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Abstract Objective To evaluate whether age-related differences exist in clinical characteristics, diagnostic approach and management strategies in patients with Cushing’s syndrome included in the European Registry on Cushing’s Syndrome (ERCUSYN). Design Cohort study. Methods We analyzed 1791 patients with CS, of whom 1234 (69%) had pituitary-dependent CS (PIT-CS), 450 (25%) adrenal-dependent CS (ADR-CS) and 107 (6%) had an ectopic source (ECT-CS). According to the WHO criteria, 1616 patients (90.2%) were classified as younger (<65 years) and 175 (9.8%) as older (>65 years). Results Older patients were more frequently males and had a lower BMI and waist circumference as compared with the younger. Older patients also had a lower prevalence of skin alterations, depression, hair loss, hirsutism and reduced libido, but a higher prevalence of muscle weakness, diabetes, hypertension, cardiovascular disease, venous thromboembolism and bone fractures than younger patients, regardless of sex (p<0.01 for all comparisons). Measurement of UFC supported the diagnosis of CS less frequently in older patients as compared with the younger (p<0.05). An extra-sellar macroadenoma (macrocorticotropinoma with extrasellar extension) was more common in older PIT-CS patients than in the younger (p<0.01). Older PIT-CS patients more frequently received cortisol-lowering medications and radiotherapy as a first-line treatment, whereas surgery was the preferred approach in the younger (p<0.01 for all comparisons). When transsphenoidal surgery was performed, the remission rate was lower in the elderly as compared with their younger counterpart (p<0.05). Conclusions Older CS patients lack several typical symptoms of hypercortisolism, present with more comorbidities regardless of sex, and are more often conservatively treated. From https://academic.oup.com/ejendo/advance-article-abstract/doi/10.1093/ejendo/lvad008/7030701?redirectedFrom=fulltext&login=false

The following is the summary of “Increased Risk of Ocular Hypertension in Patients With Cushing’s Disease” published in the December 2022 issue of Glaucoma by Ma, et al. Ocular hypertension was more common in people with Cushing’s illness. The usage of steroids in the body is a major contributor to high intraocular pressure (IOP). Topical or systemic glucocorticoid use may increase the prevalence of ocular hypertension in the general population from 30–40%. The prevalence of ocular hypertension in endogenous hypercortisolemia and the ophthalmological consequences following endocrine remission after surgical resection are unknown. During the period of January 2019 through July 2019, all patients with Cushing’s disease (CD) who were hospitalized at a tertiary pituitary facility for surgical intervention had their intraocular pressure (IOP), vision field, and peripapillary retinal nerve fiber layer thickness recorded. Nonfunctioning pituitary adenoma (NFPA) patients and acromegaly patients from the same time period were used as comparison groups. Researchers showed postoperative changes in IOP, estimated the odds ratio (OR), and identified risk variables for the development of ocular hypertension. About 52 patients with CD were included in the study (mean age 38.4±12.4 years). Patients with CD had an IOP that was 19.4±5.4 mm Hg in the left eye and 20.0±7.1 mm Hg in the right eye, which was significantly higher than that of patients with acromegaly (17.5±2.3 mm Hg in the left eye and 18.6±7.0 mm Hg in the right eye, P=0.033) and NFPA (17.8±2.6 mm Hg in the left eye and 17.4±2.4 mm Hg in the right eye, Ocular hypertension was diagnosed in 21 eyes (20.2%) of CD patients, but only 4 eyes (4.7%) of acromegaly patients and 4 eyes (4.5%) of NFPA patients. Patients with CD had an odds ratio (OR) of 5.1 [95% CI, 1.3-25.1, P=0.029] and 6.6 [95% CI, 1.8-30.3, P=0.007] for developing ocular hypertension compared with the 2 control groups. Higher levels of urine-free cortisol were associated with an increased risk of ocular hypertension in CD patients (OR=19.4, 95% CI, 1.7-72.6). Patients with CD saw a decrease in IOP at 1 month following surgery, and this improvement was maintained for another 2 months. Researchers conclude that endogenous hypercortisolemia should be included as part of the glaucoma assessment due to the increased risk of ocular hypertension in CD. Ophthalmologists and neuroendocrinologists should use their judgment in light of this finding. Source: journals.lww.com/glaucomajournal/Fulltext/2022/12000/Increased_Risk_of_Ocular_Hypertension_in_Patients.3.aspx

The study covered in this summary was published on researchsquare.com as a preprint and has not yet been peer reviewed. Key Takeaways The hypothalamic-pituitary-adrenal (HPA) axis recovered in nearly three quarters of patients with Cushing disease (CD) within 2 years after successful trans-sphenoidal surgery (TSS), with a median recovery time of 12 months. Preoperative total triiodothyronine (TT3) level appears to be an independent predictor of central adrenal insufficiency (CAI) in CD patients with biochemical remission post surgery. Why This Matters Transient CAI typically occurs after successful TSS, requiring physiologic hydrocortisone replacement until HPA recovery. Inadequate replacement may result in glucocorticoid withdrawal symptoms, including adrenal crisis, while overreplacement could lead to glucocorticoid side effects. Findings have been inconsistent regarding recovery time in CD patients and factors predicting HPA axis recovery. The new findings could help clinicians predict HPA axis-function recovery time and adjust cortisone replacement treatment in postoperative CD patients. Study Design The retrospective study included 140 patients with biochemical remission following CD surgery at a single institution from 2014–2020. Key Results The HPA axis in 103 patients (73.6%) recovered during 2 years' postsurgical follow-up. In 57 patients (55% of this subgroup), it recovered within 12 months. Patients were considered to have recovered if they achieved central adrenal sufficiency (CAS). These patients were significantly younger and had significantly lower midnight levels of adrenocorticotrophic hormone at baseline than those with persistent CAI. The researchers found no significant differences in gender, disease duration, maximal tumor diameter, or history of surgery between the two groups at the time of their diagnosis with CD. Both TT3 and free triiodothyronine levels were significantly lower in patients with persistent CAI vs CAS. There were no significant differences between the two groups in other laboratory parameters, surgical approach, or extended compared with nonextended resection, but more patients in the persistent CAI group underwent partial hypophysectomy. In a multiple logistic regression analysis, TT3 levels at diagnosis independently and significantly predicted HPA recovery at 2-year follow-up post surgery after adjustment for gender, age, duration at diagnosis, maximum tumor diameter, history of surgery, surgical approach (endoscopic or microscopic transsphenoidal surgery), adenomectomy range, and the minimal serum cortisol level within the first 7 postoperative days. Among the 37 patients with persistent CAI at 2 years, 23 (62%) had multiple pituitary axis dysfunctions, including hypothyroidism (19 patients), hypogonadism (19), and central diabetes insipidus (5). Limitations This retrospective study could not prove the causality of TT3 level for influencing recovery of the HPA axis. However, the number of enrolled patients was relatively large, and follow-up was regular ― factors that make the conclusion credible and representative, the authors said. Disclosures The study received no commercial funding. The authors had no disclosures. This is a summary of a preprint research study, "The Recovery Time of Hypothalamic-Pituitary-Adrenal Axis After Curative Surgery in Cushing’s Disease and Its Predictor," by researchers at Huashan Hospital Fudan University, Shanghai, China, published on Research Square and provided to you by Medscape. This study has not yet been peer reviewed. The full text of the study can be found on researchsquare.com. Abstract Objective Patients with Cushing’s disease (CD) experienced transient central adrenal insufficiency (CAI) after successful surgery. However, the reported recovery time of hypothalamic-pituitary-adrenal (HPA) axis varied and the risk factors which could affect recovery time of HPA axis had not been extensively studied. This study aimed to analyze the duration of CAI and explore the risk factors affecting HPA axis recovery in post-operative CD patients with biochemical remission. Design and methods Medical records of diagnosis with CD in Huashan Hospital were reviewed between 2014 and 2020. 140 patients with biochemical remission and regular follow-up after surgery were enrolled in this retrospective cohort study according to the criteria. Demographic details, clinical and biochemical information at baseline and each follow-up (within 2 years) were collected and analyzed. Results Overall, 103 patients (73.6%) recovered from transient CAI within 2 years follow-up and the median recovery time was 12 months [95% confidence intervals (CI): 10–14]. The age and midnight ACTH at baseline were significantly lower, while the TT3 and FT3 levels were significantly higher in patients with recovered HPA compared to patients with CAI at 2-year follow-up(p < 0.05). In persistent CAI group, more patients underwent partial hypophysectomy. TT3 at diagnosis was an independent predictor of the recovery of HPA axis, even after adjusting for gender, age, duration, surgical history, maximum tumor diameter, surgical strategy, and postoperative nadir serum cortisol level (p = 0.04, OR: 6.03, 95% CI: 1.085, 22.508). Among patients with unrecovered HPA axis at 2-year follow-up, 23 CAI patients (62%) were accompanied by multiple pituitary axis dysfunction besides HPA axis, including hypothyroidism, hypogonadism, or central diabetes insipidus. Conclusion HPA axis recovered in 73.6% of CD patients within 2 years after successful surgery, and the median recovery time was 12 months. TT3 level at diagnosis was an independent predictor of postoperative recovery of HPA axis in CD patients. Moreover, patients coexisted with other hypopituitarism at 2-year follow-up had a high probability of unrecovered HPA axis. total triiodothyronine Cushing’s disease central adrenal insufficiency Read more at

