evaluation was performed at least 3 months after the cessation of the therapy. Tumor size was measured on magnetic resonance imaging (MRI) before RT and annually after the completion of RT. Any reduction in or stabilization of tumor size was considered as tumor control. Tumor recurrence was defined as an increase of 2 millimeters in 2 dimensions comparing to MRI before RT, or from invisible tumor to a visible tumor on MRI (9). Anterior pituitary function was assessed before RT and every 6 months during the follow-up after RT. RT-induced hypopituitarism was defined as the development of new onset hormone deficiency after RT. The diagnostic criteria for growth hormone deficiency (GHD), central hypothyroidism and hypogonadotropic hypogonadism (HH) refer to previous literature (10–12). Panhypopituitarism referred to three or more anterior pituitary hormone deficiencies (13). Statistical analysis Statistical analysis was performed with SPSS version 25.0. Longitudinal analysis was performed with Kaplan-Meier method. For time-dependent variable, Log rank test was used for univariate analysis and Cox regression for multivariate analysis. The cut-off of F, ACTH and 24hUFC were defined as their median value. All variants in the univariate analysis were included in the model of multivariate analysis. P value < 0.05 was considered statistically significant. Plot was created with GraphPad Prism version 9.4. Results Patient characteristics Of 70 cases included in the study, the median age was 32 years (range, 11-66 years). 60 (85.7%) were female and 10 (14.3%) were male (F:M= 6:1). The median follow-up time was 36.8 months (range, 3.0-111.0 months). 68 patients received RT as a second-line treatment because of incomplete tumor resection, failure to achieve complete endocrine remission or recurrence postoperative, and 2 were treated with RT alone because of contraindication of surgery. The frequency of surgical treatment was 1 for 42 patients, 2 for 21 and more than 3 for 5. A total of 8 patients received medical treatment before RT. 5 of them used pasireotide, 2 used ketoconazole and 1 used mifepristone. The median ACTH level was 58.7 pg/ml (range 14.9-265 pg/ml), F, 26.2μg/dl (range 11.8-72.6 μg/dl) and 24hUFC, 355.7 μg/24hr (range 53.5-3065 μg/24hr) before RT. Tumor size evaluation was performed in all 70 patients before RT. Among them, 36 patients showed no visible residual tumor identified on MRI and only 5 patients showed tumor size more than 1 cm. Hypopituitarism was found in 31 patients (38.8%) before RT. HH was the most common (21 patients, 26.3%), followed by central hypothyroidism (13 patients, 16.3%) and GHD (9 patients, 11.3%). Panhypopituitarism was found in 4 patients (5.0%). (Table 1). Table 1 Table 1 Patient characteristics. Endocrine remission Endocrine remission was achieved in 37 of 70 patients during the follow-up. Six of them were evaluated by 1mg DST. The hormonal remission rate at 1, 2, 3 and 5 years were 28.5%, 50.2%, 62.5% and 74.0%, respectively, gradually increasing with follow-up time (Figure 1). The median time to remission was 24.0 months (95%CI: 14.0-34.0 months). Univariate analysis indicated that only higher Ki-67 index (P=0.044) was significant favorable factors for endocrine remission. There was no significant correlation between remission and age, sex, tumor size, the frequency of surgery, medication prior RT. The hormone levels (F, ACTH and 24hUFC prior RT) were divided into high and low groups by the median value, and were also not found to be associated with endocrine remission (Table 2). Since only Ki-67 was significant in the univariate analysis and all other parameters were far from significant, a multivariate analysis was no longer performed. Figure 1 Figure 1 Endocrine remission rate during the follow-up after RT. Table 2 Table 2 Univariate predictors of endocrine remission. Endocrine recurrence was found in 5 patients till the last follow-up, with an overall recurrence rate of 13.5% (5/37). The median time to recurrence after reaching endocrine remission was 22.5 months. The recurrence-free rate at 1, 2, 3 and 5 years after endocrine remission was 98.2%, 93.9%, 88.7% and 88.7%, respectively (Figure 2). Figure 2 Figure 2 Recurrence free rate after endocrine emission. Tumor control A total of 51 patients had repeated MRI examinations before and after treatment. During the follow-up, 20 patients showed reduction and 30 patoents remained stable in tumor size, with a tumor control rate of 98%. Only 1 patient showed enlargement tumor 1 year after RT, with F, ACTH and 24hUFC increase continuously. Complications At the last follow-up, 16 patients developed new onset hypopituitarism after RT. The overall incidence of RT-induced hypopituitarism was 22.9%. Hypothyroidism was the most common of hypopituitarism (8 patients), followed by HH (7 patients), adrenal insufficiency (4 patients) and GHD (3 patients). Only 1 patient (1.3%) with systemic lupus erythematosus (SLE) comorbidity complained of progressively worsening visual impairment during the follow up. No cerebrovascular event or radiation associated intracranial malignancy was found in our cohort. Discussion Efficacy and radiotherapy techniques RT has been emerged as an effective second-line treatment for CD for many years. Although conventional fractionated RT has been used for a long experience in patients with CD, study on the modern precise radiotherapy, particularly IMRT, is rare and reports limited evidence on its long-term treatment outcome. IMRT can be implemented in many different techniques, such as fixed-field intensity-modulated radiotherapy (FF-IMRT), volumetric-modulated arc therapy (VMAT) and tomotherapy. Compared with conventional RT, IMRT allows a better target volume conformity while preserves adequate coverage to the target (14, 15). Our study reported that IMRT for CD has an endocrine remission rate of 74.0% at 5 years, with a median time to remission of 24.0 months (95%CI: 14.0-34.0 months). The endocrine remission rate at 5 years was comparable to those reported in previous series of FRT, with a median time to remission within the reported range (4.5-44 months) (9, 16–18) (Table 3). Compared with SRS in treating CD, the endocrine remission rate and median time to remission were also similar. Pivonello et al (19) summarized 36 studies of SRS for CD between 1986 to 2014, the mean endocrine remission rate was 60.8% and the median time to remission was 24.5 months. Tumor control rate was 98% in our cohort, only one patient showed enlargement tumor with elevating hormones. This local control rate was also comparable to that reported in a series of pituitary adenoma treated with FRT (93-100%) and SRS(92-96%) (9, 16–18, 20, 21). Indeed, despite the lack of controlled studies about SRS and FRT in treating CD, many reviews that summarize the biochemical control and tumor contral of both are similar (2, 6, 19). Table 3 Table 3 Literature review of FRT and SRS in patients with CD published in recent years. The overall endocrine recurrence rate in our study was 13.5%, with a median time to recurrence of 22.5 months. We, for the first time, reported the actuarial recurrence free rate at 1, 2, 3 and 5 years in CD patients treated with IMRT. The recurrence free rate at 3 and 5 years was 88.7% in our study. Outcomes were comparable to those reported in patients treated with conventional RT or SRS, with a mean recurrence rate and a median recurrence time of 15.9% (range, 0-62.5%) and 28.1 months, or 12.3% (range, 0-100%) and 33.5 months, according to a review conducted by Pivonello et al (19). At 2020, we reported the outcomes of pituitary somatotroph adenomas treated with IMRT at our institution (20). Compared with pituitary somatotroph adenomas, CD has a similar 5-year remission rate (74.0% vs 74.3%) but a shorter median time to remission (24.0m vs 36.2m) (Figure 3). The tumor contral rates were similar, at 98% and 99%, respectively. The endocrine recurrence rate was significantly different, with CD being about one-fold higher than the pituitary somatotroph adenoma (13.8% vs 6.1%). This may be due to the majority of microadenomas in CD and that of macroadenomas in pituitary somatotroph adenomas. Figure 3 Figure 3 Endocrine remission rate of CD and pituitary somatotroph adenoma. Predictors of endocrine remission In the univariate analysis, we found that only Ki-67 index ≥ 3% was correlated with better endocrine remission (p=0.044). Cortisol levels before RT and tumor size were not predictors of endocrine remission. For surgery in treating CD, higher preoperative ACTH level was considered as unfavorable prognostic factor for endocrine remission in a few studies (22, 23). For radiotherapy, some previous studies also have reported a faster endocrine remission in patients with lower serum cortisol level. Minniti et al. reported that hormone level was normalized faster in patients with lower urinary and plasma cortisol level at the time of RT (16). Apaydin also reported that low postoperative cortisol and 1mg DST was a favorable factors for faster remission in patients treated with gamma knife surgery (GKS) and hypofractionated radiotherapy (HFRT), although no significant relationship was found between remission rate and plasma cortisol level prior RT in both studies (9, 16). Castinetti et al. found that initial 24hUFC was a predicative factor of endocrine remission in patients treated with GKS, which was not reported in our cohort treated with IMRT (24). However, the discrepancy between the results can be attributed to various factors, including selection bias of retrospective study, duration of follow-up, endocrine remission criteria and cut-off value. Tumor size before RT was considered as a significant predictor for endocrine remission in some published series of patients treated with SRS. Jagannathan et al. reported a significant relationship between preoperative tumor volume and endocrine remission in patients with CD treated with GKS (25). However no significant correlation between tumor size and endocrine remission was found in series of patients treated with FRT (5, 9, 16, 17). But our study found no significant correlation between tumor size (visible or no-visible residual tumor on MRI) before RT and endocrine remission. The frequency of surgery before RT was also not found to be associated with endocrine remission in our study, which reached a similar conclusion with some previous studies (9, 17, 18, 26). Abu Dabrh et al. reported a higher remission rate in patients receiving TSS prior RT in their meta-analysis (5). Similar result was also reported in a review on the treatment outcome of GKS in patients with CD, that postoperative GKS was more effective than primary GK (19). However, analysis on this parameter was difficult in our cohort considering the low number of patients who received IMRT as the first-line treatment. Reports on the effect of medical treatment on endocrine remission have been controversial. Some studies reported a negative effect of medical treatment at the time of SRS on endocrine remission in patients with CD. Castinetti et al. showed a significant higher rate of endocrine remission in patients who were not receiving ketoconazole at the time of GKS, compared to those who were (27). Sheehan et al. also found a significantly shorter time to remission in patients who discontinued ketoconazole at the time of GKS (28). However, no such correlation was found in patients treated with FRT (9, 17). Like previous studies on FRT, we also noted no significant relationship between preradiation use of medication and endocrine remission, but our statistical analysis may be hindered by the low proportion of patients undergoing medical treatment before RT. Moreover, the anticortisolic drugs used in previous studies were mainly ketoconazole or cabergoline, while most of our patient have received pasireotide, whose effect have not been well-studied yet. Further studies are necessary to understand the effect of somatostatin receptor ligands on the outcome of radiotherapy in patients with CD. Complications Hypopituitarism is the most common complication secondary to radiotherapy, with the rate of new-onset hypopituitarism ranging widely in previous report. Pivonello et al. reviewed series of CD patients who were treated with conventional RT with a follow-up of at least 5 years (19). The reported mean and median rates of hypopituitarism were 50% and 48.3%, respectively (range, 0-100%). As regards FRT, the overall rate of new-onset hypopituitarism was 22.2-40% at a median follow-up ranging from 29-108 months, with both incidence and severity increasing with longer follow-up (9, 16–19). The incidence of hypopituitarism in our series was 22.9%, which was within the reported range of new onset hypopituitarism after FRT. Lower rate of hypopituitarism after SRS compared to conventional RT has been recognized in previous reviews (2, 6). Our study showed that new onset hypopituitarism was less prevalent after IMRT than after conventional RT. This can be attributed to a higher precision in contouring the target volume and OARs, allowing these modern radiotherapy techniques to provide a better protection to hypothalamus-pituitary axes. In previous studies, potential risk factors for new onset hypopituitarism included suprasellar extension, higher radiation dose to the tumor margin and lower isodose line prescribed (29, 30). Sensitivity of individual hormonal axes to RT varies in different series. In our study, central hypothyroidism was the most common individual axis deficiency, followed by HH, adrenal insufficiency and GHD. This sequence was similar to that reported by Sheehan et al., whose series included 64 CD patients treated with SRS, as well as some other series (29, 31). It is noted in some studies that GHD is the most vulnerable axes (19, 32, 33). Limited number of patients undergoing stimulation test may underestimate the prevalence of GHD in our study and some previous series, and longer follow-up is needed to generate a more accurate, time-dependent rate of new onset hypopituitarism. In our study, only one patient complained of mild visual impairment, which was comparable to the rate ranging from 0-4.5% in previous series of FRT treating pituitary adenoma (9, 16–18, 26, 32, 34, 35). This patient had concomitant SLE and the associated microangiopathy may render the optic nerve intolerant to radiotherapy. Cranial nerve damage was acknowledged as an uncommon complication, with an estimated risk of vision deterioration below 1% if single radiation dose was no more than 2.0 Gy and total dose no more than 45-50 Gy (2, 36). The actuarial rate of optic neuropathy at 10 years was 0.8% in a series containing 385 patients with pituitary adenoma (37). No patient in our cohort developed cerebrovascular accident or secondary brain tumor. This finding was consistent with the low actuarial prevalence of these complications reported in other published series of FRT. Secondary brain tumor was extremely rare after SRS, with an overall incidence of 6.80 per patients-year, or a cumulative incidence of 0.00045% over 10 years in a multicenter cohort study containing 4905 patients treated with GKS (38). Ecemis et al. reviewed cohort studies of conventional RT in treating pituitary adenoma from 1990 to 2013 and found that 1.42% of patients developed secondary brain tumor, with a latency period of 19.6 years for meningioma, 11 years for glioma and 9 years for astrocytoma (39). As for cerebrovascular accident, Minniti et al. reported two patients (in a total of 40 patients) who had stroke 6 and 8 years after FRT (16). Data was still limited for FRT. Considering the low incidence and long latency period, large, controlled cohort study with long follow-up of FRT is still needed to accurately evaluate these complications. Limitations Our study has several limitations. First, not all patients rigorously followed regular follow-up time points, making time-dependent statistical analysis less accurate. In addition, the excessively low number of cases with 1mg DST as the endocrine remission criterion may affect the accuracy of the remission rate.Moreover, a median follow-up time of about 3 years hampered evaluation on some late complications, including cerebrovascular events and secondary brain tumor. In conclusion, our study revealed that IMRT was a highly effective second-line therapy with low side effect profile for CD patients, and it’s endocrine remission, tumor control and recurrence rates were comparable to previous reports on FRT and SRS. Data availability statement The original contributions presented in the study are included in the article/supplementary material. Further inquiries can be directed to the corresponding author. Author contributions 1. Conceptualization: FZ and HZ 2. Data curation: XL and ZX. 3. Funding acquisition: FZ. 4. Investigation: XL and ZX 5. Methodology: WW 6. Resources: XL, SS and XH 7. Validation: LL and HZ. 8. Writing – original draft: ZX 9. 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This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. *Correspondence: Fuquan Zhang, zhangfq@pumch.cn †These authors have contributed equally to this work Disclaimer: All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article or claim that may be made by its manufacturer is not guaranteed or endorsed by the publisher. From https://www.frontiersin.org/articles/10.3389/fendo.2023.1241669/full

Bridget Houser felt despairing. In the months before her 2018 wedding, Houser, who had never struggled with her weight, noticed that it inexplicably began to creep up. In response she doubled the length of her runs to eight miles, took back-to-back high intensity workout classes and often consumed only water, coffee and fruit during the day before a spartan, mostly vegetable, dinner. Yet no matter what Houser did, her weight stubbornly increased and her oval face grew round, a transformation that was glaringly obvious in comparison with her identical twin sister. Houser wondered whether the five pounds she gained despite her herculean effort was a corollary of other problems. For the previous two years she had battled a string of maladies: first daily headaches, then crippling anxiety, followed by insomnia, hair loss and acne, something she’d never endured as a teenager. “Stress was the universal explanation,” recalled Houser, a controller for a small business in Chicago. When doctors suggested that her upcoming marriage might be a cause of her problems, Houser considered, then rejected, the theory. It just didn’t jibe with her feelings. In early 2019, about six months after her wedding, Houser insisted that her doctors perform several tests. They ultimately revealed that her symptoms weren’t the result of stress or marital misgivings but of a serious illness that had been smoldering for years. After successful treatment followed by a long recovery Houser, now 34, feels far better than she did during those miserable years in her late 20s. “I wish I’d been nicer to myself and not blamed myself for what was going on,” she said. Getting through the wedding In 2016 Houser began experiencing daily pain in the back of her head, a common spot for tension headaches. When the headaches failed to improve with dietary changes or nonprescription pain relievers, she consulted her primary care doctor, followed by a neurologist who told her she had migraines. Houser, then 27, noticed that the headaches were worse when she wore contact lenses. “It was affecting my daily life and I talked myself into thinking the problem was my contacts,” she said. She decided Lasik surgery might help and in October 2017 underwent the procedure, which uses a laser to reshape the cornea, reducing or eliminating dependence on contacts or glasses. Her vision improved and the pain disappeared — briefly. A week after eye surgery, her headaches returned. “I wasn’t overly concerned,” Houser said. “I know a lot of people have headaches.” A few months later for no apparent reason Houser developed “really bad anxiety. It wasn’t just like I was anxious,” she recalled. “I couldn’t function. I’m Type A so I knew what anxiety is, but not to this degree.” One weekday morning in early 2018 she felt so overwhelmed that she took a sick day, then called her twin, Molly, and their mother and told them she needed help immediately. They managed to schedule a same-day appointment with a psychiatrist whom Houser began seeing regularly, along with a therapist. The psychiatrist zeroed in on her impending wedding and told Houser that the event can cause “huge anxiety.” She began taking an antidepressant along with Ativan, an anti-anxiety drug she used when things got really bad. She also ramped up her yoga practice, hoping it might calm her. Houser vividly remembers riding the escalator to her office one morning “and in my head I kept saying, ‘I’m in trouble, I’m in trouble,’” although she didn’t know what was wrong. Her changing appearance had become a source of great unhappiness. Although her weight remained in the normal range, Houser couldn’t figure out why she was gaining weight after drastically slashing her food intake and dramatically ramping up exercise. Her normally thick hair had thinned so noticeably that her hairdresser gently advised her to consult a doctor. Houser’s psychiatrist thought her hair loss might be caused by her antidepressant and switched medications. That didn’t seem to help. Houser was particularly bothered by her newly chubby face. “It was like a joke in my family,” she said, adding that she was teased about being overly sensitive. Even her wedding day was colored by unhappiness about her appearance and the intense amorphous anxiety that seemed omnipresent. “Rather than think about how excited I was,” Houser recalled, “it was ‘How can I get through this day?’” Normal thyroid After her wedding Houser felt worse. She developed severe insomnia, night sweats and acne. In February 2019 a nurse practitioner in her primary care practice ordered tests of her thyroid, which were normal. When Houser pressed for additional testing, she was referred to an endocrinologist. He told her she was stressed. Dissatisfied, she saw a second endocrinologist who agreed with the first. “She said ‘I don’t think there’s anything wrong with you’” metabolically, Houser recalled. The second endocrinologist’s nurse even revisited the marriage question in the presence of Houser’s husband, Doug, who had accompanied her to the appointment. “She said ‘I knew on my honeymoon I shouldn’t have gotten married,’” Houser remembered her saying. “‘Are you in a happy marriage?’ I couldn’t believe it.” Months earlier, the nurse practitioner who ordered the thyroid tests briefly mentioned measuring levels of cortisol, a hormone involved in the body’s response to stress and other functions. Elevated levels of cortisol can indicate Cushing’s syndrome, an uncommon hormonal disorder that occurs when the body produces too much of the hormone over a prolonged period. “She had thrown cortisol testing out there and I think it was always in the back of my mind,” Houser said. She asked the second endocrinologist to order cortisol tests. The doctor agreed, but not before telling Houser that she didn’t think she had Cushing’s because she lacked the classic symptoms: major weight gain, purple stretch marks and a fatty hump between the shoulders. Houser did have the “moon face” characteristic of Cushing’s that is also seen in people who take high doses of steroids for long periods to treat various illnesses — but Houser wasn’t taking steroids. Insomnia, headaches, acne and anxiety can be symptoms of Cushing’s. There are several forms of Cushing’s syndrome, which typically results from a tumor — usually benign but sometimes cancerous — in the pituitary or adrenal gland that pumps out excess cortisol. Sometimes tumors develop elsewhere in the body such as the lungs or pancreas. Cushing’s affects roughly five times as many women as men and typically occurs between the ages of 30 and 50. If left untreated, it can be fatal. A trio of tests measuring cortisol levels in Houser’s blood, urine and saliva were significantly elevated; the amount in her urine was eight times higher than normal. The formerly skeptical Chicago endocrinologist told Houser she had Cushing’s and referred her to James Findling, a Milwaukee endocrinologist who is internationally recognized for his treatment of the disease. “I was just so happy to have a diagnosis,” Houser recalled. Revealing photos Findling asked Houser to bring photographs taken several years earlier to her October 2018 appointment. It is a request he makes of patients as a way of spotting telltale physical manifestations. In Houser’s case, the facial change was particularly striking because she is an identical twin. Findling noted that delayed diagnosis is typical, because physical changes and other symptoms tend to occur gradually and insidiously. Houser, he added, “didn’t look like the typical Cushing’s patient. She wasn’t obese and she didn’t have diabetes or hypertension. It was more subtle than many cases.” The next step was determining the location of the tiny tumor. Tests found nothing in Houser’s pituitary or adrenal glands, and CT scans of her pelvis, chest and abdomen were clean. Findling ordered a dotatate PET scan, a highly sensitive CT scan that can find tumors that elude conventional imaging. The scan revealed a nodule in Houser’s left lung. Houser sought a second opinion from a thoracic surgeon in Chicago. While Findling and a thoracic surgeon at Milwaukee’s Froedtert Hospital strongly recommended that she undergo surgery to remove the tumor, the Chicago doctor disagreed. He said he didn’t think the lung nodule was causing Cushing’s and recommended that Houser continue therapy and anti-anxiety medication. “Do you know what it’s like to wake up from surgery and to not be better?” she remembers him asking her. After deliberating with her husband and conferring with her Milwaukee doctors, Houser opted for surgery performed Oct. 30, which removed part of her left lung. A pathologist determined that the nodule was a rare, slow-growing neuroendocrine lung cancer known as a bronchial carcinoid, which can cause Cushing’s. The Stage 2 cancer had spread to a nearby lymph node. “Fortunately I think we got it early,” Findling said. “She’s had a sustained remission and a cure of her Cushing’s.” “The cancer didn’t rock my world,” said Houser, who had previously had a melanoma skin cancer removed. (Doctors have told her they don’t think the cancers are related.) “It was about not having Cushing’s anymore, which was more important.” So why didn’t Houser’s doctors, among them endocrinologists, suspect Cushing’s? Findling, who estimates he has treated as many as 2,000 people with the disease in his 40-year career, said that while doctors are taught that Cushing’s is rare, it’s not. He cites a 2016 study, which that found that 26 of 353 endocrinology patients were found to have the disease. Textbook descriptions, which include the presence of purple stretch marks and a hump, are “almost a caricature,” Findling observed. “It’s pretty well recognized that Cushing’s is more subtle than that … and can cause neuropsychiatric and neurocognitive problems.” Houser’s normal weight and the fact that she didn’t have high blood pressure or diabetes may have misled doctors. “I think we’ve moved the needle a little bit, especially among endocrinologists,” he continued, adding that “the threshold for screening has got to change. Once you tell a primary care doctor that it’s a rare disorder, it goes in one ear and out the other. They think they’ll never see it.” “When you make this diagnosis it can have fabulous outcomes,” he added, citing Houser’s case. “That’s why I’m still doing this at my age.” Houser considers Findling to be her “literal lifesaver.” She spent the next year seeing him as she was slowly weaned off medications to normalize her hormone levels and recover her strength. She is monitored for Cushing’s annually, remains cancer-free and, other than residual fatigue, feels well. In October 2021 she gave birth to a daughter. Her son was born eight weeks ago. Houser regards the help provided by her family, particularly her husband whom she called “my biggest supporter,” as essential. That seems especially ironic because stress about their marriage had been blamed for symptoms that were actually caused by a cancer. “He was a huge help in calling doctors and making the necessary appointments when I didn’t have the energy to fight anymore.” His unwavering love, she said, was “a testament to our strong marriage.” From https://www.washingtonpost.com/wellness/2023/10/07/weight-anxiety-wedding-medical-mysteries/

