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Over the years, we went on several Windjammer Barefoot Cruises. We liked them because they were small, casual and were fairly easy on the wallet. They sailed around the Caribbean to a variety of islands, although they sometimes changed itineraries depending on weather, crew, whatever. One trip we were supposed to go to Saba but couldn't make port. A lot of people got off at the next port and flew home. The captains were prone to "Bedtime Stories" which were often more fiction than true but they added to the appeal of the trip. We didn't care if we missed islands or not - we were just there to sail over the waves and enjoy the ride. The last trip we took with them was about two years before I started having Cushing's problems. (You wondered how I was going to tie this together, right?) The cruise was uneventful, other than the usual mishaps like hitting docks, missing islands and so on. Until it was a particularly rough sea one day. I was walking somewhere on deck and suddenly a wave came up over the deck making it very slippery. I fell and cracked the back of my head on the curved edge of a table in the dining area. I had the next-to-the-worse headache I have ever had, the worst being after my pituitary surgery. At least after the surgery, I got some morphine. We asked several doctors later if that hit could have contributed to my Cushing's but doctors didn't want to get involved in that at all. The Windjammer folks didn't fare much better, either. In October 1998, Hurricane Mitch was responsible for the loss of the s/v Fantome (the last one we were on). All 31 crew members aboard perished; passengers and other crew members had earlier been offloaded in Belize. The story was recorded in the book The Ship and the Storm: Hurricane Mitch and the Loss of the Fantome by Jim Carrier. The ship, which was sailing in the center of the hurricane, experienced up to 50-foot (15 m) waves and over 100 mph (160 km/h) winds, causing the Fantome to founder off the coast of Honduras. "In October 1998, the majestic schooner Fantome came face-to-face with one of the most savage storms in Atlantic history. The last days of the Fantome are reconstructed in vivid and heartbreaking detail through Jim Carrier's extensive research and hundreds of personal interviews. What emerges is a story of courage, hubris, the agony of command, the weight of lives versus wealth, and the advances of science versus the terrible power and unpredictability of nature." This event was similar to the Perfect Storm in that the weather people were more interested in watching the hurricane change directions than they were in people who were dealing with its effects. I read this book and I was really moved by the plight of those crew members. I'll never know if that hit on my head contributed to my Cushing's but I have seen several people mention on the message boards that they had a traumatic head injury of some type in their earlier lives.

Abstract Cushing's syndrome (CS) arises from an excess of endogenous or exogenous cortisol, with Cushing's disease specifically implicating a pituitary adenoma and exaggerated adrenocorticotropic hormone (ACTH) production. Typically, Cushing's disease presents with characteristic symptoms such as weight gain, central obesity, moon face, and buffalo hump. This case report presents an unusual manifestation of CS in a 48-year-old male with a history of hypertension, where severe hypokalemia was the primary presentation. Initial complaints included bilateral leg swelling, muscle weakness, occasional shortness of breath, and a general feeling of not feeling well. Subsequent investigations revealed hypokalemia, metabolic alkalosis, and an abnormal response to dexamethasone suppression, raising concerns about hypercortisolism. Further tests, including 24-hour urinary free cortisol and ACTH testing, confirmed significant elevations. Brain magnetic resonance imaging (MRI) identified a pituitary macroadenoma, necessitating neurosurgical intervention. This case underscores the rarity of CS presenting with severe hypokalemia, highlighting the diagnostic challenges and the crucial role of a collaborative approach in managing such intricate cases. Introduction Cushing's syndrome (CS), characterized by excessive cortisol production, is well-known for its diverse and often conspicuous clinical manifestations. Cushing's disease is a subset of CS resulting from a pituitary adenoma overproducing adrenocorticotropic hormone (ACTH), leading to heightened cortisol secretion. The classic presentation involves a spectrum of symptoms such as weight gain, central