Abstract Cushing’s syndrome (CS) is a diagnosis used to describe multiple causes of serum hypercortisolism. Cushing’s disease (CD), the most common endogenous subtype of CS, is characterized by hypercortisolism due to a pituitary tumor secreting adrenocorticotropic hormone (ACTH). A variety of tests are used to diagnose and differentiate between CD and CS. Hypercortisolism has been found to cause many metabolic abnormalities including hypertension, hyperlipidemia, impaired glucose tolerance, and central adiposity. Literature shows that many of the symptoms of hypercortisolism can improve with a low carb (LC) diet, which consists of consuming <30 g of total carbohydrates per day. Here, we describe the case of a patient with CD who presented with obesity, hypertension, striae and bruising, who initially improved some of his symptoms by implementing a LC diet. Ultimately, as his symptoms persisted, a diagnosis of CD was made. It is imperative that practitioners realize that diseases typically associated with poor lifestyle choices, like obesity and hypertension, can often have alternative causes. The goal of this case report is to provide insight on the efficacy of nutrition, specifically a LC diet, on reducing metabolic derangements associated with CD. Additionally, we will discuss the importance of maintaining a high index of suspicion for CD, especially in those with resistant hypertension, obesity and pre-diabetes/diabetes. Keywords: low carb; carnivore; ketogenic; Cushing syndrome; Cushing disease; glucose intolerance; hypertension; obesity; metabolic health 1. Introduction Cushing’s syndrome (CS) is a rare disorder of hypercortisolism related to exposure to high levels of cortisol (>20 mcg/dL between 0600–0800 or >10 mcg/dL after 1600) for an extended period [1,2]. CS affects 10 to 15 people per million and is more common among those with diabetes, hypertension, and obesity [3]. The metabolic derangements associated with CS include visceral obesity, elevated blood pressure, dyslipidemia, type II diabetes mellitus (T2DM) and insulin resistance [4]. CS physical exam findings include round face, dorsal fat pad, central obesity, abdominal striae, acne, and ecchymosis [3]. Other symptoms associated with CS include low libido, headache, change in menses, depression and lethargy [2,3,5]. The most common features of CS are weight gain, which is found in 82% of cases, and hypertension, which is found in 50–85% of cases [6]. CS can be caused by exogenous glucocorticoids, known as iatrogenic CS, ectopic ACTH secretion (EAS) from sources like a small cell lung cancer or adrenal adenoma, known as EAS CS, or excess production of ACTH from a pituitary tumor, known as CD [3]. In CD, ACTH subsequently causes increased production of cortisol from the adrenal glands. CD accounts for 80–85% of endogenous cases of CS [3]. Other conditions including alcoholism, depression, severe obesity, bulimia and anorexia nervosa can lead to a Cushing-like state, although are not considered true CS [3]. Many studies have demonstrated that LC diets can ameliorate some of the most common metabolic derangements seen in CD, namely hyperglycemia, weight gain, hypertension and insulin resistance. A LC diet is a general term for diets which lower the total carbohydrates consumed per day [4]. A ketogenic diet is a subtype of LC that is described as having even fewer carbohydrates, typically less than 30 g/day. By reducing carbohydrate intake and thus limiting insulin production, the body achieves ketosis by producing an elevated number of ketones including β-hydroxybutyric acid, acetoacetic acid, and acetone, in the blood [7]. A carnivore diet, a specific type of a ketogenic diet, is defined as mainly eating animal food such as meat, poultry, eggs and fish. Contrarily, a standard American diet (SAD) is defined as a diet high in processed foods, carbs, added sugars, refined fats, and highly processed dairy products [8]. There are several therapeutic applications for LC diets that are currently supported by strong evidence. These include weight loss, cardiovascular disease, T2DM, and epilepsy. LC diets have clinical utility for acne, cancer, polycystic ovary syndrome (PCOS), and neurologic deficits [9]. In this case report, the patient endorsed initially starting a LC diet to address weight gain and high blood sugars that he noted on a glucometer. The patient noted a 35 pounds (lbs.) weight loss over the first 1.5 years on his LC diet, as well as improved blood pressure and in his overall health. He then adopted a carnivore diet but found that weight loss was difficult to maintain, although his body composition continued to improveand his clothes fit better. Later, he noted that his blood pressure would at times be poorly controlled despite multiple medications and strict dietary adherence. The patient reported “being in despair” and “not trusting his doctors” because they did not understand how much his diet had helped him. Despite strict adherence, his symptoms of insulin resistance and hypertension persisted. In this report, we will describe how his symptoms of CD were ameliorated by the ketogenic diet. This case report also highlights that when patients are unable to overcome hormonal pathology, clinicians should not blame patients for lack of adherence to a diet, but instead understand the need to evaluate for complex pathology. 2. Detailed Case Description A male patient in his thirties, of Asian descent, had a past medical history of easy bruising, central obesity, headaches, hematuria, and hypertension and past family medical history of hypertension in his father and brother. In 2015, he was at his heaviest weight of 179 lbs. with a body mass index (BMI) of 28 kg/m2, placing him in the overweight category (25.0–29.9 kg/m2). At that time the patient reported he was following a SAD diet and was active throughout the day. The patient stated he ate a diet of vegetables, fruits and carbohydrates, but he was not able to lose weight. The patient stated that he switched to a LC diet, to address weight gain and hyperglycemia, and he reported that he lost approximately 35 lbs. in 1.5 years. The patient described his LC diet as eating green leafy vegetables, low carb fruits, fish, poultry, beef and dairy products. The patient then later switched to a carnivore diet. He noted despite aggressively adhering to his diet, that his weight-loss had plateaued, although his waist circumference continued to decrease. The patient noted his carnivore diet consisted of eating a variety of different meats, poultry, fish and eggs. The metabolic markers seen in Table 1 were obtained after the patient had started a carnivore diet. The patient’s blood glucose levels decreased overtime despite impaired glucose metabolism being a known side effect of hypercortisolism [4]. The patient’s high-density lipoprotein (HDL) remained in a healthy range (40–59 mg/dL) and his triglycerides stayed in an optimal range (<100 mg/dL), despite dyslipidemia being a complication of CD [4]. When the patient was consuming a SAD diet, he was not under the care of a physician and was unable to provide us with previous biomarkers. Table 1. Patient’s metabolic markers on a carnivore diet. Glucose (70 to 99 mg/dL), total cholesterol (desirable <200 mg/dL, borderline high 200–239 mg/dL, high >239 mg/dL), triglycerides (optimal: <100 mg/dL), HDL (low male: <40 mg/dL), low density lipoprotein (LDL) (Optimal: <100 mg/dL). Despite strict adherence to his diet and initial improvement in his weight, his blood pressure and his blood sugar levels, in October of 2021 the patient was admitted to the hospital for hypertensive urgency, with a blood pressure of 216/155. His complaints at the time were unexplained ecchymosis, hematuria and significant headaches that were resistant to Excedrin (acetaminophen-aspirin-caffeine) use. At the hospital, the patient underwent a computed tomography (CT) scan of the head and radiograph of the chest, and both images were negative for acute pathology. During his hospital admission, the patient denied any changes in vision, chest pain or edema of the legs. Ultimately, the patient was told to eat a low-salt diet and to follow-up with a cardiologist. At discharge, the patient was placed on hydrochlorothiazide, labetalol, amlodipine and lisinopril. The patient was then seen by his primary care physician in November of 2021 and his urinalysis at that time showed 30 mg/mL (Negative/Trace) of protein in his urine, without hematuria. The patient’s primary care physician discontinued his hydrochlorothiazide and started the patient on furosemide. Additionally, the primary care physician reinforced cutting out salt and limiting his calories to prevent any further weight gain, which his physician explained would contribute further to his hypertension. He was referred to hematology and oncology in November of 2021 for his symptoms of hematuria and abnormal ecchymosis to his abdomen, thighs and arms. The patient’s coagulation and platelet counts were normal, and his symptoms were noted to be improving. His hematuria and ecchymosis were attributed to his significant Excedrin use from the past 1–2 months, secondary to his headaches, and their anti-platelet effect. It was noted that the patient had significant hemolysis during his hospital admission. However, in his follow up examination, there were no signs of hemolysis, and it was attributed to his hypertensive urgency. Again, a low-salt, calorie-limited diet was recommended. The patient was referred to cardiology where he was evaluated for secondary hypertension, because despite his weight loss and his strict adherence to his diet, his blood pressure was still uncontrolled on multiple medications. He had a normal echocardiogram and renal ultrasound which showed no signs of renal artery stenosis bilaterally. At that time the patient’s serum renin, aldosterone and urine metanephrine levels were all normal. His cardiologist increased his lisinopril, and continued him on amlodipine, furosemide and labetalol and reinforced the recommendations of lowering his salt and preventing weight gain. The patient first contacted our office in January of 2022. At that time his blood pressure was noted to be 160/120 despite being compliant with current blood pressure medications. The patient reported strict adherence to his carnivore diet by sharing his well-documented meals on his social media accounts. Given the persistent symptoms, despite his significant change in diet and weight loss, we were concerned that a hormonal etiology may be driving his symptoms. The patient was seen in-person, in our office, in March of 2022. At the request of the patient, we again reviewed his social media profile to assess his meal choices and diet. While the patient was eager to show us his carnivore meals, what we incidentally noted in his photos was despite weight loss and strict diet adherence, he had developed moon facies (Figure 1a,b). On the physical exam, we noted his prominent abdominal striae (Figure 2). Several screening tests for Cushing’s syndrome were ordered. A midnight salivary cortisol was ordered, with values of 0.884 ug/dL (<0.122 ug/dL) and 0.986 ug/dL (<0.122 ug/dL) and a urinary free cortisol excretion (UFC) was ordered, with values of 8.8 ug/L (5–64 ug/L). At this point our suspicion was confirmed that the patient had inappropriately elevated cortisol. Figure 1. The patient’s progression of moon facies, (a) photo from 2019 after initial weight loss (b) photo from office visit in 2022. Figure 2. The arrows demonstrate early striae visualized on the lower abdomen bilaterally, unclear in image due to poor office lighting. Based on screening tests and significant physical exam findings, we referred the patient to endocrinology for a low dose dexamethasone suppression test (DST). They performed a low dose DST revealing a dehydroepiandrosterone (DHEA) of 678 ug/dL (89–427 ug/dL) and ACTH of 23.9 pg/mL (7.2–63.3 pg/mL). The low dose DST and midnight salivary cortisol were both positive indicating hypercortisolism. To begin determining the source of hypercortisolism, the plasma ACTH was evaluated and was 27.2 pg/mL (7.2–63.3 pg/mL). While ACTH was within normal range, a plasma ACTH > 20 pg/mL is suggestive of ACTH-dependent CS, so a magnetic resonance imaging (MRI) of the brain was ordered [2]. The MRI revealed a 4 mm heterogeneous lesion in the central pituitary gland which is suspicious of a cystic microadenoma. To confirm that a pituitary tumor was the cause of the patient’s increased cortisol, the patient was sent for inferior petrosal sinus sampling (IPSS). The results of the IPSS indicated an increase in ACTH in both inferior petrosal sinuses and peripheral after corticotropin-releasing hormone (CRH) stimulation (Figure 3a–c), which was consistent with hypercortisolism. Figure 3. (a) Right IPS venous sampling values for ACTH and prolactin after CRH stimulation over multiple time intervals. (b) Left IPS venous sampling values for ACTH and prolactin after CRH stimulation over multiple time intervals. (c) Peripheral sampling values for ACTH and prolactin after CRH stimulation over multiple time intervals. Lab results from the patient’s IPSS venous sampling can be seen above. The graphs depict the lab values of ACTH (7.2–63.3 pg/mL) and prolactin (PRL) (2.1–17.7 ng/mL) before and after CRH stimulation during IPSS. PRL acts as a baseline to indicate successful catheterization in the procedure [10]. Using the ACTH levels from our patient’s IPSS we calculated a ratio of inferior petrosal sinus to peripheral (IPS:P). These results can be seen below (Table 2). The right IPS:P was calculated as 3.60 at 10 min and the left IPS:P as 7.65 at 10 min. These ratios confirmed that the hypercortisolism was due to the pituitary tumor, as it is higher than the 3:1 ratio necessary for diagnosis of CD [11]. The patient is currently scheduled to undergo surgical resection of the pituitary microadenoma. Table 2. Right and left petrosal sinus to peripheral serum ACTH ratios. 3. Clinical Evaluation for CS In this case, the patient presented with uncontrolled hypertension, weight gain despite a strict diet, hyperglycemia, abdominal striae and moon facies. Despite evaluation, both inpatient and outpatient, a diagnosis of CS was not yet explored. When CS is suspected based on clinical findings, the use of exogenous steroids must first be excluded as it is the most common cause of hypercortisolism [3]. If there is still concern for CS, there are three screening tests that can be done which are sensitive but not specific for hypercortisolism. The screening tests include: a 24-h UFC, 2 late night salivary cortisol tests, low dose (1 g) DST [3]. To establish the preliminary diagnosis of hypercortisolism two screening tests must be abnormal [2]. The first step to determine the cause of hypercortisolism is to measure the plasma level of ACTH. Low values of ACTH < 5 pg/mL indicate the cause is likely ACTH-independent CS and imaging of the adrenal glands is warranted as there is a high suspicion of an adrenal adenoma [2,3]. When the serum ACTH is elevated >/20 pg/mL it is likely an ACTH-dependent form of CS [2]. To further evaluate an ACTH-dependent hypercortisolism, an MRI should be obtained as there is high suspicion that the elevated cortisol is coming from a pituitary adenoma. If there is a pituitary mass >6 mm there is a strong indication for the diagnosis of CD [2]. However, pituitary tumors can be quite small and can be missed on MRIs in 20–58% of patients with CD [2]. If there is still a high suspicion of CD with an inconclusive MRI, a high dose DST (8 g) is done. Patients with CD should not respond and their ACTH and DHEA, a steroid precursor, should remain high. Similarly, CRH stimulation test is done and patients with CD should have an increase in ACTH and/or cortisol within 45 min of CRH being given. If the patient has a positive high-dose DST, CRH-stimulation test and an MRI with a pituitary tumor >6 mm no further testing is needed as it is likely the patient has CD [2]. If either of those tests are abnormal, the MRI shows a pituitary tumor < 6 mm, or there is diagnostic ambiguity, the patient should undergo IPSS with ACTH measurements before and after the administration of CRH [4]. IPSS is the gold standard for determining the source of ACTH secretion and confirming CD. In this invasive procedure, ACTH, prolactin, and cortisol levels are sampled prior to CRH stimulation and after CRH stimulation. PRL acts as a baseline to indicate successful catheterization in the procedure [12]. To confirm CD, a ratio of IPS:P is calculated for values prior to and after CRH stimulation. A peak ratio greater than 2.0 before CRH stimulation or a peak ratio greater than 3.0 after CRH stimulation is indicative of CD. In comparing the right and left petrosal sinus sample, an IPS:P ratio greater than 1.4 suggests adenoma lateralization. However, due to high variability, IPSS should not be used for diagnosing lateralization [13]. 4. Discussion Surgical intervention remains the primary treatment for CD [4]. However, remission is not guaranteed as symptoms and metabolic diseases have been shown to persist afterwards. In the literature it has been shown that nutrition can have a powerful impact on suppressing, or even reversing metabolic disorders and comorbidities associated with CD. A LC diet has been shown to promote significant weight loss, reduce hypertension, improve dyslipidemia, reverse T2DM and improve cortisol levels (2, 14–15, 18–21). There are reports of weight loss on a LC diet in the literature. A LC significantly reduced weight and BMI of 30 male subjects [14]. In a group of 120 participants over 24 weeks who followed a LC versus low fat (LF) diet, showed a greater weight loss in the LC group vs. the LF group [15]. Patients diagnosed and treated for CD found that their weight remained largely unchanged even after treatment [6]. In many cases, surgical treatment does not always resolve the associated comorbidity of central adiposity in CD. In such cases, a LC diet can be used before, during and after treatment, as an adjunct, to decrease associated weight gain and comorbidities. Nutritional intervention can be a powerful adjunct to reduce comorbidities associated with CD. As seen in this case report, the patient’s symptoms of CD, especially hypertension and weight gain, improved with dietary changes despite him having a pituitary microadenoma. Multiple studies showed that a LC diet was able to decrease blood pressure parameters. In a group of 120 participants over 24 weeks who followed a LC versus a LF diet showed a greater decrease in both systolic and diastolic blood pressure in the LC group vs. the LF group [15]. Other literature which studied the effect of a LC diet on hypertension demonstrated the reduction of blood pressure and is thought to be due to ketogenesis. It is thought the production of ketones have a natriuretic effect on the body therefore lowering systemic blood pressure [16]. A LC diet improves lipid profiles and inflammatory markers associated with metabolic syndrome [14]. Literature shows that a LC diet has a greater impact on decreasing triglyceride levels and increasing HDL levels, when compared to a LF diet [15]. Triglyceride levels in patients in CD remission remained high [17]. Therefore, it can be hypothesized that a LC diet would be beneficial, in addition to standard CD treatment, to lower the associated comorbidity of hypertriglyceridemia and metabolic syndrome. Insulin resistance, a precursor to T2DM, is a common comorbidity of hypercortisolism which can be treated with a LC diet. One study showed that in subjects with T2DM, a decrease in A1c and a reduction in antidiabetic therapy were seen with consumption of a LC diet [18]. Additionally, a cohort of 9 participants following a LC diet were able to collectively lower their A1c on average by 1% while concurrently discontinuing various antidiabetic therapies including insulin [19]. Literature shows that a LC diet can minimize systemic cortisol levels through various mechanisms. Current treatment of CD includes medications which block cortisol production and/or cortisol secretion [2]. LC can imitate similar results seen through medication intervention for CD. Carbohydrate restriction can lower cortisol levels, as carbohydrates stimulate adrenal cortisol secretion and extra-adrenal cortisol regeneration [4]. A ketogenic diet can lower the level of ghrelin, a peptide produced in the stomach that has orexigenic properties [20,21]. Literature shows that ghrelin increases levels of serum cortisol [22]. Therefore, implementing a ketogenic diet would decrease ghrelin, and subsequently minimize the effects of increased ghrelin on serum cortisol. A LC diet decreases visceral fat which itself is an endocrine organ and can increase the synthesis of cortisol [14]. Therefore, decreasing visceral fat also decreases the production of cortisol. A LC was shown to significantly reduced weight, BMI and cortisol levels of 30 obese male subjects [14]. Further, a LC diet excludes foods with a high glycemic index which cause increased stress on the body which subsequently leads to the activation of the hypothalamic-pituitary-axis which causes increased levels of cortisol [14]. This case report illustrated how a LC diet was initially successful at ameliorating the patient’s associated symptoms of hypertension and obesity, making his diagnosis of CD go undetected. Literature shows that while the prevalence of CS on average is a fraction of a percent, it is much higher among patients with poorly controlled diabetes, hypertension and early onset osteoporosis [3]. Two hundred patients with diabetes mellitus were studied and 5.5% were found to have CS [23]. Another study discovered that in subjects with CD, 36.4% were found to have hyperlipidemia, 73.1% with hypertension, and 70.2% with impaired glucose metabolism [17]. It can be concluded that a higher index of suspicion and lower threshold for screening for CS may be necessary in obese and diabetic patient populations. A lower threshold for screening can allow for earlier diagnosis for many patients, and therefore provide better outcomes for those diagnosed with CS. It is important for clinicians to consider alternative pathology for patients combating metabolic derangements. As depicted in this case, the patient lost 35 lbs. while on a LC diet, despite having hypercortisolism, presumably for months to years prior to the diagnosis of his condition. The patient noted a tendency to gain weight, have elevated blood sugar and blood pressure which prompted him to begin self-treatment with increasingly strict carbohydrate restriction. The patient was able to keep his symptoms of hypercortisolism managed, potentially making the diagnosis difficult for his team of clinicians. From a diagnostic perspective, it’s important to understand that strict dietary adherence can have profound impacts on even the most severe hormonal pathology. Ultimately, this case serves as a reminder of the power of nutrition to address metabolic derangements and simultaneously as a reminder to diagnosticians to never rely on lack of dietary adherence as a reason for persistent metabolic symptoms. The reflexive advice to “not gain weight” and “lower salt intake” in retrospect appears both dogmatic and careless. In this case, the patient had seen several doctors and was even hospitalized and yet his disease state remained unclear and the dietary messaging cursory. 5. Conclusions Many chronic diseases, including diabetes, hypertension and obesity, are generally thought to be caused by dietary and lifestyle choices. However, as exemplified in this report underlying medical problems, such as endocrine disorders, can be the cause of such metabolic derangements. It is critical that practitioners consider other causes of metabolic derangements, as assuming that they are due to poor dietary adherence, can allow them to go undiagnosed. While there is extensive literature on LC diets and their effect on the metabolic derangements associated with hypercortisolism, there needs to be further research on LC as an adjunctive therapy to conventional CD treatment. Ultimately, nutrition can have a powerful impact on suppressing, or even reversing metabolic disorders. As depicted in this case study, a LC diet is powerful enough to temporarily suppress symptoms of CD. Author Contributions M.K.D., E.-C.P.-M. and T.K. equally contributed to this case report. All authors have read and agreed to the published version of the manuscript. Funding This research received no external funding. Institutional Review Board Statement Not applicable. Informed Consent Statement Written informed consent has been obtained from the patient to publish this paper. Data Availability Statement The data presented in this study are available in article. Acknowledgments We would like to thank our patients and the Society of Metabolic Health Practitioners. Conflicts of Interest T.K. is an unpaid member of the Board of Directors of the Society of Metabolic Health Practitioners and a producer of podcasts on health and nutrition, with all proceeds donated to humanitarian charities; his spouse has ownership interest in a food company. The other author reports no conflicts of interest. References Nieman, L.K. UpToDate. Available online: https://www.uptodate.com/contents/measurement-of-cortisol-in-serum-and-saliva?search=cortisol%20level&source=search_result&selectedTitle=1~150&usage_type=default&display_rank=1 (accessed on 27 September 2022). Feelders, R.; Sharma, S.; Nieman, L. Cushing’s Syndrome: Epidemiology and Developments in Disease Management. Clin. Epidemiol. 2015, 7, 281. [Google Scholar] [CrossRef] [PubMed] Guaraldi, F.; Salvatori, R. Cushing Syndrome: Maybe Not so Uncommon of an Endocrine Disease. J. Am. Board Fam. 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Objective: The first-line treatment for Cushing’s disease is transsphenoidal surgery, after which the rates of remission are 60 to 80%, with long-term recurrence of 20 to 30%, even in those with real initial remission. Drug therapies are indicated for patients without initial remission or with surgical contraindications or recurrence, and ketoconazole is one of the main available therapies. The objective of this study was to evaluate the safety profile of and the treatment response to ketoconazole in Cushing’s disease patients followed up at the endocrinology outpatient clinic of a Brazilian university hospital. Patients and methods: This was a retrospective cohort of Cushing’s disease patients with active hypercortisolism who used ketoconazole at any stage of follow-up. Patients who were followed up for less than 7 days, who did not adhere to treatment, or who were lost to follow-up were excluded. Results: Of the 172 Cushing’s disease patients who were followed up between 2004 and 2020, 38 received ketoconazole. However, complete data was only available for 33 of these patients. Of these, 26 (78%) underwent transsphenoidal surgery prior to using ketoconazole, five of whom (15%) had also undergone radiotherapy; seven used ketoconazole as a primary treatment. Ketoconazole use ranged from 14 days to 14.5 years. A total of 22 patients had a complete response (66%), three patients had a partial response (9%), and eight patients had no response to treatment (24%), including those who underwent radiotherapy while using ketoconazole. Patients whose hypercortisolism was controlled or partially controlled with ketoconazole had lower baseline 24-h urinary free cortisol levels than the uncontrolled group [times above the upper limit of normal: 0.62 (SD, 0.41) vs. 5.3 (SD, 8.21); p < 0.005, respectively] in addition to more frequent previous transsphenoidal surgery (p < 0.04). The prevalence of uncontrolled patients remained stable over time (approximately 30%) despite ketoconazole dose adjustments or association with other drugs, which had no significant effect. One patient received adjuvant cabergoline from the beginning of the follow-up, and it was prescribed to nine others due to clinical non-response to ketoconazole alone. Ten patients (30%) reported mild adverse effects, such as nausea, vomiting, dizziness, and loss of appetite. Only four patients had serious adverse effects that warranted discontinuation. There were 20 confirmed episodes of hypokalemia among 10/33 patients (30%). Conclusion: Ketoconazole effectively controlled hypercortisolism in 66% of Cushing’s disease patients, being a relatively safe drug for those without remission after transsphenoidal surgery or whose symptoms must be controlled until a new definitive therapy is carried out. Hypokalemia is a frequent metabolic effect not yet described in other series, which should be monitored during treatment. Introduction Cushing’s disease (CD) results from a pituitary tumor that secretes adrenocorticotropic hormone (ACTH), which leads to chronic hypercortisolism. It is a potentially fatal disease with high morbidity and a mortality rate of up to 3.7 times than that of the general population (1–4) associated to several clinical–metabolic disorders caused by excess cortisol and/or loss of circadian rhythm (5). In general, its management is a challenge even in reference centers (6, 7). Transsphenoidal surgery (TSS), the treatment of choice for CD, results in short-term remission in 60 to 80% of patients (8). However, recurrence rates of 20 to 30% are found in long-term follow-up, even in those with clear initial remission (9). Drug therapies can help control excess cortisol in patients without initial remission, in cases of recurrence, and in those with contraindications or high initial surgical risk (10). Nevertheless, specific drugs that act on the pituitary adenoma, which could directly treat excess ACTH, have a limited effect, and only pasireotide is approved for this purpose in Brazil (11, 12). In this scenario, adrenal steroidogenesis blockers are important. One such off-label medication is the antifungal drug ketoconazole, a synthetic imidazole derivative that inhibits the enzymes CYP11A1, CYP17, CYP11B2, and CYP11B1. Because of its hepatotoxicity and the availability of other drugs, it has been withdrawn from the market in several countries (13). In Europe, it is still approved for use in CD, although in the United States, it is recommended for off-label use almost in CD (14–16). Due to the potential benefits for hypercortisolism, ketoconazole has been replaced by levoketoconazole, which the European Union has recently approved for CD with a lower expected hepatotoxicity (17). Thus, when adrenal inhibitors are used as an alternative treatment for CD, information about the outcomes of drugs such as ketoconazole are important. Clinical studies on these effects in CD are scarce, mostly retrospective, multicenter, or from developed countries (14, 18). A recent meta-analysis on the therapeutic modalities for CD included only four studies (246 patients) that evaluated urinary cortisol response as a treatment outcome and eight studies (366 patients) describing the prevalence of some side effects: change in transaminase activity, digestive symptoms, skin rash, and adrenal insufficiency. Hypokalemia was not mentioned in this meta-analysis (19). The objective of this study was to evaluate the safety profile of and treatment response to ketoconazole in CD patients followed during a long term in the endocrinology outpatient clinic of a Brazilian university hospital. Patients and methods Patients We retrospectively evaluated 38 patients (27 women) diagnosed with CD. These patients, whose treatment included ketoconazole at any time between 2004 and 2020, are part of a prospective cohort series from the Hospital de Clínicas de Porto Alegre neuroendocrinology outpatient clinic. The diagnostic criteria for hypercortisolism were based on high 24-h urinary free cortisol levels (24-h UFC) in at least two samples, non-suppression of serum cortisol after low-dose dexamethasone testing (>1.8 µg/dl), and/or loss of cortisol rhythm (midnight serum cortisol >7.5 µg/dl or midnight salivary cortisol >0.208 nmol/L). CD was diagnosed by normal or elevated ACTH levels, evidence of pituitary adenoma >0.6 cm on magnetic resonance image (MRI), and ACTH central/periphery gradient on inferior petrosal sinus catheterization when MRI was normal or showed an adenoma <0.6 cm. CD was considered to be in remission after the improvement of hypercortisolism symptoms or clinical signs of adrenal insufficiency, associated with serum cortisol within reference values, normalization of 24-h UFC and/or serum cortisol <1.8 μg/dl at 8 am after 1 mg dexamethasone overnight, and/or normalization of midnight serum or salivary cortisol. In patients with active disease, to evaluate the ketoconazole treatment response, 24-h UFC was used as a laboratory parameter, as recommended in similar publications (14, 16, 20, 21), but in some cases, we considered elevated late night salivary cortisol and/or 1 mg dexamethasone overnight cortisol (even with normal 24-h UFC), given the greater assessment sensitivity seen through these two methods in the detection of early recurrence when compared with 24-h UFC (22). Inclusion criteria We included patients with CD and active hypercortisolism who used ketoconazole either as primary treatment, after TSS without hypercortisolism remission, or after a recurrence. Exclusion criteria We excluded patients with CD and active hypercortisolism who used ketoconazole but had <7 days of follow-up, irregular outpatient follow-up, treatment non-adherence, and incomplete medical records or those who were lost to follow-up. Evaluated parameters Prior to ketoconazole treatment, all patients underwent an assessment of pituitary function and hypercortisolism, including serum cortisol, ACTH, 24-hour UFC, cortisol suppression after 1 mg dexamethasone overnight, midnight serum cortisol, and/or midnight salivary cortisol. The evaluated parameters were sex, age at diagnosis, weight, height, prevalence and severity of hypertension and DM, pituitary tumor characteristics, prior treatment (surgery, radiotherapy, or other medications), symptoms at disease onset, biochemical tests (renal function, hepatic function, and lipid profile), number of medications used to treat associated comorbidities, data on medication tolerance, and reasons for discontinuation, when necessary. The clinical parameters observed during treatment were control of blood pressure and hyperglycemia, anthropometric measurements (weight, height, and body mass index), jaundice, and any other symptoms or adverse effects reported by patients. The biochemical evaluation included fasting glucose, glycated hemoglobin, lipid profile (total cholesterol, high-density lipoprotein, low-density lipoprotein, and triglycerides), markers of liver damage (transaminases, bilirubin, gamma-glutamyl transferase, and alkaline phosphatase), electrolytes (sodium and potassium), and renal function (creatinine and urea). Hypecortisolism was accessed preferentially by 24-h UFC, however, late-night salivary cortisol and cortisol after 1 mg overnight dexamethasone could also be used. Study design This retrospective cohort study included patients with CD who were followed up at the Hospital de Clínicas de Porto Alegre Endocrinology Division, with their medical records from the first outpatient visit and throughout clinical follow-up collected. This study was approved by the Hospital de Clínicas de Porto Alegre Research Ethics Committee (number 74555617.0.0000.5327). Outcomes Hypercortisolism was considered controlled when the 24-h UFC and/or late-night salivary cortisol (LNSC) and/or overnight 1 mg dexamethasone suppression test (DST) levels were normalized in at least two consecutive assessments. Hypercortisolism was considered partially controlled when there was a 50% over-reduction in 24-h UFC and/or LNSC and/or DST levels but still above normal. A reduction lower than 50% in these parameters was considered as non-response. We also assessed the ketoconazole doses that resulted in 24-h UFC normalization, maximum dose, medication tolerance, adverse effects, and changes in liver, kidney, and biochemical function. Due to the characteristics of this study, these outcomes were periodically evaluated in all patient consultations, which occurred usually every 2 to 4 months. Data collection This retrospective cohort evaluated outpatient medical records and any tests indicated by the attending physician as a pragmatic study. Ketoconazole use followed the department’s care protocol, which is based on national and international guidelines (4), and all patients received a similar care routine: the recommended initial prescription was generally taken in two to six doses at 100 to 300 mg/day. It was then increased by 200 mg every 2 to 4 months until hypercortisolism was controlled or side effects developed, especially those related to liver function. The maximum prescription was 1,200 mg/day. Clinical follow-up of these patients was performed 30 days after starting the medication and every 2–4 months thereafter (23). Clinical, anthropometric, laboratory, and other exam data were collected through a review of the hospital’s electronic medical records for the entire follow-up period. Data from the first and last consultation were considered in the final analysis of all parameters. Statistical analysis Baseline population characteristics were described as mean and standard deviation (SD) or median with interquartile ranges (25–75) for continuous variables. The chi-square test was used to compare qualitative variables, and Student’s t-test or ANOVA was used to compare the quantitative variables. The Mann–Whitney U-test was used for unpaired data. P-values <0.05 were considered significant. Statistical analysis was performed in SPSS 18.0 (SPSS Inc., Chicago, IL, USA) and R package geepack 1.3-1. Results Treatment with ketoconazole was indicated for 41 of the 172 CD patients. In 3/41 patients, ketoconazole was unallowed due to concomitant liver disease, and 38 received ketoconazole during CD treatment between 2004 and 2020. Of these, five were excluded due to insufficient data to determine the response to ketoconazole (short treatment time, irregular follow-up, incomplete medical records, or lost to follow-up). The baseline characteristics of every sample are shown in Table 1. Thus, 33/41 patients were included in the final analysis. The patients were predominantly women (84.2%) and white (89.5%); 11 had microadenoma, 15 had macroadenoma, and 11 had no adenoma visualized. In 12/33 patients, pituitary imaging was not performed immediately before starting ketoconazole. Hypertension was observed in 26 patients (78%) and DM in 12 patients (36%). The mean age at CD diagnosis was 31.7 years. Table 1 TABLE 1 Baseline clinical data of Cushing’s disease patients treated with ketoconazole. Of the 33 patients with complete data, 26 (78%) underwent TSS prior to starting ketoconazole, five of whom (15%) had also undergone radiotherapy. Thus, seven patients used ketoconazole as primary treatment since performing a surgical procedure was impossible at that time. Of these, four had no response to ketoconazole, one had a partial response, and two had a complete response. At follow-up, four of these patients underwent their first TSS, and three continued the ketoconazole therapy, achieving full UFC control. Among those who used ketoconazole after TSS (n = 26), 20 had a complete response, two had a partial response, and four had no response. Figure 1 shows the study flow chart and patient distribution throughout the treatment. Figure 1 FIGURE 1 Flowchart of ketoconazole treatment in Cushing's disease patients. Individual patient data are described in Table 2. The duration of ketoconazole use ranged from 14 days (in one patient who used it pre-TSS) to 14.5 years. The total follow-up time of the 22 patients with controlled CD ranged from 3 months to 14.5 years, with a mean of 5.33 years and a median of 4.8 years. Table 2 TABLE 2 Individual data. Therapeutic response Relative therapeutic response data are described in Table 3. Patients whose hypercortisolism was controlled or partially controlled with ketoconazole had lower baseline 24-h UFC than the uncontrolled group [times above the upper limit of normal: 0.62 (SD, 0.41) vs. 5.3 (SD, 8.21); p < 0.005, respectively], in addition to more frequent prior TSS (p < 0.04). In some patients (4/33), 24-h UFC was in the normal range at the beginning of ketoconazole therapy, but they were prescribed with the medication due to the clinical recurrence of CD associated to cortisol non-suppression after 1 mg dexamethasone overnight and/or abnormal midnight salivary or serum cortisol. Table 3 TABLE 3 Baseline characteristics of Cushing’s disease patients according to therapeutic response to ketoconazole. Figure 2 shows that the prevalence of uncontrolled patients remained stable over time (approximately 30%) despite dose adjustments or association with other drugs, which led to no differences. When analyzing only the results of the last follow-up visit (eliminating fluctuations during follow-up), 22 patients had a complete response (66%), three patients had a partial response (9%), and eight patients had no response to ketoconazole treatment (24%), which includes patients who underwent radiotherapy during ketoconazole treatment. Figure 2 FIGURE 2 Prevalence of controlled hypercortisolism during follow-up of Cushing's disease patients treatesd with ketoconazole. During follow-up, no significant differences were found in blood pressure control or in dehydroepiandrosterone sulfate, cortisol, ACTH, or glucose levels. Worsening of hypertension control was observed in association with hypokalemia in some cases, as described in side effects. The ketoconazole doses ranged from 100 to 1,200 mg per day, and there were no significant dose or response differences between the groups (Table 4). Figure 3 shows the patients, their dosages, and 24-h UFC control at the first and last consultation, showing a trend toward hypercortisolism reduction in approximately 70% of the cohort (25 of 33). Only four patients used doses lower than 300 mg at the end of follow-up. One of them used before TSS and suspended its use after surgery. One patient, who has already undergone radiotherapy, discontinued ketoconazole due to intolerance, despite adequate control of hypercortisolism. Another one, who had also undergone radiotherapy, was lost to follow-up when it was controlled using 100 mg daily, and one remained controlled using 200 mg, without previous radiotherapy. Table 4 TABLE 4 Final dose of ketoconazole used in patients with Cushing’s disease. Figure 3 FIGURE 3 First and last consultation 24çhour UFC results vs. ketoconazole dosage in Cushing's disease patients. Side effects Regarding adverse effects (Table 5), there was no significant difference between the controlled/partially controlled group and the uncontrolled group regarding liver enzyme changes or drug intolerance. Mild adverse effects, including nausea, vomiting, dizziness, and loss of appetite, occurred in 10 patients (30%). Only four patients had serious adverse effects that warranted discontinuing the medication. In two cases, ketoconazole was discontinued due to a significantly acute increase in liver enzymes (drug-induced hepatitis) during the use of 400 and 800 mg of ketoconazole. Non-significant elevation of transaminases (up to three times the normal value) was observed in three cases. A slight increase in gamma-glutamyltransferase occurred in six patients. In these nine patients with elevated liver markers, the daily dose ranged from 400 to 1,200 mg. None of those with mild increases in liver markers needed to discontinue ketoconazole. Table 5 TABLE 5 Adverse effects of ketoconazole in Cushing’s disease patients treated with ketoconazole. One female patient developed pseudotumor cerebri syndrome, which was treated with acetazolamide. She did not need to discontinue ketoconazole, having used it for more than 10 years without new side effects and achieving complete control of hypercortisolism (24). Another patient became pregnant during follow-up while using the medication, but no maternal or fetal complications occurred (25). Hypokalemia was also observed during follow-up. Twenty episodes of reduced potassium levels occurred in 10 patients over the course of treatment. Of these episodes, six occurred in controlled patients, three in partially controlled patients, and 11 in uncontrolled patients (Table 6). The hypokalemia was managed with spironolactone (25 to 100 mg) and oral potassium supplementation. Table 6 TABLE 6 Characteristics of Cushing’s disease patients who developed hypokalemia during ketoconazole treatment. Ketoconazole and associations Of the patients who used an association of cabergoline and ketoconazole, one did so since the beginning of follow-up, while another nine were prescribed cabergoline during follow-up due to non-response to ketoconazole alone. Of these 10 patients, two did not start the medication due to problems in obtaining the drug. Thus, in two of the nine patients on the maximum tolerated dose of ketoconazole or who could not tolerate a higher dose due to hepatic enzymatic changes, 1.5–4.5 mg of cabergoline per week was associated. In patients not controlled with ketoconazole plus cabergoline, mitotane (two patients) or pasireotide (two patients) was added. Only two of nine patients responded to the combination of cabergoline and ketoconazole. Data on these associations are shown in Table 7. Table 7 TABLE 7 Effects of associating cabergoline with ketoconazole in Cushing’s disease patients. Considering that one of the indications for the treatment of hypercortisolism may be complementary to radiotherapy, we analyzed the eight patients who underwent radiotherapy after transsphenoidal surgery. In these patients, doses of ketoconazole from 200 to 1,200 mg were used, and in six patients there was a normalization of the UFC in 1 to 60 months of treatment. Thus, the association of ketoconazole with radiotherapy was effective in normalizing the 24-h UFC in 75% of cases. Clinical follow-up New therapeutic approaches were attempted in some patients during follow-up: radiotherapy (eight patients), new TSS (five patients), and bilateral adrenalectomy (four patients). At the end of this analysis, 11 patients remained on ketoconazole, all with controlled hypercortisolism. Among the 11 patients who were not fully controlled by the last visit, five were using ketoconazole as pre-TSS therapy and underwent TSS as soon as possible, while three others underwent radiotherapy and two underwent bilateral adrenalectomy. One patient was lost to follow-up. Discussion According to the current consensus about CD, drug treatment should be reserved for patients without remission after TSS, those who cannot undergo surgical treatment, or those awaiting the effects of radiotherapy (4, 16). Drugs available in this context may act as adrenal steroidogenesis blockers (ketoconazole, osilodrostat, metyrapone, mitotane, levoketoconazole, and etomidate), in pituitary adenoma (somatostatinergic receptor ligands—pasireotide), dopamine receptor agonists (cabergoline), or glucocorticoid receptor blockers (mifepristone) (16, 26). Among these alternatives, the drug of choice still cannot be determined. Thus, the best option must be established individually, considering aspects such as remission potential, safety profile, availability, cost, etc. (16, 27, 28). For over 30 years, ketoconazole has been prescribed off-label for CD patients with varied rates of remission of hypercortisolism, and it can be used in monotherapy or associated with other drugs (29, 30). The Brazilian public health system does not provide drugs for the treatment of CD, and among medications with a better profile for controlling hypercortisolism, such as osilodrostat, levoketoconazole, and pasireotide, only pasireotide has been approved by the national regulatory authority (ANVISA). Due to such pragmatic considerations, ketoconazole is among the most commonly used drugs in our health system, whether recently associated or not with cabergoline (7). In this cohort, the most prevalent response type was complete (66%). Since 75% of the CD patients who used ketoconazole had a complete or partial response, there was a clear trend towards improvement in hypercortisolism. When only those who used ketoconazole post-TSS were evaluated, the rate of control increased to 76%. We found that patients with a higher initial 24-h UFC tended to have less control of excess cortisol, a difference that was not observed when analyzing ketoconazole dose or follow-up time. In our series and at the prescribed doses, the combination of cabergoline and ketoconazole was not effective in the management of hypercortisolism since only two of nine patients (22%) had their 24-hour UFC normalized. However, it should be observed that this association was used in patients who had more severe CD and, consequently, were less likely to have a favorable response. The effects of cabergoline in CD patients remain controversial, although some studies have shown promising responses (31, 32). Previous reviews found that the efficacy of ketoconazole for hypercortisolism control was quite heterogeneous, ranging from 14 to 100% in 99 patients (33, 34). Our cohort’s response rate was lower than that of Sonino et al. (89%) (20) but higher than that of a multicenter cohort by Castinetti et al. (approximately 50%) (14). Regarding other smaller series (35–37) our results reinforce some findings that demonstrate a percentage of control greater than 50% of the cases. Our analyses showed a trend toward a response that continued, with some oscillations, over time. The rate of uncontrolled patients remained stable over time (approximately 30%), regardless of association with other drugs (cabergoline, mitotane, or pasireotide) or dose adjustments. Speculatively, it would appear that patients who respond to ketoconazole treatment would show some type of response as soon as therapy begins. Our cohort has the longest follow-up time of any study on ketoconazole use in CD, nearly 15 years. Our results demonstrate that patients who benefit from ketoconazole (i.e., control of hypercortisolism and associated comorbidities) can safely use it for a long term since those who did not experience liver enzyme changes at the beginning of treatment also had no long-term changes. Another relevant information for clinical practice is the result of treatment with ketoconazole associated with radiotherapy, which demonstrated normalizing the 24-h UFC in 75% of cases, a finding that reinforces the use of this therapeutic combination, especially in cases that are more resistant to different treatment modalities. As described in the literature, adverse effects, such as nausea, vomiting, dizziness, headache, loss of appetite, and elevated transaminases, are relatively frequent (38). In our cohort, 10 patients (30%) had mild adverse effects, and four (12%) had more serious adverse effects requiring discontinuation. In other studies, up to 20% of patients required discontinuation due to side effects (14). We documented 20 episodes of hypokalemia during ketoconazole treatment, some with worsening blood pressure control. In most cases, hypokalemia has occurred in association with the use of diuretic drugs, which may have potentiated potassium spoliation, reinforcing the need of stringent surveillance in hypertensive Cushing’s disease patients using this combination. It can also result from the enzymatic blockade that could lead to the elevation of adrenal mineralocorticoid precursors (pex. deoxycorticosterone), with consequent sodium retention and worsening hypertension. Although it has not been analyzed in other series with ketoconazole, this side effect has been observed in patients who received other adrenal-blocking drugs, such as osilodrostat and metyrapone (16). This alteration seems to be transient in some patients; in our series, it was managed by suspending drugs that could worsen hypokalemia and introducing spironolactone and/or potassium supplementation. Hypokalemia may also result from continuing intense adrenal stimulation by ACTH and changes in the activity of the 11-beta-hydroxysteroid dehydrogenase enzyme, which increase the mineralocorticoid activity of cortisol, as observed in patients with severe hypercortisolism in uncontrolled CD (39). Hypogonadism occurred in one male patient. In two adolescent patients (one female and one male), hypercortisolism was effectively controlled without altering the progression of puberty. As described in other cohorts, this effect was expected due to the high doses, which block adrenal and testicular androgen production (20). Thus, our findings confirm previous reports in the literature and add important information about the side effects and safety of long-term ketoconazole use in CD treatment. Our data reinforce the current recommendations about ketoconazole for recurrent cases or those refractory to surgery, including proper follow-up by an experienced team specializing in evaluating clinical and biochemical responses and potential adverse effects (7, 18, 40). Despite the severity of many of our CD patients, no ketoconazole-related death occurred during follow-up, including long-term observation. On the other hand, no patient progressed to definitive remission of hypercortisolism, even after many years of treatment with ketoconazole. Conclusions In our cohort of patients, ketoconazole proved to be an effective and safe alternative for CD treatment, although it can produce side effects that require proper identification and management, allowing effective long-term treatment. We found side effects that have been rarely described in the literature, including hypokalemia and worsening hypertension, which require specific care and management. Thus, ketoconazole is an effective alternative for CD patients who cannot undergo surgery, who do not achieve remission after pituitary surgery, or who have recurrent hypercortisolism. 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 the Hospital de Clínicas de Porto Alegre Research Ethics Committee. Written informed consent for participation was not required for this study in accordance with the national legislation and the institutional requirements. Author contributions CV and MAC created the research format. CV, RBM, and MCBC realized the search on medical records. CV performed the statistical analysis. MAC, ACVM, and TCR participated in the final data review and discussion. ACVM participated in the final data review and discussion as volunteer collaborator. All authors contributed to the article and approved the submitted version. Funding This work was supported by the “Coordenação de Aperfeiçoamento de Pessoal de Nı́vel Superior” (CAPES), Ministry of Health - Brazil, through a PhD scholarship; and the Research Incentive Fund (FIPE) of Hospital de Clı́nicas de Porto Alegre. Acknowledgments The authors would like to thank the HCPA Research and Graduate Studies Group (GPPG) for the statistical technical support provided by Rogério Borges. We also thank the Research Incentive Fund of Hospital de Clínicas de Porto Alegre and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), by funds applied. We also thank the Graduate Program in Endocrinology and Metabolism (PPGEndo UFRGS) for all the support in the preparation of this research. 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. 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Best Pract Res Clin Endocrinol Metab (2021) 35(1):101490. doi: 10.1016/j.beem.2021.101490 PubMed Abstract | CrossRef Full Text | Google Scholar Keywords: Cushing’s disease, Cushing’s syndrome, hypercortisolism, treatment, ketoconazole Citation: Viecceli C, Mattos ACV, Costa MCB, Melo RBd, Rodrigues TdC and Czepielewski MA (2022) Evaluation of ketoconazole as a treatment for Cushing’s disease in a retrospective cohort. Front. Endocrinol. 13:1017331. doi: 10.3389/fendo.2022.1017331 Received: 11 August 2022; Accepted: 06 September 2022; Published: 07 October 2022. Edited by: Luiz Augusto Casulari, University of Brasilia, Brazil Reviewed by: Juliana Drummond, Federal University of Minas Gerais, Brazil Monalisa Azevedo, University of Brasilia, Brazil Copyright © 2022 Viecceli, Mattos, Costa, Melo, Rodrigues 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: Mauro Antonio Czepielewski, maurocze@terra.com.br 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.2022.1017331/full

Recordati's Isturisa is expected to launch in the second or third quarter. (Getty) As part of a small 2019 deal, Italian drugmaker Recordati snagged a trio of underperforming Novartis endocrinology meds, including a late-stage candidate for Cushing's disease. Less than a year later, that drug is cleared for market after an FDA green light. The FDA on Friday approved Recordati's Isturisa (osilodrostat) to treat Cushing's disease—a rare disease in which patients' adrenal glands produce too much cortisol—in those who have undergone a prior pituitary gland surgery or are not eligible for one. Isturisa, a cortisol synthesis inhibitor, will come with the FDA's orphan drug designation, providing market exclusivity for seven years, Recordati said (PDF) in a release. The drug is expected to be commercially available in the second or third quarter. The FDA based its review on phase 3 data showing 86% of patients treated with Isturisa showed normal cortisol levels in their urine after eight weeks, compared with 29% of patients treated with placebo, the drugmaker said. Recordati is "actively building its commercial, medical, and market access teams" to accommodate Isturisa's launch through its recently created U.S. endocrinology business unit, it said. The drugmaker will launch the drug with a "comprehensive distribution model" through specialty pharmacies. Novartis, once the owner of Isturisa, turned the asset over to Recordati in 2019 as part of a $390 million offload of some of the Swiss drugmaker's endocrinology portfolio. Recordati received Signifor, long-acting sister Signifor LAR and Isturisa, positioned as a successor drug to Signifor. The purchase included milestone payments tied to Isturisa. Recordati talked up the buy of the Cushing's disease trio as a boon for its rare disease portfolio, calling it a "key and historical milestone" at the time. From https://www.fiercepharma.com/pharma/recordati-scores-fda-nod-for-cushing-s-disease-med-isturisa