Highlights The most common cause of ectopic ACTH syndrome is pulmonary carcinoid tumors and squamous cell lung cancer; however it is a relatively uncommon complication of pulmonary neoplasms. The most common cause of Cushing syndrome is iatrogenic corticosteroid use and it should be considered in all patients regardless of clinical background. Low urine cortisol levels may be associated with exogenous glucocorticoid exposure. Occult glucocorticoid exposure is rare but can be evaluated with liquid chromatography. Consumers should be aware of the potential risks of taking supplements, especially those advertised as joint pain relief products. Abstract Background Well differentiated bronchial neuroendocrine neoplasms often follow a clinically indolent course and rarely cause Ectopic ACTH syndrome. Iatrogenic corticosteroid use is the most common cause of Cushing syndrome and should be considered in all patients regardless of clinical background. Case report A 59 year old woman with an 11 year history of a 1.5 cm well differentiated bronchial carcinoid, presented with Cushingoid features. Laboratory results were not consistent with an ACTH dependent Cushing Syndrome and exogenous steroids were suspected. The patient received an FDA alert regarding a glucosamine supplement she had started 4 months prior for joint pain. Discussion Ectopic ACTH production is reported in less than 5% of patients with squamous cell lung cancer and 3% of patients with lung or pancreatic (non-MEN1) neuroendocrine tumors. Factitious corticoid exposure is rare and can be evaluated with synthetic corticosteroid serum testing. Conclusion Cushing syndrome due to supplements containing unreported corticosteroid doses should be considered in patients with typical Cushingoid features and contradictory hormonal testing. 1. Introduction Well differentiated bronchial neuroendocrine neoplasms often follow a clinically indolent course and can rarely exhibit Cushing syndrome due to ectopic production of adrenocorticotropic hormone (ACTH). However the most common cause of Cushing syndrome is iatrogenic corticosteroid use and should be considered in all patients regardless of clinical background (see Fig. 1, Fig. 2, Fig. 3, Fig. 4). Download : Download high-res image (243KB) Download : Download full-size image Fig. 1. DOTATATE PET/CT demonstrates a right upper lobe pulmonary nodule with intense uptake. Download : Download high-res image (201KB) Download : Download full-size image Fig. 2. DOTATATE PET/CT demonstrates intense uptake within a right upper lobe pulmonary nodule, consistent with biopsy-proven carcinoid tumor. There are no distant sites of abnormal uptake to suggest metastatic disease. Download : Download high-res image (399KB) Download : Download full-size image Fig. 3. Artri Ajo King Supplement (Source: FDA). The label claims that the product contains glucosamine, chondroitin, collagen, vitamin C, curcumin, nettle, omega 3, and methylsulfonylmethane. Download : Download high-res image (288KB) Download : Download full-size image Fig. 4. Artri King Supplement (Source: FDA). 2. Case report A 59–year old woman with an 11 year history of a 1.5 cm well-differentiated bronchial carcinoid, presented with 20 lb. weight gain, facial swelling, flushing, lower extremity edema and shortness of breath over 3 months. On exam, the patient was normotensive, centrally obese with mild hirsutism, facial fullness and ruddiness with evidence of a dorsocervical fat pad. Initially there was concern for hormonal activation of her known bronchial carcinoid. Testing resulted in a normal 24-hour urine 5-HIAA (6 mg/d, n < 15 mg/dL), elevated chromogranin A (201 ng/mL, n < 103 ng/mL), normal histamine (<1.5 ng/mL, n < 1.7 ng mL), low-normal 7 AM serum cortisol (5.1 μg/dL, n 3.6–19.3 μg/dL), normal 7 AM ACTH (17 pg/mL, n < 46 pg/mL) and a surprisingly low 24-hr urinary free cortisol (1.8 mcg/hr, n 4.0–50.0 mcg/hr). A late night saliva cortisol was 0.03 mcg/dL (n 3.4–16.8 mcg/dL). Testosterone, IGF-1, glucose and electrolytes were appropriate. An echocardiogram showed an ejection fraction of 60% with no evidence of carcinoid heart disease. A Dotatate PET-CT was obtained to evaluate for progression of the neuro-endocrine tumor and revealed a stable right upper lobe pulmonary nodule with no evidence of metastatic disease. Given low cortisol levels, ectopic Cushing syndrome was excluded and exogenous steroids were suspected, however the patient denied use of oral,inhaled, or injected steroids. A cosyntropin stimulation study yielded a pre-stimulation cortisol 6.2 μg/dL with an adequate post-stimulation cortisol 23.5 μg/dL. At this stage of evaluation, the patient received an FDA alert regarding a glucosamine supplement she had started 4 months prior for joint pain. The notification advised of hidden drug ingredients including dexamethasone, diclofenac, and methocarbamol contained within Artri King Glucosamine supplements not listed on the product label but verified by FDA lab analysis. The FDA had received several adverse event reports including liver toxicity and even death associated with such products. The patient's symptoms gradually improved after discontinuation of the supplement. 3. Discussion 3.1. Ectopic ACTH syndrome This patient's Cushingoid features were initially suspected to be secondary to the known bronchial neuroendocrine tumor. Ectopic ACTH production accounts for about 5–10% of all Cushing Syndrome cases [1]. The most common location of ectopic ACTH is the lungs with pulmonary carcinoid tumors being the most common cause, followed by squamous cell lung cancer [2]. Despite this patient's history of bronchial carcinoid tumor and positive chromogranin histopathological marker, her laboratory results were not consistent with an ACTH dependent Cushing Syndrome. In fact, Cushing syndrome is a relatively uncommon neuroendocrine neoplasm complication. The prevalence of ectopic ACTH production in patients with lung tumors is rare, at less than 5% in squamous cell lung cancer and about 3% in patients with lung or pancreatic (non-MEN1) neuroendocrine tumors1. Patients with ACTH dependent Cushing syndrome not suspected to originate from the pituitary, undergo further testing to evaluate for an ectopic ACTH secreting tumor. These tests include conventional imaging of the chest, abdomen and pelvis, as well as functional imaging such as octreotide scans, fluoride 18-fluorodeoxyglucose-positron emission tomography [18F-FDG PET], and gallium-68 DOTATATE positron emission tomography-computed tomography [Dotatate PET-CT] scan [3]. In our literature review, we found that there was insufficient evidence to determine the sensitivity and specificity of nuclear medicine imaging techniques [4,5]. In this case, the patient had no laboratory evidence for ACTH dependent Cushing Syndrome, but given the known bronchial carcinoid tumor, a repeat Dotatate PET-CT scan was obtained which demonstrated no indication of growth or spread of the known bronchial tumor. 3.2. Supplement induced Cushing Syndrome One of the most remarkable findings in this case was the patient's low urine cortisol level in the setting of her overt Cushingoid features. In our survey of the literature, we found that low urine cortisol levels were associated with exogenous glucocorticoid use [6,7]. The low urine cortisol levels may be reflective of intermittent glucocorticoid exposure. Indeed, this patient's Cushingoid features were determined to be secondary to prolonged use of Artri King supplement. Occult glucocorticoid use is difficult to diagnose even after performing a thorough medication reconciliation as patients may unknowingly consume unregulated doses of glucocorticoids in seemingly harmless supplements and medications. The incidence of supplement induced Cushing Syndrome is currently unknown as supplements are not regularly tested to detect hidden glucocorticoid doses. Additionally, the likelihood of developing supplement induced Cushing syndrome is dependent on dosage and duration of use. In our literature review we found nine published articles describing supplement induced Cushing Syndrome [[7], [8], [9], [10], [11], [12], [13], [14], [15]], one case report of tainted counterfeit medication causing Cushing Syndrome [16], and two cases of substances with probable glucocorticoid-like activity [17,18]. Of the nine published articles of supplement induced Cushing Syndrome, six were associated with supplements marketed as arthritic joint pain relief products including ArtriKing, Maajun, and AtriVid [[7], [8], [9], [10], [11], [12]]. These products later received government issued warnings in Mexico, Malaysia, and Colombia respectively [[19], [20], [21]]. To our knowledge there have been four published reports of ArtiKing supplement induced Cushing Syndrome [[7], [8], [9], [10]]. The first documented cases were reported in 2021 in Vera Cruz, Mexico; since then the Mexican medical community reported seeing a disproportionate increase in cases of iatrogenic Cushing Syndrome due to these supplements [7]. There have also been three American published articles describing a total of 4 cases of ArtriKing supplement induced Cushing syndrome [[8], [9], [10]]. In January 2022 the FDA issued a warning about Atri Ajo King containing diclofenac, which was not listed in the product label [22]. In April 2022 the FDA expanded its warning, advising consumers to avoid all Artri and Ortiga products after the FDA found these products contained dexamethasone and diclofenac [23]. In October 2022 the FDA issued warning letters to Amazon, Walmart, and Latin Foods market for distributing Artri and Ortiga products [24]. Many supplements are not regulated by the government and may contain hidden ingredients such as glucocorticoids. In these cases further evaluation of suspected products [25], medications [16], and patient serum [26] and urine [6] utilizing techniques such as liquid chromatography may be used to confirm occult glucocorticoid exposure. This case highlights the importance of educating patients to exercise caution when purchasing health products both online and abroad. Consumers should be aware of the potential risks of taking supplements, especially those advertised as joint pain relief products. 4. Conclusion Although the most common cause of ectopic ACTH syndrome is pulmonary carcinoid tumors and squamous cell lung cancer, it is a relatively uncommon complication of pulmonary neoplasms. Exogenous Cushing syndrome due to supplements containing unreported corticosteroid doses should be considered in patients with typical Cushingoid features and contradictory hormonal testing. Occult glucocorticoid exposure is rare but can be evaluated with liquid chromatography. This case report emphasizes the importance of teaching patients to be vigilant and appropriately research their health supplements. Patient consent Formal informed consent was obtained from the patient for publication of this case report. Declaration of competing interest The authors (Tomas Morales and Shanika Samarasinghe) of this case report declare that they have no financial conflicts of interest. Shanika Samrasinghe is an editorial member of the Journal of Clinical and Translational Endocrinology: Case Reports, and declares that she was not involved in the peer review and editorial decision making process for the publishing of this article. References [1] A.R. Hayes, A.B. Grossman The ectopic adrenocorticotropic hormone syndrome: rarely easy, always challenging Endocrinol Metab Clin N Am, 47 (2) (2018 Jun), pp. 409-425, 10.1016/j.ecl.2018.01.005 PMID: 29754641 View PDFView articleView in ScopusGoogle Scholar [2] A.M. Isidori, A. 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Pivonello, ABC Study Group Conventional and nuclear medicine imaging in ectopic cushing's syndrome: a systematic review J Clin Endocrinol Metab, 100 (9) (2015 Sep), pp. 3231-3244, 10.1210/JC.2015-1589 PMID: 26158607; PMCID: PMC4570166 View article View in ScopusGoogle Scholar [6] G. Cizza, L.K. Nieman, J.L. Doppman, M.D. Passaro, F.S. Czerwiec, G.P. Chrousos, G.B. Cutler Jr. Factitious cushing syndrome J Clin Endocrinol Metab, 81 (10) (1996 Oct), pp. 3573-3577, 10.1210/jcem.81.10.8855803 PMID: 8855803 View article View in ScopusGoogle Scholar [7] R. Patel, S. Sherf, N.B. Lai, R. Yu Exogenous cushing syndrome caused by a "herbal" supplement AACE Clin Case Rep, 8 (6) (2022 Aug 5), pp. 239-242, 10.1016/j.aace.2022.08.001 PMID: 36447831; PMCID: PMC9701910 View PDFView articleView in ScopusGoogle Scholar [8] C. Dunn, J. Amaya, P. 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Adrenal incidentalomas (AI) are associated with an increased risk of cardiometabolic complications due to adrenal hyperfunction. Obtaining accurate prevalence estimates of distinct types of functioning AIs is crucial for efficient resource allocation and effective management strategies. For a study, researchers sought to ascertain the prevalence of various forms of autonomous hormone secretion in individuals diagnosed with adrenal incidentaloma, including autonomous/possible autonomous cortisol secretion (ACS), primary aldosteronism (PA), pheochromocytoma (PHEO), and Cushing syndrome (CS). A comprehensive and systematic search was conducted across multiple databases (PubMed, Ovid MEDLINE, Web of Science) up to February 2022. Among the 1,661 publications initially screened at the title and abstract levels, 161 articles underwent full-text examination, and ultimately, 36 studies were included for analysis. Three independent reviewers meticulously extracted clinical data from these selected studies. The overarching prevalence of functioning adrenal incidentalomas was 27.5% (95% CI 23.0, 32.5). The highest prevalence was observed for ACS/possible ACS, with a rate of 11.7% (95% CI 8.6, 15.7), followed by PA at 4.4% (95% CI 3.1, 6.2). Subgroup analysis unveiled a greater prevalence of PA in patients from Asian regions than those from Europe/America. Conversely, the prevalence of ACS/possible ACS was comparatively lower in Asian countries. Meta-regression analysis elucidated that the proportion of female patients influenced the prevalence of ACS/possible ACS, while PA prevalence positively correlated with the proportion of patients with hypertension and the publication year. PHEO and CS demonstrated prevalences of 3.8% (95% CI 2.8, 5.0) and 3.1% (95% CI 2.3, 4.3), respectively. The comprehensive meta-analysis offered valuable insights into the prevalence rates of diverse types of functioning adrenal incidentalomas and identified influential factors contributing to heterogeneity in these estimates. The findings contributed significantly to understanding clinical implications and aided in devising effective management strategies for individuals diagnosed with these adrenal disorders. Source: academic.oup.com/jcem/article-abstract/108/7/1813/7015785?redirectedFrom=fulltext

Abstract Background As the population ages, the number of elderly patients with an indication for pituitary surgery is rising. Information on the outcome of patients aged over 75 is limited. This study reports a large series assessing the feasibility of surgical resection in this specific age range, focusing on surgical complications and postoperative results. Methods A retrospective cohort study of patients with pituitary adenomas and Rathke’s cleft cysts was conducted. All patients were aged 75 years or over and treated by a single expert neurosurgical team. A control population included 2379 younger adult patients operated by the same surgeons during the same period. Results Between 2008 and 2022, 155 patients underwent surgery. Indication was based on vision impairment in most patients (79%). Median follow-up was 13 months (range: 3–96). The first surgery was performed with an endoscopic transsellar approach, an extended endonasal transtuberculum approach and a microscopic transcranial approach in 96%, 3%, and 1% of patients, respectively. Single surgery was sufficient to obtain volume control in 97% of patients. From Kaplan-Meier estimates, 2-year and 5-year disease control with a single surgery were 97.3% and 86.2%, respectively. Resection higher than 80% was achieved in 77% of patients. No vision worsening occurred. In acromegaly and Cushing’s disease, endocrine remission was obtained in 90% of non-invasive adenomas. Surgical complications were noted in 5% of patients, with 30-day mortality, hematoma, cerebrospinal fluid leak, meningitis, and epistaxis occurring in 0.6%, 0.6%, 1.9%, 0.6%, and 1.3% respectively. New endocrine anterior deficits occurred in only 5%, while no persistent diabetes insipidus was noted. Compared with younger patients, the complication rate was not statistically different. 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Am J Med 135(1):39–48 Article PubMed Google Scholar Download references Author information Authors and Affiliations Department of Neurosurgery, La Pitié-Salpêtrière University Hospital, Assistance Publique-Hôpitaux de Paris, 47-83 Boulevard de L’Hôpital, 75013, Paris, France Marta Garvayo, Vincent Reina, Stephan Gaillard & Bertrand Baussart Department of Neurosurgery, University Hospital of Lausanne and University of Lausanne, Lausanne, Switzerland Marta Garvayo & Mahmoud Messerer Université Paris Cité, CNRS, INSERM, Institut Cochin, 75014, Paris, France Chiara Villa, Anne Jouinot, Jérôme Bertherat, Guillaume Assié & Bertrand Baussart Department of Neuropathology, La Pitié-Salpêtière University Hospital, AP-HP, Sorbonne University, Paris, France Chiara Villa Department of Endocrinology, Assistance Publique-Hôpitaux de Paris, Hôpital Ambroise Paré, Boulogne Billancourt, France Mirella Hage & Marie-Laure Raffin-Sanson Université de Versailles Saint-Quentin-en-Yvelines UFR Des Sciences de La Santé Simone Veil, Montigny-Le-Bretonneux, France Mirella Hage & Marie-Laure Raffin-Sanson Department of Endocrinology and Reproductive Medicine, Centre de Référence Des Maladies Endocriniennes Rares de La Croissance Et du Développement, CRMERC, Endo-ERN, Pitié-Salpêtrière Hospital, AP-HP, Sorbonne University, Paris, France Carine Courtillot & Anne Bachelot Université Paris-Saclay, Inserm, Physiologie Et Physiopathologie Endocriniennes, Assistance Publique-Hôpitaux de Paris, Hôpital Bicêtre, Service d’Endocrinologie Et Des Maladies de La Reproduction, Centre de Référence des Maladies Rares de L’Hypophyse, Le Kremlin-Bicêtre, France Peter Kamenicky & Philippe Chanson Sorbonne University, Endocrine Unit, Reproductive Medicine, Centre de Référence Des Maladies Endocriniennes Rares de La Croissance Et du Développement (CRMERC), Endo-ERN (Id 739527), Saint-Antoine Hospital, AP-HP, Paris, France Camille Vatier & Sophie Christin-Maitre Inserm UMRS938, Saint-Antoine Research Center, Sorbonne University, 75012, Paris, France Camille Vatier INSERM UMR-833, Trousseau Hospital, Paris, France Sophie Christin-Maitre Department of Endocrinology, Center of Rare Adrenal Diseases, Hôpital Cochin, Assistance Publique-Hôpitaux de Paris, Paris, France Jérôme Bertherat & Guillaume Assié Corresponding author Correspondence to Bertrand Baussart. Ethics declarations Conflict of interest The authors declare no competing interests. Additional information Publisher's Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Rights and permissions Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law. Reprints and Permissions From https://link.springer.com/article/10.1007/s00701-023-05809-x

Abstract Introduction Laparoscopic adrenalectomy is the standard treatment for adrenal tumors caused by Cushing's syndrome. However, few pregnant women have undergone adrenalectomy because of the risk of general anesthesia and surgery. Case presentation A 28-year-old woman presented with gradually worsening Cushing's signs at around 12 weeks of pregnancy. Magnetic resonance imaging displayed a 38-mm left adrenal tumor, which was the cause of the adrenal Cushing's syndrome. Metyrapone was started, which increased androgen levels. Since the management of Cushing's syndrome by medication alone is challenging, unilateral laparoscopic adrenalectomy by a retroperitoneal approach was performed at 23 weeks of the pregnancy. No perioperative complications were noted. Conclusion Adrenalectomy is considered safe in pregnant women with Cushing's syndrome. Laparoscopic adrenalectomy by retroperitoneal approach should be chosen and performed between 14 and 30 weeks of pregnancy to prevent mother and fetal complications. Abbreviations & Acronyms CS Cushing's syndrome MRI magnetic resonance imaging Keynote message We report a rare case of adrenalectomy performed via a retroperitoneal approach for Cushing's syndrome in a pregnant woman. Cushing's syndrome may affect the fetus, and surgery can be considered in addition to medical management. Adrenalectomy should be performed in the second trimester of pregnancy. Pneumoperitoneal pressure, position, and surgical approaches must receive careful attention. Introduction CS is characterized by excessive cortisol secretion and characteristic symptoms such as full moon-like facial features and central obesity. Premenopausal women with CS rarely become pregnant because excessive glucocorticoid secretion inhibits the synthesis of gonadotropins, leading to impaired ovarian and endometrial function, and causing amenorrhea or oligomenorrhea.1 Furthermore, even when women with CS become pregnant, the incidence of severe complications is high. CS can cause maternal hypertension, diabetes/glucose intolerance, osteopenia/osteoporosis, preeclampsia, pulmonary edema, heart failure, opportunistic infections, and even death. Additionally, CS can potentially cause stillbirth, prematurity, and intrauterine fetal growth restriction.1-6 Therefore, CS must be detected at an early stage in pregnancy; however, CS may go undetected because of the overlapping signs of preeclampsia and/or gestational diabetes. A cortisol-secreting adrenal tumor is the underlying cause of CS, and laparoscopic adrenalectomy is the standard treatment to it. Medical treatment of CS can include medications that inhibit 11β-hydroxylase, such as metyrapone and osilodrostat, but surgical treatment is considered if the disease is difficult to control with medical treatment. Nonobstetric surgery during pregnancy is performed in 1%–2% of pregnant women.7 Although general anesthesia is relatively safe during pregnancy, the indication for the surgery must be carefully considered because of potential risks such as neurodevelopmental delay, sudden death, etc. Herein, we present a case of a pregnant woman diagnosed with CS who underwent unilateral laparoscopic adrenalectomy by a retroperitoneal approach without any problems. Case presentation The patient was a 28-year-old primiparous woman. Since around 12 weeks of pregnancy, she has experienced facial and lower limb edema; gained 6-kg weight in 1 month; increased facial acne; and experienced subcutaneous bleeding on the forearms, red abdominal dermatitis, proximal muscle weakness, palpitations, insomnia, and decreased vision in eyes. Her symptoms gradually worsened from 14 weeks, and she was referred to our hospital to clarify the cause at 18 weeks of pregnancy. Adrenal CS was suspected on the basis of her Cushing's signs, cortisol 25 μg/dL, and adrenocorticotropic hormone <1.5 pg/mL. She had hypokalemia, hypogammaglobulinemia, and liver dysfunction, and her condition was rapidly worsening. Given her pregnant state, she was admitted for intensive testing for the case of CS from 19 weeks of pregnancy. MRI revealed a well-defined 38-mm left adrenal tumor, which was the cause of the adrenal CS (Fig. 1). She was started on metyrapone with 250 mg per day, which increased androgens (0.53–0.69 ng/mL in 1 week). We considered that the management of CS by medication alone would be challenging and performed adrenalectomy during her pregnancy. The dose of metyrapone was increased to 1000 mg per day eventually. Fig. 1 Open in figure viewerPowerPoint Magnetic resonance imaging on admission shows a left adrenal tumor with a long axis of 38 mm (arrowhead). Signal reduction was partially observed on opposed-phase images, leading to diagnosis of cortical adenoma. She was admitted to the hospital at 23 weeks and 2 days of gestation, and laparoscopic left adrenalectomy was performed via a retroperitoneal approach in the right lateral and jackknife position on the following day (Fig. S1). During the surgery, blood pressure was carefully controlled by an anesthesiologist and the patient's position and fetal heart rate were monitored by an obstetrician. The operation time, insufflation time, and general anesthesia time were 68, 59, and 123 min, respectively, and the blood loss volume was 75 mL, without any complications. Pathological findings revealed an adrenocortical adenoma. The specimen was positive for one of the nine Weiss criteria (Fig. 2). Fig. 2 Open in figure viewerPowerPoint (a) Intraoperative findings of the retroperitoneal approach. Arrowheads indicate the tumor. (b) Gross appearance of the resected adrenal tumor; a brownish-toned, substantial mass, 60 × 34 × 15 mm in size. (c, d) Hematoxylin–eosin staining showed that nodular lesion with a fibrous capsule, with foci of homogeneous cells with eosinophilic or pale, foamy sporangia and small round nuclei. Postoperatively, metyrapone was discontinued and both lower leg edema, facial acne, fatigue, and muscle weakness improved. Metyrapone was discontinued after surgery. Hydrocortisone, which had been administered at 150 mg/day during the perioperative period, was reduced every few weeks and was taken at 30 mg/day at delivery. She delivered by cesarean section at 38 weeks and 2 days of gestation, with good outcomes for the mother and her infant. Hydrocortisone was discontinued 15 weeks after delivery. We showed the changes in cortisol and ACTH from the first visit to postpartum (Fig. 3). Fig. 3 Open in figure viewerPowerPoint The transition of Cortisol and ACTH. Cortisol decreases rapidly after surgery and rises again before delivery. As cortisol improved, ACTH also increased. Discussion CS seldom occurs during pregnancy. Symptoms such as weight gain, skin striae, fatigue, and a round face can also occur in normal pregnancies. The dexamethasone suppression test can result in false positives because of ACTH produced by placenta in normal pregnancy. During pregnancy, there is a physiological state of high cortisol levels. The disappearance of diurnal rhythm is a useful indicator for diagnosis of CS in pregnancy because circadian rhythm is maintained in normal pregnancy. Useful diagnostic criteria include urine cortisol levels greater than three times the upper limit of normal, loss of diurnal cortisol rhythm, and presence of adrenal tumors on MRI. The pharmacologic treatment of endogenous cortisol is complex, and hormonal management is challenging. While the management of the cortisol levels is important, metyrapone is a risk factor for gestational hypertension and may inhibit fetal cortisol production by crossing the placenta.1-6, 8-12 In this case, because androgens were also elevated and drug management was expected to be challenging, the surgery was aggressively considered. Despite the reports of successful adrenalectomy is after 28 weeks of gestation,6, 13, 14 The surgery should be performed by an experienced team between 14 and 30 weeks of pregnancy, that is, after organogenesis phase and before the fetus grows too large.1, 13, 15 A few pregnant women with adrenal CS undergo adrenalectomy. However, the laparoscopic approach is safe, and maternal and fetal complications were higher in women who did not undergo surgery.16 Less postoperative pain, faster wound healing, and faster postoperative recovery are the main advantages of laparoscopic surgery.17 In pregnant women, pneumoperitoneal pressure should be kept <12 mmHg because increased intraabdominal pressure decreases placental blood flow and can cause fetal acidosis due to the absorption of carbon dioxide used for insufflation. Laparoscopic adrenalectomy can be safely performed through both transperitoneal and retroperitoneal approaches.18 However, in pregnant women, performing the surgery by the retroperitoneal approach in the lateral position is preferable to prevent putting pressure on the fetus during the surgery. The retroperitoneal approach is advantageous, as less pressure is placed on the uterus and adhesions are prevented. After taking the lateral position, the obstetrician is advised to check the position and confirm that the abdomen is not compressed and that the fetal heart rate is normal. Conclusions We present a case of a pregnant woman diagnosed with adrenal CS who underwent a unilateral laparoscopic adrenalectomy by a retroperitoneal approach without any problems. Adrenalectomy is a useful treatment when CS is difficult to control despite metyrapone and other medical support. Author contributions Nobuyoshi Takeuchi: Conceptualization; methodology; project administration; writing – original draft. Yusuke Imamura: Conceptualization; methodology; supervision; writing – review and editing. Kazuki Ishiwata: Data curation; supervision. Manato Kanesaka: Data curation; supervision. Yusuke Goto: Data curation; supervision. Tomokazu Sazuka: Data curation; supervision. Sawako Suzuki: Data curation; supervision. Hisashi Koide: Data curation; supervision. Shinichi Sakamoto: Data curation; supervision. Tomohiko Ichikawa: Data curation; supervision. Conflict of interest The authors declare no conflicts of interest. Approval of the research protocol by an Institutional Reviewer Board Not applicable. Informed consent Informed consent for the release of the case report and accompanying images has been obtained from the patient. Registry and the Registration No. of the study/trial Not applicable. From https://onlinelibrary.wiley.com/doi/10.1002/iju5.12637