obesity, muscle weakness, and mood alterations [1]. Despite its classic presentation, CS can demonstrate diverse and atypical features, challenging conventional diagnostic paradigms. This case report sheds light on a rare manifestation of CS, where severe hypokalemia was the primary clinical indicator. Notably, instances of CS prominently manifesting through severe hypokalemia are scarce in the literature [1,2]. Through this exploration, we aim to provide valuable insights into the diagnostic intricacies of atypical CS presentations, underscore the significance of a comprehensive workup, and emphasize the collaborative approach essential for managing such uncommon hormonal disorders. Case Presentation A 48-year-old male with a history of hypertension presented to his primary care physician with complaints of bilateral leg swelling, occasional shortness of breath, dizziness, and a general feeling of malaise persisting for 10 days. The patient reported increased water intake and urinary frequency without dysuria. The patient was diagnosed with hypertension eight months ago. He experienced progressive muscle weakness over two months, hindering his ability to perform daily activities, including using the bathroom. The primary care physician initiated a blood workup that revealed severe hypokalemia with a potassium level of 1.3 mmol/L (reference range: 3.6 to 5.2 mmol/L), prompting referral to the hospital. Upon admission, the patient was hypertensive with a blood pressure of 180/103 mmHg, a heart rate of 71 beats/minute, a respiratory rate of 18 breaths/minute, and an oxygen saturation of 96% on room air. Physical examination revealed fine tremors, bilateral 2+ pitting edema in the lower extremities up to mid-shin, abdominal distension with normal bowel sounds, and bilateral reduced air entry in the bases of the lungs on auscultation. The blood work showed the following findings (Table 1). Parameter Result Reference Range Potassium (K) 1.8 mmol/L 3.5-5.0 mmol/L Sodium (Na) 144 mmol/L 135-145 mmol/L Magnesium (Mg) 1.3 mg/dL 1.7-2.2 mg/dL Hemoglobin (Hb) 15.5 g/dL 13.8-17.2 g/dL White blood cell count (WBC) 13,000 x 103/µL 4.5 to 11.0 × 109/L Platelets 131,000 x 109/L 150-450 x 109/L pH 7.57 7.35-7.45 Bicarbonate (HCO3) 46 mmol/L 22-26 mmol/L Lactic acid 4.2 mmol/L 0.5-2.0 mmol/L Table 1: Blood work findings In order to correct the electrolyte imbalances, the patient received intravenous (IV) magnesium and potassium replacement and was later transitioned to oral. The patient was also started on normal saline at 100 cc per hour. To further investigate the complaint of shortness of breath, the patient underwent a chest X-ray, which revealed bilateral multilobar pneumonia (Figure 1). He was subsequently treated with ceftriaxone (1 g IV daily) and clarithromycin (500 mg twice daily) for seven days. Figure 1: A chest X-ray revealing (arrows) bilateral multilobar pneumonia With persistent abdominal pain and lactic acidosis, a computed tomography (CT) scan abdomen and pelvis with contrast was conducted, revealing a psoas muscle hematoma. Subsequent magnetic resonance imaging (MRI) depicted an 8x8 cm hematoma involving the left psoas and iliacus muscles. The interventional radiologist performed drainage of the hematoma involving the left psoas and iliacus muscles (Figure 2). Figure 2: Magnetic resonance imaging (MRI) depicting an 8x8 cm hematoma (arrow) involving the left psoas and iliacus muscles In light of the concurrent presence of hypokalemia, hypertension, and metabolic alkalosis, there arose concerns about Conn's syndrome, prompting consultation with endocrinology. Their recommended workup for Conn's syndrome included assessments of the aldosterone-renin ratio and random cortisol levels. The results unveiled an aldosterone level below 60 pmol/L (reference range: 190 to 830 pmol/L in SI units) and a plasma renin level of 0.2 pmol/L (reference range: 0.7 to 3.3 mcg/L/hr in SI units). Notably, the aldosterone-renin ratio was low, conclusively ruling out Conn's syndrome. The random cortisol level was notably elevated at 1334 nmol/L (reference range: 140 to 690 nmol/L). Furthermore, a low-dose dexamethasone suppression test was undertaken due to the high cortisol levels. Following the administration of 1 mg of dexamethasone at 10 p.m., cortisol levels were measured at 9 p.m., 3 a.m., and 9 a.m. the following day. The results unveiled a persistently elevated cortisol level surpassing 1655 nmol/L, signaling an abnormal response to dexamethasone suppression and raising concerns about a hypercortisolism disorder, such as CS. In the intricate progression of this case, the investigation