Abstract Background Endoscopic endonasal surgery is the main transsphenoidal approach for pituitary surgery in many centers, however few studies compare the endoscopic and microscopic surgical approach with regard to long-term follow-up. This single-center study aimed to compare the two techniques over 15 years. Methods Medical records and magnetic resonance images from 40 patients with primary transsphenoidal surgery for Cushing’s disease at Sahlgrenska University Hospital between 2003 and 2018 were reviewed. Fourteen patients who underwent microscopic surgery and 26 patients who underwent endoscopic surgery were included in this study. Results In the microscopic group, 12 of 14 patients achieved endocrine remission, compared to 19 of 26 patients in the endoscopic group (n. s.). Three patients in each group developed a late recurrence. Complications were seen in 5 patients in the microscopic group and in 8 patients in the endoscopic group (n. s.). No serious complications, such as carotid artery damage, cerebrovascular fluid leakage, epistaxis, or meningitis, occurred in any group. The postoperative hospital stay was shorter in the endoscopic than the microscopic group. Conclusion Endoscopic endonasal surgery for Cushing’s disease showed no difference in remission, recurrence, and complication rates compared to the microscopic approach. The endoscopic group had a shorter postoperative hospital stay than the microscopic group, which in part may be due to the minimal invasiveness of the endoscopic approach. References (0) Cited by (0) Recommended articles (6) Research article Identifying obstructive sleep apnea in patients with epilepsy: A cross-sectional multicenter study Seizure, Volume 100, 2022, pp. 87-94 Show abstract Research article Nursing Home Characteristics Associated with High and Low Levels of Antipsychotic, Benzodiazepine, and Opioid Prescribing to Residents with Alzheimer’s Disease and Related Dementias: A Cross-Sectional Analysis Journal of the American Medical Directors Association, 2022 Show abstract Research article Association between sensory impairments and restricted social participation in older adults: A cross-sectional study Collegian, 2022 Show abstract Research article Percutaneous Intervertebral-Vacuum Polymethylmethacrylate Injection for Foraminal Stenosis with Degenerative Lumbar Scoliosis World Neurosurgery, 2022 Show abstract Research article Predictors of Emergency Department service outcome for people brought in by police: A retrospective cohort study International Emergency Nursing, Volume 63, 2022, Article 101188 Show abstract Research article Interdisciplinary Care Coordination in Chronic Viral Hepatitis C The Journal for Nurse Practitioners, 2022 Show abstract Conflicts of interest The authors have no conflicts of interest. Author statements Conceptualization: D. Farahmand, E. Backlund, O. Ragnarsson and P. Trimpou Data curation: Dan Farahmand, Erica Backlund, J. Carlqvist, T. Skoglund, T. Hallén, O. Ragnarsson, P. Trimpou. Formal Analysis: D. Farahmand, E. Backlund Funding acquisition: D. Farahmand Investigation: D. Farahmand, E. Backlund, O. Ragnarsson and P. Trimpou Methodology: D. Farahmand, E. Backlund, O. Ragnarsson and P. Trimpou Project administration: D. Farahmand, E. Backlund, O. Ragnarsson and P. Trimpou Supervision: D. Farahmand Writing – original draft: Penelope Trimpou Writing – review & editing: E. Backlund, O. Ragnarsson, T. Skoglund, T. Hallén, G. Gudnadottir, J. Carlqvist and D. Farahmand. View full text From https://www.sciencedirect.com/science/article/abs/pii/S1878875022009640

Abstract Background Cushing’s disease (CD) is rare in pediatric patients. It is characterized by elevated plasma adrenocorticotropic hormone (ACTH) from pituitary adenomas, with damage to multiple systems and development. In recent years, genetic studies have shed light on the etiology and several mutations have been identified in patients with CD. Case presentation A girl presented at the age of 10 years and 9 months with facial plethora, hirsutism and acne. Her vision and eye movements were impaired. A quick weight gain and slow growth were also observed. Physical examination revealed central obesity, moon face, buffalo hump, supra-clavicular fat pads and bruising. Her plasma ACTH level ranged between 118 and 151 pg/ml, and sella enhanced MRI showed a giant pituitary tumor of 51.8 × 29.3 × 14.0 mm. Transsphenoidal pituitary debulk adenomectomy was performed and immunohistochemical staining confirmed an ACTH-secreting adenoma. Genetic analysis identified a novel germline GPR101 (p.G169R) and a somatic USP8 (p. S719del) mutation. They were hypothesized to impact tumor growth and function, respectively. Conclusions We reported a rare case of pediatric giant pituitary ACTH adenoma and pointed out that unusual concurrent mutations might contribute to its early onset and large volume. Peer Review reports Background Cushing’s disease (CD) is caused by the overproduction of adrenocorticotropic hormone (ATCH) by pituitary adenomas (PAs). It is rare in children and accounts for approximately 75% of pediatric Cushing’s syndrome from 7 to 17 years of age [1]. Weight gain and facial changes are more common in children than in adults [2]. Growth retardation is also a characteristic of children with hypercortisolemia [3]. Genetic alterations such as somatic USP8, RASD1, TP53 mutations, and germline AIP, MEN1, and CABLES1 mutations have been identified in CD patients [4]. Here we report a case of pediatric invasive pituitary ACTH macroadenoma associated with a novel germline GPR101 (p. G169R) and a somatic USP8 (p. S719del) mutation. Case presentation The girl was born at full term with a length of 48 cm and a weight of 2900 g. Her neuromotor and cognitive development was comparable to those of children of the same age. At the age of 9 years and 4 months she developed plethora, hirsutism, facial acne, rapid weight gain, and increased abdominal circumference. Her skin darkened, and purple striae appeared on thighs and in the armpits. She became dull and less talkative, as indicated by her parents. At 10 years and 3 months, the patient complained of pain around the left orbit with an intensity of 4–5 points on a numerical rating scale (NRS). Five months later bilateral blepharoptosis appeared, with significantly impaired vision of the left eye. Soon both eyes failed to rotate in all directions. On admission the patient was 10 years and 9 months, with a height of 144 cm (90–97th percentile) and a weight of 48 kg (25–50th percentile). Her weight gain was 20 kg, while the height increased by only 2–3 cm in 18 months. Her blood pressure was 115/76mmHg, and her heart rate was 80 bpm. Apart from the signs mentioned above, physical examination revealed central obesity (BMI 23.1 kg/m2), moon face, buffalo hump, supra-clavicular fat pads and bruising at the left fossa cubitalis. Her pupils were 7 mm in diameter and barely reacted to light. There was a fan-shaped visual field defect in the left eye. Her breasts were Tanner stage III and pubic hair was Tanner stage II, although menarche had not yet occurred. The parents and her younger brother at 6 years of age did not have symptoms related to Cushing syndrome, acromegaly or gigantism. There was no family history of pituitary tumor or other endocrine tumors. She had increased midnight serum cortisol (24.35 µg/dL, normal range < 1.8 µg/mL) and 24-hour urine free cortisol (24hUFC) (308.0 µg, normal range 12.3–103.5). The plasma ACTH level ranged from 118 to 151 pg/mL (< 46pg/mL). The 24hUFC was not suppressed (79.2 µg) after 48 h low-dose dexamethasone suppression test (LDDST), but suppressed to 32.8 µg (suppression rate 89.4%) after 48 h high-dose dexamethasone. Sella enhanced MRI showed a giant pituitary tumor measured 51.8 × 29.3 × 14.0 mm with heterogeneous density (Fig. 1). The mass compressed the optic chiasma and surrounded the bilateral cavernous sinus (Knosp 4). Therefore, an invasive giant pituitary ACTH adenoma was clinically diagnosed. The morning growth hormone (GH) was 1.0ng/ml (< 2 ng/ml) and insulin-like growth factor 1 416 ng/ml (88–452 ng/ml). The prolactin (PRL), luteinizing hormone (LH), follicle-stimulating hormone (FSH) and thyroid stimulating hormone (TSH) were all in normal ranges, as well as serum sodium, potassium, blood glucose and urine osmolality. Abdominal ultrasonography revealed a fatty liver. Tests concerning type 1 multiple endocrine neoplasia included serum calcium, phosphate, parathyroid hormone, gastrin and glucagon, which were all unremarkable (Table 1). Fig. 1 Contrast-enhanced coronal (A) and sagittal (B) T1-weighted MRI on admission. The sellar mass measured 51.8 × 29.3 × 14.0 cm (TD × VD × APD) with a heterogeneous density in the enhanced scan. The diaphragma sellea was dramatically elevated, with optic chiasm compressed. The sellar floor was sunken and bilateral cavernous sinus was surrounded (Knosp 4) Full size image Table 1 Laboratory data on admission Full size table Transsphenoidal pituitary debulk adenomectomy was performed immediately due to multiple cranial nerve involvement and the negative results of Sandostatin loading test. A decompression resection was done. The plasma ACTH level declined to 77 pg/ml and serum cortisol 30.2 µg/dl three days after the operation. Vision, pupil dilation, eye movements and blepharoptosis also partially improved. Histopathology and immunohistochemical staining confirmed a densely–granulated corticotroph adenoma (Fig. 2, NanoZoomer S360 digital slide scanner and NDP.view 2.9.25 software, Hamamatsu, Japan). Neither necrosis nor mitotic activity was observed. The immunostaining for somatostatin receptor SSTR2A was positive with a cytoplasmic pattern, while GH, PRL, TSH, FSH, LH and PIT were all negative. The Ki 67 index was found to be 10%. One month after the operation the ACTH level increased to 132 pg/mL again, and the parents agreed to refer their child for radiotherapy to control the residual tumor. Fig. 2 Histopathology and immunohistochemistry staining results of the pituitary tumor. By light microscopy, the tumor cells were mostly basophilic and arranged in papillary architecture. Neither necrosis nor mitotic activity was observed (A hematoxylin-eosin, ×200). Immunohistochemistry staining was positive for ACTH (B immunoperoxidase, ×200) and transcription factor T-PIT (C immunoperoxidase, ×200). Cytoplasmic staining of SSTR2A was observed in around 1/3 tumor cells besides the strong staining of endothelial cells (D immunoperoxidase, ×200). The Ki-67 index was 10% (E immunoperoxidase, ×200). Cytokeratin CAM5.2 was diffusely positive in the cytoplasm (F immunoperoxidase, ×200). The positive control for ACTH and T-PIT was the human anterior pituitary gland, and for SSRT2, Ki-67 and CAM5.2 were cerebral cortex, tonsil and colonic mucosa, respectively Full size image The early onset and invasive behavior of this tumor led to the consideration of whether there was a genetic defect. Genetic studies were recommended for the families and they all agreed and signed the written informed consent forms. Whole exome sequencing (WES) was performed on the patient’s blood sample using an Illumina HiSeq sequencer to an average read depth of at least 90 times per individual. Raw sequence files were mapped to the GRCH37 human reference genome and analyzed using the Sentieon software. The results revealed a germline heterozygous GPR101 gene mutation c.505G > C (p.Gly169Arg), which was subsequently confirmed to be of maternal origin by Sanger sequencing. Meanwhile WES of the tumor tissue identified an additional somatic heterozygous c.2155_2157delTCC (p.S719del) mutation of the USP8 gene . Discussion and conclusions In this report, we described an extremely giant and invasive pituitary ACTH adenoma in a 10-year-old girl. According to Trouillas et al., invasive and proliferative pituitary tumors have a poor prognosis [5]. CD is rare among children, and the fast-growing and invasive nature of the tumor in this case led to the investigation of genetic causes. The somatic USP8 gene mutation has been recently reported to be associated with the pathogenesis of CD [6, 7]. This gene encodes ubiquitin-specific protease 8 (USP8). S718, S719 and P720 are hotspots in different studies [6,7,8,9,10,11,12,13,14]. They are located at the 14-3-3 binding motif, and the mutations disrupt the binding between USP8 and 14-3-3 protein, which leads to increased deubiquitination and EGFR signaling. High levels of EGFR consequently trigger proopiomelanocortin (POMC) transcription and ACTH secretion [6, 7]. The p.S719del mutation has been previously reported and its pathogenicity has been confirmed [7]. Thus, we speculate the p.S719del mutation plays a role in this patient with CD. It is noteworthy that in our case, the pituitary corticotrophin adenoma was extremely giant and bilaterally invasive. USP8 mutations have been found in 31% of pediatric CD patients [10]. It is well known that microadenomas are most common in adult and pediatric CD patients. Previously, the Chinese and Japanese cohorts observed smaller sizes of USP8-mutated PAs than wild-type PAs [7, 9]. The Chinese cohort also reported a lower rate of invasive adenomas in USP8-mutated PAs [7]. This may be explained by the finding that UPS8 mutations did not significantly promote cell proliferation more than the wild-type ones [6]. Other cohorts suggested no difference in tumor size or invasiveness between USP8-mutated and wild-type PAs [8, 10, 12,13,14], which may be partially explained by the differences in sample sizes and ethnic backgrounds. Owing to the lack of evidence of USP8 mutations significantly contributing to tumor growth and invasiveness, additional pathogenesis should be investigated in this case. The p.Gly169Arg mutation of the GPR101 gene has not been reported in patients with pituitary tumors. In silico predictions were performed using Polyphen-2, Mutation Taster and PROVEAN, and all of the programs reported it to be pathogenic. The GPR101 gene encodes an orphan G protein-coupled receptor (GPCR) and microduplication encompassing the gene has been proven to be the cause of X-linked acrogigantism (XLAG) [15]. XLAG is characterized by the early onset of pituitary GH-secreting macroadenomas. Point mutations of GPR101 have been found in patients with PAs that are mostly GH-secreting [15,16,17]. Although their prevalence is very low, an in vitro study supported the pathogenic role of p.E308D, the most common mutation of GPR101. This led to increased cell proliferation and GH production in rat pituitary GH3 cells [15]. Rare cases of PRL, ACTH or TSH-secreting PAs with GPR101 variants were also documented [16, 18]. To date, there have been five cases of ACTH-secreting PAs with four different germline GPR101 mutations: two cases of p.E308D, p.I122T, p.T293I and p.G31S, although in silico predictions and in vitro evaluations using AtT-20 cells have respectively determined the latter two mutations to be non-pathogenic [16, 18]. These patients were mainly children and young adults. Unlike pituitary GH-secreting tumors, the role of GPR101 mutations in the pathophysiology of CD is still questionable. Trivellin et al. demonstrated no statistically significant difference in GPR101 expression between corticotropinomas and normal human pituitaries. No significant correlation between GPR101 and POMC expression levels was found neither [18]. Given the evidences above, we hypothesize that the somatic USP8 mutation is responsible for the overexpression of ACTH in this CD girl while the germline GPR101 mutation contributes to the early onset and fast-growing nature of the tumor. Similarly, a 27-year-old woman with Nelson’s syndrome originally considered to be associated with a germline AIP variant (p.Arg304Gln) was recently reported to have a somatic USP8 mutation. The patient progressed rapidly and underwent multiple transsphenoidal surgeries [19]. Since germline AIP mutations are more commonly seen in GH-secreting PAs [20], the authors proposed that the USP8 mutation might have shifted the tumor towards ACTH-secreting [19]. Further investigations into the pathogenicity of GPR101 p.Gly169Arg and AIP p.Arg304Gln mutations are required to support the hypothesis. In summary, we report a novel germline GPR101 and somatic USP8 mutation in a girl with an extremely giant pituitary ACTH adenoma. The concurrent mutations may lead to the growth and function of the tumor, respectively. Further investigations should be carried out to verify the role of the concurrent mutations in the pathogenesis of pediatric CD. Availability of data and materials The WES data of the blood sample of the patient is available in the NGDC repository (https://ngdc.cncb.ac.cn/gsa-human/) and the accession number is HRA002396. Any additional information is available from the authors upon reasonable request. 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Eur J Endocrinol. 2018;178(1):57–63. CAS Article Google Scholar Tatsi C, Stratakis CA. The Genetics of Pituitary Adenomas. J Clin Med. 2019;9(1). Download references Acknowledgements We thanked Dr. Xiaohua Shi and Dr. Yu Xiao from the Department of Pathology, Peking Union Medical College Hospital for their expertise in pituitary pathology and critical help in accomplishment of our manuscript. Funding This research was supported by “The National Key Research and Development Program of China” (No. 2016YFC0901501), “CAMS Innovation Fund for Medical Science” (CAMS-2017-I2M–1–011). They mainly covered the fees for genetic analysis and publications. Author information Authors and Affiliations Department of Pediatrics, Peking Union Medical College Hospital, Chinese Academy of Medical Science and Peking Union Medical College, Beijing, 100730, China Xu-dong Bao Department of Endocrinology, Key Laboratory of Endocrinology of National Health Commission, Peking Union Medical College Hospital, Chinese Academy of Medical Science and Peking Union Medical College, Beijing, 100730, China Lin Lu, Hui-juan Zhu, Xiao Zhai, Yong Fu, Feng-ying Gong & Zhao-lin Lu Department of Neurosurgery, Peking Union Medical College Hospital, Chinese Academy of Medical Science and Peking Union Medical College, Beijing, 100730, China Yong Yao, Ming Feng & Ren-zhi Wang Contributions XB and LL contributed to the study design and manuscript writing. HZ and FG performed genetic analysis. XZ and YF collected the clinical data. YY, MF and RW provided the tumor tissue and histopathology data. ZL revised the manuscript. All authors have read and approved the final manuscript. Corresponding author Correspondence to Lin Lu. Ethics declarations Ethics approval and consent to participate This study was approved by the Ethics Committee of Peking Union Medical College Hospital. The parents of the patient provided written informed consent for research participation. Consent for publication The parents of the patient provided written informed consent for the publication of indirectly identifiable data in this research. Competing interests The authors declare that they have no competing interests. Additional information Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. 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Abstract Cushing's disease causes numerous metabolic disorders, cognitive decline, and sarcopenia, leading to deterioration of the general health in older individuals. Cushing's disease can be treated with transsphenoidal surgery, but thus far, surgery has often been avoided in older patients. We herein report an older woman with Cushing's disease whose cognitive impairment and sarcopenia improved after transsphenoidal surgery. Although cognitive impairment and sarcopenia in most older patients show resistance to treatment, our case indicates that normalization of the cortisol level by transsphenoidal surgery can be effective in improving the cognitive impairment and muscle mass loss caused by Cushing's disease. References (27) 1. Lindholm J, Juul S, Jorgensen JO, et al. Incidence and late prognosis of Cushing's syndrome: a population-based study. J Clin Endocrinol Metab 86: 117-123, 2001. 2. Starkman MN. 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Abstract Introduction Stress-related brain disorders can be associated with glucocorticoid disturbance and hippocampal alteration. However, it remains largely unknown how cortisol affects the structure and function of hippocampus. Cushing's disease (CD) provides a unique “hyperexpression model” to explore the effects of excessive cortisol on hippocampus as well as the relation between these effects and neuropsychological deficits. Methods We acquired high-resolution T1-weighted and resting-state functional magnetic resonance imaging in 47 CD patients and 53 healthy controls. We obtained the volume and functional connectivity of the hippocampal rostral and caudal subregions in both groups. Relationships between hippocampal alterations, neuroendocrine, and neuropsychological assessments were identified. Results Relative to control subjects, the CD patients had smaller volumes of all four hippocampal subregions. Furthermore, whole brain resting-state functional connectivity analyses with these four different hippocampal regions as seeds revealed altered hippocampal functional connectivity with high-order networks, involving the DMN, frontoparietal, and limbic networks in CD patients. The intrinsic hippocampal functional connectivity was associated with the quality of life of the CD patients. Conclusions Our findings elucidate the cumulative effect of excess cortisol on the morphology and function of hippocampus and reinforce the need for effective interventions in stress-related brain disease to halt potential hippocampal damage. 1 INTRODUCTION Converging evidence has pointed to a strong linkage between the cortisol and human brain and stress-related neuropsychiatry disorders, such as major depression disorder and posttraumatic stress disorder (de Kloet et al., 2005). However, it remains to be established how this stress hormone influences specific brain structures and functions, particularly in humans, which is of particular importance for both treatment of stress-related disorders and research on cortisol effects in the brain. Cushing's disease (CD) is caused by an adrenocorticotropic hormone pituitary adenoma and characterized by chronic hypercortisolism. This condition is therefore a unique and natural “hyperexpression model” to investigate the chronic effects of cortisol on brain physiology and cognition (Zhang et al., 2021). By applying multimodal neuroimaging techniques to CD patients, previous studies have observed that chronic hypercortisolism could cause a number of abnormalities in various brain phenotypes. Among these neural changes of CD patients, hippocampal anomalies are the most replicated findings. Studies on CD patients report hippocampal changes that converge with morphologic alterations such as reduction in volume (Burkhardt et al., 2015; Toffanin et al., 2011). Moreover, abnormal cerebral blood flow and glucose metabolism in hippocampus have also been found in CD patients. Both structural and functional alterations in the hippocampus might contribute to the psychotic symptoms in CD patients (Frimodt-Møller et al., 2019). However, it is well established that psychosis is better described as a brain connectional diaschisis rather than isolated regional dysfunctions (Matthews & Hampshire, 2016). These current hippocampus-related findings were mainly obtained by voxel-based or regional analyses of brain volume or metabolism properties, and researchers have not determined whether the organizational patterns of hippocampal functional connectivity are disrupted in CD patients. The hippocampus is easily targeted by long-term hypercortisolism because this area is a part of the stress response system and is abundant in mineralocorticoid receptors and glucocorticoid receptors (McEwen et al., 2016). Also recently, studies on macaques and humans have observed that hippocampus is an anatomically and functionally heterogeneous region along the rostral/caudal-dorsal/ventral axis (Schultz & Engelhardt, 2014). Specifically, the rostral hippocampus has connections with prefrontal regions and relates to stress, emotion, and affect. In contrast, the caudal hippocampus mainly connects to sensory cortical areas and performs primarily cognitive functions (Fanselow & Dong, 2010). Therefore, the hippocampus should be studied in a set of separate structures with rostral and caudal hippocampus. Whether the hippocampal subregions exhibit differentially altered connectivity patterns responding to chronic hypercortisolism remains largely unknown. The present study further extends this work by examining the relationship between hippocampal subregions and resting-state functional connectivity in large-scale brain networks, as measured by resting-state fMRI (rs-fMRI) (Park & Friston, 2013). We focus on default mode network (DMN), frontoparietal, and limbic networks, given their involvement in stress related psychiatric illnesses. The first is the DMN, which supports self-related cognitive functions. Complementing the DMN is the frontoparietal network, which supports the cognitive regulation of behavior and emotion. Finally, the limbic networks play a key role in emotion regulation. In this study, first, to explore the structural changes of hippocampal subregions in CD patients, we performed a volumetric MRI analysis of the four subregions (left rostral hippocampus, left caudal hippocampus, right rostral hippocampus, and right caudal hippocampus). Given the known direct neurotoxic effects of cortisol on hippocampus, we predicted that chronic hypercortisolism caused smaller hippocampal volumes in CD patients. Second, we used these four subregions as seed regions separately and mapped whole-brain functional connectivity patterns associated with each subregion to examine alterations in hippocampal functional connectivity in CD patients. Considering the psychiatric symptoms in CD patients, it is reasonable to expect the presence of altered hippocampal functional connectivity with high-order networks. 2 MATERIAL AND METHODS 2.1 Participants A total of 47 participants with a diagnosis of CD and 53 healthy control (HC) subjects were included in this study. The CD patients underwent transsphenoidal surgery at the Department of Neurosurgery, The First Medical Center of Chinese People's Liberation Army (PLA) General Hospital between May 2017 and November 2019. According to the clinical practice guideline (Nieman et al., 2015), CD was diagnosed by experienced endocrinologists and confirmed by postsurgical pathology. The detailed preoperative assessments of diagnostic criteria have been reported in our previous study. HCs were recruited from the local community and were controlled for any history of psychopathology abnormalities. All participants were right-handed and had normal vision and auditory sensation. The study was approved by the local ethics committee of the Chinese PLA General Hospital and written informed consent was obtained from each participant. The data of these 47 CD and 53 HC subjects have been partially used in our previous studies (Wang et al., 2019; Zhang et al., 2021). 2.2 Neuroendocrine and neuropsychological assessment All participants underwent biochemical evaluation to assess their cortisol level. We quantified the levels of 24-h urinary free cortisol (24hUFC, nmol/24h); serum cortisol (nmol/L) at 0:00, 8:00, and 16:00. Cortisol was detected with an ADVIA Centaur Analyzer (Siemens Healthcare Diagnostics, Tarrytown, NY, USA). Cortisol levels at 8:00 as well as 24hUFC were also measured in 51 HC subjects. All participants underwent a comprehensive neuropsychological assessment with an expert psychiatrist, including Self-Rating Depression Scale (SDS), Self-Rating Anxiety Scale (SAS), Mini-mental State Examination (MMSE), and Montreal Cognitive Assessment (MoCA). Moreover, health-related quality of life and neuropsychiatric symptoms of CD patients were evaluated with the Cushing's Quality-of-Life (CushingQoL) questionnaire (Nelson et al., 2013) and Chinese version of the neuropsychiatric inventory (CNPI) (Leung et al., 2001), respectively. 2.3 Image acquisition Structural and functional MRI data were acquired on a 3.0-Tesla MR system (Discovery MR750, General Electric) with an 8-channel head coil. High-resolution structural 3D T1-weighted images were conducted using a sagittal Fast Spoiled Gradient-Echo (FSPGR) sequence with the following parameters: repetition time = 6.7 ms, echo time = 2.9 ms, flip angle = 7°, field of view = 250 × 250 mm2, number of slices = 192, voxel size = 1 × 1 × 1 mm3 with no gap. The functional images were acquired using an echo-planar imaging (EPI) sequence with repetition time = 2000 ms, echo time = 30 ms, flip angle = 90°, thickness/gap = 3.5 mm/0.5 mm, slices = 36, field of view = 224 × 224 mm2, voxel size = 3.5 × 3.5 × 3.5 mm3, number of total volumes = 240. Soft earplugs were used to attenuate scanner noise and head motion was restrained with foam padding. During functional scanning, all participants were requested to keep their eyes closed and stay awake. 2.4 rs-fMRI data preprocessing Preprocessing of the rs-fMRI images was conducted using SPM12 and Data Processing Assistant for Resting-State fMRI (DPABI, http://www.restfmri.net/forum/DPARSF). The first 10 volume of the functional images were removed to avoid initial steady-state problems. Then functional images were spatially realigned to the first image for motion correction, and reslicing for acquisition temporal delay. The head motion of all participants in this study had no more than 2-mm translation or 2° rotation in any direction. Next, functional images were coregistered to each participant's segmented gray matter T1 image, and then spatially normalized to the MNI space, resampled to 3-mm isotropic voxels. Subsequently, the global signal, white matter signal, cerebrospinal fluid signal and 24-motion vectors were regressed from the data. Finally, linear detrending and bandpass filter (0.01−0.08 Hz) were carried out to reduce the effects of low-frequency drift and high-frequency physiological noise. 2.5 Hippocampal functional connectivity The hippocampus has been functionally parcellated into four subregions (left rostral hippocampus, left caudal hippocampus, right rostral hippocampus, and right caudal hippocampus) based on Human Brainnetome Atlas (Fan et al., 2016). On each hippocampal subregion, we performed seed-based functional connectivity analysis. Briefly, hippocampal functional connectivity maps were obtained by computing the Pearson correlation coefficient for each voxel's time course with the average time course inside the region of interest. Notably, the computation was constrained within a gray-matter mask which was generated by thresholding (a threshold of 0.2) a prior gray-matter probability map in SPM12. The resulting correlation coefficients were further converted to z scores using Fisher's r-to-z transform to improve normality. For each subject, we obtained 4 z-score maps indicative of the intrinsic functional connectivity patterns of the four hippocampal subregions. To exclude the possible confounding effect of hippocampal volume in CD patients, we performed a voxel-based morphometry analysis on structural MRI images and took the volume of hippocampal subregions as a covariate in the functional connectivity statistical analyses. 2.6 Statistical analysis All demographic and clinical variables including neuroendocrine and neuropsychological scores were compared by two-sample t-tests. Sex composition of the two groups was compared using a Pearson's chi-square test (two-tailed). To explore differences in hippocampal functional connectivity between CD patients and HCs, general linear models were performed in a voxel-wise fashion. To exclude the possible confounding effects of age, gender, education level, and volume of hippocampal subregions, we used these measures as covariates in the general linear models. Multiple comparison correction was performed using a FDR of 0.05 within the grey matter mask. In CD patients group, a linear regression analysis was further performed to explore the relationship between functional connectivity of the clusters showing significant group differences and neuropsychological scores as well as the endocrinological indicators (cortisol and 24hUFC). Multiple comparisons were also corrected using the FDR method with a corrected threshold of q < 0.05. 3 RESULTS 3.1 Demographic, endocrinological, and neuropsychological results Table 1 shows the demographic characteristics of the CD patients and the HCs. There were no significant differences in terms of age, sex distribution, and years of education between groups. Compared with HCs, CD patients had significantly lower MoCA scores and higher SDS and SAS scores (Table 1). As expected, the CD patients had significantly higher levels of serum cortisol and 24hUFC (p < .001). Moreover, we calculated the volumes of the four hippocampal subregions and found smaller volumes of all four hippocampal subregions in the CD patients. TABLE 1. Participant characteristics CDs (n = 47) HCs (n = 53) p Value Age (years) 37.38 ± 10.61 (20–59) 34.79 ± 10.72 (21–63) .113 Gender (male/female) 4/43 4/49 .859 Education (years) 11.00 ± 4.11 11.74 ± 3.10 .311 Illness duration (months) 41.62 ± 53.71 — — Neuropsychological tests MoCA 22.47 ± 3.98 (n = 45) 27.72 ± 2.00 <.001 SDS 40.18 ± 9.96 (n = 45) 27.13 ± 4.42 <.001 SAS 38.27 ± 7.90 (n = 45) 26.98 ± 4.47 <.001 CNPI 11.93 ± 9.68 (n = 45) — — Cushing QOL 37.76 ± 8.29 (n = 45) — — Endocrinological tests Serum cortisol (nmol/L) 0:00 am 633.81 ± 237.59 (n = 46) — — 8:00 am 735.34 ± 279.44 (n = 47) 358.51 ± 107.43 (n = 51) <.001 16:00 pm 671.05 ± 273.56 (n = 47) — — 24hUFC (nmol/24 h) 2381.59 ± 1653.16 (n = 41) 252.03 ± 119.47 (n = 47) <.001 Volume of hippocampal subregions (mm3) Left rostral hippocampus 343.75 ± 39.15 (257.18–423.27) 365.69 ± 27.19 (313.21–442.06) .001 Left caudal hippocampus 272.69 ± 32.74 (206.63–339.04) 296.39 ± 23.13 (249.62–347.61) <.001 Right rostral hippocampus 305.10 ± 33.71 (229.67–396.89) 336.76 ± 25.98 (274.95–415.16) <.001 Right caudal hippocampus 320.42 ± 32.60 (238.16–396.58) 347.87 ± 27.16 (294.00–415.80) <.001 Abbreviations: 24hUFC, 24-h urinary free cortisol.; CDs, Cushing's disease patients; CNPI, Chinese version of neuropsychiatric inventory; Cushing QOL, Cushing Quality of Life Scale; HCs, healthy controls; MoCA, Montreal Cognitive Assessment; SAS, Self-Rating Anxiety Scale; SDS, Self-Rating Depression Scale. Note: All values are expressed as mean ± SD. Group differences in sex between CDs and HCs were examined using chi-square test. Group differences in the other demographic and clinical characteristics between CDs and HCs were examined using two-sample t-tests (two-tailed). 3.2 Spatial distribution of hippocampal functional connectivity The hippocampal functional connectivity maps of both CD patients and HCs are presented in Figure 1. Visually, the spatial distributions of hippocampal functional connectivity were highly similar between groups, in spite of some differences in strength. We observed that the brain regions significantly positively connecting to hippocampus were primarily distributed in several limbic network regions (the orbital frontal cortex, bilateral medial temporal regions, and temporal pole) and DMN regions (bilateral medial frontal cortex, posterior cingulate gyrus/precuneus, and anterior cingulate cortex). Brain regions with negative connectivity to hippocampus were chiefly distributed in the frontoparietal network regions (dorsolateral prefrontal cortex, supramarginal gyrus, and angular gyrus). FIGURE 1 Open in figure viewerPowerPoint Between-group differences in functional connectivity of the hippocampal subregions. The first column shows the hippocampal functional connectivity subregions. The second and third columns show the hippocampal functional connectivity maps within CD and HC groups, respectively. Further between-group comparisons showed that CD patients had significantly altered hippocampal functional connectivities relative to HCs, with a corrected statistical threshold of p < .05. ROI1, left rostral hippocampus; ROI2, left caudal hippocampus; ROI3, right rostral hippocampus; ROI4, right caudal hippocampus; ROI, region of interest; CD, Cushing's disease; HC, healthy control 3.3 Altered hippocampal functional connectivity in CD patients The significant differences in functional connectivity with each hippocampal subregion between the CD patients and HCs groups are illustrated in third column of Figure 1. Both the right and left rostral hippocampus exhibited significantly decreased functional connectivity with the superior parietal lobe (SPL), a component of the frontoparietal network. Moreover, right rostral hippocampus exhibited additional increased functional connectivity with right inferior frontal gyrus, a component of DMN. For the left caudal hippocampus, significantly altered functional connectivity was found to the DMN regions, including (bilateral medial frontal cortex, angular gyrus, anterior, and posterior cingulate cortex). We also observed decreased functional connectivity between the right caudal hippocampus and anterior cingulate cortex. Additionally, the right caudal hippocampus exhibited increased functional connectivity with some limbic regions including the right orbital frontal cortex and temporal pole (Table 2). TABLE 2. Brain regions showing changed RSFC between CDs and HCs groups Peak MNI coordinate Brain regions BA Cluster size (voxels) x y z Peak T ROI-based RSFC ROI1 R IFG 48 219 57 21 —3 4.598 L angular 39 423 −27 −72 51 −5.530 RIO2 R thalamus − 114 9 −6 3 −5.905 L angular 39 195 −27 −72 54 −4.830 R angular 39 384 36 −66 48 −5.607 ROI3 R MTG 20 633 39 6 −21 4.410 L angular 39 195 −27 −72 54 −4.830 R angular 39 384 36 −66 48 −5.607 MFG/ACC 10/32 572 −3 42 −3 −4.033 PCC/PreCUN 26/23 709 12 −45 27 −4.502 ROI4 MFG/ACC 32 465 3 48 6 −4.670 R MTG/OFC 48/21 747 30 3 −21 4.208 Note: Statistical threshold was set at p < .05, corrected. Abbreviations: CDs, Cushing's disease patients; HCs, healthy controls; ROI, regions of interest; BA, Brodmann areas; MNI, Montreal Neurological Institute; RSFC, resting-state functional connectivity; SFG, superior frontal gyrus; MFG, middle frontal gyrus; dMFG, dorsal medial frontal gyrus; IPL, inferior parietal lobule; AG, angular gyrus; ROL, rolandic operculum; Ins, insular; PrCG, precentral gyrus; L, left; R, right; ROI1, left rostral hippocampus; ROI2, left caudal hippocampus; ROI3, right rostral hippocampus; ROI4, right caudal hippocampus. 3.4 Brain–behavior relationships in the CD patients In the correlation analyses of CD patients, the mean values of the functional connectivity between the left caudal hippocampus and anterior cingulate cortex correlated positively with the Cushing's QoL scores (r = .327, p < .05) (Figure 2). No other correlations were found for volumes and functional connectivity of the four hippocampal subregions with neuroendocrine and neuropsychological assessment in the CD patients. FIGURE 2 Open in figure viewerPowerPoint Significant correlations between left hippocampal functional connectivity and the quality of life in CD patients. CD, Cushing's disease; Hip, hippocampus; ACC, anterior cingulate cortex 4 DISCUSSION Using a cohort of CD patients and HCs, the present study performed a comprehensive investigation to reveal how the chronic hypercortisolism affects the morphology and connectivity of hippocampal subregions and their relationships with neuroendocrine and neuropsychological assessment. Compared with the HCs, the CD patients had smaller volumes of all four hippocampal subregions. Furthermore, CD patients exhibited differential patterns of altered hippocampal functional connectivity with high-order networks, involving the DMN, frontoparietal, and limbic networks. The intrinsic hippocampal functional connectivity was associated with the quality of life of the CD patients. Together, these findings elucidate the cumulative effect of cortisol on the morphology and function of hippocampus and provide important information to further understand the role of hippocampus in stress-related brain disease. Cortisol, the end product of the hypothalamic–pituitary–adrenal axis, plays a critical role in the body's response to stress and maintenance of homeostasis (Sapolsky et al., 2000); however, chronic hypercortisolism is known to impair neurons in the hippocampus. CD patients naturally demonstrate chronic excessive amounts of cortisol; therefore these patients serve as a natural “hyperexpression model” to investigate the chronic effects of cortisol on human hippocampus. Importantly, we showed the CD patients are associated with smaller hippocampal volumes in all four subregions. In line with our study, previous structural imaging studies have shown hippocampal volume decreases in CD patients (Frimodt-Møller et al., 2019; Toffanin et al., 2011). Furthermore, Brown et al. found that healthy volunteers were associated with a significant reduction in hippocampal volume following only 3-day stress doses of corticosteroid administration, strongly suggesting the effects of cortisol on hippocampal size. It is important to note that chronic hypercortisolism can affect the hippocampus in at least two ways: by direct neurotoxic effects on the hippocampus (Lupien et al., 2018; Uno et al., 1994) and by reduction in hippocampal neurogenesis (Saaltink & Vreugdenhil, 2014). Moreover, cortisol stimulates the release of excitatory amino acids glutamate on hippocampal cells (de Kloet et al., 2005). On the other hand, chronic elevations of cortisol also reduce neurotrophic factors that includes nerve growth factor and brain-derived neurotrophic factor (McEwen et al., 2015). The different patterns of functional connectivity in rostral hippocampus versus caudal hippocampus might be associated to the specific cytoarchitecture along the rostral/caudal hippocampus. Accumulated evidence from both animal and human studies suggests that different parts of the hippocampus display distinctive gene expression and anatomical projections patterns (Fanselow & Dong, 2010). In detail, gene expression in the rostral hippocampus correlates with regions involved in emotion and stress (amygdala and hypothalamus). Moreover, the rostral hippocampus has connections with prefrontal regions, exerts strong regulatory control of the hypothalamic–pituitary–adrenal axis with a negative feedback (Toffanin et al., 2011). Accordingly, as demonstrated in this study, chronic hypercortisolism predominantly disrupted the functional connectivity in rostral hippocampus. Another major finding in this study was altered hippocampal functional connectivity with DMN, frontoparietal, and limbic networks in CD individuals relative to that in HCs. Emerging evidence proposes that interactions within and between these large-scale brain networks play important roles on brain functions and may be affected in multiple psychiatric disorders (Menon, 2011; Sha et al., 2019). Among these brain networks, the DMN is anchored in the medial prefrontal cortex and posterior cingulate cortex and is implicated in internally directed attention and self-referential processing (Raichle, 2015), while the frontoparietal and limbic networks support the cognitive regulation of emotion, attention, and behavior (Buhle et al., 2014; Kohn et al., 2014). The engagement of these high-level functional networks may suggest the linkage of abnormal stress hormone cortisol to cognitive deficits in CD patients. In line with our study, previous studies have shown stress-induced cortisol increase was associated with altered connectivity within the major brain networks (Zhang et al., 2019, 2020, 2020). Meanwhile, structural and functional alterations in these brain systems are also found in CD patients. For example, many functional imaging studies have consistently demonstrated altered brain activities and functional connectivity involving in DMN, frontoparietal, and limbic networks (Jiang et al., 2017; Wang et al., 2019; Zhang et al., 2021), even in the patients with long-term remission of CD (van der Werff et al., 2015). Importantly, previous studies have shown that the CD patients had widespread reductions of white matter integrity, which provide further evidence for the structural substrate for the persistence of these functional deficits (Pires et al., 2015; van der Werff et al., 2014). Here, we propose that by altering hippocampal processes via the abundant glucocorticoid and mineralocorticoid receptors, exposure to hypercortisolism disrupts the interactions with DMN, frontoparietal, and limbic networks in CD patients, thus engender vulnerability for emotional and cognitive problems. In line with this view is evidence that altered hippocampal functional connectivity is associated with the quality of life in CD patients. Because impaired quality of life is a persistent complaint from CD patients (Webb et al., 2018), it is important to accurately assess which aspects of QoL are affected in order to better understand the severity of hypercortisolism on patients and the potential efficacy of treatment. CushingQoL questionnaire has proven to be a valuable resource for assessing health-related QoL in CD patients, based on the combination of psychosocial issues and physical problems (Nelson et al., 2013). A better understanding of the neuroplasticity and continuing quality of life change may in turn facilitate advances in management and intervention. Several issues need to be addressed further. First, although the sample size of this study was relatively large, the findings still need to be further replicated in an independent sample. Second, the cross-sectional, observational nature of our study design precludes any causal conclusions. Therefore, studies tracking dynamic changes in hippocampal functional connectivity following the remission of hypercortisolism are needed. We are currently following up participants as part of a longitudinal study. Finally, a combined analysis of multimodal imaging including structural and metabolic data would provide integrated information on the effect of cortisol excess on human brain. In short, we demonstrate that CD patients present atypical morphology and functional connectivity of hippocampus. Here we observed the chronic hypercortisolism caused smaller volumes of all hippocampal subregions. This volume change was in line with the preclinical research that excess cortisol cause dendritic shrinkage and loss of spines in the hippocampus. Functionally, CD patients demonstrated altered hippocampal connectivity whose nodes include key components of the DMN, frontoparietal, and limbic networks. These multimodal results reinforce the need for effective therapeutic interventions in stress-related brain disease to halt possible hippocampal damage. ACKNOWLEDGMENTS This study was supported by the National Natural Science Foundation of China (No. 82001798 and No. 81871087), Military Young Scholar Medical Research Fund of Chinese PLA General Hospital (No. QNF19071), and Medical Big Data and Artificial Intelligence Development Fund of Chinese PLA general Hospital (No. 2019MBD-039). CONFLICT OF INTEREST The authors report no biomedical financial interests or potential conflicts of interest. Read more, including references, at https://onlinelibrary.wiley.com/doi/10.1002/brb3.2507