Introduction to Endocrinology Endocrinology is a medical specialty that focuses on the diagnosis and treatment of diseases related to hormones. Endocrinologists are experts in managing and treating diseases related to the endocrine system, which includes the thyroid, pituitary, adrenal glands, and pancreas. Endocrinologists are trained to diagnose and treat conditions such as diabetes, thyroid disorders, pituitary disorders, and other conditions related to hormones. Endocrinologists also specialize in reproductive health and fertility issues, including PCOS. Endocrinology is a complex field that requires a deep understanding of the endocrine system and its role in regulating the body’s hormones. Endocrinologists must be able to interpret laboratory tests and understand the underlying causes of endocrine disorders. They must also be able to develop individualized treatment plans to address the specific needs of each patient. Diagnosing PCOS and Diabetes Endocrinologists are experts in diagnosing and managing PCOS and diabetes. PCOS is a hormonal disorder that affects the ovaries, and it is characterized by irregular menstrual cycles, excess facial and body hair, and infertility. To diagnose PCOS, an endocrinologist will perform a physical exam and order laboratory tests to measure hormone levels. The endocrinologist will also ask the patient about her symptoms and family history to determine if PCOS is the cause. Diabetes is a chronic condition that affects the body’s ability to process sugar. To diagnose diabetes, an endocrinologist will perform a physical exam and order laboratory tests to measure blood sugar levels. The endocrinologist may also order imaging tests to check for signs of diabetes-related complications. Treating PCOS and Diabetes Once the endocrinologist has diagnosed PCOS or diabetes, they will develop an individualized treatment plan to address the patient’s specific needs. For PCOS, the endocrinologist may recommend lifestyle changes such as weight loss, exercise, and dietary changes to help manage symptoms. The endocrinologist may also prescribe medications to regulate hormone levels and improve fertility. For diabetes, the endocrinologist may recommend lifestyle changes such as weight loss, exercise, and dietary changes to help manage blood sugar levels. The endocrinologist may also prescribe medications to help regulate blood sugar levels. In addition, the endocrinologist may recommend regular check-ups to monitor the patient’s progress and to adjust the treatment plan if needed. Conclusion Endocrinology plays an important role in managing PCOS and diabetes. Endocrinologists are experts in diagnosing and treating these conditions, and they are trained to develop individualized treatment plans that address the specific needs of each patient. By working with an endocrinologist, patients can get the help they need to manage their PCOS or diabetes and achieve their health goals. Endocrinology is a complex field that requires a deep understanding of the endocrine system and its role in regulating the body’s hormones. An endocrinologist can help patients with PCOS and diabetes manage their conditions and achieve their health goals. By working with an endocrinologist, patients can get the help they need to manage their PCOS or diabetes and achieve their health goals. From https://www.diabetesincontrol.com/the-role-of-endocrinology-in-managing-polycystic-ovary-syndrome-and-diabetes/

Abstract Background 18Fluorine-Fluoro-deoxy-glucose (18F-FDG) positron emission tomography (PET) is widely used for diagnosing various malignant tumors and evaluating metabolic activities. Although the usefulness of 18F-FDG PET has been reported in several endocrine diseases, studies on pituitary disease are extremely limited. To evaluate whether dexamethasone (DEX) suppression can improve 18F-FDG PET for the localization of adrenocorticotropic hormone-secreting adenomas in the pituitary gland in Cushing’s disease (CD). Methods We included 22 patients with CD who underwent PET imaging before and after DEX administration. We compared the success rates of PET before and after DEX suppression, magnetic resonance imaging (MRI), and bilateral inferior petrosal sinus sampling (BIPSS). We determined the final locations of adenomas based on intraoperative multiple-staged resection and tumor tissue identification using frozen sections. Standardized uptake value (SUV) were analyzed to confirm the change of intensity of adenomas on PET. Results Twenty-two patients were included (age at diagnosis: 37 [13–56] years), and most were women (90.91%). Pituitary adenomas compared to normal pituitaries showed increased maximum SUV after DEX suppression but without statistical significance (1.13 versus. 1.21, z=-0.765, P = 0.444). After DEX suppression, the mean and maximum SUV of adenomas showed a positive correlation with nadir cortisol levels in high-dose DEX suppression test (Rho = 0.554, P = 0.007 and Rho = 0.503, P = 0.017, respectively). In reference sites, mean SUV of cerebellum was significantly decreased (7.65 vs. 6.40, P = 0.006*), but those of the thalamus and gray matter was increased after DEX suppression (thalamus, 8.70 vs. 11.20, P = 0.010*; gray matter, 6.25 vs. 7.95, P = 0.010*). Conclusion DEX suppression did not improve 18F-FDG PET/CT localization in patients with CD. Introduction Cushing’s disease (CD) is a rare endocrine disease that results from chronic exposure to high cortisol levels because of adrenocorticotropic hormone (ACTH)-secreting pituitary tumors and is associated with increased morbidity and mortality. It represents approximately 80% of all cases of endogenous hypercortisolism [1,2,3]. Accurate localization of primary lesions in CD leads to improved remission rates and reduced adverse events following surgery [4, 5]. A biochemical remission rate of 90–100% has been reported when tumors are localized before surgery, but it can decrease to 50–60% when surgery is performed when the location of the tumor is unknown in patients with CD [6,7,8]. Currently, magnetic resonance imaging (MRI) is the gold standard for detecting pituitary adenomas. Nevertheless, modern MRI modalities, including dynamic or volumetric sequences, can reliably detect corticotrophic adenomas in 50–90% cases of CD [9,10,11,12]. This indicates that complementary imaging strategies are required to improve the localization of primary lesions in CD. One of the most characteristic features of corticotrophic adenomas is a compromised response to negative glucocorticoid feedback, which defines glucocorticoid resistance [13]. ACTH activates the adrenal glands to synthesize and secrete cortisol, which in turn negatively modulates the release of ACTH from the pituitary gland and corticotrophin-releasing hormone (CRH) and vasopressin from the hypothalamus [1]. In CD, a corticotrophic tumor is only partially sensitive to the inhibitory feedback exerted by cortisol, which in turn is not regulating its own production and secretion of ACTH, resulting in both excessive ACTH and cortisol levels. Glucocorticoid resistance is caused by multiple factors including glucocorticoid receptor availability, splice variant expression and affinity, and imbalanced glucocorticoid receptor signaling [14, 15]. Radioactive 18 F-fluorodeoxyglucose positron emission tomography/computed tomography (18F-FDG PET/CT) often demonstrates increased fluorodeoxyglucose (FDG) uptake in nonfunctioning and hormone-secreting pituitary adenomas [16,17,18]. In large observational studies of whole-body 18F-FDG positron emission tomography (PET) scans, incidental sellar 18F-FDG uptake was found in < 1% of cases, and this sign is highly specific for pituitary adenomas [19,20,21]. 18F-FDG PET imaging can detect up to 40% of corticotropinomas, some as small as 3 mm, and the rate of PET detection of corticotropinomas can be increased by CRH stimulation [9, 22]. Here, we evaluated whether DEX suppression could improve the localization of ACTH-secreting adenomas using 18F-FDG PET/CT in patients with CD. The rationale for this is as follows. FDG uptake of corticotrophic adenomas is less suppressed than that of normal pituitary glands after DEX suppression due to glucocorticosteroid resistance. Materials and methods Study design and population In this retrospective cohort study, we enrolled all patients with CD who underwent two rounds of 18F-FDG-PET/CT before and after 8-mg DEX suppression and pituitary MRI before surgery. Total 22 patients were included in this study, of which thirteen had bilateral inferior petrosal sinus sampling (BIPSS) results. All patients were diagnosed with CD by staff of the Department of Endocrinology and/or Neurosurgery at Severance Hospital between 2014 and 2015. The diagnosis of CD was confirmed based on biochemical test results, including the cortisol, 24-hour urine free cortisol (24 h UFC), and serum ACTH levels, overnight dexamethasone suppression test (ON DST) results, and high-dose dexamethasone suppression test (HD DST) results. Immediate remission was defined as hypocortisolism (serum cortisol level < 1.8 µg/dL) within the first 7 days after surgery. Delayed remission was defined as the achievement of hypocortisolism within 6 months, although immediate remission was not confirmed. If patients showed elevated postoperative cortisol levels and needed additional treatment within 6 months after surgery, we defined them as having persistent disease [23,24,25]. A serum cortisol concentration > 1.8 µg/dL for 8 h in the morning after 1 mg of DEX was given at midnight was considered to be a positive result in the ON DST [26]. Suppression of the serum cortisol level by > 50% for 6 h after 2 mg of DEX was administered for 2 days was defined as the suppression on the HD DST [26]. The final diagnosis was confirmed using surgical pathology and clinical follow-up. Endocrinological evaluation All laboratory analyses were performed at the Department of Laboratory Medicine, Severance Hospital. Preoperative cortisol and 24 h UFC were measured by chemiluminescence immunoassay using an automated UniCel DXC880i Synchron analyzer (Beckman Coulter, Pasadena, CA, USA; coefficient of variation [CV] ± 15 nmol/L at < 100 nmol/L and ± 15% at > 100 nmol/L). Preoperative ACTH levels were analyzed by electrochemiluminescence immunoassay using the Roche Cobas 6000 analyzer (Roche Diagnostics GmbH, Mannheim, Germany; CV ± 2.0 pmol/L at < 20 pmol/L and ± 10% at > 20 pmol/L). The serum cortisol concentration at 8:00 am the following day after 1 mg of DEX was administered at midnight was considered positive on the ON DST. We determined the result as “suppression” by the cortisol level of < 1.8 µg/dL. A serum cortisol level suppressed by > 50% of the original level after 6 hourly administrations of 2 mg of DEX for 48 h was defined as suppression on the HD DST [27]. 18F-FDG PET/CT evaluation PET/CT was performed using a GEADVANCE PET scanner (GE, Milwaukee, WI, USA) after the intravenous injection of 7–9 mCi of 18F-FDG. All patients fasted for at least 6 h before the test. Emission scanning was continued for 15 min (4.25-mm axial spatial resolution, 4.8-mm transaxial spatial resolution). Transmission scans were performed for 8 min using triple Ge-68 rod sources to correct attenuation. Gathered data were reconstructed in a 128 × 128 × 35 matrix with a pixel size of 1.95 × 1.95 × 4.25 mm by means of a filtered back-projection algorithm employing a transaxial 8.5-mm Hanning filter and 8.5-mm axial ramp filter. Two specialists independently interpreted the encoded baseline PET images, and after a two-week period, they interpreted the encoded post DEX suppression PET images. Each specialist was blinded to MRI imaging, clinical characteristics, and surgical outcomes of these subjects. Each was tasked with determining whether the PET image indicated a “negative” or “positive” result for pituitary adenoma and its location on a high-resolution computer screen. The scan after DEX suppression was performed 24 h after the oral administration of 8 mg of DEX using the same procedures as for the baseline PET/CT scan. 18F-FDG uptake analysis The Region of interest (ROI) was drawn using MIM software (version 6.5, Software INc., Cleveland, OH, USA) (Fig. 1). PET images were reviewed by experienced by an experienced specialist. The pituitary gland was identified and a circular ROI was drawn. A fixed ROI with a 3-mm diameter was used for all patients. The ROI was placed on the lesion with the highest FDG uptake. If there was no significantly increased FDG uptake, the same sized circular ROI was drawn on the suspected adenoma location. For the normal pituitary gland, the same sized 3 mm ROI was used. Fig. 1 Images of ROI for pituitary adenoma on18F-FDG PET scan Example of ROI definition in pituitary adenomas of 18F-FDG PET scan of the patients with CD. We draw the fixed circular ROI with a 3-mm diameter for pituitary adenomas (red circle) and normal pituitary gland (green circle) ROI, Reason of interest; 18F-FDG PET, 18 F-fluorodeoxyglucose positron emission tomography; CD, Cushing disease Full size image The mean standardized uptake value (SUVmean) and maximum SUV (SUVmax) for pituitary adenomas and normal pituitary glands were automatically measured using MIM, version 6.5 (Software Inc., Cleveland, OH, USA). The standardized uptake value (SUV) of the volume of interest was calculated as follows: (decoy-corrected activity (kBq) / volume (mL)) / (injected dose (kBq) / body weight (g)). SUVmean and SUVmax of pituitary adenomas were divided into the SUVmean of normal pituitary glands for adjustment. We used the ratio of SUVmax to SUVmean to analyze the homogeneity of the pituitary adenomas. MRI evaluation All patients underwent pituitary MRI with a 3.0-Tesla scanner (Achieva, Philips Medical Systems, Best, the Netherlands). Imaging protocols included T1-weighted imaging, T2-weighted imaging, and delayed gadolinium-enhanced T1-weighted imaging. The extent, location, and sizes of the pituitary tumors were reviewed based on official records determined by radiologists. Pituitary tumors were classified based on radiological findings using MRI of the sellar and parasellar regions. Type I refers to tumors < 1 cm in diameter limited to the sella. Type II tumors extend into the suprasellar space, < 1 cm from the diaphragm. Type III includes tumors extending into the suprasellar space > 1 cm from the diaphragm or sphenoid sinus and encroaching on the internal carotid arteries. Lastly, type IV refers to adenomas with obvious invasion into the cavernous sinus, as shown on MRI, and into the medial dural wall of the cavernous sinus, as confirmed during surgery. BIPSS Before surgery, BIPSS was performed to confirm the cause of CD and lateralize the tumors. A catheter was placed in patients using a unilateral femoral venous approach and 3 cc of blood was collected from the peripheral (P) and both inferior petrosal sinuses (IPS) [28]. CRH at a dose of 1 µg/kg was administered, and peripheral and petrosal samples were drawn after 5 and 10 min, respectively. The catheters and sheath were removed, and the groin was compressed under pressure until venous hemostasis was achieved. The IPS:P prolactin ratio was calculated at each time point to confirm the accuracy of the inferior petrosal venous sampling. A value of ≥1.8 was considered successful IPS catheterization. The prolactin-normalized ACTH ratio was calculated by dividing the dominant ACTH IPS:P ratio by the concurrent and ipsilateral IPS:P prolactin ratio. A value of ≥1.3 was considered diagnostic of CD. An intersinus ACTH ratio of ≥1.4 either at baseline or after stimulation was used for lateralization of the pituitary adenoma [29]. Location of the adenoma The final assignment of the true location of the pituitary adenoma was based on intraoperative multiple stage resection and tumor tissue identification using frozen sections. Surgically identified adenomas were histologically evaluated and stained for ACTH immunoreactivity. In cases of multiple specimens obtained during the procedure, the true location of the adenoma was assigned based on the original site of the specimen containing the adenoma [30]. Statistical analysis Data are presented as medians (ranges) or numbers (percentages). The baseline characteristics of the patients were compared using Kruskal–Wallis’ test with Dunn’s procedure for nonparametric continuous variables. Categorical variables were compared using Fisher’s exact test. Spearman’s correlation coefficients were used to determine the correlation between FDG uptake and hormone levels. Wilcoxon’s signed-rank test was used to identify changes in the SUV after DEX administration. The interobserver agreement for image analysis was assessed using κ statistics. κ values were categorized as follows: κ < 0.20 indicated poor agreement, κ of 0.21–0.40 indicated fair agreement, κ of 0.41–0.60 indicated moderate agreement, κ of 0.61–0.80 indicated good agreement, and κ > 0.81 indicated excellent agreement [31]. Statistical significance was set at a two-sided P < 0.05. All statistical analyses were performed using SPSS software (IBM Corp., Armonk, NY, USA). Results Patient characteristics We enrolled all patients with CD who underwent two rounds of the 18F-FDG PET/CT with or without DEX suppression and sellar MRI before transsphenoidal adenectomy (TSA). Twenty-two patients were included (age at diagnosis: 37 [13–56] years), and most were women (90.91%). Patients’ baseline characteristics are shown in Table 1. There were 16 microadenomas and 6 macroadenomas. Immediate remission was achieved in 81.82% of the patients and delayed remission in 13.64%; one patient showed persistent disease after TSA. The median preoperative 24 h UFC, serum ACTH, and cortisol levels were 443.35 (93.00–4452.00) µg/day, 36.16 (6.00–92.00) pg/mL, and 18.55 (6.00–40.00) µg/dL. The size of pituitary adenomas on MRI was 7.85 (2.00–28.00) mm. The Ki-67 index of 47.06% of adenomas ranged from 1 to 2, that of 35.29% was below 1, and that of 17.65% was 2 or higher. Overall, 75.00% of the adenomas were classified as Knosp grade 0, 5.00% as grade 1, 5.00% as grade 3b, and 15.00% as grade 4. In total, 77.27% (17/22) of patients had an ACTH-staining adenoma. Only one patient showed unsuppressed cortisol levels on the HD DST. Table 1 Patients’ imaging and clinical characteristics Full size table MRI negative but PET positive case Two patients showed negative MRI results, and one of them showed FDG uptakes on both 18F-FDG PET scans at baseline and after DEX suppression. A 26-year-old man visited our hospital complaining of weight gain and was diagnosed with ACTH-dependent CD. Cortisol secretion was suppressed on the HD DST; however, sellar MRI did not reveal any suspicious lesions. BIPSS revealed a central tumor (central/peripheral ACTH level of 36.25 after CRH stimulation) lateralized to the right side of the pituitary gland. The patient underwent 18F-FDG-PET/CT before and after DEX suppression to identify the primary lesions. Baseline PET/CT showed diffused FDG uptake with an SUVmax of 1.03 at the pituitary fossa but failed to localize the tumor. After DEX treatment, focal FDG uptake with an SUVmax of 1.06 remained at the right side of the pituitary fossa, which resulted in the successful localization of the corticotrophic adenoma. The MRI and PET/CT images of this case are presented in Fig. 2A–C. During TSA, the surgeon identified solid tumor-like tissues on the right side of the pituitary gland and successfully removed them. Results of pathology and ACTH immunohistochemistry were negative, but the patient achieved immediate biochemical remission and CD-related symptoms were relieved after surgery. We followed the patient for 98 months after the surgery and confirmed that he had lived without recurrence. Fig. 2 Images of a corticotroph with negative MRI but positive18 F-FDG PET/CT after DEX suppression An MRI-negative adenoma was detected on 18F-FDG PET/CT at baseline and after DEX suppression. In this patient, the pituitary adenoma was visible on PET scans at baseline (B) and after DEX suppression (C) at the same location, as confirmed by the surgeon A. Co-registered baseline 18F-FDG PET/CT and MRI images. Diffuse 18F-FDG uptake is detected in the pituitary fossa with an SUVmean of 0.86 and SUVmax of 1.03, but there was failure to localize the adenoma on baseline 18F-FDG PET/CT. B. Co-registered 18F-FDG PET/CT and MRI images after DEX suppression. 18F-FDG uptake is not suppressed in the right side of the pituitary gland with an SUVmean of 1.03 and SUVmax of 1.06. 18F-FDG PET/CT after DEX suppression was successful in localizing the right-sided corticotrophic adenoma C. MRI image. There is no suspicious lesion in the pituitary gland ACTH, adrenocorticotropic hormone; MRI, magnetic resonance imaging; 18F-FDG, 18 F-fluorodeoxyglucose; PET/CT, positron emission tomography/computed tomography; DEX, dexamethasone; SUVmean, mean standardized uptake value; SUVmax, maximum standardized uptake value Full size image Change of 18F-FDG uptake after DEX suppression We included 18 pituitary adenomas that were successfully localized using PET/CT after DEX suppression, and analyzed the change of SUV for 15 adenomas, excluding outliers with SUV over 2.00. The results are presented in Fig. 3A and B. The SUVmean of adenomas did not changed after DEX suppression compared to normal pituitary glands (SUVmean of adenoma/SUVmean of normal pituitary glands: 1.13 [0.85–1.35] vs. 1.14 [0.87–1.39], z=-1.288, P = 0.198). DEX suppression increased SUVmax of adenomas compared to normal pituitary glands but without statistical significance (SUVmax of adenoma/SUVmean of normal pituitary glands: 1.13 [0.96–1.52] vs. 1.21 [0.97–1.56], z=-0.765, P = 0.444). Fig. 3 Changes in the SUVs of corticotrophs between18F-FDG PET/CT before and after DEX suppression The SUVmean (A) and SUVmax (B) of corticotrophic adenomas are shown in this pairwise analysis. The SUVmean did not changed after DEX suppression from (z=-1.288, P = 0.198). The SUVmax of the corticotrophic adenoma increased from 1.13 to 1.21 (z=-0.765, P = 0.444). In this analysis, the SUVmean and SUVmax of pituitary adenomas were adjusted using the SUVmean of the normal pituitary gland. Colored plots and bars presented median and interquartile range in this figure. We presented the tumors with size larger than 5 mm and SUV adjusted by normal pituitary>1 for blue line SUVmean, mean standardized uptake value; SUVmax, maximum standardized uptake value; DEX, dexamethasone; 18F-FDG, 18 F-fluorodeoxyglucose; PET/CT, positron emission tomography/computed tomography Full size image In Fig. 3, the blue line indicates change in SUV of adenomas larger than 5 mm with higher FDG uptake than the surrounding pituitary parenchyma. For these adenomas, DEX suppression did not change the SUV (SUVmean of adenoma/SUVmean of normal pituitary glands: 1.31 [1.04–2.52] vs. 1.33 [1.05–2.38], z=-0.784, P = 0.433; SUVmax of adenoma/SUVmean of normal pituitary glands: 1.36 [1.02–2.61] vs. 1.40 [1.03–2.65], z=-1.022, P = 0.307). The value of SUV increased in 73.33% adenomas, while the SUVmax increased in 66.67% compared with normal pituitary glands after DEX treatment. Correlation between the hormone level and 18F-FDG uptake Table 2 shows the results of the Spearman correlation analysis of the SUV with preoperative cortisol, ACTH, and nadir cortisol levels on the HD DST. On the baseline 18F-FDG PET scan, the SUVmax of the adenomas did not show any correlation with the levels of three hormones. The SUVmean of adenomas showed a positive correlation with nadir cortisol levels on the HD DST (P = 0.014) and preoperative ACTH levels, with marginal significance (P = 0.062). After DEX suppression, the SUVmax and SUVmean of adenomas had a positive correlation with moderate degrees of nadir cortisol on the HD DST (SUVmax: Spearman Rho = 503, P = 0.017; SUVmean: Spearman Rho = 0.554, P = 0.007). Table 2 Correlation between FDG uptakes and hormone levels Full size table FDG uptake of reference sites after DEX suppression We evaluated the FDG uptake for five types of reference areas (normal pituitary gland, cerebellum, thalamus, white matter, and gray matter) (Table 3). Normal pituitary gland and white matter did not affect the unadjusted SUVmean by DEX suppression (all P >0.05). DEX significantly increased SUVmean of the thalamus and gray matter (thalamus, 8.70 [4.40–22.70] vs. 11.20 [6.40–17.5], P = 0.010*; gray matter, 6.25 [2.50–15.00] vs. 7.95 [5.00–11.90], P = 0.010*). However, SUVmean of the cerebellum significantly decreased after DEX administration (7.65 [4.50–10.80] vs. 6.40 [2.60–12.00], P = 0.006*). Table 3 The change of FDG uptake for reference sites after DEX suppression in the patients with CD Full size table Qualitative analysis by diagnostic modalities for CD The qualitative results of localizing pituitary adenomas in CD patients are shown in Table 4 and Fig. 4. Only 13 patients had BIPSS results. The success rates were 90.91% for MRI and 84.62% for BIPSS. Table 4 Qualitative analysis by diagnostic modalities for CD Full size table Fig. 4 Images for corticotroph adenomas that appear different for localization in18F-FDG PET/CT. 9 mm sized adenoma in the left lateral wing of pituitary gland. It was found in the left lateral wing of the pituitary gland, showing an 18F-FDG uptake in the pituitary fossa with an SUVmean of 1.04 and SUVmax of 1.07. However, after DEX suppression, the left side of the pituitary gland did not exhibit suppressed 18F-FDG uptake, with SUVmean 1.05 SUVmax 1.14 (A). Co-registered baseline 18F-FDG PET/CT and MRI images. (B). Co-registered 18F-FDG PET/CT and MRI images after DEX suppression. (C). MRI image 2 mm pituitary adenoma was detected at the left lateral wing, showing diffuse FDG uptake in the pituitary fossa with an SUVmean of 0.86 and SUVmax of 1.04. After DEX suppression, focal FDG uptake was observed, with SUVmean 0.87 and SUVmax 0.98. (D). Co-registered baseline 18F-FDG PET/CT and MRI images. (E). Co-registered 18F-FDG PET/CT and MRI images after DEX suppression. (F). MRI image Full size image In baseline PET scans, the specialists agreed that pituitary adenomas were visible in 17 scans and not visible in 5 scans. They reached a consensus that the tumor was evident in two scans, but there was a discrepancy in their assessments of its location. After DEX suppression, pituitary adenomas showed positive results in 16 scans and negative results in 5 scans. Specialists disagreed on the presence of pituitary adenomas in one case only. Interobserver agreement for localizing adenomas was 0.872 (95%CI: 0.711, 1.033) for baseline PET/CT and 0.938 (95%CI: 0.762, 1.056) for post dexamethasone suppression PET/CT, confirming excellent interobserver agreements, and the result was judged reliable. Among the instances where both opinions agreed, there were no lesions that showed differences in visibility between scans before and after DEX administration. This meant that lesions were either consistently visible or invisible in both scenarios. Discussion We found that DEX suppression did not improve localization of ACTH-secreting pituitary adenomas using 18F-FDG PET/CT. Further, it did not significantly affect FDG uptakes in adrenocorticotrophic adenomas or normal pituitaries in patients with CD. The decision to administer 8 mg dexamethasone was based on the standard high-dose DST, which is internationally recommended for differentiating between ectopic ACTH secretion and CD [26]. This test involved comparing serum cortisol levels at 8 am before and after a single dose of 8 mg dexamethasone administered at 11 pm. Suppression of the serum cortisol level to less than 50% of the baseline value indicated a diagnosis of CD [32, 33]. Previous studies have reported that the 8-mg DST has a sensitivity of 90%, specificity of 100%, accuracy of 96.8%, positive predictive value of 100%, and negative predictive value of 95.5% [34, 35]. Our use of 8 mg dexamethasone was based on the theory that orally administering dexamethasone at this dose can effectively suppress cortisol levels in ACTH-secreting pituitary tumors. We expected that FDG uptake by corticotrophic adenomas would not decrease after DEX administration in patients with CD, and this change may improve the ability to discriminate the tumor location from surrounding tissues on 18F-FDG PET. The SUVmax of pituitary adenomas adjusted for the normal pituitary gland increased from 1.13 to 1.21. However, this change was not statistically significant, and the success rate of localizing corticotrophic adenomas using 18F-FDG PET was not significantly improved after DEX suppression. If the FDG uptake of adenomas changed lesser compared to that of surrounding normal tissues after DEX suppression, the tumor could be more easily visualized because of the difference. In addition, we attempted to evaluate FDG uptakes in other brain areas (cerebellum, thalamus, white matter, and gray matter) according to DEX administration in CD patients. SUVmean of the cerebellum decreased significantly, but that of the thalamus and gray matter increased after DEX suppression. DEX did not change FDG uptake in pituitary adenoma, normal pituitary, or white matter. In a previous study analyzing FDG PET in CD patients, researchers observed varying correlations between FDG uptake and blood cortisol concentration across different brain regions [35, 36]. Nevertheless, the examination did not include an analysis of FDG uptake in the pituitary gland. Additionally, no previous studies have explored the effects of high-dose dexamethasone suppression on brain glucose metabolism in individuals with CD. Further studies are needed to explain the change in FDG uptake after DEX administration in patients with CD. 18F-FDG PET/CT provides information regarding glucose metabolism in the brain in vivo and has been widely used to evaluate brain metabolism in clinical and research settings [37]. Here, the nadir cortisol level on the HD DST correlated with the SUVmean and SUVmax of pituitary adenomas on PET scans after DEX suppression. Cortisol secretion activity is thought to be associated with metabolic activity, and DEX administration altered this. Cortisol levels and FDG uptake in other regions of the brain are correlated in patients with CD, but the correlation between cortisol and FDG uptake in the pituitary glands and/or corticotrophic adenomas themselves has not been discussed [35, 36]. In our study, cortisol levels did not show a correlation with FDG uptake of corticotrophic adenomas, but after DEX suppression FDG uptake showed a correlation with the nadir cortisol level on the HD DST. This indicated that tumors in which cortisol secretion was less suppressed by on the HD DST showed higher FDG uptake than tumors with lower cortisol levels on the HD DST. Although many studies have analyzed FDG uptake of brain tumors, reference sites defined in each study varied without a uniform standard. Gray matter, white matter, or adjacent tumor tissue was defined as a reference site [38,39,40]. We measured SUVmean of normal pituitary tissues, gray matter, white matter, thalamus, and cerebellum as possible references. We defined the SUVmean of normal pituitary tissues as a reference because the localization of adenomas requires an apparent difference between the adenoma and surrounding tissues. Use of fixed ROI to measure FDG uptake caused partial volume effect in this study. However, lesions smaller than 5 mm with intense FDG uptake may still show increased FDG uptake, especially in tumors, albeit with lower SUV values compared with the actual values [41]. This study was performed because pituitary adenomas smaller than 5 mm with higher FDG uptake than the surrounding pituitary parenchyma have been observed in routine clinical practice. To control for the partial volume effect, the analysis was performed again for tumors which were larger than 5 mm and had higher FDG uptake than the surrounding pituitary parenchyma, and the results remained unchanged. PET/CT has been explored as an alternative to or combined with MRI for the localization of corticotrophic adenomas. 