delved deeper with a 24-hour urinary free cortisol level, revealing a significant elevation at 521 mcg/day (reference range: 10 to 55 mcg/day). Subsequent testing of ACTH portrayed a markedly elevated level of 445 ng/L, distinctly exceeding the normal reference range of 7.2 to 63.3 ng/L. A high-dose 8 mg dexamethasone test was performed to ascertain the source of excess ACTH production. The baseline serum cortisol levels before the high-dose dexamethasone suppression test were 1404 nmol/L, which decreased to 612 nmol/L afterward, strongly suggesting the source of excess ACTH production to be in the pituitary gland. A CT scan of the adrenal glands ruled out adrenal mass, while an MRI of the brain uncovered a 1.3x1.3x3.2 cm pituitary macroadenoma (Figure 3), leading to compression of adjacent structures. Neurosurgery was consulted, and they recommended surgical removal of the macroadenoma due to the tumor size and potential complications. The patient was referred to a tertiary care hospital for pituitary adenoma removal. Figure 3: Magnetic resonance imaging (MRI) of the brain depicting a 1.3x1.3x3.2 cm pituitary macroadenoma (star) Discussion CS represents a complex endocrine disorder characterized by excessive cortisol production. While the classic presentation of CS includes weight gain, central obesity, and muscle weakness, our case highlights an uncommon initial manifestation: severe hypokalemia. This atypical presentation underscores the diverse clinical spectrum of CS and the challenges it poses in diagnosis and management [1,2]. While CS typically presents with the classic symptoms mentioned above, severe hypokalemia as the initial manifestation is exceedingly rare. Hypokalemia in CS often results from excess cortisol-mediated activation of mineralocorticoid receptors, leading to increased urinary potassium excretion and renal potassium wasting. Additionally, metabolic alkalosis secondary to cortisol excess further exacerbates hypokalemia [3,4]. Diagnosing a case of Cushing's disease typically commences with a thorough examination of the patient's medical history and a comprehensive physical assessment aimed at identifying characteristic manifestations such as central obesity, facial rounding, proximal muscle weakness, and increased susceptibility to bruising. Essential to confirming the diagnosis are laboratory examinations, which involve measuring cortisol levels through various tests, including 24-hour urinary free cortisol testing, late-night salivary cortisol testing, and dexamethasone suppression tests. Furthermore, assessing plasma ACTH levels aids in distinguishing between pituitary-dependent and non-pituitary causes of CS. Integral to the diagnostic process are imaging modalities such as MRI of the pituitary gland, which facilitate the visualization of adenomas and the determination of their size and precise location [1-4]. Treatment for Cushing's disease primarily entails surgical removal of the pituitary adenoma via transsphenoidal surgery, with the aim of excising the tumor and restoring normal pituitary function. In cases where surgical intervention is unsuitable or unsuccessful, pharmacological therapies employing medications such as cabergoline (a dopamine receptor agonist) or pasireotide (a somatostatin analogue) may be considered to suppress ACTH secretion and regulate cortisol levels. Additionally, radiation therapy, whether conventional or stereotactic radiosurgery, serves as a supplementary or alternative treatment approach to reduce tumor dimensions and mitigate ACTH production [5,6]. To assess the effectiveness of treatment, manage any problem, and assure long-term illness remission, diligent long-term follow-up and monitoring are essential. Collaborative multidisciplinary care involving specialists such as endocrinologists, neurosurgeons, and other healthcare professionals is pivotal in optimizing patient outcomes and enhancing overall quality of life [2,4]. The prognosis of CS largely depends on the underlying cause, stage of the disease, and efficacy of treatment. Early recognition and prompt intervention are essential for improving outcomes and minimizing long-term complications. Surgical resection of the adrenal or pituitary tumor can lead to remission of CS in the majority of cases. However, recurrence rates vary depending on factors such as tumor size, invasiveness, and completeness of resection [2,3]. Long-term follow-up with endocrinologists is crucial for monitoring disease recurrence, assessing hormonal function, and managing comorbidities associated with CS. Conclusions In conclusion, our case report highlights the rarity of severe