Published: May 15, 2022 (see history) DOI: 10.7759/cureus.25017 Cite this article as: Fernandez C, Bhatia S, Rucker A, et al. (May 15, 2022) Intermittent Blurry Vision: An Unexpected Presentation of Cushing’s Syndrome Due to Primary Bilateral Macronodular Adrenal Hyperplasia (PBMAH). Cureus 14(5): e25017. doi:10.7759/cureus.25017 Abstract Cushing’s syndrome (CS) is an uncommon endocrine disorder resulting from prolonged exposure to elevated glucocorticoids, with 10-15 million annual cases per the American Association of Neurological Surgeons. Exogenous and endogenous causes can further be divided into adrenocorticotropic hormone (ACTH) dependent (i.e Cushing’s Disease) or ACTH independent. ACTH-independent CS can be caused by primary bilateral macronodular adrenal hyperplasia (PBMAH) representing less than 1% cases of CS. We report a case of a woman presenting with chronic resistant hypertension, episodic blurry vision, weight gain and wasting of extremities. She was diagnosed with Cushing’s syndrome due to PBMAH. Our patient’s presentation was unusual as she presented at 40 years old, 10 years earlier than expected for PBMAH; and primarily with complaints of episodic blurry vision. Her symptoms also progressed rapidly as signs and symptoms largely presented over the course of 12 months, however responded well to surgical resection. Introduction Cushing’s syndrome (CS) is an uncommon endocrine disorder caused by prolonged exposure to elevated glucocorticoids [1]. There are exogenous or endogenous causes. The National Institute of Health’s (NIH) Genetic and Rare Diseases Information Center (GARD) estimated the prevalence of endogenous CS to be 1 in 26,000 [2]. According to a large study, the annual incidence of CS in individuals less than 65 years old was nearly 49 cases per million [3]. Cushing’s disease (CD), which is defined as Cushing’s syndrome caused by an adrenocorticotropic hormone (ACTH)-secreting pituitary tumor, accounts for approximately 80% of patients with CS; whereas ACTH-independent CS accounts for the remaining 20% [4]. Among the causes of pituitary ACTH-independent CS is bilateral macronodular adrenal hyperplasia which is rare, comprising less than 1% of patients with CS [5]. Herein is a case of rapid onset Cushing’s syndrome due to PBMAH initially presenting as episodes of bilateral blurry vision. Case Presentation The patient is a 40-year-old female with a past medical history of resistant hypertension (on four agents), and recently diagnosed type 2 diabetes mellitus (started on insulin regimen). Patient was recently seen by her primary care provider, with complaints of intermittent episodes of blurry vision going on for months. As part of evaluation in December 2020, the patient underwent a renal ultrasound as part of evaluation by the primary physician for uncontrolled hypertension. The doppler incidentally showed an indeterminate hypoechoic mass on the right kidney and presumably located within the right adrenal gland, measuring 3.4 x 5.4 cm, without sonographic evidence of renal artery stenosis. The left kidney appeared normal. She was recommended to have further evaluation with contrast enhanced MR or CT with adrenal protocol. In January 2021, the patient was sent from her PCP’s office to the ED as the patient was having blurred vision. She had a plain CT scan of the brain that was unremarkable. The patient's systolic blood pressure was in the 160s-170s mm Hg upon arrival to ED compliance with home medications of 5mg of amlodipine daily, 25mg of metoprolol succinate daily, 100mg of losartan daily, and 25mg of hydrochlorothiazide daily. Physical exam reported obesity without evidence of abdominal striae. Blood work in the ED showed elevated blood glucose level over 600 (mg/dL) despite being on a regimen of lantus 60 units, metformin 1000mg twice a day, and semaglutide SQ weekly. Hemoglobin A1c was greater than 15.5%, and vitamin D was low (15.6 ng/mL). The morning ACTH was low (<5pg/mL) (nAM levels: 7.2 - 63.3 pg/mL), AM cortisol was high at 26.1 ug/ml (normal: 5.0 - 23.0 ug/mL), plasma aldosterone was normal at 4.2 ng/dL with a normal plasma renin at 1.96 (0.25 - 5.82 ng/mL/h). 24-hour urine free cortisol (UFC) was high at 1299.5 (4.0-50.0 mcg/24h). CT of the abdomen/pelvis with and without contrast showed low-attenuation masses (less than 5 Hounsfield units) present in both adrenal glands measuring 6.9 x 5.3 cm on the right and 4.5 x 3.9 cm on the left, and did not demonstrate significant arterial enhancement (Figure 1). MR imaging of the abdomen without and with contrast was also obtained and showed the same masses of the bilateral adrenal glands, with largest on the left measured 3.6 cm and largest on the right measured 3.7 cm, as well as mild fatty infiltration of the liver. General surgery and hematology/oncology were consulted and recommendations were made for outpatient follow-up with PCP and endocrinology. Figure 1: CT of the abdomen/pelvis with contrast showing low-attenuation masses present in both adrenal glands measuring 6.9 x 5.3 cm on the right (dark gray arrow) and 4.5 x 3.9 cm on the left (light gray arrow) In early February 2021, the patient again presented to the ED complaining of recurrent episodes of bilateral blurry vision. Examination was unremarkable, including an ophthalmological exam with slit lamp exam. Blurred vision was suspected to be due to osmotic swelling in the setting of severe hyperglycemia as the patient had persistently uncontrolled blood sugars. Recommendations were for tighter control of blood glucose, and follow-up with primary care and ophthalmology. Patient followed up with the endocrinologist in mid-February to which the patient reported first noticing a difference in her energy and changes to her weight around one year prior. She communicated a weight gain of 30 to 40 lbs over the past year. Patient had a reported history of gestational hypertension diagnosed five years ago when she gave birth to her daughter, which was steadily worsening over the past year. She reported intermittent myalgias and easy bruising. Patient had no family history or any apparent features to suggest multiple endocrine neoplasia (MEN) syndrome. Blood work revealed ACTH less than 1.5 pg/mL, AM cortisol was high at 24.5 mcg/dL, and normal aldosterone at 3.6 ng/dL, with normal renin and metanephrine levels. Physical examination revealed truncal obesity as well as a round face, cushingoid in appearance, and relatively thin extremities and abdominal striae. She was then referred to a surgical specialist, and it was decided that she would undergo laparoscopic bilateral adrenalectomy due to severe Cushing’s syndrome. The surgical pathology report revealed macro-nodular cortical hyperplasia of both left and right adrenal gland masses with random endocrine atypia. The largest nodule on the left measured 4.5 cm and the largest nodule on the right measured 6.6 cm. Post-operatively she was started on hydrocortisone 20 mg every morning and 10 mg every evening, and fludrocortisone 0.1 mg twice a day as part of her steroid replacement regimen. Eventually she changed to hydrocortisone 10 mg three times a day and fludrocortisone 0.1 mg once a day. For her diabetes, her insulin glargine decreased from 60 units to 20 units. Amlodipine and hydrochlorothiazide were discontinued from her antihypertensive medications; she continued losartan and metoprolol. Follow up blood work showed stable electrolytes with potassium 4.2 mmol/L (3.5-5.2 mmol/L), sodium 137 mmol/L (134-144mmol/L), chloride 100 mmol/L (96-106 mmol/L), and carbon dioxide 23 mmol/L (20-29mmol/L). Discussion ACTH-independent Cushing’s syndrome due to bilateral cortisol-secreting nodules is rare, accounting for 2% of CS cases. The majority of causes include primary bilateral macronodular adrenal hyperplasia (PBMAH), primary pigmented nodular adrenocortical disease (PPNAD), and bilateral adrenocortical adenomas (BAA). In PBMAH, typically patients are diagnosed within the fifth or sixth decade of life [4]. The usual age of onset for PPNAD is within the first to third decade of life, with median age in the pediatric population at age 15 years [6]. BAA is such a rare entity that there exists little epidemiological data with less than 40 reported cases until 2019 [7]. A small subset of patients present with overt clinical symptoms of CS, as hypercortisolism often follows an insidious course that can delay diagnosis from years to decades, with one series reporting a diagnostic delay of approximately eight years [8]. Serum and urine hormone screening in the right clinical setting can provide clues to these endocrine disorders, however diagnosis of ACTH-independent CS often occurs incidentally wherein a radiographic study was done for reasons other than to identify adrenal disease [9]. CT or MRI alone are not able to differentiate these disease entities, requiring pathological examination for final determination [7]. Adrenal venous sampling (AVS) and I-6B-iodomethyl-19-norcholesterol (I-NP-59) can aid in identifying hormone-secreting status of each adrenal lesion, however usefulness is debated among experts [10-12]. In all cases the end goal is to normalize adrenocortical hormones, and PBMAH primarily involves surgical resection with exogenous hormone replacement. Bilateral adrenalectomy is generally the treatment of choice with overt Cushing syndrome regardless of cortisol level. These patients require lifelong steroid administration [9,13]. Another approach is unilateral adrenalectomy of the larger or more metabolically active gland, which can be identified after AVS or I-NP-59 testing. This has been proposed in order to preserve some autonomous hormonal production and prevent adrenal crisis, however remission rates of Cushing syndrome as high as 84% have been reported with eventual need for bilateral adrenalectomy [7,8,14]. Steroid enzyme inhibition to control cortisol secretion has been used as an adjunct before surgery. In some patients with identified aberrant adrenal hormone receptors, targeted pharmacological inhibition remains an alternative medical approach [8]. Despite these alternatives to surgery, surgical resection remains the optimal approach [1]. Conclusions ACTH-independent Cushing’s syndrome due to PBMAH usually presents as an indolent course, with typical diagnosis in the fifth to sixth decade. As the use of imaging for other non-endocrine related investigations becomes more utilized, PBMAH being less of a rare entity. Clinical presentation usually dictates the timing of and type of surgical intervention. Although there are some reports of unilateral resection resulting in a cure, many of these cases eventually proceed to staged bilateral resection. Our patient’s presentation as her primary complaint was recurrent episodes of blurry vision that were suspected to be due to osmotic swelling because of her uncontrolled hyperglycemia. Her case was also unusual as she presented at 40 years old, an average of 10 years earlier than is typically diagnosed for PBMAH. Her symptoms also progressed rapidly over the course of 12 months with development of resistant hypertension and insulin-dependent diabetes requiring high basal insulin. Following surgical resection, her antihypertensive regimen was de-escalated and had significant reduction in insulin requirements, and was maintained on adrenocorticoid therapy. References Nieman LK: Recent updates on the diagnosis and management of Cushing's syndrome. Endocrinol Metab (Seoul). 2018, 33:139-46. 10.3803/EnM.2018.33.2.139 Rare Disease Database: Cushing Syndrome. (2021). Accessed: 12/17/2021: https://rarediseases.org/rare-diseases/cushing-syndrome/. Broder MS, Neary MP, Chang E, Cherepanov D, Ludlam WH: Incidence of Cushing's syndrome and Cushing's disease in commercially-insured patients <65 years old in the United States. Pituitary. 2015, 18:283-9. 10.1007/s11102-014-0569-6 Lacroix A, Feelders RA, Stratakis CA, Nieman LK: Cushing’s syndrome. Lancet. 2015, 386:913-27. 10.1016/S0140-6736(14)61375-1 Tokumoto M, Onoda N, Tauchi Y, et al.: A case of adrenocoricotrophic hormone -independent bilateral adrenocortical macronodular hyperplasia concomitant with primary aldosteronism. BMC Surg. 2017, 17:97. 10.1186/s12893-017-0293-z Stratakis CA: Cushing syndrome caused by adrenocortical tumors and hyperplasias (corticotropin- independent Cushing syndrome). Endocr Dev. 2008, 13:117-32. 10.1159/000134829 Gu YL, Gu WJ, Dou JT, et al.: Bilateral adrenocortical adenomas causing adrenocorticotropic hormone-independent Cushing's syndrome: a case report and review of the literature. World J Clin Cases. 2019, 7:961-71. 10.12998/wjcc.v7.i8.961 Lacroix A: ACTH-independent macronodular adrenal hyperplasia. Best Pract Res Clin Endocrinol Metab. 2009, 23:245-59. 10.1016/j.beem.2008.10.011 Sweeney AT, Srivoleti P, Blake MA: Management of the patient with incidental bilateral adrenal nodules. J Clin Transl Endocrinol Case Rep. 2021, 20:100082. 10.1016/j.jecr.2021.100082 Lumachi F, Zucchetta P, Marzola MC, Bui F, Casarrubea G, Angelini F, Favia G: Usefulness of CT scan, MRI and radiocholesterol scintigraphy for adrenal imaging in Cushing's syndrome. Nucl Med Commun. 2002, 23:469-73. 10.1097/00006231-200205000-00007 Builes-Montaño CE, Villa-Franco CA, Román-Gonzalez A, Velez-Hoyos A, Echeverri-Isaza S: Adrenal venous sampling in a patient with adrenal Cushing syndrome. Colomb Med (Cali). 2015, 46:84-7. Guo YW, Hwu CM, Won JG, Chu CH, Lin LY: A case of adrenal Cushing's syndrome with bilateral adrenal masses. Endocrinol Diabetes Metab Case Rep. 2016, 2016:150118. 10.1530/EDM-15-0118 Wei J, Li S, Liu Q, et al.: ACTH-independent Cushing's syndrome with bilateral cortisol-secreting adrenal adenomas: a case report and review of literatures. BMC Endocr Disord. 2018, 18:22. 10.1186/s12902-018-0250-6 Osswald A, Quinkler M, Di Dalmazi G, et al.: Long-term outcome of primary bilateral macronodular adrenocortical hyperplasia after unilateral adrenalectomy. J Clin Endocrinol Metab. 2019, 104:2985-93. 10.1210/jc.2018-02204 From https://www.cureus.com/articles/90069-intermittent-blurry-vision-an-unexpected-presentation-of-cushings-syndrome-due-to-primary-bilateral-macronodular-adrenal-hyperplasia-pbmah