18F-FDG PET/CT has a limited role in CD diagnosis, but CRH stimulation can increase its success rate [22, 42]. This study is important for increasing the effectiveness of PET using DEX. In addition, data on DEX effect on brain metabolism in patients with CD will be important for future studies. Conclusions DEX suppression did not improve the localization of 18F-FDG PET/CT in patients with CD. This is considered to have sufficient significance in an effort to increase the diagnostic value of 18F-FDG PET/CT. Data Availability All datasets generated and/or analyzed during the current study are not publicly available but are available from the corresponding author upon reasonable request. Abbreviations 18F-FDG: 18F-fluorodeoxyglucose PET/CT: Positron emission tomography/computed tomography DEX: Dexamethasone MRI: Magnetic resonance imaging BIPSS: Bilateral inferior petrosal sinus sampling CD: Cushing’s disease SUV: Standardized uptake value ACTH: Adrenocorticotropic hormone CRH: Corticotrophin-releasing hormone FDG: Fluorodeoxyglucose 24hr UFC: 24-hour urine free cortisol ON DST: Overnight dexamethasone suppression test HD DST: High-dose dexamethasone suppression test SUVmean : Mean standardized uptake value SUVmax : Maximum standardized uptake value P: Peripheral IPS: Inferior petrosal sinuses TSA: Transsphenoidal adenectomy References Newell-Price J, Bertagna X, Grossman AB, Nieman LK. Cushing’s syndrome. The Lancet. 2006;367:1605–17. Article CAS Google Scholar Steffensen C, Bak AM, Rubeck KZ, Jørgensen JOL. Epidemiology of Cushing’s syndrome. Neuroendocrinology. 2010;92:1–5. Article CAS PubMed Google Scholar Lacroix A, Feelders RA, Stratakis CA, Nieman LK. 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Author information Authors and Affiliations Endocrinology, Institute of Endocrine Research, Department of Internal Medicine, Yonsei University College of Medicine, Seoul, Republic of Korea Kyungwon Kim, Cheol Ryong Ku & Eun Jig Lee Department of Radiology, Severance Hospital, Yonsei University College of Medicine, Seoul, Republic of Korea Dong Kyu Kim Department of Neurosurgery, Severance Hospital, Yonsei University College of Medicine, Seoul, Republic of Korea Ju Hyung Moon, Eui Hyun Kim & Sun Ho Kim Contributions Conception and design: EJL, CRK, KK. Acquisition of data: KK, DKK. Analysis and interpretation of data: KK. Drafting the article: KK. Administrative/technical/material support: JHM, EHK, SHK. Study supervision: EJL, CRK. Writing, review, and revision of the manuscript: KK, DKK, SHK, CRK. Final approval of the manuscript: CRK, EJL. Corresponding authors Correspondence to Cheol Ryong Ku or Eun Jig Lee. Ethics declarations Ethics approval and consent to participate The data were collected under the conditions of regular clinical care with approval from the ethics committee of our hospital, and the requirement for written informed consent was waived owing to its retrospective design (institutional review board number: 2023-0110-001). Consent for publication Not applicable. Competing interests The authors declare no conflicts of interest that could be perceived as prejudicing the impartiality of this study. Additional information Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Electronic supplementary material Additional file 1 of Dexamethasone suppression for 18F-FDG PET/CT to localize ACTH-secreting pituitary tumors Additional file 1 Supplementary Material 1 Below is the link to the electronic supplementary material. Supplementary Material 1 Supplementary Material 2 Rights and permissions Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Reprints and Permissions Cite this article Kim, K., Kim, D.K., Moon, J.H. et al. Dexamethasone suppression for 18F-FDG PET/CT to localize ACTH-secreting pituitary tumors. Cancer Imaging 23, 85 (2023). https://doi.org/10.1186/s40644-023-00600-8 Download citation Received09 May 2023 Accepted08 August 2023 Published12 September 2023 DOIhttps://doi.org/10.1186/s40644-023-00600-8 Share this article Anyone you share the following link with will be able to read this content: Get shareable link Provided by the Springer Nature SharedIt content-sharing initiative Keywords 18F-FDG PET/CT ACTH-secreting pituitary tumor Cushing’s disease Dexamethasone suppression High-dose dexamethasone suppression test From https://cancerimagingjournal.biomedcentral.com/articles/10.1186/s40644-023-00600-8

Cushing syndrome is a metabolic disease caused by chronic exposure to high levels of glucocorticoids. It can present as an endocrine emergency due to a rapid increase in circulating cortisol leading to increased risk of cardiovascular disease and infection. Etomidate rapidly reduces plasma cortisol levels by inhibiting the action of 11β-hidroxilase. We report the case of a patient with severe hypercortisolaemia accompanied by metabolic and psychiatric disorders in whom administration of etomidate reduced preoperative levels of cortisol. Introduction Cushing’s syndrome is a metabolic disease caused by chronic exposure to high levels of glucocorticoids. The main causes are ectopic ACTH secretion, adrenal tumours (adenomas or carcinomas), adrenal hyperplasia, and administration of exognous glucocorticoids—the latter being the most common aetiology.1 In most cases, Cushing’s syndrome presents an indolent course for years before diagnosis is made, although it can sometime present as an endocrine emergency due to a rapid increase in circulating cortisol levels.2 In these cases, treatment to control hypercortisolaemia must be started quickly due to the high morbidity and mortality associated with the potentially life-threatening metabolic, infectious, and neuropsychiatric alterations that occur in this syndrome.1, 2, 3, 4 The options for treating Cushing’s syndrome include surgery, radiotherapy, and pharmacological treatment. The most commonly used drugs are adrenal steroidogenesis inhibitors (ketoconazole, metyrapone),3 but this treatment is not always well tolerated and its efficacy is limited.2 Etomidate is a drug from the imidazole family that inhibits the enzyme 11β-hydroxylase, and can reduce cortisol secretion within 48−72 h.2 Section snippets Case report Our patient was a 27-year-old woman with no known drug allergies or personal history of interest. She was studied in April 2021 for anxious-depressive symptoms with rapidly evolving paranoid ideation and hirsutism. A Nugent test was performed, which was positive (46.1 mcg/dl), and cortisol in urine was measured (2715 mcg/24 h), leading to a diagnosis of Cushing's syndrome. A CT scan showed a large mass on the right adrenal gland, compatible with a primary adrenal gland tumour (Fig. 1). Discussion Endogenous Cushing's syndrome is characterized by over-production of cortisol. In patients such as ours, the syndrome presents in its most serious form, with very high hypercortisolaemia and metabolic, cardiovascular, and neuropsychiatric disorders. Cushing's syndrome is a medical emergency due to its association with several comorbidities and its high rate of mortality.5 The first therapeutic option is surgical resection of the underlying tumour; however, the accompanying hypercortisolaemia Conclusion In its severe form, Cushing's syndrome is a medical emergency that must be rapidly controlled. Etomidate is both safe and effective, and has shown promising results in the treatment of severe hypercortisolaemia. We believe that these patients should be admitted to the Anaesthesia Intensive Care Unit during etomidate therapy in order to monitor their level of consciousness, lung function, and haemodynamics, and to closely monitor cortisol and electrolyte levels. Ethical considerations Informed consent was obtained for the use of patient information for teaching and research purposes in accordance with our hospital protocol. Conflict of interests None. Funding The authors have not received any funding for this manuscript. References (8) A. Ferriere et al. Cushing’s syndrome: Treatment and new therapeutic approaches Best Pract Res Clin Endocrinol Metab (2020) Juszczak A, Morris D, Grossman A. Cushing's Syndrome [Internet]. South Dartmouth (MA): MDText.com, Inc; 2000 [revised... T.B. Carroll et al. Continuous Etomidate Infusion for the Management of Severe Cushing Syndrome: Validation of a Standard Protocol J Endocr Soc (2018) V.A. Preda et al. Etomidate in the management of hypercortisolaemia in Cushing’s syndrome: a review Eur J Endocrinol (2012) There are more references available in the full text version of this article. 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Abstract Cushing’s syndrome with concurrent primary aldosteronism (PA) is a rare presentation, and establishing an early diagnosis is imperative to preventing morbidity and long-term sequelae. The diagnosis is established by sequential lab work, showing an elevated cortisol and aldosterone level. Taking the above into consideration, it is evident that repeatedly negative results on all three tests can present an extremely challenging case. In this report, we discuss a female who presented with an adrenal incidentaloma and features suggestive of primary hyperaldosteronism as well as Cushing’s syndrome but no elevations in serum, urine, or salivary cortisol. In this study, we present a 37-year-old female with resistant hypertension and tachycardia. She had several features suggestive of Cushing’s syndrome including resistant hypertension, proximal muscle weakness, weight gain, easy bruising, hair loss, and a history of tachycardia and chest pain. Examination revealed an obese female with thin silvery abdominal striae. The patient’s labs revealed normal serum cortisol, urine-free cortisol (UFC), late-night salivary cortisol, and a normal dexamethasone suppression test. An abdominal computed tomography (CT) scan revealed a right adrenal mass measuring 2.1 x 1.5 x 2.5 cm. Due to a high index of suspicion, adrenal venous sampling was performed, which revealed high levels of cortisol and aldosterone in the right vein, confirming the diagnosis. The patient subsequently underwent a right adrenalectomy. She developed hypotension post-op, leading to the diagnosis of glucocorticoid-remediable aldosteronism. Introduction Primary aldosteronism (PA) is the excess production of aldosterone by the adrenal glands, despite a low serum renin level. The presentation of hyperaldosteronism can be vague and include symptoms such as muscle weakness, fatigue, headaches, numbness, and cramps. More specific findings include resistant hypertension, low serum potassium, and metabolic alkalosis. The etiologies are variable and can include an adrenal adenoma (Conn syndrome) or bilateral adrenal hyperplasia [1]. Cushing’s syndrome is also caused by excess hormone secretion by the adrenal glands. The etiologies include a primary adrenal adenoma, hyperplasia, carcinoma, or exogenous corticosteroid use. It can also be caused by an adrenocorticotropic hormone (ACTH)-secreting pituitary adenoma or as a result of paraneoplastic ACTH secretion. The clinical presentation is highly variable and leads to difficulties in establishing a diagnosis. The concurrent existence of primary hyperaldosteronism and Cushing’s syndrome creates additional hindrances in diagnosis, yet further obscured in a patient with a repeatedly negative workup for both conditions. Case Presentation A 37-year-old female presented to her primary care physician with complaints of proximal muscle weakness, tachycardia, and chest pain. Repeated blood pressure readings revealed that she was hypertensive, and she was started on amlodipine and benazepril, which elevated her blood pressure further. A computed tomography (CT) scan (Figure 1) of the abdomen was performed due to resistant hypertension, which revealed an adrenal incidentaloma (right adrenal gland measuring 2.1 x 1.5 x 2.5 cm). Precontract density was 5 Hounsfield units, and a 15-minute delayed washout showed 11 Hounsfield units for a 72% washout. She was thus referred to endocrinology. Figure 1: Abdominal CT scan showing a nodule in the right adrenal gland measuring 2.1 x 1.5 x 2.5 cm She presented to the endocrinology clinic on March 12, 2021. A thorough physical examination was performed, which revealed a well-appearing obese female (BMI of 38.86 kg/m2) with no acute distress. Her blood pressure was 144/108 mmHg, her pulse was 95, and she was afebrile. Thin silvery striations were present on the abdomen, and alopecia was present on the crown. A review of all other systems was unremarkable. A detailed family history revealed early-onset hypertension in her brother (age: 35 years) and her mother (age: 30 years). Personal history included elevated anxiety, weight gain, headaches (frontal band distribution), increased thirst, easy bruising as well as delayed clearance of bruises, and proximal muscle weakness presenting as difficulty in climbing stairs and inability to lift heavy objects. She reported no change in menstrual cycles. There was no history of exogenous corticosteroid use. Serum biochemistries were sent (Table 1), which showed normal levels of thyroid stimulating hormone (TSH), creatinine, liver function tests, and serum electrolytes. However, mildly elevated aldosterone (23 ng/dl), mild hypokalemia (3.3 mEq/L), and suppressed ACTH and dehydroepiandrosterone (DHEA) sulfate were discovered. The aldosterone to renin ratio was also elevated at 59.9 on spironolactone and was 71.4 three months later when spironolactone was discontinued. These findings lead to a preliminary diagnosis of primary hyperaldosteronism. Test Result Calcium 9.1 mmol/L Sodium 137 mmol/L Potassium 4.1 mmol/L Chloride 106 mmol/L CO2 27 BUN 15 mmol/L Glucose 95 mmol/L Creatinine 1.1 μmol/L AST 24 U/L ALT 20 U/L Albumin 4.4 g/L Total protein 7.0 g/L Total bilirubin 0.4 μmol/L Alkaline phosphatase 40 U/L Renin 0.44 Table 1: Patient serum biochemistries BUN: Blood urea nitrogen; AST: Aspartate transaminase; ALT: Alanine transaminase. A workup for elevated cortisol was also performed as the patient was phenotypically Cushingoid, and the following biochemistries were sent sequentially: serum cortisol, 24-hour urine-free cortisol (UFC), salivary cortisol, and a low-dose dexamethasone suppression test (Table 2). The bloodwork was hence nonconfirmatory. Endocrine workup Serum cortisol 4.5 mcg/dL Urine-free cortisol 1.57 g/24 h Salivary cortisol <0.03 μg/dL Dexamethasone suppression test 1.5 mcg/dL Aldosterone <4.0 Table 2: Patient follow-up bloodwork Despite a repeatedly negative workup for Cushing's syndrome, adrenal venous sampling was performed due to a high index of suspicion. The results revealed an inferior vena cava (IVC) cortisol of 20, left adrenal venous (LAV) cortisol of 81, and right adrenal vein (RAV) cortisol of 1280. The results of the IVC aldosterone were 24, LAV aldosterone was 660 and RAV aldosterone was 1500. The elevated levels of cortisol in the RAV were in complete contradiction to the aforementioned workup. A diagnosis of Cushing’s syndrome and concurrent PA was determined. Adrenal veinous sampling was instrumental in establishing the diagnosis but was equivocal and did not lateralize aldosterone and cortisol excess. However, the amount of aldosterone and cortisol were both significantly higher on the right side. After a panel discussion with doctors from several disciplines, a laparoscopic adrenalectomy was planned. The procedure was successful, and the patient was initially showing clinical improvement. The specimen was sent for pathological evaluation and revealed an adrenal cortical adenoma. After initial improvement, the patient developed hypotension, which was likely due to adrenal insufficiency. The patient was supplemented with 1-mg dexamethasone tablets, which stabilized her condition, and a diagnosis of glucocorticoid-remediable-aldosteronism was made. Based on a strong family history of early onset-resistant hypertension, a genetic component was suspected. Several genes associated with PA with autosomal dominant inheritance have been identified [2], such as CYP11B2, CLCN2, KCNJ5, CACNA1D, and CACNA1H. The patient was offered genetic testing but was unable to follow through due to financial reasons. Discussion This patient presented as an extremely rare example of PA and Cushing’s syndrome, with negative serum cortisol, 24-hour UFC, late-night salivary cortisol, and a dexamethasone suppression test. Despite repeatedly negative lab results, the patient presented with a markedly elevated cortisol on adrenal venous sampling. In our literature search, we found an instance of a patient with several negative UFCs [3]; however, to the best of our knowledge, there have been no reported instances of a completely negative workup in a patient who is positive for Cushing’s syndrome. In fact, in the practice guidelines published by the Journal of Clinical Endocrinology & Metabolism [4], it is recommended that patients with a suspected diagnosis of Cushing’s syndrome or an adrenal incidentaloma and two concordant negative test results need not undergo further investigations. One proposed mechanism for the misleading workup could be assay interference. Interference occurs when a substance or process falsely alters an assay result [5]. This can lead to incorrect diagnosis and subsequent treatment and poses a threat to the patient. Another suggested mechanism causing false negative test results could be the hook effect [6]. The hook effect is described as a phenomenon that leads to falsely low results due to the presence of excessive analyte. In a study by Friedman et al. [7], it was noted that patients with “episodic Cushing’s syndrome” or those with mild symptoms had a negative workup. The study recommended serial monitoring for the disease. The interesting fact is that our patient had several features suggestive of active Cushing’s syndrome, and the hypotension seen postoperatively was a testament to the fact that there was in fact a cortisol excess, which led to adrenal insufficiency. In light of the above, a consistently negative workup is perplexing. Zhang et al. suggested performing a low-dose dexamethasone suppression test in individuals presenting with PA, prior to adrenal vein sampling (AVS) and surgery due to the high prevalence of Cushing’s syndrome in patients with PA [8]. A positive test result can lead to a straightforward diagnosis; however, in this rare case where the patient had severe negative tests, it can present as a challenge in diagnosis and treatment. Conclusions The presence of PA and concurrent Cushing’s syndrome can present as a diagnostic challenge. It is recommended to follow up on the signs of Cushing's syndrome with preliminary tests and to presume its absence if two concordant tests are negative. Our patient, however, was an exceptional case. This case highlighted the importance of maintaining a high index of suspicion for patients presenting with several signs and symptoms of the disease and a negative workup. More attention should be paid to the patient's history, and a thorough physical examination should be conducted. In those with an uncertain diagnosis, adrenal venous sampling can provide a clearer picture and lead to a more accurate understanding of the case. References Reincke M, Bancos I, Mulatero P, Scholl UI, Stowasser M, Williams TA: Diagnosis and treatment of primary aldosteronism. Lancet Diabetes Endocrinol. 2021, 9:876-92. 10.1016/S2213-8587(21)00210-2 Dutta RK, Söderkvist P, Gimm O: Genetics of primary hyperaldosteronism. Endocr Relat Cancer. 2016, 23:R437-54. 10.1530/ERC-16-0055 Moloney KJ, Mercado JU, Ludlam WH, Broyles FE: Diagnosis of Cushing's disease in a patient with consistently normal urinary free cortisol levels: a case report. Clin Case Rep. 2016, 4:1181-3. 10.1002/ccr3.647 Nieman LK, Biller BM, Findling JW, Newell-Price J, Savage MO, Stewart PM, Montori VM: The diagnosis of Cushing's syndrome: an Endocrine Society Clinical Practice Guideline. J Clin Endocrinol Metab. 2008, 93:1526-40. 10.1210/jc.2008-0125 Dimeski G: Interference testing. Clin Biochem Rev. 2008, 29:S43-8. The hook effect. (2014). Accessed: June 19, 2023: https://www.aacc.org/science-and-research/clinical-chemistry-trainee-council/trainee-council-in-english/pearls-of-lab.... Friedman TC, Ghods DE, Shahinian HK, et al.: High prevalence of normal tests assessing hypercortisolism in subjects with mild and episodic Cushing's syndrome suggests that the paradigm for diagnosis and exclusion of Cushing's syndrome requires multiple testing. Horm Metab Res. 2010, 42:874-81. 10.1055/s-0030-1263128 Zhang Y, Tan J, Yang Q, et al.: Primary aldosteronism concurrent with subclinical Cushing's syndrome: a case report and review of the literature. J Med Case Rep. 2020, 14:32. 10.1186/s13256-020-2353-8 From https://www.cureus.com/articles/170896-rare-challenges-in-diagnosing-cushings-syndrome-and-primary-aldosteronism-a-case-report-of-a-female-with-a-negative-workup#!/

Abstract Background: Cushing syndrome (CS) is a rare disease caused by excess cortisol levels with high cardiovascular morbidity and mortality. Hypertension in CS promotes hypercortisolism-associated cardiovascular events. Adipose tissue is a highly plastic tissue with most cell types strongly affected by the excess cortisol exposure. We hypothesized that the molecular and cellular changes of periadrenal adipose tissue in response to cortisol excess impact systemic blood pressure levels in patients with CS. Methods: We investigated gene expression signatures in periadrenal adipose tissue from patients with adrenal CS collected during adrenal surgery. Results: During active CS we observed a downregulation of gene programs associated with inflammation in periadrenal adipose tissue. In addition, we observed a clustering of the patients based on tissue gene expression profiles into 2 groups according to blood pressure levels (CS low blood pressure and CS high blood pressure). The 2 clusters showed significant differences in gene expression pattens of the renin-angiotensin-aldosterone-system. Renin was the strongest regulated gene compared with control patients and its expression correlated with increased blood pressure observed in our patients with CS. In the CS high blood pressure group, systemic renin plasma levels were suppressed indicative of an abnormal blood pressure associated with periadrenal adipose tissue renin-angiotensin-aldosterone-system activation. Conclusions: Here, we show for the first time a relevant association of the local renin-angiotensin-aldosterone-system and systemic blood pressure levels in patients with CS. Patients from the CS high blood pressure group still had increased blood pressure levels after 6 months in remission, highlighting the importance of local tissue effects on long-term systemic effects observed in CS. Footnotes *U. Stifel and F. Vogel contributed equally. For Sources of Funding and Disclosures, see page xxx. Supplemental Material is available at https://www.ahajournals.org/doi/suppl/10.1161/HYPERTENSIONAHA.123.21185. Correspondence to: Martin Reincke, Department of Medicine IV, University Hospital, LMU Munich, GermanyEmail martin.reincke@med.uni-muenchen.de Jan Tuckermann, Institute of Comparative Molecular Endocrinology (CME), Ulm University, GermanyEmail jan.tuckermann@uni-ulm.de eLetters eLetters should relate to an article recently published in the journal and are not a forum for providing unpublished data. Comments are reviewed for appropriate use of tone and language. Comments are not peer-reviewed. Acceptable comments are posted to the journal website only. Comments are not published in an issue and are not indexed in PubMed. Comments should be no longer than 500 words and will only be posted online. References are limited to 10. Authors of the article cited in the comment will be invited to reply, as appropriate. Comments and feedback on AHA/ASA Scientific Statements and Guidelines should be directed to the AHA/ASA Manuscript Oversight Committee via its Correspondence page. From https://www.ahajournals.org/doi/10.1161/HYPERTENSIONAHA.123.21185