hypokalemia as the initial presentation of CS. This unique presentation underscores the diverse clinical manifestations of CS and emphasizes the diagnostic challenges encountered in clinical practice. A multidisciplinary approach involving endocrinologists, neurosurgeons, and radiologists is essential for the timely diagnosis and management of CS. Early recognition, prompt intervention, and long-term follow-up are essential for optimizing outcomes and improving the quality of life for patients with this endocrine disorder. References Nieman LK, Biller BM, Findling JW, Newell-Price J, Savage MO, Stewart PM, Montori VM: The diagnosis of Cushing's syndrome: an Endocrine Society Clinical Practice Guideline. J Clin Endocrinol Metab. 2008, 93:1526-40. 10.1210/jc.2008-0125 Newell-Price J, Bertagna X, Grossman AB, Nieman LK: Cushing's syndrome. Lancet. 2006, 367:1605-17. 10.1016/S0140-6736(06)68699-6 Torpy DJ, Mullen N, Ilias I, Nieman LK: Association of hypertension and hypokalemia with Cushing's syndrome caused by ectopic ACTH secretion: a series of 58 cases. Ann N Y Acad Sci. 2002, 970:134-44. 10.1111/j.1749-6632.2002.tb04419.x Elias C, Oliveira D, Silva MM, Lourenço P: Cushing's syndrome behind hypokalemia and severe infection: a case report. Cureus. 2022, 14:e32486. 10.7759/cureus.32486 Fleseriu M, Petersenn S: Medical therapy for Cushing's disease: adrenal steroidogenesis inhibitors and glucocorticoid receptor blockers. Pituitary. 2015, 18:245-52. 10.1007/s11102-014-0627-0 Pivonello R, De Leo M, Cozzolino A, Colao A: The treatment of Cushing's disease. Endocr Rev. 2015, 36:385-486. 10.1210/er.2013-1048 From https://www.cureus.com/articles/243881-unveiling-the-uncommon-cushings-syndrome-cs-masquerading-as-severe-hypokalemia?score_article=true#!/

Abstract Cushing syndrome (CS) is a rare endocrinological disorder resulting from chronic exposure to excessive cortisol. The term Cushing disease is used specifically when this is caused by excessive secretion of adrenocorticotropic hormone (ACTH) by a pituitary tumor, usually an adenoma. This disease is associated with a poor prognosis, and if left untreated, it has an estimated 5-year survival rate of 50%. We present the case of a 66-year-old female patient who received a referral to endocrinology for an evaluation of obesity due to right knee arthropathy. Taking into consideration her age, she was screened for osteoporosis, with results that showed diminished bone density. Considering this, combined with other clinical features of the patient, suspicion turned toward hypercortisolism. Laboratory findings suggested that the CS was ACTH-dependent and originated in the pituitary gland. After a second look at the magnetic resonance imaging results, a 4-mm lesion was identified on the pituitary gland, prompting a transsphenoidal resection of the pituitary adenoma. case report, pituitary ACTH hypersecretion, Cushing syndrome, obesity Issue Section: Case Report Introduction Chronic excessive exposure to glucocorticoids leads to the diverse clinical manifestations of Cushing syndrome (CS), which has an annual incidence ranging from 1.8 to 3.2 cases per million individuals [1]. The syndrome's signs and symptoms are not pathognomonic, and some of its primary manifestations, such as obesity, hypertension, and glucose metabolism alterations, are prevalent in the general population [2], making diagnosis challenging. Endogenous CS falls into 2 categories: adrenocorticotropic hormone (ACTH)-dependent (80%-85% of cases), mostly due to a pituitary adenoma, or ACTH-independent (15%-20% of cases), typically caused by adrenal adenomas or hyperplasia [3]. Cushing disease (CD) represents a specific form of CS, characterized by the presence of an ACTH-secreting pituitary tumor [1]. Untreated CD is associated with high morbidity and mortality compared to the general population [1], with a 50% survival rate at 5 years [2]. However, surgical removal of a pituitary adenoma can result in complete remission, with mortality rates similar to those of the general population [2]. This article aims to highlight the challenges of suspecting and diagnosing CD and to discuss the current management options for this rare condition. Case Presentation A 66-year-old woman received a referral to endocrinology for an evaluation of obesity due to right knee arthropathy. During physical examination, she exhibited a body mass index of 34.3 kg/m2, blood pressure of 180/100, a history of non-insulin-requiring type 2 diabetes mellitus with glycated hemoglobin (HbA1c) of 6.9% (nondiabetic: < 5.7%; prediabetic: 5.7% to 6.4%; diabetic: ≥ 6.5%) and hypertension. Additionally, the