Abstract Corticotroph cells give rise to aggressive and rare pituitary neoplasms comprising ACTH-producing adenomas resulting in Cushing disease (CD), clinically silent ACTH adenomas (SCA), Crooke cell adenomas (CCA) and ACTH-producing carcinomas (CA). The molecular pathogenesis of these tumors is still poorly understood. To better understand the genomic landscape of all the lesions of the corticotroph lineage, we sequenced the whole exome of three SCA, one CCA, four ACTH-secreting PA causing CD, one corticotrophinoma occurring in a CD patient who developed Nelson syndrome after adrenalectomy and one patient with an ACTH-producing CA. The ACTH-producing CA was the lesion with the highest number of single nucleotide variants (SNV) in genes such as USP8, TP53, AURKA, EGFR, HSD3B1 and CDKN1A. The USP8 variant was found only in the ACTH-CA and in the corticotrophinoma occurring in a patient with Nelson syndrome. In CCA, SNV in TP53, EGFR, HSD3B1 and CDKN1A SNV were present. HSD3B1 and CDKN1A SNVs were present in all three SCA, whereas in two of these tumors SNV in TP53, AURKA and EGFR were found. None of the analyzed tumors showed SNV in USP48, BRAF, BRG1 or CABLES1. The amplification of 17q12 was found in all tumors, except for the ACTH-producing carcinoma. The four clinically functioning ACTH adenomas and the ACTH-CA shared the amplification of 10q11.22 and showed more copy-number variation (CNV) gains and single-nucleotide variations than the nonfunctioning tumors. Keywords: corticotroph; Cushing disease; ACTH-secreting carcinoma; single nucleotide variation; copy number variation; exome 1. Introduction The pathological spectrum of the corticotroph includes ACTH (adrenocorticotropic hormone)-secreting pituitary adenomas (PA), causing Cushing disease (CD), silent corticotroph adenomas (SCA), Crooke cell adenomas (CCA) and the rare ACTH-secreting carcinoma (ACTH-CA). Pituitary carcinomas account for 0.1 to 0.2% of all pituitary tumors and are defined by the presence of craniospinal or distant metastasis [1,2,3]. Most pituitary carcinomas are of corticotroph or lactotrope differentiation [3]. Although a few cases present initially as CA, the majority develop over the course of several months or years from apparently benign lesions [3,4]. CCA are characterized by the presence of hyaline material in more than 50% of the cells of the lesion, and most of them arise from silent corticotroph adenomas (SCA) or CD-provoking ACTH-secreting adenomas [5]. SCA are pituitary tumors with positive immunostaining for ACTH but are not associated with clinical or biochemical evidence of cortisol excess; they are frequently invasive lesions and represent up to 19% of clinically non-functioning pituitary adenomas (NFPA) [6]. ACTH-secreting PA represents up to 6% of all pituitary tumors and causes eloquent Cushing disease (CD), which is characterized by symptoms and signs of cortisol hypersecretion, including a two- to fivefold increase in mortality [7,8]. The 2017 World Health Organization (WHO) classification of PA considers not only the hormones these tumors synthesize but also the transcription factors that determine their cell lineage [9]. TBX19 is the transcription factor responsible for the terminal differentiation of corticotrophs [9]. All tumor lesions of corticotroph differentiation are positive for both ACTH and TBX19. ACTH-secreting PA causing CD are among the best genetically characterized pituitary tumors, with USP8 somatic variants occurring in up to 25–35% of sporadic cases [9]. Yet, information regarding the molecular pathogenesis of the lesions conforming to the whole pathological spectrum of the corticotroph is scarce. The aim of the present study is to characterize the genomic landscape of pituitary tumors of corticotroph lineage. For this purpose, we performed whole exome sequencing to uncover the mutational burden (single-nucleotide variants, SNV) and copy-number variations (CNVs) of these lesions. 2. Results 2.1. Clinical and Demographic Characteristics of the Patients A total of 10 tumor samples from 10 patients were evaluated: 4 ACTH-secreting adenomas causing clinically evident CD, three non-functioning adenomas that proved to be SCA upon immunohistochemistry (IHC), one ACTH-secreting CA with a prepontine metastasis, one rapidly growing ACTH-secreting adenoma after bilateral adrenalectomy (Nelson syndrome) in a patient with CD and one non-functioning, ACTH-producing CCA (Table 1). All except one patient were female; the mean age was 38.8 ± 16.5 years (range 17–61) (Table 1). They all harbored macroadenomas with a mean maximum diameter of 31.9 ± 13 mm (range 18–51). Cavernous sinus invasion was evident on MRI in all but one of the patients (Table 1). Homonymous hemianopia was present in seven patients, whereas right optic nerve atrophy and amaurosis were evident in patient with the ACTH-CA, and in patient with CD and pituitary apoplexy (Table 1). Detailed clinical data are included in Supplementary Table S1. Death was documented in only the patient with pituitary apoplexy, and one patient was lost during follow-up, as of October 2018. Table 1. Clinical features of the tumors analyzed and SNV present in each tumor. 2.2. General Genomic Characteristics of Neoplasms of Corticotrophic Lineage Overall, approximately 18,000 variants were found, including missense, nonsense and splice-site variants as well as frameshift insertions and deletions. Of these alterations, the majority corresponded to single-nucleotide variants, followed by insertions and deletions. The three most common base changes were transitions C > T, T > C and C > G; most of the genetic changes were base transitions rather than transversions (Figure 1). There were several genes across the whole genome affected in more than one way, meaning that the same gene presented missense and nonsense variants, insertions, deletions and splice-site variants (Figure 2). Many of these variants are of unknown pathogenicity and require further investigation. Gains in genetic material were found in 44 cytogenetic regions, whereas 72 cytogenetic regions showed loss of genetic material in all corticotroph tumors. Figure 1. Panel (A) shows the gadolinium-enhanced magnetic resonance imaging of the patient with ACTH-CA, highlighting in red the metastatic lesion in the prepontine area. Panel (B) shows the hematoxylin and eosin staining displaying the hyaline structures in the perinuclear areas denoting a Crooke cell adenoma. Panel (C,D) depict a representative corticotroph tumor with positive ACTH and TBX19 immunohistochemistry, respectively. Panel (E) shows four graphics: variant classification, variant type, SNV class and transition (ti) or transversion (tv) describing the general results of exome sequencing of the corticotroph tumors. Figure 2. Representative rainfall plots showing the SNV alterations throughout the whole genome of corticotroph tumors (A) CCA, (B) SCA, (C) CD and (D) ACTH-CA, displaying all base changes, including transversions and transitions. No kataegis events were found. Alterations across the genome were seen in all corticotroph tumors. 2.3. ACTH-Secreting Carcinoma (Tumor 1) SNV missense variants were found in the genes encoding TP53 (c.215G > C [rs1042522], p.Pro72Arg); AURKA (c.91T > A [rs2273535], p.Phe31Ile); EGFR (epidermal growth factor receptor, c.1562G > A [rs2227983], p.Arg521Lys); HSD3B1 (3-ß-hydroxisteroid dehydrogenase, c.1100C > A [rs1047303], p.Thr367Asn); CDKN1A (cyclin-dependent kinase inhibitor 1A or p21, c.93C > A [rs1801270], p.Ser31Arg); and USP8 (c.2159C > G [rs672601311], p.Pro720Arg). Interestingly, the previously reported USP48, BRAF, BRG1 and CABLES1 variants in pituitary CA cases were not found in this patient’s tumor (Figure 3). All SNV detected in WES experiments were validated by Sanger sequencing. The variants described were selected due to their potential pathogenic participation in other tumors and the allelic-risk association with tumorigenesis. Hereafter, all the mentioned variants in other corticotroph tumors are referred to by these aforementioned variants. Even though these same genes presented other variants, currently the significance of those variants is unknown. Figure 3. Panel (A) shows the oncoplot from the missense variants of the selected genes and their clinical–pathological features. Panels (B–G) depict USP8, EGFR, TP53, AURKA, CDKN1A and HSD3B1 proteins, respectively, with the changes found in DNA impacting aminoacidic changes. In general, the pituitary CA presented more CNV alterations than the benign tumors, with 27 and 32 cytogenetic regions showing gains and losses of genetic material, respectively. The cytogenetic regions showing gains were 10q11.22, 15q11.2, 16p12.3, 1p13.2 and 20p, where genes SYT15, POTEB, ARL6IP1, HIPK1 and CJD6 are coded, respectively. By contrast, 8p21.2 was the cytogenetic region showing loss of genetic material. The previously reported amplification of 1p13.2 was also detected in this tumor (Figure 4) [10]. Figure 4. Hierarchical clustering of corticotroph tumors according to their gains and losses across the whole genome (somatic chromosomes only). High contrast was used to enhance potential CNV alterations; nevertheless, there were only 44 unique cytogenetic regions that showed gains in genetic material with statistical significance, whereas only 72 unique cytogenetic regions showed loss of genetic material with statistical significance. 2.4. Crooke Cell Adenoma (Tumor 2) The CCA showed SNV in the genes encoding TP53, EGFR, HSD3B1 and CDKN1A. However, neither the genes encoding AURKA and USP8 nor those encoding USP48, BRAF, BRG1 and CABLES were affected in this tumor. In CCA, only two and fifteen gains and losses were observed in copy-number variation, respectively. CNVs only showed gains in cytogenetic regions 17q12 and 10q11.22, harboring genes CCL3L1 and NPY4R, respectively, whereas losses were found in cytogenetic regions 18q21.1, 15q12 and 2q11.2, harboring genes KATNAL2, TUBGCP5 and ANKRD36. 2.5. Silent Corticotroph Adenomas (Tumors 3–5) The three SCA shared SNVs in the genes encoding HSD3B1 and CDKN1A. SCA 4 and 5 showed SNV in the genes encoding EGFR, whereas SNV in the genes encoding AURKA and TP53 were present in SCA 3 and 5. None of the SCA were found to have SNV in the genes encoding USP8, USP48, BRAF, BRG1 or CABLES1. The SCA presented only two and eighteen gains and losses (CNV), respectively. In regard to CNV, the these clinically silent tumors presented gains of genetic material in cytogenetic regions 17q22 and 10q11.22, which harbor genes encoding CCL3L1 and NPY4R. Eighteen losses were found distributed in cytogenetic regions 18q21.1, 15q12 and 2q11.2, encompassing the genes encoding KATNAL2, TUBGCP5 and ANKRD36. This CNV pattern closely resembles the one found in the CCA, which is somewhat expected if we consider that both neoplasms are clinically non-functioning 2.6. ACTH-Secreting Adenomas (Cushing Disease) (Tumors 6–9) SNV of the genes encoding TP53 and HSD3B1 were present in tumor samples from all four CD patients, whereas none of these patients harbored adenomas with SNV in the genes encoding USP8 or CDKN1A. An SNV in the gene encoding AURKA was identified in only one of these tumors (tumor 8). EGFR SNV were found in tumors 7 and 9. None of the CD-causing ACTH-secreting adenomas showed the previously reported SNV in the genes encoding USP48, BRAF, BRG1 and CABLES1. CNV analysis in this group of eloquent-area corticotroph tumors revealed 25 gains and 55 losses of genetic material. The gains occurred in cytogenetic regions 17q12, 2p12, 9p24 and 10q11.22, where genes CCL3L1, CTNNA2, FOXD4 and NPY4R are coded, respectively. The losses were localized in cytogenetic regions 21p12, 15q11.2, and 8p23, harboring genes USP16, KLF13 and DEF130A, respectively. We also detected the previously reported 20p13 amplification [10]. 2.7. ACTH-Secreting Adenoma Causing Nelson Syndrome (Tumor 10) This patient’s tumor showed SNV in the genes encoding USP8, TP53, HSD3B1 and CDKN1A but no alterations were found in the genes encoding EGFR and AURKA. This tumor and the ACTH-CA were the only two neoplasms that harbored a USP8 variant. No SNV were identified in the genes encoding USP48, BRAF, BRG1 and CABLES1. Interestingly, CNV analysis revealed the same gains and losses of genetic material found in tumors from other patients with CD. 2.8. Tumor Phylogenic Analysis We performed a phylogenetic inference analysis to unravel a hypothetical sequential step transformation from an SCA to a functioning ACTH-secreting adenoma and finally to an ACTH-CA. The theoretical evolutive development of the ACTH CA, departing from the SCA, shows two main clades, with the smallest one comprising two of the three SCA and two of the five ACTH-adenomas causing CD. Since these four tumors have the same SNV profile, we can assume that they harbor the genes that must be altered to make possible the transition from a silent to a clinically eloquent adenoma; the gene encoding ATF7IP (c.1589A > G [rs3213764], p.K529R) characterizes this clade. The second and largest clade includes the CCA, the ACTH-CA, one of the three SCA and three of the five most aggressive ACTH adenomas causing CD, including the adenoma of the patient with Nelson syndrome. This clade represents the molecular alterations required to evolve from a CD-causing ACTH-adenoma to a more aggressive tumor, or even to a CA and is characterized by the gene encoding MSH3 (c.235A > G [rs1650697], p.I79V) (Figure 5). Figure 5. Phylogenetic analysis of the corticotroph tumors. The theoretical evolutive development of the ACTH-CA, departing from the SCA shows two main clades. The first clade, characterized by ATF7IP gene, comprises 2 of the 3 SCA and 2 of the 5 ACTH-adenomas causing CD. The second clade is characterized by the gene encoding MSH3 and includes the CCA, the ACTH-CA, one of the 3 SCA and 3 of the 5 most aggressive ACTH adenomas causing CD, including the adenoma of the patient with Nelson syndrome. Red dots represent the Cushing Disease provoking adenomas, green dots represent the silent corticotroph tumors, brown dot represent the Crooke cell adenoma and the blue dot represent the corticotroph carcinoma. 2.9. Correlation between Gene Variants and Clinicopathological Features The USP8 variant positively correlated with increased tumor mass (p = 0.019). The CDKN1A variant was significantly associated with silent tumors (p = 0.036). The rest of the genetic variants did not correlate with any of the clinicopathological features tested. The presence of the EGFR variant was not distinctly associated with any of the clinical parameters and was equally present in functional as well as non-functional tumors (p = 0.392). AURKA SNV did not correlate with any of the features, including recurrence (p = 0.524). Detailed statistical results are presented in Supplementary Table S2. 3. Discussion Corticotrophs are highly specialized cells of the anterior pituitary that synthesize and secrete hormones that are essential for the maintenance of homeostasis. In this study, we sequenced the exome of 10 corticotroph tumors, including three SCA, four ACTH adenomas causing CD, an ACTH adenoma in a patient with Nelson syndrome, a CCA and an ACTH-CA in total, representing the broad pathological spectrum of this cell. Our results portray the genomic landscape of all the neoplasms that are known to affect the corticotroph. The neoplasm with the highest number of genomic abnormalities, including SNV and CNV, was the ACTH-CA, followed by the CCA and the CD tissues. Of all the genes harboring SNVs, six were found to be present in at least two of our tumor samples: HSD3B1, TP53, CDKN1A, EGFR, AURKA and USP8. The HSD3B1 gene encodes a rate-limiting enzyme required for all pathways of dihydrotestosterone synthesis and is abundantly expressed in adrenal tumors. Gain of function of this HSD3B1 variant, which has a global allelic prevalence of 0.69678 [11], results in resistance to proteasomal degradation with the consequent accumulation of the enzyme and has been associated with a poor prognosis in patients with prostate cancer [12]. Nine of the ten corticotroph tumors in our cohort harbored an SNV of the tumor suppressor gene TP53. The TP53 variant described in our cohort has been reported to be present in 80% of non-functioning pituitary adenomas and is apparently associated with a younger age at presentation and with cavernous sinus invasion [13]. Furthermore, this TP53 variant results in a reduced expression of CDKN1A and an increased expression of vascular endothelial growth factor (VEGF) as well as an increased cellular proliferation rate [13]. CDKN1A (also known as p21) is a cyclin-dependent kinase inhibitor regulating cell cycle progression. The SNV described in our study was reported to alter DNA binding ability and expression and has a global allelic frequency of 0.086945 [14]. This cyclin-dependent kinase inhibitor SNV was found to be associated with breast carcinoma [15] and lung cancer [16]. The presence of this SNV has not been previously explored in pituitary adenomas, although CDKN1A is downregulated in clinically non-functioning pituitary adenomas of gonadotrophic lineage but not in hormone-secreting tumors [17]. EGFR encodes a transmembrane tyrosine kinase receptor, activation of which leads to mitogenic signaling [18]. This gene is upregulated in several cancers and represents a target for molecular therapies [19]. The EGFR SNV described in our corticotroph tumor series was found to be associated with the response to neoadjuvant chemotherapy in patients with breast and lung cancer [18]. EGFR is normally expressed in corticotrophs, where it participates in the regulation of POMC (proopiomelanocortin) gene transcription and cellular proliferation [20]. The EGFR rs2227983 has a 0.264334 global allelic frequency [21]. AURKA is a cell-cycle regulatory serine/threonine kinase that promotes cell cycle progression by the establishment of the mitotic spindle and centrosome separation [22]. Alterations of these gene are related to centrosomal amplification, dysfunction of cytokinesis and aneuploidy [22]; it has a global allelic frequency of 0.18078 [23]. This same SNV has been associated with overall cancer risk, particularly breast, gastric, colorectal, liver and endometrial carcinomas, but it has never been formally studied in pituitary tumors [22]. Activating somatic variants of the gene encoding USP8 were recently found in 25–40% of ACTH-secreting adenomas causing CD [24,25]. Patients harboring these variants are usually younger, more frequently females and were found to have higher long-term recurrence rates in some but not all studies [26,27]. USP8 mediates the deubiquitination of EGFR by inhibiting its interaction with protein 14-3-3, which in turn prevents its proteosomal degradation. Signaling through the recycled deubiquitinated EGFR is increased, leading to increased POMC transcription and cellular proliferation. Most activating USP8 variants are located within its 14-3-3 binding motif [24,25]. Recently, USP8 and TP53 SNV were described in corticotroph tumors as drivers of aggressive lesions [28]. To our knowledge, USP8 variants have not been evaluated in patients with pituitary carcinomas, and none of the previously mentioned studies have included patients with Nelson syndrome. In our cohort, neither the CCA nor the SCA showed variants in USP8, in concordance with previously published studies [25,29], or in the genes USP48, BRAF, BRG1 and CABLES1 [9], and none of them were present in our cohort. Genetic structural variations in the human genome can be present in many forms, from SNV to large chromosomal aberrance [30]. CNV are structurally variant regions, including unbalanced deletions, duplications and amplifications of DNA segments ranging from a dozen to several hundred base pairs, in which copy-number differences have been observed between two or more genomes [31,32]. CNV are involved in the development and progression of many tumors and occur frequently in PA [30,33]. Hormone-secreting pituitary tumors show more CNV than non-functioning tumors [34]. Accordingly, our non-functioning SCA and CCA had considerably fewer chromosomal gains and losses than the CD-causing adenomas and the ACTH-CA. Expectedly, the ACTH-CA had significantly more cytogenetic abnormalities than any other tumor in our series. Interestingly, the ACTH-adenomas causing CD, the SCA and the CCA shared the gain of genetic material in 17q12, highlighting their benign nature. The 17q12 amplification has been described in gastric neoplasms [35]. The only cytogenetic abnormality shared by all types of corticotroph tumors was the gain of genetic material in 10q11.22. Amplification of 10q11.22 was previously described in Li–Fraumeni cancer predisposition syndrome [36]. The ACTH-CA, the CCA and one SCA clustered together showing a related CNV pattern; this CNV profile could be reflective of the aggressive nature of these neoplasms, since both CCA and SCA can follow a clinically aggressive course [5,6]. Our results show that all lesions conforming to the pathological spectrum of the corticotroph share some of the SNV and CNV profiles. These genomic changes are consistent with the potential existence of a continuum, whereby silent tumors can transform into a clinically eloquent tumor and finally to carcinoma, or at least a more aggressive tumor. It can also be interpreted as the common SNV shared by aggressive tumors. It is known that silent corticotroph adenomas may switch into a hormone-secreting tumor [37] and are considered a marker for aggressiveness and a risk factor for malignancy since most of the carcinomas are derived from functioning hormone-secreting adenomas. Our phylogenetic inference analysis showed that the genes ATF7IP and MSH3 could participate in a tumor transition ending in aggressive entities or even carcinomas. ATF7IP is a multifunctional nuclear protein mediating heterochromatin formation and gene regulation in several contexts [38], while MSH3 is a mismatch-repair gene [39]. Events related to heterochromatin remodeling and maintenance have been related to aggressive pituitary adenomas and carcinomas [40]. Additionally, alterations in mismatch-repair genes are related to pituitary tumor aggressiveness and resistance to pharmacologic treatment [41,42]. The variants described in ATF7IP and MSH3 are related to prostate and colorectal cancer, respectively [43,44]. There is evidence suggesting that the ATF7IP variant could be deleterious because it leads to a negative regulation of transcription [45]. Thus, these events could be biologically relevant to corticotroph tumorigenesis, although more research is needed. 4. Conclusions We have shown genomic evidence that within the tumoral spectrum of the corticotroph, functioning ACTH-secreting lesions harbor more SNV and CNV than non-functioning ACTH adenomas. The ACTH-secreting CA shows more genomic abnormalities than the other lesions, underscoring its more aggressive biological behavior. Phylogenetic inference analysis of our data reveals that silent corticotroph lesions may transform into functioning tumors, or at least potentially, into more aggressive lesions. Alterations in genes ATF7IP and MSH3, related to heterochromatin formation and mismatch repair, could be important in corticotroph tumorigenesis. The main drawback of our study is the limited sample size. We are currently increasing the number of samples to corroborate our findings and to be able to perform a more comprehensive complementary phylogenetic analysis of our data. Finally, further research is needed to uncover the roles of these variants in corticotroph tumorigenesis. 5. Materials and Methods 5.1. Patients and Tumor Tissue Samples Ten pituitary tissues were collected: one ACTH-CA, one CCA, three SCA, and five ACTH-secreting PA causing CD, including the tumor of a patient who developed Nelson syndrome after bilateral adrenalectomy. All tumors included in the study were sporadic and were collected from patients diagnosed, treated and followed at the Endocrinology Service and the Neurosurgical department of Hospital de Especialidades, Centro Médico Nacional Siglo XXI of the Instituto Mexicano del Seguro Social, Hospital General de Mexico “Dr. Eduardo Liceaga” and Instituto Nacional de Neurologia y Neurocirugia “Manuel Velazquez”. All participating patients were recruited with signed informed consent and ethical approval from the Comisión Nacional de Ética e Investigación Científica of the Instituto Mexicano del Seguro Social, in accordance with the Helsinki declaration. CD was diagnosed according to our standard protocol. Briefly, the presence of hypercortisolism was documented based on two screening tests, namely a 24 h urinary free-cortisol level above 130 µg and the lack of suppression of morning (7:00–8:00) cortisol after administration of 1 mg dexamethasone the night before (23:00) to less than 1.8 µg/dL, followed by a normal or elevated plasma ACTH to ascertain ACTH-dependence. Finally, an overnight, high-dose (8 mg) dexamethasone test, considered indicative of a pituitary source, and a cortisol suppression > 69%, provided that a pituitary adenoma was clearly present on magnetic resonance imaging (MRI) of the sellar region. In none of the 10 patients included in the study was inferior petrosal venous sampling necessary to confirm the pituitary origin of the ACTH excess. Invasiveness was defined by the presence of tumor within the cavernous sinuses (CS). DNA was extracted from paraffin-embedded tumor tissues using the QIAamp DNA FFPE tissue kit. From frozen tumors, DNA was obtained using the Proteinase K-ammonium acetate protocol. 5.2. Construction and Sequencing of Whole Exome Libraries Exome libraries were prepared according to the Agilent SureSelect XT HS Human All exon v7 instructions. Briefly, 200 ng of DNA was enzymatically fragmented with Agilent SureSelect Enzymatic Fragmentation Kit. Fragmented DNA was end-repaired and dA-tail was added at DNA ends; then, molecular barcode adaptors were added, followed by AMPure XP bead purification. The adaptor-ligated library was amplified by PCR and purified by AMPure XP beads. DNA libraries were hybridized with targeting exon probes and purified with streptavidin-coated magnetic beads. The retrieved libraries were amplified by PCR and purified by AMPure XP beads and pooled for sequencing in NextSeq 500 using Illumina flow cell High Output 300 cycles chemistry. All quality controls of the libraries were carried out using Screen tape assays and quantified by Qubit fluorometer. Quality parameters included a DNA integrity number above 8 and a 100X sequencing depth aimed with at least 85% of coverage. 5.3. Bioinformatics Analysis The fastq files were subjected to quality control using FastQC v0.11.9, the adapters were removed using Cutadapt v3.4, the alignment was carried out with Burrows–Wheeler Alignment Tool v0.7.17 with the -M option to ensure compatibility with Picard and GRCh38 as a reference genome. The marking of duplicates as well as the sorting was carried out with Picard v2.26.4 with the AddOrReplaceReadGroups programs with the option SORT_ORDER = coordinate and MarkDuplicates, respectively. Variant calling was carried out using Genomic Analysis Toolkit (GATK) v4.2.2.0 following the Best Practices guide (available at https://gatk.broadinstitute.org/) [46] and with the parameters used by Genomic Data Commons (GDC), available at https://docs.gdc.cancer.gov/ [47]. The GATK tools used were CollectSequencingArtifactMetrics, GetPileupSummaries, CalculateContamination and Mutect2. Mutect2 was run with the latest filtering recommendations, including a Panel of Normal and a Germline Reference from the GATK database. Filtering was performed with the CalculateContamination, LearnReadOrientationModel and FilterMutectCalls tools with the default parameters. For the calculation of CNV GISTIC v2.0.23 was used with the parameters used by GDC. Catalog of Somatic Mutation in Cancer (COSMIC) was used to uncover pathogenic variants. For the analysis of variants and CNV, the maftool v2.10.0 and ComplexHeatmap 2.10.0 packages were used. All analyses were carried out on the GNU/Linux operating system under Ubuntu v20.01.3 or using the R v4.0.2 language in Rstudio v2021.09.0+351. A second bioinformatics pipeline was also used, SureCall software (Agilent) with the default parameters used for SNV variant calling. The variants found by both algorithms were taken as reliable SNV. Data were deposited in Sequence Read Archive hosted by National Center for Biotechnology Information under accession number PRJNA806516. Phylogenetic tree inference (PTI) was run by means of the default parameters using matrices for each sample. These matrices contain an identifier for each variant, mutant read counts, counts of reference reads and the gene associated with the variant. The only PTI parameter was Allele Frequency of Mutation and was used to improve the speed of the algorithm. Briefly, PTI uses an iterative process on the variants shared between the samples. First, it builds the base of the tree using the variants shared by all the samples; second, it eliminates these variants and establishes a split node; and third, it eliminates the variants of the sample that produced the division (split). PTI iteratively performs these three steps for all division possibilities. Each tree is given a score based on an aggregated variant count, and the tree with the highest score is chosen as the optimal tree. 5.4. Sanger Sequencing forConfirmation of Exome Findings Exome variant findings in exome sequencing were validated by Sanger sequencing using BigDye Terminator v3.1 Cycle Sequencing kit (ThermoFischer) in a 3500 Genetic Analyzer. Primers used for USP8 [48], TP53 [49], EGFR [50], AURKA [51], CDKN1A [52,53] and HSD3B1 sequencing have been previously reported. 5.5. Hormone and Transcription Factor Immunohistochemistry Paraffin-embedded, formalin-fixed tissue blocks were stained with hematoxylin–eosin and reviewed by a pathologist. Tumors were represented with a 2-fold redundancy. Sections (3 μm) were cut and placed onto coated slides. Immunostaining was performed by means of the HiDef detection HRP polymer system (Cell Marque, CA, USA), using specific antibodies against each pituitary hormone (TSH, GH, PRL, FSH, LH and ACTH) and the lineage-specific transcription factors TBX19, POU1F1 and NR5A1, as previously described [54]. Two independent observers performed assessment of hormones and transcription factors expression at different times. 5.6. Statistical Analysis Two-tailed Fisher exact tests and Student’s t tests were used to evaluate the relationship between the identified gene variants and clinicopathological features. A p value of <0.05 was considered statistically significant. Statistical software consisted of SPSS v28.0.1 Supplementary Materials The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/ijms23094861/s1. Author Contributions D.M.-R., K.T.-P. and M.M. conceived, designed and coordinated the project, performed experiments, analyzed, discussed data and prepared the manuscript. S.A.-E., G.S.-R., E.P.-M., S.V.-P., R.S., L.B.-A., C.G.-T., J.G.-C. and J.T.A.-S. performed DNA purification, library preparation, sequencing experiments, bioinformatics analysis and wrote the manuscript. A.-L.E.-d.-l.-M., I.R.-S., E.G.-A., L.A.P.-O., G.G., S.M.-J., L.C.-M., B.L.-F. and A.B.-L. provided biological samples and detailed patient information. All authors have read and agreed to the published version of the manuscript. Funding This work was partially supported by grants 289499 from Fondos Sectoriales Consejo Nacional de Ciencia y Tecnologia, Mexico, and R-2015-785-015 from Instituto Mexicano del Seguro Social (MM). Institutional Review Board Statement Protocol approved by the Comisión Nacional de Ética e Investigación Científica of the Instituto Mexicano del Seguro Social, in accordance with the Helsinki declaration (R-2019-785-052). Informed Consent Statement Informed consent was obtained from all subjects involved in the study. Data Availability Statement Data were deposited in Sequence Read Archive hosted by National Center for Biotechnology Information under accession number PRJNA806516. Acknowledgments Sergio Andonegui-Elguera is a doctoral student from Programa de Doctorado en Ciencias Biomédicas, Universidad Nacional Autónoma de México (UNAM) and received fellowship 921084 from CONACYT. 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Compared with placebo, levoketoconazole improved cortisol control and serum cholesterol levels for adults with endogenous Cushing’s syndrome, according to results from the LOGICS study presented here. Safety and efficacy of levoketoconazole (Recorlev, Xeris Biopharma) for treatment of Cushing’s syndrome were established in the pivotal phase 3, open-label SONICS study. The phase 3, double-blind LOGICS study sought to demonstrate the drug specificity of levoketoconazole in normalizing mean urinary free cortisol (mUFC) level. “Treatment with levoketoconazole benefited patients with Cushing’s syndrome of different etiologies and a wide range in UFC elevations at baseline by frequent normalization of UFC,” Ilan Shimon, MD, professor at the Sackler Faculty of Medicine at Tel Aviv University and associate dean of the Faculty of Medicine at Rabin Medical Center and director of the Institute of Endocrinology in Israel, told Healio. “This is a valuable Cushing’s study as it includes a placebo-controlled randomized withdrawal phase.” LOGICS participants were drawn from a cohort of 79 adults with Cushing’s syndrome with a baseline mUFC at least 1.5 times the upper limit of normal who participated in a single-arm, open-label titration and maintenance phase of approximately 14 to 19 weeks. Researchers randomly assigned 39 of those participants plus five from SONICS who had normalized mUFC levels on stable doses of levoketoconazole for at least 4 weeks to continue to receive the medication (n = 22) or to receive placebo with withdrawal of the medication (n = 22) for 8 weeks. At the end of the withdrawal period, all participants received levoketoconazole for 8 more weeks. Primary endpoint was proportion of participants who lost mUFC normalization during the randomized withdrawal period, and secondary endpoints included proportion with normalized mUFC and changes in total and LDL cholesterol at the end of the restoration period. During the withdrawal period, 95.5% of participants receiving placebo vs. 40.9% of those receiving levoketoconazole experienced loss of mUFC response, for a treatment difference of –54.5% (95% CI, –75.7 to –27.4; P = .0002). At the end of the withdrawal period, 4.5% of participants receiving placebo vs. 50% of those receiving levoketoconazole maintained normalized mUFC, for a treatment difference of 45.5% (95% CI, 19.2-67.9; P = .0015). Among participants who had received placebo and lost mUFC response, 60% regained normalized mUFC at the end of the restoration period. During the withdrawal period, participants in the placebo group had increases of 0.9 mmol/L in total cholesterol and 0.6 mmol/L in LDL cholesterol vs. decreases of 0.04 mmol/L (P = .0004) and 0.006 mmol/L (P = .0056), respectively, for the levoketoconazole group. The increases seen in the placebo group were reversed when participants restarted the medication. The most common adverse events with levoketoconazole were nausea (29%) and hypokalemia (26%). Prespecified adverse events of special interest were liver-related (10.7%), QT interval prolongation (10.7%) and adrenal insufficiency (9.5%). “This study has led to the FDA decision to approve levoketoconazole for the treatment of Cushing’s syndrome after surgical failure or if surgery is not possible,” Shimon said. From https://www.healio.com/news/endocrinology/20220512/logics-levoketoconazole-improves-cortisol-control-in-endogenous-cushings-syndrome