Objective: To evaluate the long-term efficacy and safety of osilodrostat in patients with Cushing’s disease. Methods: The multicenter, 48-week, Phase III LINC 4 clinical trial had an optional extension period that was initially intended to continue to week 96. Patients could continue in the extension until a managed-access program or alternative treatment became available locally, or until a protocol amendment was approved at their site that specified that patients should come for an end-of-treatment visit within 4 weeks or by week 96, whichever occurred first. Study outcomes assessed in the extension included: mean urinary free cortisol (mUFC) response rates; changes in mUFC, serum cortisol and late-night salivary cortisol (LNSC); changes in cardiovascular and metabolic-related parameters; blood pressure, waist circumference and weight; changes in physical manifestations of Cushing’s disease; changes in patient-reported outcomes for health-related quality of life; changes in tumor volume; and adverse events. Results were analyzed descriptively; no formal statistical testing was performed. Results: Of 60 patients who entered, 53 completed the extension, with 29 patients receiving osilodrostat for more than 96 weeks (median osilodrostat duration: 87.1 weeks). The proportion of patients with normalized mUFC observed in the core period was maintained throughout the extension. At their end-of-trial visit, 72.4% of patients had achieved normal mUFC. Substantial reductions in serum cortisol and LNSC were also observed. Improvements in most cardiovascular and metabolic-related parameters, as well as physical manifestations of Cushing’s disease, observed in the core period were maintained or continued to improve in the extension. Osilodrostat was generally well tolerated; the safety profile was consistent with previous reports. Conclusion: Osilodrostat provided long-term control of cortisol secretion that was associated with sustained improvements in clinical signs and physical manifestations of hypercortisolism. Osilodrostat is an effective long-term treatment for patients with Cushing’s disease. Clinical trial registration: ClinicalTrials.gov, identifier NCT02180217 Introduction Cushing’s disease is a rare but serious disorder resulting from an adrenocorticotropic hormone (ACTH)-producing pituitary adenoma that, in turn, promotes excess adrenal cortisol (1). Chronic exposure to excess cortisol is associated with numerous comorbidities, including hypertension, muscle weakness, hirsutism, central obesity, hypercoagulability and diabetes mellitus, all of which lead to an increased risk of mortality and poor health-related quality of life (HRQoL) (1–3). The longer the exposure to excess cortisol, the lower the chance of reversing morbidity (2). Although transsphenoidal surgery is the recommended first-line treatment, approximately one-third of patients experience persistent or recurrent disease following surgery (4), and some patients are ineligible for or refuse surgery (4–6). Steroidogenesis inhibitors are usually the first choice for medical treatment (6). The effect of medical treatment can be easily monitored by measurement of serum and urine cortisol. Owing to the unremitting nature of Cushing’s disease, patients often require continued medical therapy to maintain long-term control of cortisol excretion. To date, long-term efficacy and safety data for steroidogenesis inhibitors from prospective clinical trials are limited (7, 8). Osilodrostat is a potent oral inhibitor of 11β-hydroxylase and is approved for the treatment of adult patients with Cushing’s disease (USA) or endogenous Cushing’s syndrome (EU and Japan) who are eligible for medical therapy (9–12). The LINC 4 study was a multicenter, 48-week, Phase III clinical trial in patients with Cushing’s disease that included an upfront 12-week randomized, double-blind, placebo-controlled period. Osilodrostat led to rapid normalization of mean urinary free cortisol (mUFC) excretion and was significantly superior to placebo at week 12; normal mUFC excretion was sustained in most patients throughout the 48-week core period (13). Following the 48-week core period, patients could enter an optional open-label extension period intended to run for an additional 48 weeks. Here, we report the long-term efficacy and safety data from the extension of LINC 4. These data augment the existing efficacy and safety profile of osilodrostat (7, 8, 13, 14). Methods Patients Eligibility criteria have been described previously (13). Briefly, the study enrolled adult patients with a confirmed diagnosis of persistent or recurrent Cushing’s disease after pituitary surgery and/or irradiation, or de novo Cushing’s disease (if not surgical candidates), with mUFC >1.3 times the upper limit of normal (ULN; 138 nmol/24 h or 50 μg/24 h; calculated from three samples collected on three consecutive days, with ≥2 values >1.3 x ULN). Patients who continued to receive clinical benefit from osilodrostat, as assessed by the study investigator, could enter the extension phase. The study was conducted in accordance with the Declaration of Helsinki, with an independent ethics committee/institutional review board at each site approving the study protocol; patients provided written informed consent to participate and consented again at week 48 to taking part in the extension phase. The trial is registered at ClinicalTrials.gov (NCT02180217). Study design Data from the 48-week core period of this Phase III study, consisting of a 12-week randomized, placebo-controlled, double-blind period followed by a 36-week open-label treatment period, have been published previously (13). The optional open-label extension phase was initially planned to run for an additional 48 weeks (to week 96 for the last patient enrolled). However, patients could continue in the extension only until a managed-access program or alternative treatment became available locally, or until a protocol amendment was approved at their site that specified that patients enrolled in the optional extension phase should come for an end-of-treatment (EOT) visit within 4 weeks or by week 96, whichever occurred first. Patients still receiving clinical benefit from osilodrostat at their EOT visit were eligible to join a separate long-term safety follow-up study (NCT03606408). Consequently, the extension phase ended when all patients had transitioned to the long-term safety follow-up study, if eligible, or had discontinued from the study. Patients continued to receive open-label osilodrostat at the established effective dose from the core phase (dose adjustments were permitted based on efficacy and tolerability; the maximum dose was 30 mg twice daily [bid]). Outcomes Study outcomes assessed during the extension phase were as follows: complete (mUFC ≤ULN), partial (mUFC decrease ≥50% from baseline and >ULN) and mUFC response rate at weeks 60, 72, 84, 96 and 108, then every 24 weeks until the extension EOT visit; change in mUFC, serum cortisol and late-night salivary cortisol (LNSC) at weeks 60, 72, 84, 96 and 108, then every 24 weeks until the extension EOT visit; time to loss of mUFC control, defined as the time (in weeks) from the first collection of post-baseline normal mUFC (≤ULN) to the first mUFC >1.3 x ULN on two consecutive scheduled visits on the highest tolerated dose of osilodrostat and not related to a dose interruption or reduction for safety reasons after week 26; change in cardiovascular/metabolic-related parameters associated with Cushing’s disease (fasting plasma glucose [FPG] and glycated hemoglobin [HbA1c]) at weeks 60, 72, 84, 96 and 108, then every 24 weeks until the extension EOT visit; blood pressure, waist circumference and weight every 4 weeks until week 72, then every 12 weeks until week 108, then every 24 weeks until the extension EOT visit; change from baseline in physical manifestations of hypercortisolism at weeks 72, 96 and 108, then every 24 weeks until the extension EOT visit; changes in HRQoL (determined by Cushing’s Quality of Life Questionnaire [CushingQoL] and Beck Depression Inventory II [BDI-II]) at weeks 72 and 96 and the extension EOT visit; and proportion of patients with ≥20% decrease or increase in tumor volume. mUFC (mean of two or three 24-hour urine samples), serum cortisol (measured between 08:00 and 10:00) and LNSC (measured from two samples collected between 22:00 and 23:00) were evaluated using liquid chromatography-tandem mass spectrometry and assessed centrally. Pituitary magnetic resonance imaging with and without gadolinium enhancement was performed locally at weeks 72 and 96 and the extension EOT visit; images were assessed centrally for change in tumor size. Safety was continually assessed from core study baseline throughout the extension for all enrolled patients by monitoring for adverse events (AEs); all AEs from first patient first visit to last patient last visit are reported. AEs of special interest (AESIs) included events related to hypocortisolism, accumulation of adrenal hormone precursors, arrhythmogenic potential and QT prolongation, and enlargement of the pituitary tumor. Statistical methods Analyses presented here are based on cumulative data generated for the full analysis set (all patients enrolled at core study start who received at least one dose of osilodrostat) up to last patient last visit. Safety analyses included all enrolled patients who received at least one dose of osilodrostat and had at least one valid post-baseline safety assessment. All analyses excluded data for patients in the placebo arm collected during the placebo-controlled period. Results were analyzed descriptively, and no formal statistical testing was performed. Correlations were evaluated using the Pearson’s correlation coefficient; extreme outliers were defined as >(Q3 + 3 x IQR) or <(Q1 − 3 x IQR), where Q1 and Q3 are the first and third quartiles and IQR is the interquartile range (Q3 − Q1). Results Patient disposition and baseline characteristics LINC 4 was conducted from October 3, 2016 to December 31, 2020. Of the 73 patients who were enrolled and received treatment in the core phase, 65 completed the core phase and 60 (82.2%) opted to enter the extension; 53 (72.6%) patients completed the extension (Figure 1). At core study baseline, most patients had undergone previous pituitary surgery (87.7%) or received prior medical therapy (61.6%; Table 1). Patients had a variety of comorbidities at core study baseline, most commonly hypertension (61.6%); physical manifestations of hypercortisolism were common (Table 1). Figure 1 Figure 1 Patient disposition. *Patient was randomly allocated to osilodrostat but did not receive any study treatment because of a serious AE (grade 4 pituitary apoplexy that required hospitalization prior to receiving any study drug) that was not considered related to treatment. Table 1 Table 1 Core study patient baseline characteristics. Exposure to osilodrostat From core baseline to study end, median (range) osilodrostat exposure was 87.1 (2.0–126.6) weeks; 29 (39.7%) patients were exposed to osilodrostat for more than 96 weeks. The median (25th–75th percentiles) average osilodrostat dose received during the overall study period was 4.6 (3.7–9.2) mg/day; during the core study, median (25th–75th percentiles) average dose was 5.0 (3.8–9.2) mg/day (13). The osilodrostat dose being taken for the longest duration was most frequently 4.0 mg/day (27.4%). Following titration, daily osilodrostat dose remained stable during long-term treatment (Figure 2). Figure 2 Figure 2 (A) Mean and (B) median osilodrostat dose over time. Shaded areas indicate the randomized, double-blind period and the open-label period of the core phase. According to the study protocol, all patients restarted the open-label period on osilodrostat 2 mg bid unless they were on a lower dose at week 12. All patients on <2 mg bid osilodrostat (or matched placebo) at week 12 continued to receive the same dose, regardless of initial treatment allocation. n is the number of patients who contributed to the mean/median. Long-term efficacy of osilodrostat treatment Of patients who had received at least one dose of osilodrostat, 68.5% (n=50/73) had mUFC ≤ULN at the end of the core period, and 54.8% (n=40/73) had mUFC ≤ULN at week 72. Of patients who opted to enter the extension, 66.7% had mUFC ≤ULN (n=40/60) and 8.3% (n=5/60) had mUFC decreased by ≥50% from baseline and >ULN at week 72 (Figure 3A). Of patients with an assessment at their extension EOT visit, 72.4% (n=42/58) had mUFC ≤ULN and 8.6% (n=5/58) had mUFC decreased by ≥50% from baseline and >ULN. Figure 3 Figure 3 (A) Proportion of patients with mUFC response over time, (B) mean mUFC over time, and (C) individual patient changes in mUFC. (A) Patients with missing mUFC at any visit, including those who had discontinued treatment, were counted as non-responders. Shaded area represents the 48-week core phase; excludes data in placebo arm collected during placebo-control period. *The proportion of patients with mUFC ≤ULN at week 48 was calculated using the full analysis set (patients who had discontinued treatment were classified as non-responders). †Discontinued, n=12; missing because of the COVID-19 pandemic, n=4; mUFC not meeting response criteria, n=3; missing (any other reason), n=1. ‡mUFC not meeting response criteria, n=8; missing because of the COVID-19 pandemic, n=2; missing (any other reason), n=1. (B) Shaded areas indicate the randomized, double-blind period and the open-label period of the core phase. n is the number of patients who contributed to the mean. Analysis includes scheduled visits only. (B, C) Dashed line is the ULN for UFC (138 nmol/24 h). Mean mUFC excretion for the 48-week core period of the study has been reported previously (13); mUFC excretion normalized in patients who received osilodrostat, either during the 12-week randomized period (osilodrostat arm) or during the subsequent 36-week open-label period (all patients) (13). Mean mUFC excretion was maintained within the normal range in the extension period (week 72 (n=48), 90.5 [SD 122.6] nmol/24 h; 0.7 [0.9] x ULN; Figure 3B). Median (range) mUFC excretion is shown in Supplementary Figure 1A. Individual patient changes in mUFC from core study baseline to their last observed visit are shown in Figure 3C. There were no escape-from-response events during the extension phase following the primary analysis cut-off (February 25, 2020) (13). During the core period, mean (SD) serum cortisol levels decreased from 538.1 (182.3) nmol/L (0.9 [0.3] x ULN) at baseline to 353.9 (124.9) nmol/L (0.6 [0.2] x ULN) at week 48. Serum cortisol levels then remained stable throughout the extension period (week 72: 319.1 [129.8] nmol/L, 0.6 [0.2] x ULN; Figure 4A). LNSC also decreased and then remained stable, although >ULN, throughout the study (baseline: 10.8 [23.5] nmol/L, 4.3 [9.4] x ULN; week 48: 3.7 [2.6] nmol/L, 1.5 [1.0] x ULN; week 72: 3.8 [3.0] nmol/L, 1.5 [1.2] x ULN; Figure 4B). Median serum cortisol and LNSC are shown in Supplementary Figures 1B, C. Of patients with baseline and last observed value (LOV) measurements, 25.0% had normal LNSC at baseline (n=6/24) and 47.8% had normal LNSC at their last visit (n=11/23). Interpretation of this result is limited by the high degree of missing data (baseline: 67.1%, n=49/73; LOV: 68.5%, n=50/73). Figure 4 Figure 4 (A) Mean serum cortisol and (B) mean LNSC from baseline to the end of treatment. Shaded areas indicate the randomized, double-blind period and the open-label period of the core phase. n is the number of patients who contributed to the mean. Dashed line in (A) indicates reference serum cortisol range for males and females ≥18 years old (127–567 nmol/L). Dashed line in (B) indicates reference LNSC (22:00–23:00) range for males and females ≥18 years old (≤2.5 nmol/L). Changes in cardiovascular and metabolic parameters, physical manifestations of Cushing’s disease and patient-reported outcomes As previously reported, improvements from baseline occurred in most cardiovascular and metabolic-related parameters in the core period following osilodrostat treatment (9). This trend continued during the extension phase and included a reduction in FPG, HbA1c, cholesterol, systolic and diastolic blood pressure, waist circumference, and weight (Figure 5). Similarly, the improvements from baseline in physical features of hypercortisolism observed by week 48 were maintained for most parameters throughout the extension (Figure 6A), with either no change or improvement observed from baseline in ≥90% patients for all parameters at week 72. Facial rubor, supraclavicular fat pad, dorsal fat pad and central obesity had a favorable shift from baseline in ≥40% of patients at week 72. Few patients reported worsening from baseline of specific manifestations (Figure 6A). Figure 5 Figure 5 Changes in cardiovascular-related metabolic parameters. Shaded area indicates the core phase. n is the number of patients who contributed to the mean. Error bars indicate standard deviation. DBP, diastolic blood pressure; HDL, high-density lipoprotein; LDL, low-density lipoprotein; SBP, systolic blood pressure. Figure 6 Figure 6 Changes in (A) physical manifestations of Cushing’s disease and (B) patient-reported outcomes. Shaded area indicates the core phase. n is the number of patients who contributed to the mean. Improvements were also observed in scores for patient quality of life (QoL). Both standardized CushingQoL and BDI-II scores improved steadily during the core phase. QoL scores continued to improve further during the extension. At week 72 and EOT, mean (SD) standardized CushingQoL score was 66.4 (19.6) and 69.0 (20.9), and mean (SD) BDI-II score was 6.5 (7.0) and 6.2 (7.1), representing a mean (SD) change from baseline of 15.2 (19.0) and 17.1 (17.1) and −4.1 (9.3) and −4.5 (7.9), respectively (Figure 6B). Adverse events AEs that occurred in >20% of patients, irrespective of study-drug relationship, during the entire study period (median [range] osilodrostat exposure for all patients: 87.1 [2.0–126.6] weeks; excluding data collected in the placebo arm during the placebo-controlled period) are shown in Table 2. The most common AEs were decreased appetite (46.6%), arthralgia (45.2%) and fatigue (39.7%). Most AEs were mild or moderate; 60.3% were reported as grade 1/2 (Table 2). Table 2 Table 2 Summary of adverse events during LINC 4 core and extension periods. Overall, 10 AEs (adrenal insufficiency, n=3; hyperbilirubinemia, hypokalemia, headache, arthralgia, pituitary tumor, benign pituitary tumor, and depression, n=1 each) in nine patients (12.3%; one patient experienced both arthralgia and headache) led to treatment discontinuation. For two patients (2.7%), those AEs were reported as grade 3 (hyperbilirubinemia and hypokalemia). One patient discontinued following the primary analysis cut-off date (February 25, 2020). The most common AESIs in both the core and extension periods were those related to adrenal hormone precursors. However, the proportion of patients reporting these AESIs was lower in the extension than in the core period (Figure 7). AESIs related to hypocortisolism were most frequent during the core period but did occur throughout the remainder of the study, albeit at lower frequency (Figure 7). Hypocortisolism-related AEs were most frequently managed with temporary osilodrostat interruption (n=20) or dose adjustment (n=6), and with concomitant glucocorticoids (n=15). There were no new occurrences of AESIs related to arrhythmogenic potential and QT prolongation, or to pituitary tumor enlargement, in the extension (Figure 7). During the entire study period from core baseline to the end of the extension, AESIs led to osilodrostat discontinuation in six (8.2%) patients (n=1, related to accumulation of adrenal hormone precursors [hypokalemia]; n=3, related to hypocortisolism [all adrenal insufficiency]; n=2, related to pituitary tumor enlargement [pituitary tumor and pituitary tumor benign]). Figure 7 Figure 7 Occurrence of AESIs by time interval. The denominator for each time period only included patients who had at least one scheduled visit, or at least one observed AE, during that period. From baseline to week 12, the denominator only included patients randomized to osilodrostat. A patient with multiple occurrences of an AE within the same period is counted only once in that period. However, if an AE ends and occurs again in a different period, it is then counted in both periods. Shaded areas indicate the randomized, double-blind period and the open-label period of the core phase. *Maximum duration of follow-up was 127 weeks. Following an increase in 11-deoxycortisol and 11-deoxycorticosterone during the core study, levels tended to decrease during longer-term treatment (Figure 8). From baseline to LOV, the proportion of patients with elevated 11-deoxycorticosterone and 11-deoxycortisol levels increased from 10.0% (n=1/10) to 90.0% (n=9/10) and from 57.9% (n=33/57) to 86.7% (n=5 and 2/60), respectively. In female patients, mean (SD) testosterone levels increased from 1.1 (0.6) nmol/L at baseline to 2.5 (2.6) nmol/L at the end of the core phase, then decreased to within the normal range (0.7−2.6 nmol/L for females) by the extension phase end-of-treatment visit (1.9 [1.7] nmol/L; Figure 8). The proportion of females with an elevated testosterone level increased from 15.0% (n=9/61) at baseline to 63.2% (n=24/61) at week 72 and then reduced to 41.7% (n=25/61) at LOV. In males, testosterone levels increased and remained within the normal range throughout osilodrostat treatment (Figure 8). The proportion of male patients with testosterone levels below the lower limit of normal decreased from 58.3% (n=7/12) at baseline to 33.3% (n=4/12) at LOV. The proportion of patients experiencing AEs potentially related to increased testosterone (increased blood testosterone, acne and hirsutism) was lower during the extension than during the core study (Supplementary Figure 2). Mean serum potassium levels remained stable and within the normal range (3.5–5.3 mmol/L) throughout osilodrostat treatment (Figure 8). The proportion of patients with a normal potassium level was similar between baseline (98.6%, n=72/73) and LOV (94.4%, n=68/72). Figure 8 Figure 8 Mean (± SD) levels up to the end-of-treatment visit in the extension phase for 11-deoxycortisol, 11-deoxycorticosterone, potassium and testosterone (in males and females). Shaded area indicates the core phase. n is the number of patients who contributed to the mean. Reference ranges: 11-deoxycortisol ULN, 3.92 nmol/L in males and 3.1 nmol/L in females, or lower depending on age; 11-deoxycorticosterone ULN, 455 pmol/L in males and 696 pmol/L in females (mid-cycle); potassium, 3.5–5.3 mmol/L; testosterone, 8.4–28.7 nmol/L in males and 0.7–2.6 nmol/L in females. At baseline, median (range) tumor volume was 82.0 (12.0–2861.0) mm3; 28.8% (n=21/73) of patients had a macroadenoma (≥10 mm) and 68.5% (n=51/73) had a microadenoma (<10 mm). At week 72, median (range) tumor volume was 68.0 (10.0–3638.0) mm3 (Figure 9A). Of the 27 patients with measurements at both baseline and week 72, 29.6% (n=8/27) had a ≥20% decrease in tumor volume and 37.0% (n=10/27) had a ≥20% increase (Figure 9B). Notably, mean (SD) plasma ACTH increased steadily between baseline (17.1 [32.1] pmol/L, n=73) and week 72 (65.0 [96.9] pmol/L, n=45; Figure 9C); mean ACTH levels appeared to stabilize after week 72. All patients experienced an increase in ACTH levels from baseline to week 72 (n=45) and LOV (n=73); of these, 34/45 (75.6%) and 47/73 (64.4%) experienced an increase in ACTH of ≥2 × baseline levels to week 72 and to LOV, respectively. There was no correlation between change in tumor volume and change in ACTH from baseline to week 72 (r=0.1; calculated without two extreme outliers). Figure 9 Figure 9 (A) Mean and median tumor volume over time, (B) number of patients with a change in tumor volume from baseline, and (C) mean ACTH over time. Shaded areas indicate the core phase. n is the number of patients who contributed to the mean. Dashed lines in (C) indicate reference morning (07:00–10:00) plasma ACTH ranges for males and females ≥18 years old (1.3–11.1 pmol/L). Discussion Following transsphenoidal surgery, approximately one-third of patients experience persistence or recurrence of disease and subsequently require further treatment to control excess cortisol secretion (4). It is therefore essential that clinical studies evaluating the long-term safety and efficacy of potential new treatments, such as osilodrostat, are performed. The data presented here from the LINC 4 extension reinforce previous reports demonstrating that osilodrostat is effective and well tolerated during long-term treatment of Cushing’s disease (7, 8, 13, 14). The normalization of mUFC excretion, observed from as early as week 2 in some patients (13), was sustained to the end of the optional open-label extension phase. Overall, the response rate was durable and remained ≥60% throughout the study, with 72.4% of patients maintaining mUFC ≤ULN at their extension EOT visit. Considering the range in baseline mUFC values (21.4–2607.3 nmol/24 h), this indicates that patients can benefit from osilodrostat treatment regardless of their baseline mUFC level. This also suggests that baseline mUFC is not an indicator of whether a patient will respond to osilodrostat treatment. Notably, there were no escape events during the extension period. Additionally, the improvements in most cardiovascular and metabolic parameters, physical manifestations and QoL previously reported during the 48-week core phase were maintained or further improved with long-term treatment (13). Collectively, these results demonstrate the ability of osilodrostat to reduce the burden of disease and comorbidities frequently experienced by patients with Cushing’s disease. mUFC excretion is commonly assessed in clinical trials and during routine clinical practice to evaluate response to treatment. It is also important to monitor the recovery of the circadian cortisol rhythm in response to treatment by measuring serum cortisol and LNSC (6, 15–17). Elevated LNSC levels have been linked to dysregulation in glucose tolerance, insulin sensitivity and insulin secretion (18). As such, one potential explanation for persistent comorbidities in some patients with normalized mUFC excretion is that LNSC, although reduced, remains just above the ULN. Assessment of LNSC during treatment with other medical therapies has been reported, although differences in treatment duration and patient population type and size limit meaningful comparisons between therapies (15–17). In LINC 4, mean serum cortisol levels remained within the normal range. Mean LNSC improved considerably from baseline but remained above the ULN throughout the study; 47.8% (n=11/23) of patients achieved normalized LNSC at their LOV visit. A numerically large decrease in LNSC, but with mean levels remaining above the ULN, is consistent with previous reports during long-term osilodrostat treatment (8); the mechanism underlying this observation is currently unknown. In real-life clinical practice, the osilodrostat label allows flexible dosing (9, 11), which may help achieve normalization of LNSC. Furthermore, the number of patients with available LNSC assessments was limited, particularly during the extension; therefore, the data should be interpreted with caution. Future studies should examine whether patients with normalization of both UFC and LNSC have better outcomes than patients with only normalized UFC. Overall, the safety findings reported here for the extension period were consistent with those reported in the primary analysis (13) and previous clinical trials (7, 8, 14). Osilodrostat was generally well tolerated throughout the study; most reported AEs were mild or moderate in severity and manageable. Only nine of 73 (12.3%) patients discontinued osilodrostat at any time because of an AE (3/73 [4.1%] prior to week 48; 6/60 [10.0%] after week 48). Given that osilodrostat is a potent inhibitor of 11β-hydroxylase, AEs related to hypocortisolism or increased levels of adrenal hormone precursors are expected. The frequency of these AEs was lower in the extension period than in the core period, although events did still occur, highlighting the importance of monitoring patients regularly throughout long-term osilodrostat use. AEs potentially related to arrhythmogenic potential and QT prolongation remained infrequent throughout the study. Furthermore, the clinical benefit and tolerability of osilodrostat is supported by the high proportion of patients who chose to continue into the extension period: 92.3% who completed the core phase continued into the optional extension phase, with 88.3% of those completing the extension. Although dose adjustments were allowed in the open-label phase, the dose of osilodrostat remained stable over long-term treatment, with 4 mg/day adequate for most patients to achieve and sustain control of mUFC excretion. Most AEs related to hypocortisolism occurred during the dose-escalation periods of both LINC 4 (27%) and LINC 3 (51%) (19); the lower occurrence in LINC 4 than LINC 3 may have been related to the more gradual dose-escalation schedule of LINC 4 (every 3 weeks) relative to that of LINC 3 (every 2 weeks) (13, 14, 19). As such, an increased dose-titration interval could be considered when there is a need to mitigate the potential for glucocorticoid withdrawal syndrome or hypocortisolism-related AEs following a rapid decrease in cortisol. Dose-increase decisions should be informed by regular cortisol assessments, the rate of decrease in cortisol, and the individual’s clinical response and tolerability to osilodrostat. Furthermore, as with all steroidogenesis inhibitors, patients should be educated on the expected effects of treatment and dose increases, with a particular focus on the symptoms of hypocortisolism and the advice to contact their physician if they occur. As expected, levels of 11-deoxycortisol, 11-deoxycorticosterone and, in women, testosterone increased during osilodrostat treatment. These then decreased during long-term treatment; notably, testosterone levels in women returned to within the normal range and to near baseline levels. These observations are consistent with the findings of LINC 3, which also demonstrated that these increases were reversible following discontinuation of osilodrostat (14). Compared with the primary analysis, there were no new AEs of increased testosterone in the extension phase of LINC 4; these findings are consistent with both LINC 2 and LINC 3 long-term analyses (7, 8). In general, osilodrostat did not adversely affect pituitary tumor volume, with similar proportions of patients reporting either a ≥20% decrease, ≥20% increase or stable tumor volume throughout the study. Although ACTH levels increased during osilodrostat treatment, there was no apparent correlation between the change in ACTH and the change in tumor volume after 72 weeks of treatment; however, longer-term data are needed to evaluate this further. As ACTH-producing pituitary adenomas are the underlying drivers of hypercortisolism, in turn responsible for the high morbidity and poor QoL associated with the disease, tumor stability is of great clinical importance in patients with Cushing’s disease, especially those for whom surgery has failed or is not a viable option. In addition to LINC 4, other studies have assessed the long-term efficacy and safety of other medical therapies (20–24); however, there is a paucity of prospective, long-term data. For metyrapone, an oral steroidogenesis inhibitor that is given three or four times daily (25), prospective data are currently only available for 36 weeks of treatment in the Phase III/IV PROMPT study (22, 23). Normalization of mUFC excretion was observed in 48.6% (n=17/35) of patients at week 36 (23), and gastrointestinal, fatigue and adrenal insufficiency AEs were the most commonly reported during the first 12 weeks of treatment (22). Current data for levoketoconazole, an oral steroidogenesis inhibitor that is a ketoconazole stereoisomer taken twice daily, are available for 12 months (median duration of exposure 15 months, n=60) following the extended open-label extension of the Phase III SONICS study (26). Of patients with data, 40.9% (n=18/44) had normal mUFC excretion at month 12 (26). During the extension, no patient experienced alanine aminotransferase or aspartate aminotransferase >3 x ULN, suggesting that the potentially clinically important events relating to liver toxicity may be more likely to occur early during treatment, although periodic monitoring during long-term treatment is advisable (26). Pasireotide is a second-generation somatostatin receptor ligand that is administered subcutaneously twice daily (27, 28) or intramuscularly once a month (29–31). In a 12-­month extension of a Phase III study evaluating the long-term efficacy of long-acting pasireotide, 53.1% of patients had normalized mUFC at study completion (median treatment duration 23.9 months), with the most common AEs being related to hyperglycemia (21). The differences in duration and design of these studies prevent a meaningful comparison of the long-term efficacy of medical treatments for Cushing’s disease. The extension period of LINC 4 was initially planned to run to week 96; however, in agreement with the FDA, a protocol amendment was approved that resulted in approximately half of the patients completing the extension phase between weeks 72 and 96. We also acknowledge the potential for selection bias for patients who experienced the greatest clinical benefit during the 48-week core study; however, over 80% of patients chose to continue osilodrostat treatment after consenting to take part in the extension. Conclusions During the LINC 4 extension period, osilodrostat provided long-term control of cortisol excretion, accompanied by sustained improvements in clinical symptoms, physical manifestations of hypercortisolism and QoL. The safety profile was favorable. These data provide further evidence of the durable clinical benefit of long-term osilodrostat treatment in patients with persistent, recurrent or de novo Cushing’s disease. Data availability statement The datasets generated and analyzed during the current study are not publicly available but are available from the corresponding author on reasonable request. Recordati Rare Diseases will share the complete de-identified patient dataset, study protocol, statistical analysis plan, and informed consent form upon request, effective immediately following publication, with no end date. Ethics statement The studies involving human participants were reviewed and approved by an independent ethics committee/institutional review board at each study site. The patients/participants provided their written informed consent to participate in this study. Author contributions The study steering committee (PS, AH, RF, and RA), AP, and the funder designed the study. AH, MG, MB, PW, ZB, AT, and PS enrolled patients in the study. Data were collected by investigators of the LINC 4 Study Group using the funder’s data management systems. MP and the funder’s statistical team analyzed the data. A data-sharing and kick-off meeting was held with all authors and an outline prepared by a professional medical writer based on interpretation provided by the authors. Each new draft of the manuscript subsequently prepared by the medical writer was reviewed and revised in line with direction and feedback from all authors. All authors contributed to the article and approved the submitted version. Funding This study was funded by Novartis Pharma AG; however, on July 12, 2019, osilodrostat became an asset of Recordati. Financial support for medical editorial assistance was provided by Recordati. Acknowledgments We thank all the investigators, nurses, study coordinators and patients who participated in the trial. We thank Catherine Risebro, PhD of Mudskipper Business Ltd for medical editorial assistance with this manuscript. Conflict of interest Author MG has received speaker fees from Recordati, Ipsen, Crinetics Pharmaceuticals, and Novo Nordisk and attended advisory boards for Novo Nordisk, Recordati, Ipsen, and Crinetics Pharmaceuticals. Author PS reports consultancy for Teva Pharmaceuticals. Author PW reports receiving travel grants and speaker fees from Novartis, Ipsen, Recordati, Novo Nordisk, Strongbridge Biopharma now Xeris Pharmaceuticals, and Lilly. Author MB reports receiving travel grants from Novartis, Ipsen, and Pfizer and consultancy for Novartis. Author ZB has nothing to disclose. Author AT reports consultancy for CinCor and PhaseBio. Author RF reports consultancy for HRA Pharma and Recordati and a research grant from Corcept Therapeutics. Author AH reports speaker fees from Chiasma and Ipsen and has been an advisor to Strongbridge Biopharma now Xeris Pharmaceuticals, Novo Nordisk, and Lundbeck Pharma. Author MP is employed by the company Novartis Pharma AG. Author AP was employed by the company Recordati AG at the time of manuscript development. Author RA reports grants and personal fees from Xeris Pharmaceuticals, Spruce Biosciences, Neurocrine Biosciences, Corcept Therapeutics, Diurnal Ltd, Sparrow Pharmaceuticals, and Novartis and personal fees from Adrenas Therapeutics, Janssen Pharmaceuticals, Quest Diagnostics, Crinetics Pharmaceuticals, PhaseBio Pharmaceuticals, H Lundbeck A/S, Novo Nordisk, and Recordati Rare Diseases. Publisher’s note All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher. Supplementary material The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fendo.2023.1236465/full#supplementary-material References 1. Lacroix A, Feelders RA, Stratakis CA, Nieman LK. Cushing's syndrome. Lancet (2015) 386:913–27. doi: 10.1016/S0140-6736(14)61375-1 PubMed Abstract | CrossRef Full Text | Google Scholar 2. Feelders RA, Pulgar SJ, Kempel A, Pereira AM. The burden of Cushing's disease: clinical and health-related quality of life aspects. Eur J Endocrinol (2012) 167:311–26. doi: 10.1530/eje-11-1095 PubMed Abstract | CrossRef Full Text | Google Scholar 3. 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Efficacy and safety of once-monthly pasireotide in Cushing's disease: a 12 month clinical trial. Lancet Diabetes Endocrinol (2018) 6:17–26. doi: 10.1016/S2213-8587(17)30326-1 PubMed Abstract | CrossRef Full Text | Google Scholar Keywords: Cushing’s disease, osilodrostat, hypercortisolism, 11β-hydroxylase, long-term treatment Citation: Gadelha M, Snyder PJ, Witek P, Bex M, Belaya Z, Turcu AF, Feelders RA, Heaney AP, Paul M, Pedroncelli AM and Auchus RJ (2023) Long-term efficacy and safety of osilodrostat in patients with Cushing’s disease: results from the LINC 4 study extension. Front. Endocrinol. 14:1236465. doi: 10.3389/fendo.2023.1236465 Received: 07 June 2023; Accepted: 28 July 2023; Published: 23 August 2023. Edited by: Fabienne Langlois, Centre Hospitalier Universitaire de Sherbrooke, Canada Reviewed by: Filippo Ceccato, University of Padua, Italy Kevin Choong Ji Yuen, Barrow Neurological Institute (BNI), United States Copyright © 2023 Gadelha, Snyder, Witek, Bex, Belaya, Turcu, Feelders, Heaney, Paul, Pedroncelli and Auchus. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. *Correspondence: Mônica Gadelha, mgadelha@hucff.ufrj.br †Present address: Alberto M. Pedroncelli, Camurus AB, Lund, Sweden Disclaimer: All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article or claim that may be made by its manufacturer is not guaranteed or endorsed by the publisher. From https://www.frontiersin.org/articles/10.3389/fendo.2023.1236465/full

October 7 @ 9:00 am – 1:00 pm The annual Pituitary Patient Education Day is a free event that features presentations from Johns Hopkins pituitary experts. To RSVP, please email pituitaryday@jhmi.edu. Space is limited. One member per family is encouraged to attend. Presentations Pituitary lesions: acromegaly, Cushing, prolactinomas, non-functioning masses and hypophysitis (Roberto Salvatori, M.D.) How pituitary tumors can affect your vision (Amanda Henderson, M.D.) Medications for pituitary disease: what you should know (Amir Hamrahian, M.D.) The nose as the door to the pituitary gland (Masaru Ishii, M.D., Ph.D.) Surgery for small and large pituitary tumors: images from the Johns Hopkins operating room (Gary Gallia, M.D., Ph.D.) Radiation: when is it needed and how (Lawrence Kleinberg, M.D.) Chevy Chase Auditorium 1800 Orleans Street Baltimore, Maryland 21287+ Google Map More info at https://events.hopkinsmedicine.org/event/johns-hopkins-pituitary-patient-education-day/

Key takeaways: Cushing’s syndrome symptoms moderately impact quality of life for adults with the condition. Weight gain, muscle fatigue and menstrual changes decline in severity from diagnosis to follow-up. Adults with endogenous Cushing’s syndrome reported that the condition moderately affects their quality of life and causes them to have symptoms about 16 days in a given month, according to findings published in Pituitary. “Our study aimed to evaluate the ongoing burden of Cushing’s syndrome in order to identify areas of unmet need,” Eliza B. Geer, MD, medical director of the Multidisciplinary Pituitary and Skull Base Tumor Center and associate attending of endocrinology and neurosurgery at Memorial Sloan Kettering Cancer Center, told Healio. “We found that patients with treated Cushing’s continue to experience ongoing symptoms more than half of the days in a given month, miss about 25 workdays per year and need twice the average number of outpatient visits per year, indicating a significant impact on daily function and work productivity. Some of these symptoms, like fatigue and pain, have not been well studied in Cushing’s patients, and need more attention.” Geer and colleagues administered a cross-sectional survey to 55 adults aged 21 years and older who had been diagnosed with Cushing’s syndrome at least 6 months before the survey and were receiving at least one pharmacologic therapy for their disease (85% women; mean age, 43.4 years). The survey was conducted online from June to August 2021. Five patient-reported outcome scales were included. The CushingQoL was used to analyze quality of life, a visual analog scale was included to assess pain, the Brief Fatigue Inventory was used to measure fatigue, the Sleep Disturbance v1.0 scale assessed perceptions of sleep and the PROMIS Short Form Anxiety v1.0-8a scale was used to measure fear, anxious misery, hyperarousal and somatic symptoms related to arousal. Participants self-reported the impact of Cushing’s syndrome on daily life and their physician’s level of awareness of Cushing’s syndrome. Some symptoms decline in severity over time Of the study group, 81% had pituitary or adrenal tumors, and 20% had ectopic adrenocorticotropic hormone-producing tumors; 80% of participants underwent surgery to treat their Cushing’s syndrome. The frequency of reported symptoms did not change from Cushing’s syndrome diagnosis to the time of the survey. The most frequently reported symptoms were weight gain, muscle fatigue and weakness and anxiety. Participants reported a decline in symptom severity for weight gain, muscle fatigue and weakness and menstrual changes from diagnosis to the survey. Though symptom severity declined, none of the three symptoms were entirely eliminated. Adults did not report declines in severity for other symptoms. Hirsutism and anxiety were reported by few participants, but were consistently scored high in severity among those who reported it. There were no changes in patient satisfaction with medications from their first appointment to the time of the survey. “It was surprising that anxiety and pain did not improve with treatment,” Geer said. “A quarter of patients at baseline reported anxiety and this percentage was exactly the same after treatment. Same for pain — nearly a quarter of patients reported pain despite treatment. While the presence of anxiety has been well-documented in Cushing’s patients, pain has not, and needs further study.” Nearly half of primary care providers unable to diagnose Cushing’s syndrome All participants reported having at least one challenge with being diagnosed with Cushing’s syndrome. Of the respondents, 49% said their primary care provider was unable to diagnose their Cushing’s syndrome and 33% initially received the wrong diagnosis. Physicians referred 49% of participants to a specialist, and 39% of adults said their doctor lacked knowledge or understanding of their condition. The study group had a moderate level of quality of life impairment as assessed through the CushingQoL scale. The mean pain score was 3.6 of a possible 10, indicating low levels of pain. Moderate to severe levels of fatigue were reported by 69% of participants. Self-reported sleep and anxiety scores were similar to what is observed in the general population. Participants said sexual activity, self-confidence and life satisfaction were most impacted by a Cushing’s syndrome diagnosis. Adults experienced symptoms a mean 16 days in a typical month and saw their outpatient physician an average of six times per year. Those who were employed said they miss 2 days of work per month, or about 25 days per year, due to Cushing’s syndrome. “Longitudinal assessment of clinically relevant patient-reported outcomes based on validated measures and coupled with biochemical and treatment data is needed in a large cohort of Cushing’s patients,” Geer said. “This will allow us to identify clinically meaningful changes in symptom burden within each patient, as well as predictors of outcomes — which patients improve on which symptoms, and which patients do not feel better despite biochemical normalization. We need to improve our ability to help our patients feel better, not just achieve normal cortisol levels.” For more information: Eliza B. Geer, MD, can be reached at geere@mskcc.org. From https://www.healio.com/news/endocrinology/20230830/adults-with-cushings-syndrome-report-high-burden-of-illness-despite-ongoing-treatment

Introduction: The differential diagnosis between Cushing’s disease (CD) and ectopic ACTH syndrome (EAS) is complex, and bilateral inferior petrosal sinus sampling (BIPSS) is considered the gold-standard test. However, BIPSS with corticotropin-releasing hormone (CRH) stimulation is rarely available. Objective: This retrospective cohort study aimed to assess the accuracy of the inferior petrosal sinus to peripheral ACTH gradient (IPS:P) before and after desmopressin stimulation for the differential diagnosis of ACTH-dependent Cushing’s syndrome (CS), applying different cutoff values. Methods: A total of 50 patients (48 with CD and 2 with EAS) who underwent BIPSS were included in this study. The sensitivity and specificity of IPS:P in BIPSS before and after desmopressin stimulation were evaluated. Various cutoff values for IPS:P were examined to determine their diagnostic accuracy. Results: Using the traditional IPS:P cutoff, the sensitivity was 85.1% before stimulation, 89.6% after stimulation, and a combined sensitivity of 91.7%. Applying cutoff values of IPS:P >1.4 before and >2.8 after stimulation, the sensitivity was 87.2% and 89.6%, respectively, with a combined sensitivity of 91.7%. Receiver operating characteristic (ROC) curve analysis determined optimal cutoff values of 1.2 before stimulation and 1.57 after stimulation, resulting in a sensitivity of 93.6% and 93.8%, respectively, with a combined sensitivity of 97.9%. Specificity remained at 100% throughout all analyses. Among the 43 patients who responded positively to stimulation, 42 (97.7%) did so within the first three minutes, and all 43 (100%) did so within the first five minutes. None of the assessed clinical variables predicted the ACTH response to stimulation in BIPSS with statistical significance. Discussion: ACTH stimulation with desmopressin during BIPSS improves the accuracy of IPS:P, making it a valuable tool for investigating ACTH-dependent Cushing’s syndrome. Considering the low risk of complications, we recommend the use of desmopressin stimulation during BIPSS for the differential diagnosis of ACTH-dependent CS. Introduction Cushing Syndrome (CS) is a rare disease that results from chronic exposure to elevated cortisol levels. It can be caused by either endogenous or exogenous factors, and its incidence is estimated to be 0.7-3.2 cases per million per year (1, 2). The mortality rate for CS is elevated and may remain higher than the general population even after remission of hypercortisolism (3, 4). The causes of endogenous CS are traditionally classified into two categories: ACTH-dependent (about 80-85% of cases) and ACTH-independent (15-20% of cases) (5). The most common cause of ACTH-dependent CS (75-80% of cases) is Cushing Disease (CD), which is characterized by a corticotropic pituitary adenoma. The remaining cases (15-20%) of ACTH-dependent CS are caused by ectopic ACTH syndrome (EAS), which occurs when tumors of various sites, histological differentiation, and aggressiveness produce ACTH. There are also exceptionally rare cases (<1%) of ectopic CRH-producing tumors (5, 6). CS diagnosis is a complex and challenging pathway due to the variable pattern of hormonal findings, the non-specificity of clinical presentation, particularly in mild hypercortisolism states (7), and the technical limitations of diagnostic tests. Once CS is confirmed, it should be differentiated between ACTH-dependent or -independent cases (8). ACTH levels <10 pg/ml suggest an adrenal cause; ACTH levels >20 pg/ml suggest ACTH-dependent causes; and levels between 10-20 pg/ml are considered indeterminate, requiring additional tests to establish the etiology (5, 8). When ACTH-dependency is confirmed, the next diagnostic step is the differentiation between CD and EAS. In this step, non-invasive tests are initially recommended, such as the CRH test (CRH-t), the 8 mg dexamethasone suppression test (DST-8 mg), and a pituitary magnetic resonance imaging (MRI) (5, 8). These tests, however, presents heterogenous results, depend on the availability of CRH, restricted in many countries including Brazil, and present low discriminatory power (9, 10). An alternative to CRH-t is the use of desmopressin, which stimulates ACTH release in most patients harboring ACTH-secreting pituitary adenomas. The use of this stimulus for the differential diagnosis of CD vs EAS is controversial, since studies have demonstrated that EAS patients may present ACTH elevation following desmopressin administration (11–14). The DST-8 mg is widely available; however it also presents limitation due to the variability of criteria used; furthermore, it has shown insufficient discriminatory capacity in some studies (15, 16). Pituitary MRI fails to detect adenomas in CD patients in about 30-50% of cases even with modern technology equipment (17); moreover, it may also generate false-positive results since pituitary incidentalomas are common in the population, including macroadenomas (18). In cases of conflicting non-invasive test results and unavailability of other methods, bilateral inferior petrosal sinus sampling (BIPSS) should be performed to detect a central-to-peripheral ACTH gradient that allows the localization of the ACTH production (5). Some authors and guidelines recommend performing BIPSS in all patients with pituitary lesions < 6 mm demonstrated on MRI (5, 8, 19), whereas others suggest BIPSS should routinely be performed, especially to guide surgical therapy of CD (20–23). Thus, the procedure is considered the gold-standard in the differential diagnosis of ACTH-dependent CS, preferentially performed with CRH or, less frequently, with desmopressin. The use of CRH is a limiting factor since it is unavailable in many countries. On the other hand, although used in some medical centers, desmopressin as a stimulus for BIPSS is still poorly debated and assessed in the literature, and its utility in this setting remains uncertain since studies validating it in different populations and in larger series are still lacking (8, 24–26). A recent study evaluating desmopressin in a large cohort of patients proposed new diagnostic criteria, questioning the need of stimulus with the new cut-offs (27). Thus, the aim of this study is to assess the role of central-to-peripheral ACTH gradient after stimulus with desmopressin during BIPSS for the differential diagnosis of ACTH-dependent CS in a cohort of patients followed-up in a referral center for CS in Brazil. Patients and methods Patients Between 1998 and 2020, 107 patients with ACTH-dependent CS were retrospectively evaluated at the Neuroendocrinology clinic of a tertiary center in Southern Brazil for BIPSS under desmopressin stimulation during initial diagnostic evaluation or after recurrence. Of these, 58 patients underwent BIPSS with desmopressin, 50 of which for the initial diagnostic evaluation, 7 after recurrence and 1 after emergency adrenalectomy. Eight patients who underwent BIPSS were excluded for insufficient data regarding final etiologic diagnosis (lack of histopatological confirmation, lack of biochemical remission 6 months after surgery, or lack of remission after radiotherapy). Finally, 50 patients were included in the analysis. The present study was conducted in compliance with the principles laid down in the Declaration of Helsinki and was approved by the Hospital de Clínicas de Porto Alegre Ethics Committee. Diagnosis of CS and ACTH-dependency status After exhaustive screening for exogenous glucocorticoid administration, CS diagnosis was based on the presence of at least two of the following conditions: cortisol after low-dose dexamethasone suppression test (either 1 mg overnight or 0.5 mg 6/6 hours for 48h) > 1.8 µg/dL (DST-1mg); 24-h urinary free cortisol (UFC) or late night salivary cortisol consistently elevated in at least two samples (8). Additionally, late night serum cortisol > 7.5 µg/dL (8) and a desmopressin test (DES-t) with a peak ACTH > 71.8 pg/mL or an increase in ACTH ≥ 37 pg/mL from baseline (28) were also considered suggestive of CS. After clinical and biochemical diagnostic confirmation of CS, plasma ACTH measurement classified CS into ACTH-dependent (ACTH > 20 pg/dL) or ACTH-independent (ACTH < 10 pg/dL). Values between 10-20 pg/dL were considered indeterminate and new samples were obtained for correct classification (8). Next, patients diagnosed with ACTH-dependent CS underwent pituitary MRI for the identification of an adenoma. Due to the unavailability of CRH-t, it was rarely performed. The DES-t for the differential diagnosis of CD and EAS was considered predictive of CD when the increase was > 20% in cortisol or >35% in ACTH after stimulus. In virtue of its low accuracy, DST-8 mg was only performed in a few cases. Patients with inconclusive or negative imaging, those with adenomas < 6 mm or those with adenomas > 6 mm but discordant non-invasive tests were submitted to BIPSS with sampling of ACTH at baseline and after desmopressin stimulus. After investigation, patients with a suggestive diagnosis of CD underwent transsphenoidal surgery. Histological confirmation of a pituitary adenoma staining positive for ACTH was considered the gold-standard for diagnosis. Additionally, patients with inconclusive or absent histological specimen who exhibited clinical and biochemical remission 6 months after surgery or who remitted after pituitary radiotherapy were also considered diagnosed for CD. The EAS cases were confirmed based on surgical excision or biopsy of tumoral lesions confirming the presence of ACTH-staining neoplastic cells. Bilateral inferior petrosal sinus sampling The procedure was performed in the presence of documented hypercortisolism, in an angiography room, under sedation with fentanyl and midazolam, and by a qualified professional in interventional radiology. Initially, bilateral common femoral venipuncture was performed, maintained with 6 French (F) introducers. Then, ascending catheterization of the superior vena cava and internal jugular veins was performed with a 5F vertebral catheter and hydrophilic guidewire, with final positioning of the catheter tip at the level of the inferior petrosal sinuses. Angiographic confirmation was performed after injection of 10 ml of diluted nonionic contrast under digital subtraction, demonstrating bilateral sinus and sellar region opacification. In situations of fine-caliber inferior petrosal sinuses, a coaxial microcatheter was used for a better distal reach of the required topography. Heparinization was not usually necessary in this technique, only sequential washing of the catheters was performed between the sampling times with saline solution with 2 ml of heparin for each 1000 ml of solution. Samples were collected after washing the catheters at baseline. Then, 10 µg of desmopressin was administered intravenously and samples were collected after one, three, five, and 15 minutes. In some cases, the sampling times were slightly different, but always with one sampling at baseline and at least 3 samplings after stimulation. All samples were collected in ice-cold tubes, kept on ice and then centrifuged in a refrigerated centrifuge and frozen at -8°C until ACTH measurement, which occurred immediately after the end of the procedure. After the samplings, the catheters and introducers were removed, followed by manual compression of the inguinal region at the puncture site for 10 minutes, until complete hemostasis. After compression, a compressive dressing was placed at the puncture site and the patients remained at bed rest without flexing the thigh for 6 h. Our routine protocol in performing the BIPSS did not include the concomitant measurement of prolactin as suggested in some previous studies in the literature. Hormone assays Until April 2004, cortisol was measured using a commercially available radioimmunoassay (RIA) kit (Diagnostic Systems Laboratories, Webster, TX, USA). From May 2004 to March 2010, the method was modified to an electrochemiluminescence immunoassay (ECLIA) kit (Modular Analytics E 170; Roche, Mannheim, Germany). From March 2010 to February 2014, cortisol was measured by chemiluminescence immunoassay (ADVIA Centaur XP Immunoassay System, Tarrytown, NY, USA). From February 2014 to October 2019, the method was Competitive Electrochemiluminescence. (Roche e602 equipment line). From October 2019 until the end of the study, the method was Microparticle Chemiluminescent Immunoassay. (Abbott equipment line). ACTH measurements up to February 2000 were performed by commercially available RIA. From February 2000 to April 2015, the method was chemiluminescence with the Immulite 1000 equipment. From May 2015 to April 2018, the method was electrochemiluminescence with the Roche e602 equipment. From May 2018 to August 2019, the method was sandwich electrochemiluminescence using the Roche e602 equipment. From August 2019 until the end of the study, the method was chemiluminescent immunoassay in the Immulite 2000 equipment. These assay differences do not show a large variation from normal values and as samples collected from the same patient were always analyzed with the same assay, the calculations of different indexes of central versus peripheral samplings did not change as a result of the trials. Of the cases studied, ACTH was measured by RIA in 1 patient, by Immulite 1000 in 35 patients, by Roche e602 via electrochemiluminescence in 9 patients, by Roche e602 via sandwich electrochemiluminescence in 4 patients and by Immulite 2000 in 1 patient. The basal ACTH and UFC values, therefore, are presented according to the percentage above the ULN according to each methodology used at each moment. For the calculation of the ACTH inferior petrosal sinus to peripheral gradient (IPS:P), however, absolute values were used since the ratios are calculated