patient complained of proximal weakness in all 4 limbs. Diagnostic Assessment Upon admission, densitometry revealed osteoporosis with T scores of −2.7 in the lumbar spine and −2.8 in the femoral neck. Hypercortisolism was suspected due to concomitant arterial hypertension, central obesity, muscle weakness, and osteoporosis. Physical examination did not reveal characteristic signs of hypercortisolism, such as skin bruises, flushing, or reddish-purple striae. Late-night salivary cortisol (LNSC) screening yielded a value of 8.98 nmol/L (0.3255 mcg/dL) (reference value [RV] 0.8-2.7 nmol/L [0.029-0.101 mcg/dL]) and ACTH of 38.1 pg/mL (8.4 pmol/L) (RV 2-11 pmol/L [9-52 pg/mL]). A low-dose dexamethasone suppression test (LDDST) was performed (cutoff value 1.8 mcg/dL [49 nmol/L]), with cortisol levels of 7.98 mcg/dL (220 nmol/L) at 24 hours and 20.31 mcg/dL (560 nmol/L) at 48 hours. Subsequently, a high-dose dexamethasone suppression test (HDDST) was conducted using a dose of 2 mg every 6 hours for 2 days, for a total dose of 16 mg, revealing cortisol levels of 0.0220 nmol/L (0.08 ng/mL) at 24 hours and 0.0560 nmol/L (0.0203 ng/mL) at 48 hours, alongside 24-hour urine cortisol of 0.8745 nmol/L (0.317 ng/mL) (RV 30-145 nmol/24 hours [approximately 11-53 μg/24 hours]) [4]. These findings indicated the presence of endogenous ACTH-dependent hypercortisolism of pituitary origin. Consequently, magnetic resonance imaging (MRI) was requested, but the results showed no abnormalities. Considering ectopic ACTH production often occurs in the lung, a high-resolution chest computed tomography scan was performed, revealing no lesions. Treatment Upon reassessment, the MRI revealed a 4-mm adenoma, prompting the decision to proceed with transsphenoidal resection of the pituitary adenoma. Outcome and Follow-Up The histological analysis revealed positive staining for CAM5.2, chromogranin, synaptophysin, and ACTH, with Ki67 staining at 1%. At the 1-month follow-up assessment, ACTH levels were 3.8 pmol/L (17.2 pg/mL) and morning cortisol was 115.8621 nmol/L (4.2 mcg/dL) (RV 5-25 mcg/dL or 140-690 nmol/L). Somatomedin C was measured at 85 ng/mL (RV 70-267 ng/mL) and prolactin at 3.5 ng/mL (RV 4-25 ng/mL). At the 1-year follow-up, the patient exhibited a satisfactory postoperative recovery. However, she developed diabetes insipidus and secondary hypothyroidism. Arterial hypertension persisted. Recent laboratory results indicated a glycated hemoglobin (HbA1c) level of 5.4%. Medications at the time of follow-up included prednisolone 5 milligrams a day, desmopressin 60 to 120 micrograms every 12 hours, losartan potassium 50 milligrams every 12 hours, and levothyroxine 88 micrograms a day. Discussion CD is associated with high mortality, primarily attributable to cardiovascular outcomes and comorbidities such as metabolic and skeletal disorders, infections, and psychiatric disorders [1]. The low incidence of CD in the context of the high prevalence of chronic noncommunicable diseases makes early diagnosis a challenge [2]. This case is relevant for reviewing the diagnostic approach process and highlighting the impact of the availability bias, which tends to prioritize more common diagnoses over rare diseases. Despite the absence of typical symptoms, a timely diagnosis was achieved. Once exogenous CS is ruled out, laboratory testing must focus on detecting endogenous hypercortisolism to prevent misdiagnosis and inappropriate treatment [5]. Screening methods include 24-hour urinary free cortisol (UFC) for total cortisol load, while circadian rhythm and hypothalamic-pituitary-adrenal (HPA) axis function may be evaluated using midnight serum cortisol and LNSC [5]. An early hallmark of endogenous CS is the disruption of physiological circadian cortisol patterns, characterized by a constant cortisol level throughout the day or no significant decrease [2]. Measuring LNSC has proven to be useful in identifying these patients. The LNSC performed on the patient yielded a high result. To assess HPA axis suppressibility, tests such as the overnight and the standard 2-day LDDST [5] use dexamethasone, a potent synthetic corticosteroid with high glucocorticoid receptor affinity and prolonged action, with minimal interference with cortisol measurement [6]. In a normal HPA axis, cortisol exerts negative feedback, inhibiting the secretion of corticotropin-releasing-hormone (CRH) and ACTH. Exogenous corticosteroids suppress CRH and ACTH secretion, resulting in decreased synthesis and secretion of cortisol. In pathological hypercortisolism, the HPA axis becomes partially or entirely resistant to feedback inhibition by exogenous