Osilodrostat is associated with rapid normalization of mean urinary free cortisol (mUFC) excretion in patients with Cushing disease and has a favorable safety profile, according to the results of a study published in the Journal of Clinical Endocrinology & Metabolism. The phase 3 LINC-4 study (ClinicalTrials.gov Identifier: NCT02697734) evaluated the safety and efficacy of osilodrostat, a potent, orally available 11β­-hydroxylase inhibitor, compared with placebo in patients with Cushing disease. The trial, which was conducted at 40 centers in 14 countries, included a 12-week, randomized, double-blind, placebo-controlled period that was followed by a 36-week, open-label osilodrostat treatment period with an optional extension. Eligible patients were aged 18 to 75 years with a confirmed diagnosis of persistent or recurrent Cushing disease after pituitary surgery and/or irradiation or de novo disease, as well as an mUFC level greater than 1.3 times the upper limit of normal (ULN). The patients were randomly assigned 2:1 to osilodrostat 2 mg twice daily or matching placebo, stratified by prior pituitary irradiation. The primary endpoint was the proportion of patients who achieved mUFC ≤ULN at week 12. The key secondary endpoint was the proportion of patients who achieved mUFC ≤ULN at week 36. A total of 73 patients (median age, 39.0 years; 83.6% women) were randomly assigned to either osilodrostat (n=48) or placebo (n=25) and received at least 1 study drug dose from November 2016 to March 2019. The participants had a median (interquartile range [IQR]) time since diagnosis of Cushing disease of 67.4 (26.4-93.8) months. The median treatment duration in the randomized, placebo-controlled period was 12.0 weeks in both the osilodrostat group (IQR, 2.0-13.0 weeks) and the placebo group (IQR, 11.7-13.7 weeks). The proportion of patients who achieved mUFC ≤ULN (≤138 nmol/24 h) at week 12 was significantly increased in those who received osilodrostat (n=37, 77.1%) vs those who received placebo (n=2, 8.0%), with an estimated odds ratio of 43.4 (95% CI, 7.1-343.2) in favor of osilodrostat (P <.0001). A total of 59 patients (80.8%; 95% CI, 69.9-89.1) also achieved the key secondary endpoint of mUFC ≤ULN at week 36, after 24 weeks of open-label osilodrostat. The most frequently occurring adverse events in the placebo-controlled period in the osilodrostat and placebo groups, respectively, were decreased appetite (37.5% vs 16.0%), arthralgia (35.4% vs 8.0%), nausea (31.3% vs 12.0%), and fatigue (25.0% vs 16.0%). A potential study limitation is that although osilodrostat exposure was greater than 1 year among the participants, some adverse effects may take longer to be observed. “This randomized, placebo-controlled trial demonstrates that osilodrostat is a highly effective treatment for Cushing disease, normalizing UFC excretion in 77% of patients after 12 weeks’ treatment,” stated the investigators. “Cortisol reductions were maintained throughout 48 weeks of treatment and were accompanied by improvements in clinical signs of hypercortisolism and quality of life. The safety profile was favorable.” Disclosure: This study was funded by Novartis Pharma AG. Some of the study authors declared affiliations with biotech, pharmaceutical, and/or device companies. Please see the original reference for a full list of disclosures. Reference Gadelha M, Bex M, Feelders RA, et al. Randomized trial of osilodrostat for the treatment of Cushing’s disease. J Clin Endocrinol Metab. Published online March 23, 2022. doi:10.1210/clinem/dgac178 From https://www.endocrinologyadvisor.com/home/topics/general-endocrinology/osilodrostat-effective-for-cushing-disease/

Abstract Cushing’s syndrome (CS) secondary to ectopic adrenocorticotrophic hormone (ACTH)-producing prostate cancer is rare with less than 50 cases reported. The diagnosis can be challenging due to atypical and variable clinical presentations of this uncommon source of ectopic ACTH secretion. We report a case of Cushing’s syndrome secondary to prostate adenocarcinoma who presented with symptoms of severe hypercortisolism with recurrent hypokalaemia, limb oedema, limb weakness, and sepsis. He presented with severe hypokalaemia and metabolic alkalosis (potassium 2.5 mmol/L and bicarbonate 36 mmol/L), with elevated 8 am cortisol 1229 nmol/L. ACTH-dependent Cushing’s syndrome was diagnosed with inappropriately normal ACTH 57.4 ng/L, significantly elevated 24-hour urine free cortisol and unsuppressed cortisol after 1 mg low-dose, 2-day low-dose, and 8 mg high-dose dexamethasone suppression tests. 68Ga-DOTANOC PET/CT showed an increase in DOTANOC avidity in the prostate gland, and his prostate biopsy specimen was stained positive for ACTH and markers for neuroendocrine differentiation. He was started on ketoconazole, which was switched to IV octreotide in view of liver dysfunction from hepatic metastases. He eventually succumbed to the disease after 3 months of his diagnosis. It is imperative to recognize prostate carcinoma as a source of ectopic ACTH secretion as it is associated with poor clinical outcomes, and the diagnosis can be missed due to atypical clinical presentations. 1. Introduction Ectopic secretion of adrenocorticotropic hormone (ACTH) is responsible for approximately 10–20% of all causes of Cushing syndrome [1]. The classic sources of ectopic ACTH secretion include bronchial carcinoid tumours, small cell lung carcinoma, thymoma, medullary thyroid carcinoma (MTC), gastroenteropancreatic neuroendocrine tumours (NET), and phaeochromocytomas [2]. Ectopic adrenocorticotropic syndrome (EAS) is diagnostically challenging due to its variable clinical manifestations; however, prompt recognition and treatment is critical. Ectopic ACTH production from prostate carcinoma is rare, and there are less than 50 cases published to date. Here, we report a case of ectopic Cushing’s syndrome secondary to prostate adenocarcinoma who did not present with the typical physical features of Cushing’s syndrome, but instead with features of severe hypercortisolism such as hypokalaemia, oedema, and sepsis. 2. Case Presentation A 61-year-old male presented to our institution with recurrent hypokalaemia, lower limb weakness, and oedema. He had a history of recently diagnosed metastatic prostate adenocarcinoma, for which he was started on leuprolide and finasteride. Other medical history includes poorly controlled diabetes mellitus and hypertension of 1-year duration. He presented with hypokalaemia of 2.7 mmol/L associated with bilateral lower limb oedema and weakness, initially attributed to the intake of complementary medicine, which resolved with potassium supplementation and cessation of the complementary medicine. One month later, he was readmitted for refractory hypokalaemia of 2.5 mmol/L and progression of the lower limb weakness and oedema. On examination, his blood pressure (BP) was 121/78 mmHg, and body mass index (BMI) was 24 kg/m2. He had no Cushingoid features of rounded and plethoric facies, supraclavicular or dorsocervical fat pad, ecchymoses, and no purple striae on the abdominal examination. He had mild bilateral lower limb proximal weakness and oedema. His initial laboratory findings of severe hypokalaemia with metabolic alkalosis (potassium 2.5 mmol/L and bicarbonate 36 mmol/L), raised 24-hour urine potassium (86 mmol/L), suppressed plasma renin activity and aldosterone, central hypothyroidism, and elevated morning serum cortisol (1229 nmol/L) (Table 1) raised the suspicion for endogenous hypercortisolism. Furthermore, hormonal evaluations confirmed ACTH-dependent Cushing’s syndrome with inappropriately normal ACTH (56 ng/L) and failure of cortisol suppression after 1 mg low-dose, 2-day low-dose, and 8 mg high-dose dexamethasone suppression tests (Table 2). His 24-hour urine free cortisol (UFC) was significantly elevated at 20475 (59–413) nmol/day. Table 1 Investigations done during his 2nd admission. Table 2 Diagnostic workup for hypercortisolism. To identify the source of excessive cortisol secretion, magnetic resonance imaging (MRI) of the pituitary fossa and computed tomography (CT) of the thorax, abdomen, and pelvis were performed. Pituitary MRI was unremarkable, and CT scan showed the known prostate lesion with extensive liver, lymph nodes, and bone metastases (Figure 1). To confirm that the prostate cancer was the source of ectopic ACTH production, gallium-68 labelled somatostatin receptor positron emission tomography (PET)/CT (68Ga-DOTANOC) was done, which showed an increased DOTANOC avidity in the inferior aspect of the prostate gland (Figure 2). Immunohistochemical staining of his prostate biopsy specimen was requested, and it stained positive for ACTH and markers of neuroendocrine differentiation (synaptophysin and CD 56) (Figures 3 and 4), establishing the diagnosis of EAS secondary to prostate cancer. Figure 1 CT thorax abdomen and pelvis showing prostate cancer (blue arrow) with liver metastases (red arrow). Figure 2 Ga68-DOTANOC PET/CT demonstrating increased DOTANOC avidity seen in the inferior aspect of the right side of the prostate gland (red arrow). Figure 3 Hematoxylin and eosin staining showing acinar adenocarcinoma of the prostate featuring enlarged, pleomorphic cells infiltrating as solid nests and cords with poorly differentiated glands (Gleason score 5 + 4 = 9). Figure 4 Positive ACTH immunohistochemical staining of prostate tumour (within the circle). The patient was started on potassium chloride 3.6 g 3 times daily and spironolactone 25 mg once daily with normalisation of serum potassium. His BP was controlled with the addition of lisinopril and terazosin to spironolactone and ketoconazole, and his blood glucose was well controlled with metformin and sitagliptin. To manage the hypercortisolism, he was treated with ketoconazole 400 mg twice daily with an initial improvement of serum cortisol from 2048 nmol/L to 849 nmol/L (Figure 5). Systemic platinum and etoposide-based chemotherapy was recommended for the treatment of his prostate cancer after a multidisciplinary discussion, but it was delayed due to severe bacterial and viral infection. With the development of liver dysfunction, ketoconazole was switched to intravenous octreotide 100 mcg three times daily as metyrapone was not readily available in our country. However, the efficacy was suboptimal with marginal reduction of serum cortisol from 3580 nmol/L to 3329 nmol/L (Figure 5). The patient continued to deteriorate and was deemed to be medically unfit for chemotherapy or bilateral adrenalectomy. He was referred to palliative care services, and he eventually demised due to cancer progression within 3 months of his diagnosis. Figure 5 The trend in cortisol levels on pharmacological therapy. 3. Discussion Ectopic ACTH secretion is an uncommon cause of Cushing’s syndrome accounting for approximately 9–18% of the patients with Cushing’s syndrome [3]. Clinical presentation is highly variable depending on the aggressiveness of the underlying malignancy, but patients typically present with symptoms of severe hypercortisolism such as hypokalaemiaa, oedema, and proximal weakness which were the presenting complaints of our patient [4]. The classical symptoms of Cushing’s syndrome are frequently absent due to the rapid clinic onset resulting in diagnostic delay [5]. Prompt diagnosis and localisation of the source of ectopic ACTH secretion are crucial due to the urgent need for treatment initiation. The usual sources include small cell lung carcinoma, bronchial carcinoid, medullary thyroid carcinoma, thymic carcinoid, and pheochromocytoma. CT of the thorax, abdomen, and pelvis should be the first-line imaging modality, and its sensitivity varies with the type of tumour ranging from 77% to 85% [6]. Functional imaging such as 18-fluorodeoxyglucose-PET and gallium-68 labelled somatostatin receptor PET/CT can be useful in localising the source of occult EAS, determining the neuroendocrine nature of the tumour or staging the underlying malignancy [3, 6]. As prostate cancer is an unusual cause of EAS, we proceeded with 68Ga-DOTANOC PET/CT in our patient to localise the source of ectopic ACTH production. The goals of management in EAS include treating the hormonal excess and the underlying neoplasm as well as managing the complications secondary to hypercortisolism [3]. Prompt management of the cortisol excess is paramount as complications such as hyperglycaemia, hypertension, hypokalaemia, pulmonary embolism, sepsis, and psychosis can develop especially when UFC is more than 5 times the upper limit of normal [3]. Ideally, surgical resection is the first-line management, but this may not be feasible in metastatic, advanced, or occult diseases. Pharmacological agents are frequently required with steroidogenesis inhibitors such as ketoconazole and metyrapone, which reduce cortisol production effectively and rapidly [3, 6], the main drawback of ketoconazole being its hepatic toxicity. The efficacy of ketoconazole is reported to be 44%, metyrapone 50–75%, and ketoconazole-metyrapone combination therapy 73% [3, 7]. Mitotane, typically used in adrenocortical carcinoma, is effective in controlling cortisol excess but has a slow onset of action [3, 8]. Etomidate infusion can be used for short-term rapid control of severe symptomatic hypercortisolism and can serve as a bridge to definitive therapy [9]. Mifepristone, a glucocorticoid receptor antagonist, is indicated mainly in difficult to control hyperglycaemia secondary to hypercortisolism [8]. Somatostatin analogue has been proposed as a possible pharmacological therapy due to the expression of somatostatin receptors by ACTH secreting tumours [8, 10]. Bilateral adrenalectomy should be considered in patients with severe symptomatic hypercortisolism and life-threatening complications who cannot be optimally managed with medical therapies, especially in patients with occult EAS or metastatic disease [3, 8]. Bilateral adrenalectomy results in immediate improvement in cortisol levels and symptoms secondary to hypercortisolism [11]. However, surgical complications, morbidity, and mortality are high in patients with uncontrolled hypercortisolism [8], and our patient was deemed by his oncologist and surgeon to have too high a risk for bilateral adrenalectomy. For the treatment of prostate carcinoma, platinum and etoposide-based chemotherapies have been used, but their efficacy is limited with a median survival of 7.5 months [4, 12]. The side effects of chemotherapy can be severe with an enhanced risk of infection due to both cortisol and chemotherapy-mediated immunosuppression. Prompt control of hypercortisolism prior to chemotherapy and surgical procedure is strongly suggested to attenuate life-threatening complications such as infection, thrombosis, and bleeding with chemotherapy or surgery as well as to improve prognosis [3, 13]. There are rare reports of ectopic ACTH secretion from prostate carcinoma. These tumours were predominantly of small cell or mixed cell type, and pure adenocarcinoma with neuroendocrine differentiation are less common [4, 5]. There is a strong correlation between the prognosis and the types of malignancy in patients with EAS, and patients with prostate carcinoma have a poor prognosis [4]. These patients had metastatic disease at presentation, and the median survival was weeks to months despite medical treatment, chemotherapy, and even bilateral adrenalectomy [4], as seen with our patient who passed away within 3 months of his diagnosis. In conclusion, adenocarcinoma of the prostate is a rare cause of EAS. The diagnosis and management are complex and challenging requiring specialised expertise with multidisciplinary involvement. The presentation can be atypical, and it is imperative to suspect and recognise prostate carcinoma as a source of ectopic ACTH secretion. Prompt initiation of treatment is important, as it is a rapidly progressive and aggressive disease associated with intense hypercortisolism resulting in high rates of mortality and morbidity. Data Availability The data used to support the findings of this study are included within the article. Conflicts of Interest The authors declare that there are no conflicts of interest. Acknowledgments The authors would like to thank the Pathology Department of Changi General Hospital for their contribution to this case. References I. Ilias, D. J. Torpy, K. Pacak, N. Mullen, R. A. Wesley, and L. K. Nieman, “Cushing’s syndrome due to ectopic corticotropin secretion: twenty years’ experience at the national institutes of health,” Journal of Clinical Endocrinology & Metabolism, vol. 90, no. 8, pp. 4955–4962, 2005.View at: Publisher Site | Google Scholar J. Newell-Price, P. Trainer, M. Besser, and A. Grossman, “The diagnosis and differential diagnosis of cushing’s syndrome and pseudo-cushing’s states,” Endocrine Reviews, vol. 19, no. 5, pp. 647–672, 1998.View at: Publisher Site | Google Scholar J. Young, M. Haissaguerre, O. Viera-Pinto, O. Chabre, E. Baudin, and A. 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Brower, “Management of patients with small cell carcinoma and the syndrome of ectopic corticotropin secretion,” Cancer, vol. 73, no. 5, pp. 1361–1367, 1994.View at: Google Scholar Copyright Copyright © 2022 Wanling Zeng and Joan Khoo. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. From https://www.hindawi.com/journals/crie/2022/3739957/

More than three-quarters of adults with Cushing’s disease assigned osilodrostat had a normalized mean urinary free cortisol level at 12 weeks and maintained a normal level at 36 weeks, according to data from the LINC 4 phase 3 trial. In findings published in The Journal of Clinical Endocrinology & Metabolism, 77% of adults with Cushing’s disease randomly assigned to osilodrostat (Isturisa, Recordati) had mean urinary free cortisol (UFC) levels reduced to below the upper limit of normal at 12 weeks compared with 8% of adults assigned to placebo. Most adults with Cushing's disease taking 2 mg twice daily osilodrostat had normalized mean UFC levels at 12 weeks compared with placebo. Data were derived from Gadelha M, et al. J Clin Endocrinol Metab. 2022;doi:10.1210/clinem/dgac178. “Osilodrostat is a highly effective treatment for Cushing’s disease, normalizing urinary free cortisol excretion in 77% of patients after 12 weeks’ treatment,” Mônica Gadelha, MD, professor of endocrinology at The Federal University of Rio de Janeiro, and colleagues wrote. “Cortisol reductions were maintained throughout 48 weeks of treatment and were accompanied by improvements in clinical signs of hypercortisolism and quality of life.” Gadelha and colleagues enrolled 73 adults aged 18 to 75 years with Cushing’s disease from 40 centers in 14 countries into the LINC 4 phase 3 trial. Participants were randomly assigned to 2 mg osilodrostat twice daily (n = 48) or placebo (n = 25) for 12 weeks. Urinary samples were collected at weeks 2, 5 and 8 to measure mean UFC, and dosage was adjusted based on efficacy and tolerability. After 12 weeks, participants from both groups received osilodrostat in a 36-week open-label treatment period. All participants restarted the open-label portion of the trial at 2 mg osilodrostat unless they were on a lower dose at week 12. Dose adjustments in the open-label phase were made using the same guidelines in the randomized, double-blind, placebo-controlled trial. The primary endpoint was the efficacy of osilodrostat at achieving a mean UFC below the upper limit of normal of 138 nmol per 24 hours at 12 weeks vs. placebo; the key secondary endpoint was the percentage of participants achieving a normal mean UFC at 36 weeks. At 12 weeks, the percentage of adults with a normalized mean UFC level was higher in the osilodrostat group compared with placebo (77.1% vs. 8%; P < .0001). At 36 weeks, 80.8% of all participants had a normal mean UFC level. The overall response rate was 79.5% at 48 weeks. Median time to first controlled mean UFC response was 35 days for those randomly assigned to osilodrostat as well as those randomly assigned to placebo who crossed over to osilodrostat for the open-label phase. At 48 weeks, 84% of participants were receiving 10 mg or less of osilodrostat per day, including 56% receiving 4 mg or less daily. At 12 weeks, the osilodrostat group had several cardiovascular and metabolic-related improvements, including systolic and diastolic blood pressure, HbA1c, HDL cholesterol, body weight and waist circumference. No changes were observed in the placebo group. “The improvements in cardiovascular and metabolic parameters were sustained throughout osilodrostat treatment and have the potential to alleviate the burden of comorbidities in many patients with Cushing’s disease,” the researchers wrote. At 12 weeks, 52.5% of those receiving osilodrostat had a reduction in supraclavicular fat pad and 50% had a reduction in dorsal fat pad. At least 25% of participants also had improvements in facial redness, striae, proximal muscle atrophy and central obesity. Improvements were sustained through week 48. During the placebo-controlled trial, grade 3 and 4 adverse events occurred for about 20% of participants in both groups. For the entire study, 38.4% of adults reported grade 3 and 4 adverse events, with the most common being hypertension. Eight participants discontinued the study due to adverse events. From https://www.healio.com/news/endocrinology/20220408/osilodrostat-normalizes-urinary-free-cortisol-in-most-adults-with-cushings-disease

Highlights • Cushing syndrome (CS) is a rare disorder with a variety of underlying etiologies. • CS is expected to affect 0.2 to 5 people per million per year. • Adrenal-dependent CS is an uncommon variant of CS. • This study reports a rare occurrence of pituitary and adrenal adenoma with CS. Abstract Introduction Cushing syndrome is a rare disorder with a variety of underlying etiologies, that can be exogenous or endogenous (adrenocorticotropic hormone (ACTH)-dependent or ACTH-independent). The current study aims to report a case of ACTH-independent Cushing syndrome with adrenal adenoma and nonfunctioning pituitary adenoma. Case report A 37–year–old female presented with amenorrhea for the last year, associated with weight gain. She had a moon face, buffalo hump, and central obesity. A 24-hour urine collection for cortisol was performed, revealing elevated cortisol. Cortisol level was non-suppressed after administering dexamethasone. MRI of the pituitary revealed a pituitary microadenoma, and the CT scan of the abdomen with adrenal protocol revealed a left adrenal adenoma. Discussion Early diagnosis may be postponed due to the variety of clinical presentations and the referral of patients to different subspecialists based on their dominant symptoms (gynecological, dermatological, cardiovascular, psychiatric); it is, therefore, critical to consider the entire clinical presentation for correct diagnosis. Conclusion Due to the diversity in the presentation of CS, an accurate clinical, physical and endocrine examination is always recommended. Keywords Cushing syndrome Cushing's disease Adrenal adenoma Pituitary adenoma Urine free cortisol 1. Introduction Cushing syndrome (CS) is a collection of clinical manifestations caused by an excess of glucocorticoids [1]. CS is a rare disorder with a variety of underlying etiologies that can be exogenous due to continuous corticosteroid therapy for any underlying inflammatory illness or endogenous due to either adrenocorticotropic hormone (ACTH)-dependent or ACTH-independent [2], [3]. Cushing syndrome is expected to affect 0.2 to 5 people per million per year. Around 10% of such cases involve children [4], [5]. ACTH-dependent glucocorticoid excess owing to pituitary adenoma accounts for the majority (60–70%) of endogenous CS, with primary adrenal causes accounting for only 20–30% and ectopic ACTH-secreting tumors accounting for the remaining 5–10% [6]. Adrenal-dependent CS is an uncommon variant of CS caused mostly by benign (90%) or malignant (8%) adrenal tumors or, less frequently, bilateral micronodular (1%) or macronodular (1%) adrenal hyperplasia [7]. The current study aims to report a case of ACTH-independent Cushing syndrome with adrenal adenoma and nonfunctioning pituitary adenoma. The report has been arranged in line with SCARE guidelines and includes a brief literature review [8]. 2. Case report 2.1. Patient's information A 37–year–old female presented with amenorrhea for the last year, associated with weight gain. She denied having polyuria, polydipsia, headaches, visual changes, dizziness, dryness of the skin, cold intolerance, or constipation. She had no history of chronic disease and denied using steroids. She visited an internist, a general surgeon, and a gynecologist and was treated for hypothyroidism. She was put on Thyroxin 100 μg daily, and oral contraceptive pills were given for her menstrual problems. Last time, the patient was referred to an endocrinology clinic, and they reviewed the clinical and physical examinations. 2.2. Clinical examination She had a moon face, buffalo hump, central obesity, pink striae over her abdomen, and proximal weakness of the upper limbs. After reviewing the history and clinical examination, CS was suspected. 2.3. Diagnostic assessment Because the thyroid function test revealed low thyroid-stimulating hormone (TSH), free T3, and freeT4, the patient was sent for a magnetic resonance imaging (MRI) of the pituitary, which revealed a pituitary microadenoma (7 ∗ 6 ∗ 5) mm (Fig. 1). Since the patient was taking thyroxin and oral contraceptive pills, the investigations were postponed for another six weeks due to the contraceptive pills' influence on the results of the hormonal assessment for CS. After six weeks of no medication, a 24-hour urinary free cortisol (UFC) was performed three times, revealing elevated cortisol levels (1238, 1100, and 1248) nmol (normal range, 100–400) nmol. A dexamethasone suppression test was done (after administering dexamethasone tab 1 mg at 11 p.m., serum cortisol was measured at 9 a.m.). The morning serum cortisol level was 620 nmol (non-suppressed), which normally should be less than 50 nmol. The ACTH level was below 1 pg/mL. Download : Download high-res image (103KB) Download : Download full-size image Fig. 1. Contrast enhanced T1W weighted MRI (coronal section) showing small 7 mm hypo-enhanced microadenoma (yellow arrow) in right side of pituitary gland with mild superior bulge. Based on these findings, ACTH independent CS was suspected. The computerized tomography (CT) scan of the abdomen with adrenal protocol revealed a left adrenal adenoma (33 mm × 25 mm) without features of malignancy (Fig. 2). Download : Download high-res image (168KB) Download : Download full-size image Fig. 2. Computed tomography scan of the abdomen with IV contrast, coronal section, showing 33 mm × 25 mm lobulated enhanced left adrenal tumor (yellow arrow), showing absolute washout on dynamic adrenal CT protocol, consistent with adrenal adenoma. 2.4. Therapeutic intervention The patient was referred to the urologist clinic for left adrenalectomy after preparation for surgery and perioperative hormonal management. She underwent laparoscopic adrenalectomy and remained in the hospital for two days. The histopathology results supported the diagnosis of adrenal adenoma. 2.5. Follow-up She was released home after two days on oral hydrocortisone 20 mg in the morning and 10 mg in the afternoon. After one month of follow-up, serum cortisol was 36 nmol, with the resolution of some features such as weight reduction (3 kg) and skin color (pink striae became white). 3. Discussion Cushing's syndrome is a serious and well-known medical condition that results from persistent exposure of the body to excessive glucocorticoids, either from endogenous or, most frequently, exogenous sources [9]. The average age of diagnosis is 41.4 years, with a female-to-male ratio of 3:1 [10]. ACTH-dependent CS accounts for almost 80% of endogenous CS, while ACTH-independent CS accounts for nearly 20% [10]. This potentially fatal condition is accompanied by several comorbidities, including hypertension, diabetes, coagulopathy, cardiovascular disease, infections, and fractures [11]. Exogenous CS, also known as iatrogenic CS, is more prevalent than endogenous CS and is caused by the injection of supraphysiologic glucocorticoid dosages [12]. ACTH-independent CS is induced by uncontrolled cortisol release from an adrenal gland lesion, most often an adenoma, adrenocortical cancer, or, in rare cases, ACTH-independent macronodular adrenal hyperplasia or primary pigmented nodular adrenal disease [13]. The majority of data suggests that early diagnosis is critical for reducing morbidity and mortality. Detection is based on clinical suspicion initially, followed by biochemical confirmation [14]. The clinical manifestation of CS varies depending on the severity and duration of glucocorticoid excess [14]. Some individuals may manifest varying symptoms and signs because of a rhythmic change in cortisol secretion, resulting in cyclical CS [15]. The classical symptoms of CS include weight gain, hirsutism, striae, plethora, hypertension, ecchymosis, lethargy, monthly irregularities, diminished libido, and proximal myopathy [16]. Neurobehavioral presentations include anxiety, sadness, mood swings, and memory loss [17]. Less commonly presented features include headaches, acne, edema, abdominal pain, backache, recurrent infection, female baldness, dorsal fat pad, frank diabetes, electrocardiographic abnormalities suggestive of cardiac hypertrophy, osteoporotic fractures, and cardiovascular disease from accelerated atherosclerosis [10]. The current case presented with amenorrhea, weight gain, moon face, buffalo hump, and skin discoloration of the abdomen. Similar to the current case, early diagnosis may be postponed due to the variety of clinical presentations and the referral of patients to different subspecialists based on their dominant symptoms (gynecological, dermatological, cardiovascular, psychiatric); it is, therefore, critical to consider the entire clinical presentation for correct diagnosis [18]. Weight gain may be less apparent in children, but there is frequently an arrest in growth with a fall in height percentile and a delay in puberty [19]. The diagnosis and confirmation of the etiology can be difficult and time-consuming, requiring a variety of laboratory testing and imaging studies [20]. According to endocrine society guidelines, the initial assessment of CS must include one or more of the three following tests: 24-hour UFC measurement; evaluation of the diurnal variation of cortisol secretion by assessing the midnight serum or salivary cortisol level; and a low-dose dexamethasone suppression test, typically the 1 mg overnight test [21]. Although UFC has sufficient sensitivity and specificity, it does not function well in milder cases of Cushing's syndrome [22]. In CS patients, the typical circadian rhythm of cortisol secretion is disrupted, and a high late-night cortisol serum level is the earliest and most sensitive diagnostic indicator of the condition [23]. In the current case, the UFC was elevated, and cortisol was unsuppressed after administration of dexamethasone. All patients with CS should have a high-resolution pituitary MRI with a gadolinium-based contrast agent to prove the existence or absence of a pituitary lesion and to identify the source of ACTH between pituitary adenomas and ectopic lesions [24]. Adrenal CT scan is the imaging modality of choice for preoperatively localizing and subtyping adrenocortical lesions in ACTH-independent Cushing's syndrome [9]. MRI of the pituitary gland of the current case showed a microadenoma and a CT scan of the adrenals showed left adrenal adenoma. Surgical resection of the origin of the ACTH or glucocorticoid excess (pituitary adenoma, nonpituitary tumor-secreting ACTH, or adrenal tumor) is still the first-line treatment of all forms of CS because it leaves normal adjacent structures and results in prompt remission and inevitable recovery of regular adrenal function [12], [25]. Laparoscopic (retroperitoneal or transperitoneal) adrenalectomy has become the gold standard technique for adrenal adenomas since it is associated with fewer postoperative morbidity, hospitalization, and expense when compared to open adrenalectomy [17]. In refractory cases, or when a patient is not a good candidate for surgery, cortisol-lowering medication may be employed [26]. The current case underwent left adrenalectomy. Symptoms of CS, such as central obesity, muscular wasting or weakness, acne, hirsutism, and purple striae generally improve first and may subside gradually over a few months or even a year; nevertheless, these symptoms may remain in 10–30% of patients [27]. Glucocorticoid replacement is essential after adrenal-sparing curative surgery until the pituitary-adrenal function returns, which might take up to two years, especially if adrenal adenomas have been resected [25]. Chronic glucocorticoid excess causes lots of new co-morbidities, lowering the quality of life and increasing mortality. The most common causes of mortality in CS are cardiovascular disease and infections [28]. After one month of follow-up, serum cortisol was 36 nmol, and several features, such as weight loss (3 kg) and skin color, were resolved (pink striae became white). In conclusion, the coexistence of adrenal adenoma and pituitary adenoma with CS is a rare possibility. Due to the diversity in the presentation of CS, an accurate clinical, physical and endocrine examination is always recommended. Laparoscopic adrenalectomy is the gold standard for treating adrenal adenoma. Consent Written informed consent was obtained from the patient's family for publication of this case report and accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal on request. Provenance and peer review Not commissioned, externally peer-reviewed. Ethical approval Approval is not necessary for case report (till 3 cases in single report) in our locality. The family gave consent for the publication of the report. Funding None. Guarantor Fahmi Hussein Kakamad, Fahmi.hussein@univsul.edu.iq. Research registration number Not applicable. CRediT authorship contribution statement Abdulwahid M. Salh: major contribution of the idea, literature review, final approval of the manuscript. Rawa Bapir: Surgeon performing the operation, final approval of the manuscript. Fahmi H. Kakamad: Writing the manuscript, literature review, final approval of the manuscript. Soran H. Tahir, Fattah H. Fattah, Aras Gh. Mahmood, Rawezh Q. Salih, Shaho F. Ahmed: literature review, final approval of the manuscript. Declaration of competing interest None to be declared. References [1] S.M. Ahmed, S.F. Ahmed, S. Othman, B.A. Abdulla, S.H. Mohammed, A.M. Salih, et al. 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This is one of the suggestions from the Cushing’s Awareness Challenge post: Our “Official mascot” is the zebra. Our mascot In med school, student doctors are told “When you hear hoofbeats, think horses, not zebras“. According to Wikipedia: “Zebra is a medical slang term for a surprising diagnosis. Although rare diseases are, in general, surprising when they are encountered, other diseases can be surprising in a particular person and time, and so “zebra” is the broader concept. The term derives from the aphorism ‘When you hear hoofbeats behind you, don’t expect to see a zebra’, which was coined in a slightly modified form in the late 1940s by Dr. Theodore Woodward, a former professor at the University of Maryland School of Medicine in Baltimore. Since horses are the most commonly encountered hoofed animal and zebras are very rare, logically you could confidently guess that the animal making the hoofbeats is probably a horse. A zebra cup my DH bought me 🙂 By 1960, the aphorism was widely known in medical circles.” Why? Because those of us who DO have a rare disorder know from personal experience what it feels like to be dismissed by a doctor or in many cases, multiple doctors. Many physicians have completely lost the ability to even imagine that zebras may exist! Cushing’s is too rare – you couldn’t possible have that. Well… rare means some people get it. Why couldn’t it be me? Although one of my signature images has a zebra, many have rainbows or butterflies in them so I guess that I consider those my own personal mascots. I posted this in 2010 in 40 Days of Thankfulness: Days Twenty-Two through Thirty Butterflies are something else again. I like them because I would like to think that my life has evolved like a butterfly’s, from something ugly and unattractive to something a big easier on the eye. My Cushie self was the caterpillar, post-op is more butterfly-ish, if not in looks, in good deeds. From July, 2008