for the same patient using the same assay. Statistical analysis The Kolmogorov-Smirnov test was used to assess the distribution of variables. Continuous variables with normal distribution are presented as mean ± standard deviation (SD). Continuous variables with asymmetric distribution are shown as median and interquartile range (IQR). Categorical variables were compared using Fischer’s exact test. The comparison of continuous variables was performed using the Mann-Whitney test. ROC curves were used to assess the ability of the IPS:P gradient to discriminate between CD and EAS, and the Youden index was used to define optimal cutoffs. Sensitivity and specificity were calculated for the different criteria analyzed. Statistical analyzes were performed using the SPSS 24.0 program (statistical package software, SPSS Incorporation, Chicago, IL, USA). Differences were considered significant when p<0.05. Results Patient characteristics are shown in Table 1. During the study period, 50 patients with a confirmed diagnosis of ACTH-dependent CS whose etiology could be confirmed through histopathological or biochemical data (remission after 6 months of surgery or after radiotherapy) who had undergone the BIPSS were included. The mean age (SD) at diagnosis was 38.22 (15.56) years, 39 patients (78%) were female, and 48 patients had CD and 2 EAS. Table 1 Table 1 Characteristics of studied patients. In the imaging results, 23 (46%) were microadenomas, among which 15 were < 0.6cm (65.2% of microadenomas), 8 were macroadenomas (16%), and 19 had negative or inconclusive imaging (38%). One of the patients with EAS had an image suggestive of a 0.4 cm microadenoma on MRI. Regarding macroadenomas, the indication for BIPSS was proposed based on the following situations: 3 presented with a clinical picture of EAS, including 2 with systemic lesions suspicious for neoplasia, 3 presented imaging characteristics that were somewhat atypical for adenomas, 1 was associated with a brainstem vascular lesion and one was a recurrent disease with postsurgical alteration and residual lesion. BIPSS was performed in 44 patients who had not yet undergone investigation or treatment and in 6 patients who had been previously treated for CD but had relapsed during follow-up. No complications were recorded in any of the cases submitted to BIPSS. There were no thromboembolism events related to the procedure. At baseline (before stimulation), 49 patients were evaluated (1 patient with CD had samples collected, but his results were not properly recorded). The median IPS:P gradient at baseline was 6.62 (IQR 2.46-11.36) in patients with CD and 1.14 (IQR 1.10-1.14) in patients with EAS (p=0.01). Using the IPS:P>2 gradient criteria, 40 of 47 patients with CD were positive and none of the 2 patients with EAS were positive, resulting in 85.1% sensitivity (95% confidence interval (CI) 71.1-93.3%) and 100% specificity. After stimulation with desmopressin, all 50 patients were evaluated. The median SPI:P gradient after stimulation was 29.46 (IQR 15.39-61.50) in patients with CD and 1.26 (min-max 1.25-1.28) in patients with EAS (p=0.01). In patients with EAS, the highest ACTH peak was 537 pg/mL (109.5% increase from baseline), while in patients with CD, the lowest increase from baseline was 19.48%. Using the IPS:P gradient criteria > 3, 43 of 48 patients with CD were positive, and none of the 2 patients with EAS were positive, resulting in 89.6% sensitivity (95%CI 76.5-96.1%) and 100% specificity. When evaluating patients who were positive at baseline and/or after stimulation in a combined manner, 44 of 48 CD patients were positive, whereas no EAS patients were positive. The overall sensitivity, therefore, was 91.7% (95%CI 79.1-97.3%), and the specificity was 100%. Of the 9 negative patients at baseline, 3 (33.33%) became positive after stimulation. Among the 43 patients who tested positive after the stimulus, 42 (97.7%) had already tested positive up to the third minute, and 100% of the patients were positive up to the fifth minute (Figure 1), totaling 86% of the total sample. Of the 3 patients whose stimulation was necessary, 2 had microadenomas and 1 had macroadenomas. In the two patients with EAS, the time of peak of ACTH was at 1 minute for patient 1 (31.1% increase from baseline) and at 3 minutes for patient 2 (109.5% increase from baseline). Figure 1 Figure 1 Time (minutes) until obtaining IPS:P gradient values of ACTH considered positive response of BIPSS after stimulation with desmopressin. When assessing only the 23 patients with microadenoma, 20 of 22 patients with CD were positive at baseline, and the patient with EAS and 0.4 cm microadenoma was negative, resulting in 90.9% sensitivity (95%CI 69.37-98.4%), while maintaining 100% specificity. After stimulation, all 22 patients with CD were positive and the only patient with EAS and microadenoma was negative, resulting in 100% sensitivity (95%CI 81.5-100%) while maintaining 100% specificity. When only microadenomas < 0.6 cm were evaluated, 12 of 14 CD patients were positive at baseline, and the patient with EAS and 0.4 cm microadenoma was negative, resulting in 85.7% sensitivity (95%CI 56.2-97.5), with 100% specificity. After stimulation, all 14 patients with CD were positive, and the patient with EAS and microadenoma was negative, resulting in a sensitivity of 100% (95%CI 73.2-100%) while maintaining 100% specificity. All eight patients with microadenomas >0.6cm were already positive at baseline and remained positive after stimulation (100% sensitivity and 100% specificity). Thus, only patients with microadenoma <0.6 cm improved sensitivity after stimulation. Among the 8 patients with macroadenoma, sensitivity was 75% at baseline and remained the same after stimulation. However, when assessed for need for stimulation, only one patient with macroadenoma benefited, but sensitivity did not increase because a patient who was positive at baseline became negative after stimulation. Assessing all patients with positive imaging on MRI (micro or macroadenomas, n = 31), 26 of 30 CD patients were positive at baseline, and the patient with EAS and microadenoma was negative, resulting in 86.7% sensitivity and 100% specificity. After stimulation, 28 of 30 CD patients were positive and the patient with EAS and microadenoma remained negative, resulting in 93.3% sensitivity and maintaining 100% specificity. The combined sensitivity (baseline or after stimulus) in this group of patients was 96.7%. Among the 19 patients with negative imaging, 18 had baseline results and were evaluated. Baseline sensitivity was 82.4%. After stimulation, data from 19 patients were evaluated and resulted in a sensitivity of 83.3%. When the patients with negative imaging (n=19) and those with microadenomas <0.6 cm (n=15) were analyzed together, which represent the most difficult cases in clinical practice, we observed that the IPS:P gradient >2 at baseline resulted in sensitivity of 83.9% and 100% specificity. After stimulation, the IPS:P >3 gradient had a sensitivity of 90.6% while maintaining 100% specificity. After assessing the traditionally proposed criteria, the analysis was performed using the criteria proposed by Chen et al. (27). Using the IPS:P gradient at baseline > 1.4, 41 of 47 CD patients were positive and none of the EAS patients were positive, resulting in 87.2% sensitivity (95%CI 73.5-94.7%) while maintaining 100% specificity. After stimulation, using the IPS:P>2.8 gradient criteria, 43 of 48 patients with CD were positive, resulting in 89.6% sensitivity (95%CI 76.5-96.1%), strictly the same as the traditional criteria maintaining 100% specificity. When evaluating patients who were positive at baseline and/or after stimulation, 44 of 48 patients with CD were positive, and no patient with EAS was positive, resulting in 91.7% overall sensitivity (95%CI 79.1-97.3%), the same as the traditional criteria. Finally, only 2 of 49 patients who were negative at baseline became positive after stimulation. To establish institution-specific cut-off points, a ROC curve was performed to assess the accuracy of the central/peripheral ACTH gradient in BIPSS in our cohort of patients. For the IPS:P gradient at baseline, the cut-off point with the highest accuracy was 1.2, whereas for the IPS:P gradient after stimulation, the cut-off point with the highest accuracy was 1.57 (Figure 2). Using these cut-off points, 44 of 47 CD patients were positive at baseline and no EAS patients were positive, resulting in 93.6% sensitivity (95%CI 81.4-98.3%), while maintaining 100% specificity. After stimulation, 45 of 48 CD patients were positive and no EAS patients were positive, resulting in 93.8% sensitivity (95%CI 81.8-98.4%), with 100% specificity (Figure 3). When evaluating patients who were positive at baseline and/or after stimulation, 47 of 48 CD patients were positive and no EAS patients were positive, resulting in an overall sensitivity of 97.9% (95%CI 87.5-99.9%) With 100% specificity. Finally, only 2 patients who were negative at baseline became positive after stimulation. Figure 2 Figure 2 ROC curve of baseline IPS:P values in BIPSS in the investigation of ACTH-dependent CS. Figure 3 Figure 3 ROC curve of IPS:P values after stimulation with desmopressin in BIPSS in the investigation of ACTH-dependent CS. In the comparison between the traditional criterion and our study criterion, the baseline sensitivity changed from 85.1 to 93.6%. After stimulation, baseline sensitivity changed from 89.6 to 93.8%, respectively. A summary of the sensitivity results with the different diagnostic criteria is presented in Table 2. Table 2 Table 2 Sensitivity of BIPSS with traditional criteria and with present study criteria. Technical difficulties or anatomical variations were found in 6 patients undergoing BIPSS. Among the 43 cases with a positive IPS:P gradient, 3 had anatomical variations and 1 had some technical difficulty. Of the 5 cases in which the IPS:P gradient did not occur (false-negatives), 1 presented anatomical variation and 1 presented some technical difficulty during the test. Among the 6 patients who underwent BIPSS after recurrence, all had a final diagnosis of CD, and only 1 was negative on BIPSS. Of the 50 patients evaluated, 43 had undergone DES-t as part of the diagnostic workup, of which 41 were later diagnosed with CD and 2 with EAS. Forty patients were considered responsive in DES-t, 38 patients with CD and 2 patients with EAS. Among the 40 responsive patients, 34 (85%) were also positive in BIPSS, all with a final diagnosis of CD. The 3 non-responsive patients in DES-t presented a positive response in BIPSS after desmopressin. Of the 6 patients who were positive in DES-t but negative in BIPSS, 2 were patients with EAS. Of the 4 patients with CD, 2 had normal petrosal sinus anatomy, 1 had a report of some anatomical variation, and 1 had a report of technical difficulties during BIPSS. Thus, DES-t was not able to predict response to desmopressin during BIPSS (p>0.9999). When comparing the ACTH values at baseline, 3, 5 and 10 minutes after stimulation in BIPSS, there was no significant difference between the group with positive versus negative DES-t, as well as no difference in the time to positivity between the groups, adenoma size, and number of patients with negative imaging. In addition, the clinical variables evaluated (ACTH, UFC, DST-1mg, baseline cortisol, adenoma size) were not able to significantly predict response to stimulus. Discussion In this study, the use of BIPSS with ACTH measurements at baseline and after stimulation with desmopressin in the differential diagnosis of the ACTH-producing source in a sample of 50 patients with ACTH-dependent CS and inconclusive non-invasive tests resulted in 85.1% baseline sensitivity, increasing to 89.6% after stimulation, maintaining 100% specificity when applying traditional IPS:P≥2 criteria at baseline and ≥3 after stimulation (29). When combined, the baseline and/or stimulated sensitivity results were 91.7%. Results of meta-analyses that combined studies performed with CRH stimulation and desmopressin indicate that the sensitivity of BIPSS ranges from 86-97% and the specificity from 89-100% (27, 30). Published studies with desmopressin are generally small, with a variable number of cases of EAS, different indications for BIPSS, and variable diagnostic criteria. In a study with a sample of 56 patients with ACTH-dependent CS and negative imaging, using the criterion of IPS:P≥2 at baseline and IPS:P≥3 after stimulation with desmopressin, the combined sensitivity was 92.1% and 100% specificity, similar to the findings of the present study (25). Smaller studies that also used desmopressin stimulation found similar (26, 31–33) or slightly higher sensitivities (34, 35). Studies performed exclusively in pediatric patients were less uniform, with one of them reporting similar results to studies that included adults (36) and another study demonstrating lower sensitivity in adult population (37). Our institution’s optimal cut-off points, determined by analyzing the ROC curve, were IPS:P≥1.2 at baseline and ≥1.57 after stimulation. This resulted in 93.6% baseline sensitivity (it was 85.1% with IPS:P≥2), and 93.8% after stimulation (was 89.6% with IPS:P≥3), and a combined sensitivity of 97.9% (it was 91.7%), maintaining specificity at 100%. Despite the increased sensitivity, these criteria should be used with caution, since the number of cases with EAS was small. The IPS:P gradient at baseline and after stimulation achieved in patients with EAS in some studies with desmopressin would exceed the cutoffs found by us (24, 25, 27), which would incorrectly classify these patients as CD. Before adopting the new values in our institution, therefore, more patients with EAS are necessary to validate these criteria. Also using the ROC curve, Castinetti et al. evaluated 43 patients with ACTH-dependent SC (36 DC and 7 EAS) and established the criteria of IPS:P>2 at baseline or after stimulation, obtaining a sensitivity of 86% at baseline and 97% after stimulation with desmopressin, not mentioning the combined sensitivity. The study, however, showed 85% specificity at baseline, given that a patient with EAS had a 3.33 gradient (24). In addition to applying the traditional criteria, Machado et al. also used ROC curve analysis to establish cut-off points, finding an IPS:P≥1.45 at baseline (88.2% sensitivity) and ≥ 2.04 after stimulation (92.2% sensitivity) as optimal, both with 100% specificity, although the authors did not recommend the use of these new values (25). The results of these studies using the ROC curve suggest that lower cutoff points, both at baseline and after stimulation, can improve sensitivity without compromising specificity. However, a study that performed a ROC curve in patients stimulated with CRH found an optimal 2.10 baseline cut-off, slightly higher than the traditional one of 2, although the post-stimulation cut-off point was 2.15, lower than the one usually used (38). A study with desmopressin, in turn, found values in the ROC curve of 1.76 at baseline, lower than the traditional one, but ≥3.9 after stimulation, higher than the gradient of three usually used, increasing baseline sensitivity but keeping the sensitivity after stimulation unchanged (32). The largest published study evaluating BIPSS with desmopressin stimulation evaluated 226 patients with CD and 24 with EAS (27). Applying the IPS:P>2 criteria at baseline and >3 after stimulation, the sensitivity was 87.2 and 94.2%, respectively, while maintaining 100% specificity. The combined sensitivity was 96.5%. In this series, 3 cases of EAS reached gradients greater than 2 after stimulation, which suggests that cut-off points equal to or lower than this may decrease specificity. The authors also performed an ROC curve, determining the cutoff point of >1.4 at baseline and >2.8 after stimulation. In this analysis, the sensitivity at baseline was 94.7% and 96% after stimulation, resulting in a combined 97.8% sensitivity, higher than that found with the traditional criteria. According to the authors, with these cut-off points, only 7 patients benefited from the stimulus. After this publication, no other studies have tested these new cutoffs. Our study was the first, therefore, to assess the new values. In our series, using the cutoff point of >1.4 at baseline and >2.8 after stimulation, the sensitivity was 87.2 and 89.3%, respectively, and the combined sensitivity was 91.7%, thus slightly improving the sensitivity at baseline with little change after stimulation. In an attempt to identify predictors of need for stimulation, Chen et al. found that patients requiring stimulation had adenomas < 0.6 cm or negative imaging. In addition, patients who required stimulation had lower IPS ACTH levels and did not lateralize. These data, however, are obtained only after performing the BIPSS, which makes their use in practice unfeasible (27). In our series, among patients with microadenomas, only those with lesions <0.6 cm benefited from the stimulus. Patients with negative imaging had a small increase in sensitivity. A patient with a macroadenoma also benefited from the stimulus, although the sensitivity of the cases with macroadenoma did not change, as a positive patient at baseline became negative after the stimulus. Despite current recommendations suggesting to perform BIPSS in patients with adenomas < 0.6 cm or with negative/inconclusive imaging results (8, 39), Chen et al. identified 2 patients with EAS and adenomas > 0.6 cm who would be misdiagnosed with CD if the 0.6 cm threshold were respected. Therefore, they suggest performing BIPSS in all patients with ACTH-dependent CS (27). Given the relevance of EAS cases in this study, a discussion about the current size criteria for indicating BIPSS should be undertaken. Of our 50 patients, 43 (41 CD and 2 EAS) underwent DES-t prior to BIPSS, and 40 were considered responsive, including the two cases of EAS. Among the responders, 34 patients also responded to the stimulus during the BIPSS, all of them with CD. The 3 patients who did not respond to the peripheral stimulus were, however, positive in the BIPSS. The lack of correlation between the DES-t results and the BIPSS may be related to the different sampling intervals in the two exams (short intervals in the BIPSS and long intervals in the peripheral test). Considering that the majority (86%) of our patients performed both tests, it is possible to conclude that the DES-t did not help in the prediction of response to the central stimulus, which makes the use of peripheral test results debatable for this purpose. Of the BIPSS studies with desmopressin, only one described the results of DES-t, although it did not perform any specific analysis of the relationship with BIPSS (36). The study differs from ours, also, as it only evaluated pediatric patients. Although BIPSS is still considered the gold standard in the differential diagnosis of ACTH-dependent CS, some authors have suggested that the procedure should be indicated only in cases in which t-CRH was negative (40, 41). Recent studies have evaluated non-invasive strategies combining t-CRH, DES-t, TSD-8mg, and imaging to reduce the need for BIPSS. Strategies that resulted in a positive predictive value of 100%, however, included t-CRH as part of the diagnostic process (42, 43), which makes adherence to this diagnostic modality inapplicable in many countries due to the unavailability of CRH. In one of these studies, the combination of TSD-8mg with DES-t, which would be possible in Brazil, was inferior to the combination of DES-t with t-CRH or t-CRH with TSD-8mg (43). The low number of patients undergoing TSD-8mg in our study did not allow the evaluation of this strategy. Although not recommended as a test in the differential diagnosis of the etiology of ACTH-dependent SC, DES-t seems promising as a marker of long-term postoperative outcome and as an early marker of recurrence (44), which encourages further studies in these circumstances. Despite there have been reports of thromboembolic events related to BIPSS that occurred heparin (45, 46), it is a very rare complication. The administration of desmopressin, which increases coagulation factor VIII and von Willebrand factor (47), has raised concerns about the potential for increased incidence of thromboembolic events during BIPSS. This is due to the fact that desmopressin is associated with the hypercoagulable state of CS (48) and may also interfere with VIII and von Willebrand factors. The study by Chen et al, the largest published with desmopressin to date, did not record any case of thromboembolism, even without routine anticoagulation during the procedure (27). In our study, performed without routine anticoagulation, there were also no thromboembolic events. The only desmopressin BIPSS study that recorded thromboembolic events routinely used heparin during the procedure (25). Thromboembolic events, therefore, do not appear to be an additional concern when using desmopressin, with or without the use of heparin during the procedure. The decision regarding the use or not of anticoagulants during BIPSS should be a decision of each institution and based on the usual anticoagulation recommendations. In our study, we did not perform the concomitant dosage of prolactin in samples collected from the inferior petrosal sinuses, a procedure that potentially reduces false negatives, as advised by some authors based on studies with CRH (49–51) and a study with desmopressin (31). These findings, however, were not confirmed by all groups, both with CRH (52) and with desmopressin (32), and their applicability depends on further studies to define its role. In our study, a total of 3 patients who were negative at baseline benefited from the stimulus, As they became positive, 2 of them with microadenomas and one with macroadenoma. The study by Chen et al. questions the use of routine stimulation in all patients to reduce the risks and the duration of the procedure, potentially reducing complications. The authors argue that, when using the IPS:P>1.4 criterion at baseline, the sensitivity was high enough to classify most patients, with the exception of 7 patients with adenoma <0.6 cm who needed stimulation (27). Our study, however, would have misclassified a case with CD and macroadenoma as EAS if the stimulus had not been performed. The assessment of the need for stimulation in cases of CS with macroadenoma is limited since most studies performed the BIPSS only in patients with lesions < 0.6 cm or negative imaging, preventing a more comprehensive assessment. Considering that BIPSS is currently still the gold standard in the differential diagnosis of ACTH-dependent CS, even small gains in sensitivity should be considered important since incorrect classification of patients can lead to inappropriate treatments and potentially fatal delays in the resolution of hypercortisolism. Considering that BIPSS is generally well tolerated and the rate of serious complications is low (53), other strategies to reduce the risks of the procedure that do not involve avoiding the stimulus seem necessary. In this context, it is important to evaluate the time interval between the infusion of the secretagogue and the positive test result. In our study, 97.7% of the patients who tested positive after stimulation were already positive in the third minute and 100% of the patients were positive until the fifth minute, demonstrating that there seems to be no benefit in prolonging the test beyond this period. All of the few studies on BIPSS with desmopressin have directly or indirectly reported a similar time to positivity and for peak ACTH (i.e., positive up to 3-5 minutes) (26, 33, 35, 37). Stimulating patients for a maximum time of 5 minutes considerably reduces the procedure time without neglecting the sensitivity gain resulting from the stimulation and may, therefore, be a strategy to potentially reduce the risk of complications. Our study evaluated a sample of patients whose BIPSS indication was more comprehensive since the unavailability of t-CRH and the low accuracy of TSD-8mg limited the use of non-invasive tests. The wide heterogeneity existing in the BIPSS studies regarding the characteristics of the evaluated patients (primary diagnosis or recurrence), the BIPSS technique (sampling times, anticoagulant use, material used, laboratory assays, cut-off points, type of secretagogue) makes direct comparisons difficult. Conducting multicenter prospective studies with a greater sample of EAS patients is necessary to improve our understanding of the best cut-off points and procedure duration. The present study has some limitations, as expected in the complexity of CS investigation. Our main limitation is that the low prevalence of EAS that underwent BIPSS, resulting from the rarity of this condition, may explain the high specificity when applying the cutoff points indicated by the ROC curve, and the application of these new gradients of IPS:P depends on validation in larger samples of EAS. Lower specificity may result from poor responsiveness to the secretagogue (desmopressin or CRH), cyclic CS during periods of normal cortisol secretion or due to anomalous venous drainage (54). Retrospective data collection and analysis prevented access to complete information for all patients. There were differences over time in terms of sampling times, although at least 3 different samplings were always performed throughout the study period. We highlight that, in this study, we did not discuss the data regarding the eventual lateralization of the basal ACTH values and after stimulation with desmopressin to guide the location of the pituitary adenoma in the transsphenoidal surgery. This utility of the BIPSS has been less and less recommended in the literature due to the imprecision of the results, especially due to the existence of venous communications between the cavernous sinuses and the instability and intensity of blood aspiration for sample collection. In conclusion, in BIPSS with ACTH dosage, the use of stimulation with desmopressin increases the sensitivity of the test from 85.1% to 89.6%, reaching 100% in the sub-analysis of microadenomas. In spite of being small, this increase is useful in the investigation of ACTH-dependent CS, a clinical situation in which gains in diagnostic sensitivity are very important. Additionally, considering the low risk of complications and the possibility to interrupt the test within 5 minutes, as demonstrated in our study, our data recommend the use of stimulation with desmopressin in the BIPSS in the differential diagnosis of ACTH-dependent CS. Data availability statement The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation. Ethics statement The studies involving human participants were reviewed and approved by Hospital de Clínicas de Porto Alegre Ethics Committee. Written informed consent to participate in this study was provided by the participants’ legal guardian/next of kin. Author contributions TA, TR and MC conceived the study and designed the research. TA conducted the data collection and database management. TA performed the data analysis. LS, MF and FG performed the BIPSS procedures. TA, TR, FC and MC contributed to the interpretation of the results. TA and MC drafted the manuscript. FC critically revised the manuscript. All authors read and approved the final version of the manuscript. All authors contributed to the article and approved the submitted version. Funding This work was supported by the Research Incentive Fund (FIPE) of Hospital de Clínicas de Porto Alegre and the Postgraduate Program in Medical Sciences: Endocrinology (PPG ENDO) from Universidade Federal do Rio Grande do Sul. Acknowledgments The authors would like to acknowledge the contributions of Guilherme Alcides Flores Soares Rollin, Arthur Boschi, and Camila Viecceli to the data collection process. Conflict of interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Publisher’s note All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher. Abbreviations BIPSS, bilateral inferior petrosal sinus sampling; CD, Cushing Disease; CRH-t, CRH test; CS, Cushing Syndrome; DES-t, desmopressin test; DST-1 mg, 1 mg dexamethasone suppression test; DST-8 mg, 8 mg dexamethasone suppression test; EAS, Ectopic ACTH Syndrome; ECLIA, electrochemiluminescence immunoassay; F, French; IPS:P, inferior petrosal sinus to peripheral gradient; IQR, interquartile range; MRI, magnetic resonance imaging; RIA, radioimmunoassay; SD, standard deviation; UFC, urinary free cortisol; ULN, upper limit of normal. References 1. Newell-Price J, Bertagna X, Grossman AB, Nieman LK. Cushing’s syndrome. 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Second-line tests in the diagnosis of adrenocorticotropic hormone-dependent hypercortisolism. Ann Lab Med (2021) 41(6):521–31. doi: 10.3343/alm.2021.41.6.521 PubMed Abstract | CrossRef Full Text | Google Scholar Keywords: Cushing’s syndrome, Cushing’s disease, ectopic ACTH syndrome, bilateral inferior petrosal sinus sampling, ACTH, desmopressin Citation: Almeida TSd, Rodrigues TdC, Costenaro F, Scaffaro LA, Farenzena M, Gastaldo F and Czepielewski MA (2023) Enhancing Cushing’s disease diagnosis: exploring the impact of desmopressin on ACTH gradient during BIPSS. Front. Endocrinol. 14:1224001. doi: 10.3389/fendo.2023.1224001 Received: 17 May 2023; Accepted: 11 July 2023; Published: 03 August 2023. Edited by: Fabienne Langlois, Centre Hospitalier Universitaire de Sherbrooke, Canada Reviewed by: Filippo Ceccato, University of Padua, Italy Matthieu St-Jean, Université de Sherbrooke, Canada Copyright © 2023 Almeida, Rodrigues, Costenaro, Scaffaro, Farenzena, Gastaldo and Czepielewski. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. *Correspondence: Tobias Skrebsky de Almeida, tsalmeid@gmail.com Disclaimer: All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article or claim that may be made by its manufacturer is not guaranteed or endorsed by the publisher. From https://www.frontiersin.org/articles/10.3389/fendo.2023.1224001/full