Researchers published the study covered in this summary on Research Square as a preprint that has not yet been peer reviewed. Key Takeaways Among women who underwent pituitary surgery to treat Cushing disease subsequent pregnancy had no apparent effect on Cushing disease recurrence, in a single-center review of 113 women treated over a 30-year period. Why This Matters No single factor predicts the recurrence of Cushing disease during long-term follow-up of patients who have undergone pituitary surgery. This is the first study to assess the effect of pregnancy on Cushing disease recurrence in a group of reproductive-age women who initially showed post-surgical remission. Study Design Retrospective study of 355 patients with confirmed Cushing disease who were admitted to a single tertiary hospital in Brazil between 1990 and 2020. All patients had transsphenoidal surgery, with a minimum follow-up of 6 months and median follow-up of 83 months. Remission occurred in 246 of these patients. The current analysis focused on 113 of the patients who achieved remission, were women, were 45 years old or younger at time of surgery (median 32 years old), and had information available on their obstetric history. Ninety-one of these women (81%) did not become pregnant after their surgery, and 22 (19%) became pregnant after surgery. Key Results Among the 113 women in the main analysis 43 (38%) had a Cushing disease recurrence, a median of 48 months after their pituitary surgery. Following surgery, 11 women in each of the two subgroups (recurrence, no recurrence) became pregnant. Although the subgroup with recurrence had a higher incidence of pregnancy (11/43; 26%) compared with those with no recurrence (11/70; 16%) Kaplan-Meier analysis showed that survival free of Cushing disease recurrence was similar and not significantly different in the women with a postsurgical pregnancy and those who did not become pregnant (P=.531). The review also showed that, of the women who became pregnant, several obstetrical measures were similar between patients who had a recurrence and those who remained in remission, including number of pregnancies per patient, maternal weight gain, type of delivery (normal or cesarean), delivery time (term or premature), neonatal weight, and neonatal size. The review also showed roughly similar rates of maternal and fetal complications in these two subgroups of women who became pregnant. Limitations The study was retrospective and included a relatively small number of patients. The authors collected information on obstetric history for some patients by telephone or email contacts. Disclosures The study received no commercial funding. None of the authors had disclosures. This is a summary of a preprint research study , " Pregnancy After Pituitary Surgery Does Not Influence the Recurrence of Cushing's Disease, " written by researchers at the Sao Paulo (Brazil) University Faculty of Medicine on Research Square provided to you by Medscape. This study has not yet been peer reviewed. The full text of the study can be found on researchsquare.com.

The study covered in this summary was published on Research Square as a preprint and has not yet been peer reviewed. Key Takeaways A study of 78 patients who underwent elective transsphenoidal adenomectomy to remove a pituitary tumor or other lesions within the pituitary fossa at a single center in the UK suggests that postoperative plasma levels of copeptin — a surrogate marker for levels of arginine vasopressin (antidiuretic hormone) — can rule out development of central (neurogenic) diabetes insipidus caused by a deficiency of arginine vasopressin following pituitary surgery. The researchers suggest using as a cutoff a copeptin level of >3.4 pmol/L at postoperative day 1 to rule out diabetes insipidus. Such a cutoff yields the following: A high sensitivity of 91% for ruling out diabetes insipidus. A negative predictive value of 97%. Only 1 of 38 patients with a copeptin value >3.4 pmol/L at day 1 postoperatively developed diabetes insipidus. A low specificity of 55%, meaning that copeptin level is not useful for diagnosing diabetes insipidus Why This Matters An estimated 1% to 67% of patients who undergo pituitary gland surgery develop diabetes insipidus, often soon after surgery, although it is often transient. Diagnosing diabetes insipidus in such patients requires a combination of clinical assessment, the monitoring of fluid balance, and determining plasma and urine sodium and osmolality. Currently, clinical laboratories in the UK do not have assays for arginine vasopressin, which has a short half-life in vivo and is unstable ex vivo, even when frozen, and is affected by delayed or incomplete separation from platelets. Copeptin, an arginine vasopressin precursor, is much more stable and measurable by commercial immunoassays. The findings suggest that patients who have just undergone pituitary gland surgery and are otherwise healthy and meet the copeptin cutoff for ruling out diabetes insipidus could be discharged 24 hours after surgery and that there is no need for additional clinical and biochemical monitoring. This change would ease demands on the healthcare system. Study Design The study reviewed 78 patients who underwent elective transsphenoidal adenomectomy to remove a pituitary tumor from November 2017 to June 2020 at the John Radcliffe Hospital in Oxford, United Kingdom. Patients remained in hospital for a minimum of 48 hours after their surgery. Clinicians collected blood and urine specimens preoperatively and at day 1, day 2, day 8, and week 6 post surgery. The patients were restricted to 2 L of fluid a day postoperatively to prevent masking of biochemical abnormalities through compensatory drinking. Diabetes insipidus was suspected when patients' urine output was >200 mL/h for 3 consecutive hours or >3 L/d plus high plasma sodium (>145 mmol/L) and plasma osmolality (> 295 mosmol/kg) plus inappropriately low urine osmolality. Definitive diagnosis of diabetes insipidus was based on clinical assessment, urine and plasma biochemistry, and need for treatment with desmopressin (1-deamino-8-D-arginine vasopressin). Key Results The median age of the patients was 55, and 53% were men; 92% of the lesions were macroadenomas; the most common histologic type was gonadotroph tumor (47%). Among the 78 patients, 11 (14%) were diagnosed with diabetes insipidus postoperatively and required treatment with desmopressin; of these, seven patients (9%) continued taking desmopressin after 6 weeks (permanent diabetes insipidus), but the other four did not need to take desmopressin for more than a week. Patients who developed diabetes insipidus were younger than other patients (mean age, 46 vs 56), and 8 of the 11 patients who developed diabetes insipidus (73%) were women. Preoperative copeptin levels were similar in the two groups. At day 1, day 8, and 6 weeks postoperatively, copeptin levels were significantly lower in the diabetes insipidus group; there were no significant differences at day 2, largely because of an outlier result. An area under the receiver operating characteristic curve (AUC; C-statistic) analysis showed that on day 1 after surgery, copeptin levels could account for 74.22% of the incident cases of diabetes insipidus (AUC, 0.7422). On postop day 8, the AUC for copeptin was 0.8015, and after 6 weeks, the AUC associated with copeptin was 0.7321. Limitations Blood samples for copeptin tests from patients who underwent pituitary surgery were collected at specified times and were frozen for later analysis; performing the test in real time might alter patient management. The study may have missed peak copeptin levels by not determining copeptin levels sooner after pituitary gland surgery, inasmuch as other researchers have reported better predictive values for diagnosing diabetes insipidus from samples taken 1 hour after extubation or <12 hours after surgery. Disclosures The study did not receive commercial funding. The authors report no relevant financial relationships. This is a summary of a preprint research study, "Post-Operative Copeptin Analysis Predicts Which Patients Do Not Develop Diabetes Insipidus After Pituitary Surgery," by researchers from John Radcliffe Hospital, Oxford University Hospitals NHS Foundation Trust, in the United Kingdom. Preprints from Research Square are provided to you by Medscape. This study has not yet been peer reviewed. The full text of the study can be found on researchsquare.com. Read the article here: https://www.medscape.com/viewarticle/970357#vp_1

Abstract Summary The pandemic caused by severe acute respiratory syndrome coronavirus 2 is of an unprecedented magnitude and has made it challenging to properly treat patients with urgent or rare endocrine disorders. Little is known about the risk of coronavirus disease 2019 (COVID-19) in patients with rare endocrine malignancies, such as pituitary carcinoma. We describe the case of a 43-year-old patient with adrenocorticotrophic hormone-secreting pituitary carcinoma who developed a severe COVID-19 infection. He had stabilized Cushing’s disease after multiple lines of treatment and was currently receiving maintenance immunotherapy with nivolumab (240 mg every 2 weeks) and steroidogenesis inhibition with ketoconazole (800 mg daily). On admission, he was urgently intubated for respiratory exhaustion. Supplementation of corticosteroid requirements consisted of high-dose dexamethasone, in analogy with the RECOVERY trial, followed by the reintroduction of ketoconazole under the coverage of a hydrocortisone stress regimen, which was continued at a dose depending on the current level of stress. He had a prolonged and complicated stay at the intensive care unit but was eventually discharged and able to continue his rehabilitation. The case points out that multiple risk factors for severe COVID-19 are present in patients with Cushing’s syndrome. ‘Block-replacement’ therapy with suppression of endogenous steroidogenesis and supplementation of corticosteroid requirements might be preferred in this patient population. Learning points Comorbidities for severe coronavirus disease 2019 (COVID-19) are frequently present in patients with Cushing’s syndrome. ‘Block-replacement’ with suppression of endogenous steroidogenesis and supplementation of corticosteroid requirements might be preferred to reduce the need for biochemical monitoring and avoid adrenal insufficiency. The optimal corticosteroid dose/choice for COVID-19 is unclear, especially in patients with endogenous glucocorticoid excess. First-line surgery vs initial disease control with steroidogenesis inhibitors for Cushing’s disease should be discussed depending on the current healthcare situation. Keywords: Adult; Male; Other; Belgium; Pituitary; Adrenal; Neuroendocrinology; Oncology; Insight into disease pathogenesis or mechanism of therapy; February; 2022 Background The pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has had a significant impact on the health care systems to date. The clinical presentation of coronavirus disease 2019 (COVID-19) is diverse, ranging from asymptomatic illness to respiratory failure requiring admission to the intensive care unit (ICU). Risk factors for severe course include old age, male gender, comorbidities such as arterial hypertension, diabetes mellitus, chronic lung-, heart-, liver- and kidney disease, malignancy, immunodeficiency and pregnancy (1). Little is known about the risk of COVID-19 in patients with rare endocrine malignancies, such as pituitary carcinoma. Case presentation This case concerns a 43-year-old man with adrenocorticotrophic hormone (ACTH)-secreting pituitary carcinoma (with cerebellar and cervical drop metastases) with a severe COVID-19 infection. He had previously received multiple treatment modalities including surgery, radiotherapy, ketoconazole, pasireotide, cabergoline, bilateral (subtotal) adrenalectomy and temozolomide chemotherapy as described elsewhere (2). His most recent therapy was a combination of immune checkpoint inhibitors consisting of ipilimumab (3 mg/kg) and nivolumab (1 mg/kg) (anti-CTLA-4 and anti-PD-1, respectively) every 3 weeks for four cycles, after which maintenance therapy with nivolumab (240 mg) every 2 weeks was continued. Residual endogenous cortisol production was inhibited with ketoconazole 800 mg daily. He had stabilized disease with a decrease in plasma ACTH, urinary free cortisol and stable radiological findings (2). Surgical resection of the left adrenal remnant was planned but was not carried out due to the development of a COVID-19 infection. In March 2021, he consulted our emergency department for severe respiratory complaints. He had been suffering from upper respiratory tract symptoms for one week, with progressive dyspnoea in the last three days. He tested positive for SARS-CoV-2 the day before admission. On examination, his O2 saturation was 72%, with tachypnoea (40/min) and bilateral pulmonary crepitations. His temperature was 37.2°C, blood pressure 124/86 mmHg and pulse rate 112 bpm. High-flow oxygen therapy was initiated but yielded insufficient improvement (O2 saturation of 89% and tachypnoea 35/min). He was urgently intubated for respiratory exhaustion. Investigation Initial investigations showed type 1 respiratory insufficiency with PaO2 of 52.5 mmHg (normal 75–90), PaCO2 of 33.0 mmHg (normal 36–44), pH of 7.47 (normal 7.35–7.45) and a P/F ratio of 65.7 (normal >300). His inflammatory parameters were elevated with C-reactive protein level of 275.7 mg/L (normal <5·0) and white blood cell count of 7.1 × 10⁹ per L with 72.3% neutrophils. His most recent morning plasma ACTH-cortisol level (measured using the Elecsys electrochemiluminescence immunoassays on a Cobas 8000 immunoanalyzer [Roche Diagnostics]) before his admission was 213 ng/L (normal 7.2–63) and 195 µg/L (normal 62–180) respectively, while a repeat measurement 3 weeks after his admission demonstrated increased cortisol levels of 547 µg/L (possibly iatrogenic due to treatment with high-dose hydrocortisone) and a decreased ACTH of 130 ng/L. Treatment On admission, he was started on high-dose dexamethasone therapy for 10 days together with broad-spectrum antibiotics for positive sputum cultures containing Serratia, methicillin-susceptible Staphylococcus aureus and Haemophilus influenzae. Thromboprophylaxis with an intermediate dose of low molecular weight heparin (tinzaparin 14 000 units daily for a body weight of 119 kg) was initiated. A ‘block-replacement’ regimen was adopted with the continuation of ketoconazole (restarted on day 11) in view of his endocrine treatment and the supplementation of hydrocortisone at a dose depending on the current level of stress. The consecutive daily dose of hydrocortisone and ketoconazole is shown in Fig. 1. View Full Size Figure 1 ‘Block-replacement’ therapy with ketoconazole and hydrocortisone/dexamethasone. Dexamethasone 10 mg daily was initially started as COVID-19 treatment, followed by hydrocortisone at a dose consistent with current levels of stress. Ketoconazole was restarted on day 11 and titrated to a dose of 800 mg daily to suppress endogenous glucocorticoid production. Citation: Endocrinology, Diabetes & Metabolism Case Reports 2022, 1; 10.1530/EDM-21-0182 Download Figure Download figure as PowerPoint slide Outcome and follow-up He developed multiple organ involvement, including metabolic acidosis, acute renal failure requiring continuous venovenous hemofiltration, acute coronary syndrome type 2, septic thrombophlebitis of the right jugular vein, and critical illness polyneuropathy. He was readmitted twice to the ICU, for ventilator-associated pneumonia and central line-associated bloodstream infection respectively. He eventually recovered and was discharged from the hospital to continue his rehabilitation. Discussion We describe the case of a patient with severe COVID-19 infection with active Cushing’s disease due to pituitary carcinoma, who was treated with high-dose dexamethasone followed by ‘block-replacement’ therapy with hydrocortisone in combination with off-label use of ketoconazole as a steroidogenesis inhibitor. His hospitalization was prolonged by multiple readmissions to the ICU for infectious causes. Our case illustrates the presence of multiple comorbidities for a severe and complicated course of COVID-19 in a patient with active Cushing’s disease. Dexamethasone was initially chosen as the preferred corticosteroid therapy, in analogy with the RECOVERY trial, in which dexamethasone at a dose of 6mg once daily (oral or i.v.) resulted in lower 28-day mortality in hospitalized patients with COVID-19 requiring oxygen therapy or invasive mechanical ventilation (3). However, the optimal dose/choice of corticosteroid therapy is unclear, especially in a patient population with pre-existing hypercortisolaemia. A similar survival benefit for hydrocortisone compared to dexamethasone has yet to be convincingly demonstrated. This may be explained by differences in anti-inflammatory activity but could also be due to the fact that recent studies with hydrocortisone were stopped early and were underpowered (4, 5). Multiple risk factors for a complicated course of COVID-19 are present in patients with Cushing’s syndrome and might increase morbidity and mortality (6, 7). These include a history of obesity, arterial hypertension and impaired glucose metabolism. Prevention and treatment of these pre-existing comorbidities are essential. Patients with Cushing’s syndrome also have an increased thromboembolic risk, which is further accentuated by the development of severe COVID-19 infection (6, 7). Thromboprophylaxis with low molecular weight heparin is associated with lower mortality in COVID-19 patients with high sepsis‐induced coagulopathy score or high D-dimer levels (8) and is presently widely used in the treatment of severe COVID-19 disease (9). Subsequently, this treatment is indicated in hospitalized COVID-19 patients with Cushing’s syndrome. It is unclear whether therapeutic anticoagulation dosing could provide additional benefits (6, 7). An algorithm based on the International Society on Thrombosis and Hemostasis-Disseminated Intravascular Coagulation score was proposed to evaluate the ideal anticoagulation therapy in severe/critical COVID-19 patients, with an indication for therapeutic low molecular weight heparin dose at a score ≥5 (9). Furthermore, the chronic cortisol excess induces suppression of the innate and adaptive immune response. Patients with Cushing’s syndrome, especially when severe and active, should be considered immunocompromised and have increased susceptibility for viral and other (hospital-acquired) infections. Prophylaxis for Pneumocystis jirovecii with trimethoprim/sulfamethoxazole should therefore be considered (6, 7). Additionally, there is a particular link between the pathophysiology of COVID-19 and Cushing’s syndrome. The SARS-CoV-2 virus (as well as other coronaviruses) enter human cells by binding the ACE2 receptor. The transmembrane serine protease 2 (TMPRSS2), expressed by endothelial cells, is additionally required for the priming of the spike-protein of SARS-CoV-2, leading to viral entry. TMPRSS2 was studied in prostate cancer and found to be regulated by androgen signalling. Consequently, the androgen excess frequently associated with Cushing’s syndrome might be an additional risk factor for contracting COVID-19 via higher TMPRSS2 expression (10), especially in women, in whom the effect of excess androgen would be more noticeable compared to male patients with Cushing’s syndrome. Treating Cushing’s syndrome with a ‘block-replacement’ approach, with suppression of endogenous steroidogenesis and supplementation of corticosteroid requirements, is an approach that should be considered, especially in severe or cyclic disease. The use of this method might decrease the need for monitoring and reduce the occurrence of adrenal insufficiency (7). Our patient was on treatment with ketoconazole, which was interrupted at initial presentation and then restarted under the coverage of a hydrocortisone stress regimen. Ketoconazole was chosen because of its availability. Advantages of ketoconazole over metyrapone include its antifungal activity with the potential for prevention of invasive pulmonary fungal infections, as well as its antiandrogen action (especially in female patients) and subsequent inhibition of TMPRSS2 expression (10). Regular monitoring of the liver function (every month for the first 3 months, at therapy initiation or dose increase) is necessary. Caution is needed due to its inhibition of multiple cytochrome P450 enzymes (including CYP3A4) and subsequently greater risk of drug-drug interactions vs metyrapone (7, 10). Another disadvantage of ketoconazole is the need for oral administration. In our patient, ketoconazole was delivered through a nasogastric tube. i.v. etomidate is an alternative in case of an unavailable enteral route. Finally, as a general point, the first-line treatment of a patient with a novel diagnosis of Cushing’s disease is transsphenoidal surgery. Recent endocrine recommendations pointed out the possibility of initial disease control with steroidogenesis inhibitors in patients without an indication for urgent intervention during a high prevalence of COVID-19 (7). This would allow the optimalization of metabolic parameters; emphasizing that the short-to mid-term prognosis is related to the cortisol excess and not its cause. Surgery could then be postponed until the health situation allows for safe elective surgery (7). This decision depends of course on the evolution of COVID-19 and the healthcare system in each country and should be closely monitored by policymakers and physicians. Declaration of interest The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported. Funding This work did not receive any specific grant from any funding agency in the public, commercial, or not-for-profit sector. Patient consent Written informed consent for publication of their clinical details and/or clinical images was obtained from the patient. Author contribution statement J M K de Filette is an endocrinologist-in-training and was the main author. All authors were involved in the clinical care of the patient. All authors contributed to the reviewing and editing process and approved the final version of the manuscript. References 1↑ Gao Y-D, Ding M, Dong X, Zhang J-J, Kursat Azkur A, Azkur D, Gan H, Sun Y-L, Fu W, Li W, et al.Risk factors for severe and critically ill COVID-19 patients: a review. Allergy 2021 76 428–455.(https://doi.org/10.1111/all.14657) Search Google Scholar Export Citation 2↑ Sol B, de Filette JMK, Awada G, Raeymaeckers S, Aspeslagh S, Andreescu CE, Neyns B, Velkeniers B. Immune checkpoint inhibitor therapy for ACTH-secreting pituitary carcinoma: a new emerging treatment? European Journal of Endocrinology 2021 184 K1–K5. (https://doi.org/10.1530/EJE-20-0151) Search Google Scholar Export Citation 3↑ The RECOVERY Collaborative Group. Dexamethasone in hospitalized patients with Covid-19. 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https://doi.org/10.1002/ccr3.5337 Abstract A 50-year-old woman with adrenal Cushing's syndrome and chronic hepatitis C developed an acute exacerbation of chronic hepatitis C before adrenectomy. After administration of glecaprevir/pibrentasvir was started, her transaminase levels normalized promptly and a rapid virological response also was achieved. Laparoscopic left adrenectomy was then performed safely. 1 INTRODUCTION Reports of reactivation of hepatitis C virus (HCV) and acute exacerbation of chronic hepatitis C associated with immunosuppressive therapy and cancer drug therapy are rarer than for hepatitis B virus (HBV) but have been made occasionally. In HBV infection, viral reactivation and acute hepatitis caused by an excess of endogenous cortisol due to Cushing's syndrome have been reported, but no acute exacerbation of chronic hepatitis C has been reported so far. Here, we report a case of acute exacerbation of chronic hepatitis C during the course of adrenal Cushing's syndrome. 2 CASE REPORT A woman in her 50s underwent a CT scan at a nearby hospital to investigate treatment-resistant hypertension and was found to have a left adrenal mass. Her blood tests showed low ACTH and HCV antibody positivity, and she was referred to our hospital because she was suspected of having Cushing's syndrome and chronic hepatitis C. There is nothing special to note about her medical or family history. She had never smoked and drank very little. Her physical findings on admission were 164.5 cm tall, 92.6 kg in weight, and a BMI of 34.2 kg/m2. Her blood pressure was 179 / 73 mmHg, pulse 64 /min (rhythmic), body temperature 36.8°C, and respiratory rate 12 /min. She had findings of central obesity, moon face, buffalo hump, and red skin stretch marks. Her blood test findings (Table 1) showed an increase in ALT, HCV antibody positivity, and an HCV RNA concentration of 4.1 log IU/mL. The virus was genotype 2. Cortisol was within the reference range, but ACTH was as low, less than 1.5 pg/mL. Her bedtime cortisol level was 7.07 μg/dL, which was above her reference of 5 μg/dL, suggesting the loss of diurnal variation in cortisol secretion. Testing showed the amount of cortisol by 24-hour urine collection was 62.1 μg/day, and this level of cortisol secretion was maintained. In an overnight low-dose dexamethasone suppression test, cortisol after loading was 6.61 μg/dL, which exceeded 5 μg/dL, suggesting that cortisol was autonomously secreted. Her contrast-enhanced CT scan (Figure 1) revealed a tumor with a major axis of about 30 mm in her left adrenal gland. MRI scans showed mild hyperintensity in the “in phase” (Figure 2A) and decreased signal in the “out of phase” (Figure 2B), suggesting her adrenal mass was an adenoma. Based on the above test results, she was diagnosed with chronic hepatitis C and adrenal Cushing's syndrome. She agreed to receive treatment with direct acting antiviral agents (DAAs) after resection of the left adrenal tumor. However, two months later, she had liver dysfunction with AST 116 U/L and ALT 213 U/L (Figure 3). HBV DNA was undetectable at the time of liver injury, but the HCV RNA concentration increased to 6.4 logIU/mL. Therefore, an acute exacerbation of chronic hepatitis C was suspected, and a percutaneous liver biopsy was performed. The biopsy revealed an inflammatory cell infiltration, mostly composed of lymphocytes and plasma cells and mainly in the portal vein area (Figure 4). Fibrosis and interface hepatitis were also observed, and spotty necrosis was evident in the hepatic lobule. No clear fat deposits were found in the hepatocytes, ruling out NASH or NAFLD. According to the New Inuyama classification, hepatitis equivalent to A2-3/F1-2 was considered. Because HBV DNA was not detected, no new drug was used, and no cause of liver damage, such as biliary atresia, was found; the patient was diagnosed with liver damage due to reactivation of HCV, with acute exacerbation of chronic hepatitis C. The treatment policy was changed, in order to treat hepatitis C before the left adrenal resection, and administration of glecaprevir/pibrentasvir was started. A blood test two weeks after the start of treatment confirmed normalization of AST and ALT, and a rapid virological response was achieved (Figure 3). Subsequently, HCV RNA remained negative, no liver damage was observed, and laparoscopic left adrenectomy was safely performed nine months after the initial diagnosis. The pathological findings were adrenal adenoma, and no atrophy was observed in the attached normal adrenal cortical gland. After the operation, hypertension improved and weight loss was obtained (92.6 kg (BMI: 34.2 kg/m2) before the operation, but 77.0 kg (BMI: 28.5 kg/m2) one year after the operation). ACTH increased, and the adrenal Cushing's syndrome was considered to have been cured. Regarding HCV infection, the sustained virological response has been maintained to date, more than 2 years after the completion of DAA therapy, and the follow-up continues. TABLE 1. Laboratory data on admission Hematology Chemistry WBC 6100 /μL TP 8.2 g/dL DHEA-S 48 /μL RBC 526 x 104 /μL Alb 3.4 g/dL PRA 0.7 ng/mL/h Hb 15.8 g/dL T-Bil 0.3 mg/dL ALD 189 pg/mL Ht 49.1 % AST 33 U/L PLT 25.5 x 104 /μL ALT 46 U/L Serological tests LDH 201 U/L CRP <0.10 mg/dL ALP 292 U/L HBsAg (-) γ-GTP 77 U/L anti-HBs (-) Coagulation BUN 13 mg/dL anti-HBc (+) PT 126.1 % Cr 0.63 mg/dL HBeAg (-) APTT 27.5 sec HbA1c 6.2 % anti-HBe (+) Cortisol 7.46 μg/dL anti-HCV (+) ACTH <1.5 pg/mL FBS 82 mg/dL Genetic tests Na 138 mmol/L HBV DNA Undetectable Cl 105 mmol/L HCV RNA 4.1 LogIU/Ml K 3.6 mmol/L HCV genotype 2 Ca 9.0 mg/dL Abbreviations: Hematology: WBC, white blood cells; RBC, red blood cells; Hb, hemoglobin; Ht, hematocrit; PLT, platelets. Coagulation: PT, prothrombin time; APTT, activated partial thromboplastin time. Chemistry: TP, total protein; Alb, albumin; T-Bil, total bilirubin; AST, aspartate transaminase; ALT, alanine aminotransferase; LDH, lactate dehydrogenase; ALP, alkaline phosphatase; γGTP, γ-glutamyl transpeptidase; BUN, blood urea nitrogen; Cr, creatinine; HbA1c, Hemoglobin A1c; FBS, fasting blood sugar; Na, sodium; Cl, chlorine; K, potassium; Ca, calcium; DHEA-S, dehydroepiandrosterone sulfate; PRA, plasma renin activity; ALD, aldosterone. Serological tests: CRP, C-reactive protein; HBsAg, hepatitis B surface antigen; anti-HBs, hepatitis B surface antibody; anti-HBc, hepatitis B core antibody; HBeAg, hepatitis B e antigen; anti-HBe, hepatitis B e antibody; anti-HCV, hepatitis C virus antibody. Genetic tests: HBV DNA, hepatitis B virus deoxyribonucleic acid; HCV RNA, hepatitis C virus ribonucleic acid. FIGURE 1 Open in figure viewerPowerPoint Contrast-enhanced CT examination. Contrast-enhanced CT examination revealed a tumor (arrow) with a major axis of about 30 mm in the left adrenal gland FIGURE 2 Open in figure viewerPowerPoint MRI image of the adrenal lesion. MRI showed mild hyperintensity in the "in phase" (A) and decreased signal in the "out of phase" (B), suggesting adrenocortical adenoma (arrow) FIGURE 3 Open in figure viewerPowerPoint Changes in serum transaminase and HCV RNA levels. All showed rapid improvement by administration of direct acting antivirals. ALT: alanine aminotransferase, AST: aspartate transaminase, HCV RNA: hepatitis C virus ribonucleic acid FIGURE 4 Open in figure viewerPowerPoint Pathological findings of tissues obtained by percutaneous liver biopsy. Infiltration of inflammatory cells, which was mostly composed of lymphocytes and plasma cells and a small number of neutrophils, was observed mainly in the portal vein area. This was accompanied by fibrous enlargement and interface hepatitis. Although the arrangement of hepatocytes was maintained in the hepatic lobule, spotty necrosis was observed in some parts. No clear fat deposits were found in the hepatocytes, and NASH or NAFLD was a negative finding. According to the New Inuyama classification, hepatitis equivalent to A2-3/F1-2 was considered (a; ×100, b; ×200, scale bar = 500 µm) 3 DISCUSSION Reactivation of HBV can cause serious liver damage. Therefore, it is recommended to check the HBV infection status before starting anticancer chemotherapy or immunotherapy and to continue monitoring for the presence or absence of reactivation thereafter.1, 2 On the other hand, there are fewer reports of the reactivation of HCV, and many aspects of the pathophysiology of HCV reactivation remain unclear. In this case, it is possible that chronic hepatitis C was acutely exacerbated due to endogenous cortisol secretion in Cushing's syndrome. Although the definition of HCV reactivation has not been defined, several studies3-5 have defined an increase of HCVRNA of 1.0 log IU/ml or more as HCV reactivation. In addition, the definition of acute exacerbation of chronic hepatitis C is that ALT increases to more than three times the upper limit of the reference range.3, 4, 6 Mahale et al. reported a retrospective study in which acute exacerbation of chronic hepatitis C due to cancer medication was seen in 11% of 308 patients.3 Torres et al. also reported that, in a prospective study of 100 patients with cancer medication, HCV reactivation was found in 23%.4 Given these reports, HCV reactivation potentially could occur quite frequently. However, Torres et al. reported that only 10% of all patients had acute exacerbations, none of which led to liver failure.4 Such data suggest that HCV reactivation may often be overlooked in actual cases without aggravation. Thus, the frequency of aggravation due to hepatitis virus reactivation is thought to be lower for HCV than for HBV. However, there are some reports of deaths from acute exacerbation of chronic hepatitis C.7-10 In addition, if severe hepatitis develops following viral reactivation, mortality rates have been reported to be similar for HBV and HCV.8, 11 Thus, reactivation of HCV is considered to be a pathological condition that requires caution, similar to HBV. Torres et al. reported that administration of rituximab or corticosteroids is a significant independent risk factor.4 In addition, there are reports of acute exacerbation of chronic hepatitis C due to corticosteroids administered as antiemetics and as immunosuppressive therapy.12-14 Therefore, excess cortisol can reactivate not only HBV but also HCV. The mechanism by which HCV is reactivated with cortisol is assumed to be decreased cell-mediated immunity due to rapid apoptosis of circulating T cells caused by glucocorticoids,4 enhancement of HCV infectivity by upregulation of viral receptor expression on the hepatocyte surface,15 and enhanced viral replication.16 In addition, there is a report that genotype 2 is more common in cases with acute exacerbation of chronic hepatitis C,4, 13 which is consistent with this case. Regarding HBV reactivation due to Cushing's syndrome, three cases of acute exacerbation of chronic hepatitis B have been reported.17-19 It is believed that Cushing's syndrome caused a decrease in cell-mediated immunity and humoral immunity due to an endogenous excess of cortisol, resulting in an acute exacerbation of chronic hepatitis B.13 As described above, because an excess of cortisol can cause reactivation of HCV, it is considered that a decrease in immunocompetence due to Cushing's syndrome, which is an excess of endogenous cortisol, can also cause reactivation of HCV and acute exacerbation of chronic hepatitis. However, as far as we can determine, no cases of Cushing's syndrome causing HCV reactivation or acute exacerbation of chronic hepatitis C have been reported and similar cases may be latent. Among the reports of acute exacerbation of hepatitis B due to adrenal Cushing's syndrome, there is a case in which the liver damage and viral load were improved only by adrenalectomy.17 Therefore, it is also possible that hepatitis C was improved by adrenal resection in this case. However, general anesthesia associated with adrenalectomy and the use of various drugs used for postoperative physical management should be avoided, if possible, in situations where some severe liver damage is present. In addition, reactivation of immunity due to rapid depletion of glucocorticoid, following resection of an adrenal tumor, may lead to exacerbation of liver damage. In this case, the amount of HCV and hepatic transaminase levels were improved rapidly by glecaprevir/pibrentasvir treatment, and the operation could be performed safely. If Cushing's syndrome is complicated by an acute exacerbation of hepatitis C, clinicians should consider including treatment strategies such as in this case. Summarizing the above, when liver damage appears in HCV-infected patients with Cushing's syndrome, it will be necessary to distinguish the acute exacerbation and reactivation of chronic hepatitis C. Treatment with DAAs may then be considered to be effective for reactivation of HCV and acute exacerbation of chronic hepatitis. 4 CONCLUSION We report a case of chronic hepatitis C with acute exacerbation during the course of Cushing's syndrome. At the time of cancer drug therapy and in the state of endogenous and extrinsic corticosteroid excess, it is necessary to pay attention not only to acute exacerbation of chronic hepatitis B but also to hepatitis C. ACKNOWLEDGEMENTS All authors would like to thank the patient and his family for allowing this case study. CONFLICT OF INTEREST The authors have no conflict of interests. AUTHOR CONTRIBUTIONS TO and KM were collected and analyzed the data and wrote and edited the manuscript. KH, ST, HO, KT, KM, and JK were involved in the patient's care and provided advice on the preparation of this case report. ETHICAL APPROVAL This study complied with the standards of the Declaration of Helsinki and the current ethical guidelines. CONSENT Written informed consent was obtained from the patient to publish this report in accordance with the journal's patient consent policy. From https://onlinelibrary.wiley.com/doi/10.1002/ccr3.5337

Join our Rare Disease Day virtual panel discussion as BioNews columnists from a variety of our rare communities participate in a lively conversation with fellow patient advocate Liza Bernstein. This window into often overlooked aspects of life with a rare disease will provide a variety of patient perspectives. Topics will include awareness and advocacy, equity, mental health, empowerment, and more. We invite everyone to join us for this signature event and look forward to your participation in the Q and A! Panelists: Paris Dancy, Columnist, Cushing’s Disease News Michelle Gonzaba, Columnist, Myasthenia Gravis News Claire Richmond, Columnist, Porphyria News Sherry Toh, Columnist, SMA News Today Hosted by Liza Bernstein, Patient Advocate & Sr. Director Patient & Community Engagement Time Feb 28, 2022 02:00 PM in Central Time (US and Canada) Register at https://us06web.zoom.us/webinar/register/WN_dylme0wBRCyH8TfQ7B6x-w

Abstract Summary Here, we describe a case of a patient presenting with adrenocorticotrophic hormone-independent Cushing’s syndrome in a context of primary bilateral macronodular adrenocortical hyperplasia. While initial levels of cortisol were not very high, we could not manage to control hypercortisolism with ketoconazole monotherapy, and could not increase the dose due to side effects. The same result was observed with another steroidogenesis inhibitor, osilodrostat. The patient was finally successfully treated with a well-tolerated synergitic combination of ketoconazole and osilodrostat. We believe this case provides timely and original insights to physicians, who should be aware that this strategy could be considered for any patients with uncontrolled hypercortisolism and delayed or unsuccessful surgery, especially in the context of the COVID-19 pandemic. Learning points Ketoconazole–osilodrostat combination therapy appears to be a safe, efficient and well-tolerated strategy to supress cortisol levels in Cushing syndrome. Ketoconazole and osilodrostat appear to act in a synergistic manner. This strategy could be considered for any patient with uncontrolled hypercortisolism and delayed or unsuccessful surgery, especially in the context of the COVID-19 pandemic. Considering the current cost of newly-released drugs, such a strategy could lower the financial costs for patients and/or society. Keywords: Adult; Male; White; France; Adrenal; Adrenal; Novel treatment; December; 2021 Background Untreated or inadequately treated Cushing’s syndrome (CS) is a morbid condition leading to numerous complications. The latter ultimately results in an increased mortality that is mainly due to cardiovascular events and infections. The goal of the treatment with steroidogenesis inhibitors is normalization of cortisol production allowing the improvement of comorbidities (1). Most studies dealing with currently available steroidogenesis inhibitors used as monotherapy reported an overall antisecretory efficacy of roughly 50% in CS. Steroidogenesis inhibitors can be combined to better control hypercortisolism. To the best of our knowledge, we report here for the first time a patient treated with a ketoconazole–osilodrostat combination therapy. Case presentation Here, we report the case of Mr D.M., 53-years old, diagnosed with adrenocorticotrophic hormone (ACTH)-independent CS 6 months earlier. At diagnosis, he presented with resistant hypertension, hypokalemia, diabetes mellitus, easy bruising, purple abdominal striae and major oedema of the lower limbs. Investigations A biological assessment was performed, and the serum cortisol levels are depicted in Table 1. ACTH levels were suppressed (mean levels 1 pg/mL). Mean late-night salivary cortisol showed a four-fold increase (Table 2), and mean 24 h-urinary cortisol showed a two-fold increase. Serum cortisol was 1000 nmol/L at 08:00 h after 1 mg dexamethasone dose at 23:00 h. The rest of the adrenal hormonal workup was within normal ranges (aldosterone: 275 pmol/L and renin: 15 mIU/L). An adrenal CT was performed (Fig. 1) and exhibited a 70-mm left adrenal mass (spontaneous density: 5 HU and relative washout: 65%) and a 45-mm right adrenal mass (spontaneous density: −2 HU and relative washout: 75%). The case was discussed in a multidisciplinary team meeting, which advised to perform 18F-FDG PET-CT and 123I-Iodocholesterol scintigraphy before considering surgery. A genetic screening was performed, testing for ARMC5 and PRKAR1A pathogenic variants. View Full Size Figure 1 Adrenal CT depicting the bilateral macronodular adrenocortical hyperplasia. Citation: Endocrinology, Diabetes & Metabolism Case Reports 2021, 1; 10.1530/EDM-21-0071 Download Figure Download figure as PowerPoint slide Table 1 Serum cortisol levels at diagnosis (A), using ketoconazole monotherapy (B), using osilodrostat monotherapy (C) and using osilodrostat–ketoconazole combination therapy (D). Serum cortisol (nmol/L) 08:00 h 24:00 h 16:00 h 20:00 h 12:00 h 16:00 h A. At diagnosis 660 615 716 566 541 561 B. Ketoconazole monotherapy 741 545 502 224 242 508 C. Osilodrostat monotherapy 658 637 588 672 486 692 D. Osilodrostat–ketoconazole combination 436 172 154 103 135 274 Table 2 Salivary cortisol levels at diagnosis (A), using ketoconazole monotherapy (B), using osilodrostat monotherapy (C) and using osilodrostat-ketoconazole combination therapy (D). Salivary cortisol (nmol/L) 23:00 h 12:00 h 13:00 h Mean A. At diagnosis 47 62 38 49 B. Ketoconazole monotherapy 20 15 21 18 C. Osilodrostat monotherapy 85 90 56 77 D. Osilodrostat–ketoconazole combination 10 14 9 11 Treatment As this condition occurred during the COVID-19 pandemic, it was decided to first initiate steroidogenesis inhibitors to lower the patient’s cortisol levels. Initially, ketoconazole was initiated and uptitrated up to 1000 mg per day based on close serum cortisol monitoring, with a three-fold increase of liver enzymes and poor control of cortisol levels (Table 1). In the absence of biological efficacy, ketoconazole was replaced by osilodrostat, which was gradually increased up to 30 mg per day (10 mg at 08:00 h and 20 mg at 20:00 h) without reaching normal cortisol levels (Table 1) and with slightly increased blood pressure levels. Considering the lack of efficacy of anticortisolic drugs used as monotherapy, we combined osilodrostat (30 mg per day) to ketoconazole (600 mg per day), that is, at the last maximal tolerated dose as monotherapy of each drug. Outcome This combination of steroidogenesis inhibitors achieved a good control in cortisol levels, mimicking a physiological circadian rhythm (Table 1D). The patient did not exhibit any side effect and the control of cortisol levels resulted in a rapid improvement of hypertension, kalemia, diabetes control and disappearance of lower limbs oedema. The patient underwent a 18F-FDG PET-CT that did not exhibit any increased uptake in both adrenal masses and a 123I-Iodocholesterol scintigraphy exhibiting a highly increased uptake in both adrenal masses, predominating in the left adrenal mass (70 mm). Unilateral adrenalectomy of the larger mass was then performed, and as the immediate post-operative serum cortisol level was 50 nmol/L, hydrocortisone was administered at a dose of 30 mg per day, with a stepwise decrease to 10 mg per day over 3 months. Pathological examination exhibited macronodular adrenal hyperplasia with a 70-mm adreno cortical adenoma (WEISS score: 1 and Ki67: 1%). The genetic screening exhibited a c.1908del p.(Phe637Leufs*6) variant of ARMC5 (pathogenic), located in exon 5. The patient has no offspring and is no longer in contact with the rest of his family. Discussion The goal of the treatment with steroidogenesis inhibitors is normalization of cortisol production allowing the improvement of comorbidities (1). Most studies dealing with currently available steroidogenesis inhibitors used as monotherapy reported an overall antisecretory efficacy of roughly 50% in CS. This rate of efficacy was probably underestimated in retrospective studies due to the lack of adequate uptitration of the dose; For example, the median dose reported in the French retrospective study on ketoconazole was only 800 mg/day, while 50% of the patients were uncontrolled at the last follow-up (2). Steroidogenesis inhibitors can be combined to better control hypercortisolism. Up to now, such combinations, mainly ketoconazole and metyrapone, were mainly reported in patients with severe CS (median urinary-free Cortisol (UFC) 30- to 40-fold upper-limit norm (ULN)) and life-threatening comorbidities (3, 4). Normal UFC was reported in up to 86% of these patients treated with high doses of ketoconazole and metyrapone. Expected side effects (such as increased liver enzymes for ketoconazole or worsened hypertension and hypokalemia for metyrapone) were reported in the majority of the patients. The fear of these side effects probably explains the lack of uptitration in previous reports. Combination of steroidogenesis inhibitors has previously been described by Daniel et al. in the largest study reported on the use of metyrapone in CS; 29 patients were treated with metyrapone and ketoconazole or mitotane, including 22 in whom the second drug was added to metyrapone monotherapy because of partial efficacy or adverse effects. The final median metyrapone dose in patients controlled with combination therapy was 1500 mg per day (5). Combination of adrenal steroidogenesis inhibitors should not be reserved to patients with severe hypercortisolism. In the case shown here, the association was highly effective in terms of secretion, using lower doses than those applied as a single treatment, but without the side effects previously observed with higher doses of each treatment used as a monotherapy. To our knowledge, the association of ketoconazole and osilodrostat had never been reported. Ketoconazole blocks several enzymes of the adrenal steroidogenesis such as CYP11A1, CYP17, CYP11B2 (aldosterone synthase) and CYP11B1 (11-hydroxylase), leading to decreased cortisol and occasionally testosterone concentrations. Though liver enzymes increase is not dose-dependent, it usually happens at doses exceeding 400–600 mg per day (2). Osilodrostat blocks CYP11B1 and CYP11B2; a combination should thus allow for a complete blockade of these enzymes that are necessary for cortisol secretion. Short-term side effects such as hypokalemia and hypertension are similar to those observed with metyrapone, due to increased levels of the precursor deoxycorticosterone, correlated with the dose of osilodrostat (6). As for our patient, the occurrence of side effects should not lead to immediately switch to another drug, but rather to decrease the dose and add another cortisol-lowering drug. Moreover, considering the current cost of newly-released drugs such a strategy could lower financial costs for patients and/or society. Another point to take into account is the current COVID-19 pandemic, for which, as recently detailed in experts’ opinion (7), the main aim is to reach eucortisolism, whatever the way. Indeed patients presenting with CS usually also present with comorbidities such as obesity, hypertension, diabetes mellitus and immunodeficiency (8). Surgery, which represents the gold standard strategy in the management of CS (1, 9), might be delayed to reduce the hospital-associated risk of COVID-19, with post-surgical immunodepression and thromboembolic risks (7). Because immunosuppression and thromboembolic diathesis are common CS features (9, 10), during the COVID-19 pandemic, the use of steroidogenesis inhibitors appears of great interest. In these patients, combing steroidogenesis inhibitors at intermediate doses might allow for a rapid control of hypercortisolism without risks of major side effects if a single uptitrated treatment is not sufficient. Obviously, the management of associated comorbidities would also be crucial in this situation (11). To conclude, we report for the first time a case of CS, in the context of primary bilateral macronodular adrenocortical hyperplasia successfully treated with a well-tolerated combination of ketoconazole and osilodrostat. While initial levels of cortisol were not very high, we could not manage to control hypercortisolism with ketoconazole monotherapy, and could not increase the dose due to side effects. The same result was observed with another steroidogenesis inhibitor, osilodrostat. This strategy could be considered for any patient with uncontrolled hypercortisolism and delayed or unsuccessful surgery, especially in the context of the COVID-19 pandemic. Declaration of interest F C and T B received research grants from Recordati Rare Disease and HRA Pharma Rare Diseases. Frederic Castinetti is on the editorial board of Endocrinology, Diabetes and Metabolism case reports. Frederic Castinetti was not involved in the review or editorial process for this paper, on which he is listed as an author. Funding This work did not receive any specific grant from any funding agency in the public, commercial or not-for-profit sector. Patient consent Informed written consent has been obtained from the patient for publication of the case report. Author contribution statement V A was the patient’s physician involved in the clinical care and collected the data. T B and F C supervised the management of the patient. F C proposed the original idea of this case report. V A drafted the manuscript. F C critically reviewed the manuscript. T B revised the manuscript into its final version. References 1↑ Nieman LK, Biller BMK, Findling JW, Murad MH, Newell-Price J, Savage MO, Tabarin A & Endocrine Society. Treatment of Cushing’s syndrome: an Endocrine Society clinical practice guideline. Journal of Clinical Endocrinology and Metabolism 2015 100 2807–2831. (https://doi.org/10.1210/jc.2015-1818) Search Google Scholar Export Citation 2↑ Castinetti F, Guignat L, Giraud P, Muller M, Kamenicky P, Drui D, Caron P, Luca F, Donadille B & Vantyghem MC et al.Ketoconazole in Cushing’s disease: is it worth a try? Journal of Clinical Endocrinology and Metabolism 2014 99 1623–1630. (https://doi.org/10.1210/jc.2013-3628) Search Google Scholar Export Citation 3↑ Corcuff JB, Young J, Masquefa-Giraud P, Chanson P, Baudin E, Tabarin A. Rapid control of severe neoplastic hypercortisolism with metyrapone and ketoconazole. European Journal of Endocrinology 2015 172 473–481. (https://doi.org/10.1530/EJE-14-0913) Search Google Scholar Export Citation 4↑ Kamenický P, Droumaguet C, Salenave S, Blanchard A, Jublanc C, Gautier JF, Brailly-Tabard S, Leboulleux S, Schlumberger M & Baudin E et al.Mitotane, metyrapone, and ketoconazole combination therapy as an alternative to rescue adrenalectomy for severe ACTH-dependent Cushing’s syndrome. Journal of Clinical Endocrinology and Metabolism 2011 96 2796–2804. (https://doi.org/10.1210/jc.2011-0536) Search Google Scholar Export Citation 5↑ Daniel E, Aylwin S, Mustafa O, Ball S, Munir A, Boelaert K, Chortis V, Cuthbertson DJ, Daousi C & Rajeev SP et al.Effectiveness of metyrapone in treating Cushing’s syndrome: a retrospective multicenter study in 195 patients. Journal of Clinical Endocrinology and Metabolism 2015 100 4146–4154. (https://doi.org/10.1210/jc.2015-2616) Search Google Scholar Export Citation 6↑ Pivonello R, Fleseriu M, Newell-Price J, Bertagna X, Findling J, Shimatsu A, Gu F, Auchus R, Leelawattana R & Lee EJ et al.Efficacy and safety of osilodrostat in patients with Cushing’s disease (LINC 3): a multicentre phase III study with a double-blind, randomised withdrawal phase. Lancet: Diabetes and Endocrinology 2020 8 748–761. (https://doi.org/10.1016/S2213-8587(2030240-0) Search Google Scholar Export Citation 7↑ Newell-Price J, Nieman LK, Reincke M, Tabarin A. ENDOCRINOLOGY IN THE TIME OF COVID-19: Management of Cushing’s syndrome. European Journal of Endocrinology 2020 183 G1–G7. (https://doi.org/10.1530/EJE-20-0352) Search Google Scholar Export Citation 8↑ Kakodkar P, Kaka N, Baig MN. A comprehensive literature review on the clinical presentation, and management of the pandemic coronavirus disease 2019 (COVID-19). Cureus 2020 12 e7560. (https://doi.org/10.7759/cureus.7560) Search Google Scholar Export Citation 9↑ Pivonello R, De M, Cozzolino A, Colao A. The treatment of Cushing’s disease. Endocrine Reviews 2015 36 385–486. (https://doi.org/10.1210/er.2013-1048) Search Google Scholar Export Citation 10↑ Hasenmajer V, Sbardella E, Sciarra F, Minnetti M, Isidori AM, Venneri MA. The immune system in Cushing’s syndrome. Trends in Endocrinology and Metabolism 2020 31 655–669. (https://doi.org/10.1016/j.tem.2020.04.004) Search Google Scholar Export Citation 11↑ Pivonello R, Ferrigno R, Isidori AM, Biller BMK, Grossman AB, Colao A. COVID-19 and Cushing’s syndrome: recommendations for a special population with endogenous glucocorticoid excess. Lancet: Diabetes and Endocrinology 2020 8 654–656. (https://doi.org/10.1016/S2213-8587(2030215-1) Search Google Scholar Export Citation From https://edm.bioscientifica.com/view/journals/edm/2021/1/EDM21-0071.xml?body=fullHtml-9967

Cushing’s disease is a progressive pituitary disorder in which there is an excess of cortisol in the body. While the disease can be treated surgically, this option is not possible for all patients. This is one of the approved medications that assist in controlling cortisol levels in people with Cushing’s disease. Recorlev Recorlev was approved by the FDA in December 2021 to treat those Cushing’s patients for whom surgery is not a choice or has failed to lower cortisol levels. The medication is an oral cortisol synthesis inhibitor that prevents the adrenal glands — sitting atop the kidneys — from producing too much cortisol, thereby easing Cushing’s symptoms. Recorlev (levoketoconazole) is a treatment that Strongbridge Biopharma — now acquired by Xeris Pharmaceuticals — developed for endogenous Cushing’s syndrome. Endogenous Cushing’s is a form of the disease in which symptoms occur because the body produces too much cortisol. Abnormally high cortisol levels in Cushing’s syndrome may be primarily due to a tumor in the brain’s pituitary gland — a type of the condition called Cushing’s disease. The first treatment option is surgery to remove those tumors. However, in some patients, this procedure is not an option or is ineffective at lowering cortisol levels. Recorlev was approved by the U.S. Food and Drug Administration (FDA) in December 2021 to treat those Cushing’s patients for whom surgery is not a choice or has failed to lower cortisol levels. How does Recorlev works? Cortisol plays several important roles in the body, including regulating salt and sugar levels, blood pressure, inflammation, breathing, and metabolism. Too much cortisol, however, throws the body off balance, causing a wide range of symptoms, such as obesity, high blood sugar levels, bone problems, and fatigue. Recorlev is an oral cortisol synthesis inhibitor that prevents the adrenal glands — sitting atop the kidneys — from producing too much cortisol, thereby easing Cushing’s symptoms. Recorlev in clinical trials Recorlev’s approval was mainly supported by data from two Phase 3 clinical trials: one called SONICS (NCT01838551) and the other LOGICS (NCT03277690). SONICS was a multicenter, open-label, three-part trial that evaluated the safety and effectiveness of Recorlev in 94 patients with endogenous Cushing’s syndrome who were not candidates for radiation therapy or surgery, and whose cortisol levels in the urine were at least 1.5 times higher than normal. Top-line data from SONICS revealed that nearly a third of patients saw their urinary cortisol levels drop to a normal range after six months of maintenance treatment with Recorlev, without requiring any dose increments in that period of time. A subgroup analysis of the study also showed Recorlev helped control cortisol and blood sugar levels in patients with both Cushing’s and diabetes. The study also showed that Recorlev was able to lessen symptoms, ease depression, and improve patients’ quality of life. LOGICS was a double-blind, randomized, withdrawal and rescue study that assessed the safety, efficacy, and pharmacological properties of Recorlev in patients with endogenous Cushing’s syndrome who had previously participated in SONICS, or who had never been treated with Recorlev. After a period of taking Recorlev, some participants were switched to a placebo while others remained on the medication. This design allowed researchers to assess the effects of treatment withdrawal. According to patients who stopped using Recorlev and moved to a placebo saw their urine cortisol levels rise in response to the lack of treatment, compared with those who remained on Recorlev. Additional data from the study also showed that patients who switched to a placebo lost Recorlev’s cholesterol-lowering benefits. Safety data from an ongoing open-label Phase 3 extension study called OPTICS (NCT03621280) also supported Recorlev’s approval. This trial, which is expected to conclude in June 2023, is designed to assess the long-term effects of Recorlev in patients who completed one or both previous studies, for up to three years. Other details Recorlev’s starting dose is 150 mg twice daily and should be taken orally with or without food. The maximum recommended dose is 1,200 mg per day, given as 600 mg twice daily. The most common side effects associated with Recorlev include nausea, vomiting, increased blood pressure, abnormally low blood potassium levels, fatigue, headache, abdominal pain, and unusual bleeding. Liver enzymes should be monitored before and during the treatment since this therapy can cause hepatotoxicity, or liver damage, in some individuals. For this reason, it is contraindicated in people with liver diseases such as cirrhosis. Recorlev should be immediately stopped if signs of hepatotoxicity are observed. Recorlev also can influence heartbeat. As such, patients with certain heart conditions should be closely monitored before and during treatment. Hypocortisolism, or lower-than-normal levels of cortisol, also may occur during treatment with Recorlev. For this reason, patients should have their cortisol levels closely monitored, and lessen or interrupt treatment if necessary. Recorlev interacts with medicines on which certain liver enzymes act, such as CYP3A4. Treatment also is an inhibitor of P-gp, OCT2, and MATE1, which are transporters of certain medicines. The use of Recorlev with these medicines may increase the risk of adverse reactions.