Background: Café-au-lait skin macules, Cushing syndrome (CS), hyperthyroidism, and liver and cardiac dysfunction are presenting features of neonatal McCune–Albright syndrome (MAS), CS being the rarest endocrine feature. Although spontaneous resolution of hypercortisolism has been reported, outcome is usually unfavorable. While a unified approach to diagnosis, treatment, and follow-up is lacking, herein successful treatment and long-term follow-up of a rare case is presented. Clinical case: An 11-day-old girl born small for gestational age presented with deterioration of well-being and weight loss. Large hyperpigmented macules on the trunk, hypertension, hyponatremia, hyperglycemia, and elevated liver enzymes were noted. ACTH-independent CS due to MAS was diagnosed. Although metyrapone (300 mg/m2/day) was started on the 25th day, complete remission could not be achieved despite increasing the dose up to 1,850 mg/m2/day. At 9 months, right total and left three-quarters adrenalectomy was performed. Cortisol decreased substantially, ACTH remained suppressed, rapid tapering of hydrocortisone to physiological dose was not tolerated, and supraphysiological doses were required for 2 months. GNAS analysis from the adrenal tissue showed a pathogenic heterozygous mutation. During 34 months of follow-up, in addition to CS due to MAS, fibrous dysplasia, hypophosphatemic rickets, and peripheral precocious puberty were detected. She is still regularly screened for other endocrinopathies. Conclusion: Neonatal CS due to MAS is extremely rare. Although there is no specific guideline for diagnosis, treatment, or follow-up, addressing side effects and identifying treatment outcomes will improve quality of life and survival. Introduction McCune–Albright syndrome (MAS) is a rare mosaic disorder of remarkable complexity with an estimated prevalence of 1/100,000 and 1/1,000,000 (1). Timing of postzygotic missense gain of function mutation of GNAS encoding stimulatory Gαs determines the extent of tissue involvement, imposing a unique clinical phenotype. Although a combination of two or more classical features, such as fibrous dysplasia of bone (FD), café-au-lait skin macules, and hyperfunctioning endocrinopathies (gonadotropin-independent gonadal function, nonautoimmune hyperthyroidism, growth hormone excess, and neonatal hypercortisolism), are diagnostic, renal, hepatobiliary, and cardiac involvement have also been reported (2–4). Adrenocorticotropic hormone (ACTH)-independent adrenal Gαs activation results in the rarest endocrine feature of MAS, which almost invariably presents in the neonatal period: Cushing syndrome (CS). Due to greater burden of Gαs-mutation-bearing cells, the presence of CS is correlated with increased number of accompanying features of MAS and a poorer outcome. Although there is spontaneous resolution in 33% of cases with neonatal CS, mortality occurs with a high rate of 20% (4). A dilemma for the clinician is that most publications to date have been case reports, and there is as yet no guideline for diagnosis, treatment, or follow-up. Here, a rare case of severe CS due to MAS, underlining the unique clinical phenotype specific to the neonatal period, is presented. Our goal is to offer a practical approach based on 3 years of clinical experience of this rare disorder that will help navigate challenges during follow-up. Case presentation A baby girl, born small for gestational age with a birthweight of 2,340 g (−2.1 SDS) and a head circumference of 32.6 cm (−1.61 SDS) was admitted to the neonatal intensive care unit in the first day of life for respiratory distress. She was the second child of a healthy non-consanguineous Caucasian couple, born 38 weeks of gestation via cesarean section following an uneventful pregnancy. Alanine aminotransferase [ALT, 2,376 U/L (normal, 0–40)] and aspartate aminotransferase [AST, 875 U/L (normal, 0–40)] were elevated; gamma-glutamyl transferase and bilirubin were normal. Antibiotics were administered intravenously after a diagnosis of possible neonatal sepsis. Respiratory distress resolved, and liver enzymes decreased (ALT, 687 U/L; AST, 108 U/L). As soon as the antimicrobial treatment was completed, she was discharged in the seventh day of life. She was referred to our center, 4 days later, for failure to thrive (2,315 g), difficulty in feeding, and deterioration of general health. On physical examination, round facies, elongated philtrum and retro-micrognatia, hyperpigmented macules both at the front and back of the trunk and on labia majora, which do not cross midline, and hypertrichosis on the forehead and extremities were noted (Supplementary Figure S1). Newborn reflexes were hypoactive, blood pressure was 100/70 mmHg, and second-degree cardiac murmur was also detected. Systems were normal otherwise. Laboratory findings revealed hyponatremia, impaired renal and liver function tests, tubulopathy, and proteinuria, while blood count was normal (hemoglobin, 10.4 g/dl; leukocyte, 25.0 × 103/μl; platelet count, 449×103/μl) (Table 1). Hyponatremia resolved with fluid treatment, while liver enzymes, blood urea nitrogen, and creatinine remained elevated. Further endocrine evaluation revealed an elevated serum basal cortisol [225.68 g/dl (N, 6.7–22.6 µg/dL)] and 24-h urinary free cortisol [1,129 μg/day (N, 1.4–20 μg/day)]. Serum cortisol was not suppressed during overnight high-dose dexamethasone suppression test (Table 2) (5). Thyroid hormones were consistent with non-thyroidal illness. Table 1 Table 1 Laboratory investigations on admission, prior to medical treatment (19 days), after medical treatment (6 months), and post-adrenalectomy. Table 2 Table 2 Endocrine evaluation prior to medical treatment (19 days), after medical treatment (6 months), and post-adrenalectomy. ACTH-independent CS and café-au-lait spots suggested MAS. Hypercortisolism-related complications emerged. On the 11th day, hyperglycemia (blood glucose, 250 mg/dl) was seen, and it persisted after cessation of intravenous fluids in the exclusively breastfed neonate; thus, 0.5 U subcutaneous neutral protamine Hagedorn insulin (NPH) (three times a day) was initiated on the 16th day of life when blood glucose was 340 mg/dl, and serum insulin was 18.10 μIU/ml. Hypertension (110/90 mmHg) and hypokalemia were triggered by mineralocorticoid action of excessive cortisol on 20th day. Spironolactone (2 mg/kg/day) was started, and nifedipine (0.5 mg/kg/day) was added in order to control blood pressure (Supplementary Figure S2). Since immunosuppressive effects of excess cortisol may increase the risk for opportunistic infections, Pneumocystis jirovecii prophylaxis was started and live vaccines were postponed. Features of MAS and accompanying hyperfunctioning endocrinopathies were screened (Table 2). On ultrasonography, adrenal glands were hypertrophic; kidneys showed increased parenchymal echogenicity, loss of separation between the cortex and medulla, and enhanced medullary echogenicity; and size and echogenicity of the liver were normal. Magnetic resonance imaging of the abdomen confirmed that adrenal glands were hypertrophic (right and left adrenal gland were 24×22×18 mm and 18×19×20 mm in size, respectively) and lobulated. Echocardiogram revealed left ventricular hypertrophy. Bone survey verified generalized decrease in bone mass and revealed areas of irregular ossification and radiolucency in radius, ulna, and distal tibia, which were interpreted as osteoporosis due to hypercortisolism (Supplementary Figure S1). Medical treatment Metyrapone (300 mg/m2/day, per oral, in four doses) was started on the 25th day (Supplementary Figure S2) (6). Since liver function tests were impaired, metyrapone was preferred over ketoconazole. Soon after metyrapone was started, hyperglycemia and hypertension improved, enabling the discontinuation of insulin and nifedipine. Spironolactone was also gradually tapered and discontinued after 13 days of metyrapone treatment, and she was discharged. The dose of metyrapone was adjusted frequently, according to clinical findings and serum cortisol levels during regular visits. However, even after gradually increasing metyrapone dose to 1,850 mg/m2/day over the course of 6 months, total biochemical suppression of serum cortisol could not be achieved (Supplementary Figure S3A), and the patient had progressive loss of bone mineral density, persistent left ventricular hypertrophy, and a lack of catch-up growth. In addition to that, café-au-lait macules became darker, dehydroepiandrosterone sulfate (DHEA-S) gradually increased (Table 2), and previously non-existent marked clitoromegaly was noted as a side effect of high-dose metyrapone. She was also prescribed ursodeoxycholic acid (15 mg/kg/day); however, liver enzymes remained high (Table 1). Right total and left three-quarters adrenalectomy Right total and left three-quarters adrenalectomy was carried out at 9 months of age in light of the patient’s continued clinical findings of hypercortisolism, the existence of unfavorable prognostic markers (high cortisol levels upon admission and heart and liver problems), and the adverse effects of high-dose metyrapone. The patient was administered 100 mg/m2/day glucocorticoids (GC) perioperatively; however, she developed symptoms of adrenal insufficiency. The required GC dose to attain euglycemia, restore general well-being, and resolve adrenal insufficiency was 300 mg/m2/day. Fludrocortisone (0.05 mg/day) was also started. Following surgery, supraphysiological doses of GC were required, as she suffered frequent symptoms of adrenal insufficiency (hypoglycemia, malaise, and loss of appetite). GC dose could be tapered very slowly, and a daily dose of 15 mg/m2/day could be attained in 2 months. As liver function tests, serum cortisol levels and left ventricular hypertrophy all improved following adrenalectomy (Table 1). Bilateral nodular adrenal hyperplasia was observed in the pathological evaluation of surgical specimen, while the findings of liver wedge biopsy were non-specific (Supplementary Figure S4). Sequence analysis of GNAS from the surgical sample of adrenal gland revealed a heterozygous, previously described missense mutation in exon 8 (c.2530C>A, p.Arg844Ser), while the sequence analysis of the GNAS gene from peripheral blood sample was normal. Lymphocyte activation was normal 3 months post-adrenalectomy, and immunization schedule for live vaccines was established. Other findings of MAS She had breast development and vaginal bleeding that lasted 2 days when she was 7 months old, which repeated five more times after the adrenalectomy till 26 months of age. Breast development was Tanner stage 3, and bone age was markedly advanced (4 years and 2 months), despite severe hypercortisolism. On pelvic ultrasonography, uterus was enlarged to 34×22×24 mm; thus, letrozole (0.625 mg, per oral) was started at 26 months of age. She also developed marked hypophosphatemia at the age of 6 months (Table 1). Radiological investigations since birth demonstrated severe osteopenia and lytic lesions, which were attributed to severe hypercortisolism; however, overt lesions of FD were not confirmed. When she was 9 months old, FGF-23 was elevated [122 pg/ml (normal <52)], which suggested hypophosphatemic rickets associated with FD. Oral phosphate (8 mg/kg) and calcitriol (18 ng/kg) were started. At the age of 23 months, bone survey revealed sclerosis of the base of the skull and maxilla and FD in the lower extremities. She has been on oral phosphate (58.7 mg/kg/day), while calcitriol was ceased. She is now 34 months old with severe short stature [height, 81 cm (−3.5 SDS); weight, 9,580 g (−3.7SDS)] (Supplementary Figure S3B). She had been under regular clinic visits and has been on 15 mg/m2/day hydrocortisone and fludrocortisone 0.025 mg/day, letrozole (1×6.25 mg/day), phosphate (58 mg/kg), and ursodeoxycholic acid (100 mg/day) (Supplementary Figure S2). She has six words, cannot form two-word sentences, shows body parts, cannot stand up from supine position without support, and takes a few steps with support. Despite regular physiotherapy and ergotherapy, developmental delay is evident (Bayley Scales of Infant and Toddler Development III language scale, 13/79; motor scale, 2/46). Discussion ACTH-independent CS and café-au-lait macules suggested MAS in this case. Interestingly, this patient was admitted for hyponatremia and hyperglycemia requiring insulin treatment. Neonatal MAS and CS are rare conditions, and presentation of this case is quite unique (4). The earlier the timing of somatic mutation, the greater the burden of Gsα-mutation-bearing cells leading to widespread tissue involvement in MAS. In the current case, adrenal, hepatic, cardiac, renal, and bone tissue involvement were evident in first weeks of life, while precocious puberty and hypophosphatemic rickets were observed later. A lifetime risk of additional tissue involvement is being acknowledged. CS is the rarest endocrine manifestation of MAS, which appears in <5%–7.1%. It presents exclusively within the first year of life (median age, 3.1 months) where features may develop as early as in utero (2–4, 7). The fact that our case was SGA and had moon facies and hirsutism with impaired linear growth, weight gain, hyperglycemia, hypertension, and nephrocalcinosis detected in the neonatal period, suggested severe, in utero onset CS. Upon suspicion, both comorbidities (hyperthyroidism, excess growth hormone, FD, and cardiac and hepatobiliary function) of MAS and complications of GC excess (hypertension, hyperglycemia, hyperlipidemia, nephrocalcinosis, decreased bone mineral density, and muscle atrophy) were assessed (1, 3). Since the initial description of MAS, only 20 neonates with CS have been described with various initial basal serum cortisol ranging from 9.6 to 80.1 µg/dl, and data regarding long-term follow-up and outcome are still developing (1, 2, 8–11). Disease course is heterogenous, and spontaneous resolution of hypercortisolism has been reported (30%) since Gs-bearing cells are mostly located in the fetal adrenal zone, which normally undergoes apoptosis after birth. However, the outcome is mostly unfavorable in cases with extensive endocrine and extra-endocrine manifestations (1, 2, 8–15). Brown et al. reported poorer prognosis and a lower likelihood of spontaneous remission of adrenal disease in patients with cardiac (cardiomyopathy) and liver involvement (hepatocellular adenomas, inflammatory adenomas, choledochal cysts, neonatal cholestasis, and hepatoblastoma). It was hypothesized that these patients have a greater burden of Gsα mutation (3, 4). Treatment of neonatal CS is a long and challenging path where both cortisol excess and its complications should be targeted. Marked hypercortisolism that precipitate neonatal diabetes requiring insulin treatment like our patient is rare and was previously reported only in six patients with CS (4). Until hypercortisolism is managed, hyperglycemia should be treated with insulin. Hypertension is due to mineralocorticoid effect of excess cortisol; thus, blood pressure lowering agents of choice should be aldosterone antagonists (spironolactone) or potassium-sparing diuretics. The treatment strategy of hypercortisolism is determined by disease severity. In a mildly affected case, medical treatment with an expectation of spontaneous resolution (due to previously stated apoptosis of fetal adrenal zone) may be of choice (3, 4, 16–19). Metyrapone, ketoconazole, and mitotane are medical options for lowering cortisol (20–23). Since our patient had impaired liver function, metyrapone, a potent, rapid acting relatively selective inhibitor of 11-hydroxylase was preferred over ketoconazole for its low risk of hepatotoxicity. Reports reviewing adult data suggest an initial dose of 500–750 mg/day and achievement of biochemical control with 1,500 mg/day (23). However, the initial and maximum dose of metyrapone in neonates is unclear; some authors recommend 300 mg/m2/day in four equal doses (6). In our case, adequate biochemical and clinical suppression of cortisol with metyrapone was not achieved despite an increase in dose from 300 to 1,850 mg/m2/day. There are important issues to be considered while using a steroidogenesis inhibitor like metyrapone. Monitoring biochemical response is essential, not only for dose titration and management of cortisol excess but also for adrenal insufficiency due to possible overtreatment. Clinical signs of adrenal insufficiency should always be questioned and assessed. The 24-h urinary free cortisol is the commonly used method; however, it may be impractical due to difficulties in the collection of urine in infants. Alternative methods may be the measurement of early morning serum cortisol and ACTH (23). Low ACTH level may indicate hypercortisolism or may be a sign of suppression due to long-term exposure to hypercortisolism. However, there are deadlocks to be considered in the evaluation of these measurements. A high cortisol level measured by immunoassays does not always indicate an actual elevation. It should be kept in mind that cortisol immunoassays exhibit significant cross-reactivity with cortisol precursors that may be elevated in patients treated with a steroidogenesis inhibitor (especially with metyrapone, which is known to increase 11-deoxycortisol). Such cross-reactivity can be a cause for overestimation of cortisol and may lead to risk of overtreatment (24, 25). It has been suggested that the patients on metyrapone should be biochemically monitored via specific methods, such as mass spectrometry (24–26). Metyrapone is a relatively selective inhibitor of 11-hydroxylase and 18-hydroxylase. Recent in vitro studies indicate greater inhibitory action of metyrapone on aldosterone synthase, resulting in significant reversible reduction in both cortisol and aldosterone. The loss of negative feedback leads to an increase in ACTH, which causes an accumulation of cortisol and aldosterone precursors resulting in an increase in adrenal androgens (23). Although we could not serologically prove an increase in ACTH, hyperpigmentation and the increase in adrenal androgens confirm this mechanism. As far as we know, an increase in DHEA-S causing virilization was an unreported side effect of metyrapone. Clinical (clitoromegaly and hirsutism) and laboratory (DHEA-S) signs of hyperandrogenism should be monitored when higher doses of metyrapone are required. In the severely affected case with CS, where medical treatment is inadequate and the chance of spontaneous resolution is subsiding, adrenalectomy is indicated when medically feasible. Brown et al. suggested that the presence of comorbid cardiac and liver disease like in our case should prompt consideration for early adrenalectomy (4). Although a previous correlation with initial serum cortisol level and prognosis was not established, it may be speculated that excessively high serum cortisol level is associated with increased number of Gsα-mutation-bearing adrenal cells. Thus, we suggest that in neonatal CS due to MAS, initial very high serum cortisol levels, like our case, may be a negative prognostic factor both for spontaneous resolution and clinical response to medical treatment. In infants with severe CS, bilateral adrenalectomy is generally performed. Alternatives like unilateral adrenalectomy and one-side total, other-side three-quarters adrenalectomy may be considered to avoid the requirement for lifelong GC and mineralocorticoid replacement. Unilateral adrenalectomy was reported to successfully improve clinical symptoms and endocrinological status in adult studies; nevertheless, recurrence during follow-up was 23.1%, while 17.5% required contralateral adrenalectomy (27–29). Since the causes of CS in adult series are variable and different from pediatric CS due to MAS, it should be borne in mind that reproducibility of adult data is poor. In CS due to MAS, Gsα-mutation-bearing adrenal gland cells are heterogeneously distributed, and partial adrenalectomy may carry the risk of inadequate management and recurrence. Only a few pediatric case reports addressed this issue. Unilateral adrenalectomy of the larger gland was performed in two neonates with CS due to MAS; remission was achieved for 2 years (30, 31). Itonaga et al. reported a 6-month-old neonate with MAS-associated CS treated with right-sided total adrenalectomy and left-sided half adrenalectomy with remission for 2 years (32). Although these cases were less severe [basal serum cortisol: 16.9, 18.5, and 23.4 µg/dl, respectively (N: 6.2–18.0 µg/dL)], we preferred to perform partial adrenalectomy (right total and left three-quarters adrenalectomy) and succeeded. Our patient has been in remission for more than 2 years. In the largest case–control analysis of CS in patients with MAS, overall mortality was 20% (six cases) where four of them were deceased following bilateral adrenalectomy (66.7% of all deaths) (4). Anaphylaxis (or adrenal insufficiency), sudden cardiac arrest, sepsis, and sudden death were listed as causes of mortality in those four cases where GC dose and process of GC tapering were not clearly described. The fact that our patient required high-dose GC during peri- and postoperative period to restore well-being, tapering to maintenance dose was very slow, and she is still on maintenance dose GC, suggests that rapid tapering of GCs should be avoided and, although being speculative, may explain sudden death following adrenalectomy. Gross motor developmental delay may be caused by prenatal exposure to excess GCs. Prenatal GC treatment for possible congenital adrenal hyperplasia or risk of premature birth have been shown to result in cognitive deficits after birth. Furthermore, children who develop CS later in life may experience a decline in cognitive and school performance where the younger the age of onset, the greater the deterioration in IQ scores (3, 4, 33, 34). Since transgenic mice with Gsα mutation was shown to have short- and long-term memory deficits and impaired associative and spatial learning, it may also be speculated that Gsα mutation may also be present in the central nervous system (35, 36). The establishment of diagnosis of FD follows a characteristic and predictable time course. Although GNAS mutations are acquired early in embryogenesis, skeletal development appears to be relatively normal in utero, without frank clinical signs of FD at birth. Boyce et al. affirmed that FD lesions become apparent over the first several years of life and expand during childhood and adolescence, like our case. Previous case reports have also stated severe osteoporosis, rickets, polyostotic irregular lucencies, pathological fractures, and biopsy-proven FD during infancy (1, 2, 8–15). The exact pathophysiological mechanism is unclear, and Gsα activation in abnormally differentiated osteocytes is accused. FGF-23 overproduction is an inherent feature of FD, and most patients have elevated circulating levels of FGF-23, but frank hypophosphatemia is rare. The increase in FGF-23 is linked to substantial skeletal involvement. Although FGF-23 levels may wax and wane over time, an increase in FGF-23 usually occurs during periods of rapid growth like infancy and adolescence. Concurrent hyperfunctioning endocrinopathies like hyperthyroidism or CS may also adversely affect bone health. Peripheral precocious puberty (PP) is the most frequent presenting feature in female patients with MAS (85%) (6). To date, a safe, effective, and long-term treatment for PP in girls with MAS has not been established. The benefits of current interventions on the ultimate outcome of interest, adult height, have not been well-established due to the rarity of the condition and heterogeneous nature of the disease. Despite the small sample size, studies have concluded that letrozole resulted in a statistically significant decrease in the bone age/chronological age ratio, growth velocity, hence increasing predicted adult height (37). Growth outcome in MAS is not only dependent on timing of pubertal onset but on several other disease components (skeletal involvement and endocrinopathies) as well. Hyperthyroidism and growth hormone excess may accelerate growth, while CS may decelerate it (37, 38). Lack of consensus on both medical and surgical treatment strategies were major obstacles while navigating this case of severe neonatal MAS. The eminence of this report is that it presents current literature with clinical experience on this rare case of neonatal CS due to MAS. High index of suspicion for MAS in a neonate with extensive café-au-lait macules and symptoms of hypercortisolism is the key for early recognition and intervention. Initial excessive cortisol in neonatal CS may be a negative prognostic factor for spontaneous resolution and response to medical treatment, indicating early right total and left three-quarters adrenalectomy. Post-adrenalectomy survival may be related to close supervision during GC tapering. Data availability statement The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/Supplementary Material. Ethics statement Written informed consent was obtained from the individual(s), and minor(s)’ legal guardian/next of kin, for the publication of any potentially identifiable images or data included in this article. Author contributions YU collected and analyzed data, drafted the initial manuscript, and reviewed and revised the manuscript. OG collected data. İU, HH, BG, SE, and TK collected data and reviewed and revised the manuscript. ZO and EG analyzed data, conceptualized the work, and revised and critically reviewed the manuscript for important intellectual and medical content. All authors approved the final manuscript as submitted and agree to be accountable for all aspects of the work. Acknowledgments We thank our patient’s family for providing consent for publication of this work. Conflict of interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Publisher’s note All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher. Supplementary material The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fendo.2023.1209189/full#supplementary-material Supplementary Figure 1 | (A) The findings of physical and radiologic examination. Notice cushingoid facies, hyperpigmented macules that does not cross the midline at the front of the trunk. (B) Anteroposterior radiographs reveal irregularities in radius, ulna and femur. Although generalized osteopenia improves at 34 months, FD lesions become prominent over months. Supplementary Figure 2 | Timeline of the course of symptoms in neonatal McCune Albright Syndrome noting adjustments made in treatment. Grey box denotes age in days for the first month of life then in months. NPH: Neutral Protamine Hagedorn insulin, CS: Cushing syndrome, PP: precocious puberty. 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(2018) 7(12):1424–31. doi: 10.1530/EC-18-0344 PubMed Abstract | CrossRef Full Text | Google Scholar Keywords: McCune Albright syndrome, neonatal Cushing syndrome, metyrapone, adrenalectomy, follow-up Citation: Unsal Y, Gozmen O, User İR, Hızarcıoglu H, Gulhan B, Ekinci S, Karagoz T, Ozon ZA and Gonc EN (2023) Case Report: Severe McCune–Albright syndrome presenting with neonatal Cushing syndrome: navigating through clinical obstacles. Front. Endocrinol. 14:1209189. doi: 10.3389/fendo.2023.1209189 Received: 20 April 2023; Accepted: 04 July 2023; Published: 25 July 2023. Edited by: Martin Oswald Savage, Queen Mary University of London, United Kingdom Reviewed by: Li Chan, Queen Mary University of London, United Kingdom Sasha R Howard, Queen Mary University of London, United Kingdom Tomoyo Itonaga, Oita University, Japan Copyright © 2023 Unsal, Gozmen, User, Hızarcıoglu, Gulhan, Ekinci, Karagoz, Ozon and Gonc. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. *Correspondence: Yagmur Unsal, yagmurunsal@yahoo.com Disclaimer: All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article or claim that may be made by its manufacturer is not guaranteed or endorsed by the publisher. From https://www.frontiersin.org/articles/10.3389/fendo.2023.1209189/full