steroids [5, 6]. The LDDST involves the administration of 0.5 mg of dexamethasone orally every 6 hours for 2 days, with a total dose of 4 mg. A blood sample is drawn 6 hours after the last administered dose [6]. Following the LDDST, the patient did not demonstrate suppression of endogenous corticosteroid production. After diagnosing CS, the next step in the diagnostic pathway involves categorizing it as ACTH-independent vs ACTH-dependent. ACTH-independent cases exhibit low or undetectable ACTH levels, pointing to adrenal origin. The underlying principle is that excess ACTH production in CD can be partially or completely suppressed by high doses of dexamethasone, a response not observed in ectopic tumors [6]. In this case, the patient presented with an ACTH of 38.1 pg/mL (8.4 pmol/L), indicative of ACTH-dependent CD. Traditionally, measuring cortisol levels and conducting pituitary imaging are standard practices for diagnosis. Recent advances propose alternative diagnostic methods such as positron emission tomography (PET) scans and corticotropin-releasing factor (CRF) tests [7]. PET scans, utilizing radioactive tracers, offer a view of metabolic activity in the adrenal glands and pituitary region, aiding in the identification of abnormalities associated with CD. Unfortunately, the availability of the aforementioned tests in the country is limited. Once ACTH-dependent hypercortisolism is confirmed, identifying the source becomes crucial. A HDDST is instrumental in distinguishing between a pituitary and an ectopic source of ACTH overproduction [2, 6]. The HDDST involves administering 8 mg of dexamethasone either overnight or as a 2-day test. In this case, the patient received 2 mg of dexamethasone orally every 6 hours for 2 days, totaling a dose of 16 mg. Simultaneously, a urine sample for UFC is collected during dexamethasone administration. The HDDST suppressed endogenous cortisol production in the patient, suggesting a pituitary origin. In ACTH-dependent hypercortisolism, CD is the predominant cause, followed by ectopic ACTH syndrome and, less frequently, an ectopic CRH-secreting tumor [3, 5]. With the pretest probability for pituitary origin exceeding 80%, the next diagnostic step is typically an MRI of the pituitary region. However, the visualization of microadenomas on MRI ranges from 50% to 70%, requiring further testing if results are negative or inconclusive [5]. Initial testing of our patient revealed no pituitary lesions. Following a pituitary location, ACTH-secreting tumors may be found in the lungs. Thus, a high-resolution chest computed tomography scan was performed, which yielded negative findings. Healthcare professionals must keep these detection rates in mind. In instances of high clinical suspicion, repeating or reassessing tests and imaging may be warranted [3], as in our case, ultimately leading to the discovery of a 4-mm pituitary adenoma. It is fundamental to mention that the Endocrine Society Clinical Practice Guideline on Treatment of CS recommends that, when possible, all patients presenting with ACTH-dependent CS and lacking an evident causal neoplasm should be directed to an experienced center capable of conducting inferior petrosal sinus sampling to differentiate between pituitary and nonpituitary or ectopic cause [8]. However, in this instance, such a referral was regrettably hindered by logistical constraints. Regarding patient outcomes and monitoring in CD, there is no consensus on defining remission criteria following tumor resection. Prolonged hypercortisolism results in suppression of corticotropes, resulting in low levels of ACTH and cortisol after surgical intervention. Typically, remission is identified by morning serum cortisol values below 5 µg/dL (138 nmol/L) or UFC levels between 28 and 56 nmol/d (10-20 µg/d) within 7 days after surgical intervention. In our case, the patient's morning serum cortisol was 115.8621 nmol/L (4.2 µg/dL), indicating remission. Remission rates in adults are reported at 73% to 76% in selectively resected microadenomas and at 43% in macroadenomas [8], highlighting the need for regular follow-up visits to detect recurrence. Following the surgery, the patient experienced diabetes insipidus, a relatively common postoperative occurrence, albeit usually transient [8]. It is recommended to monitor serum sodium levels during the first 5 to 14 days postsurgery for early detection and management. Additionally, pituitary deficiencies may manifest following surgery. In this patient, prolactin levels were compromised, potentially impacting sexual response. However, postoperative somatomedin levels were normal, and gonadotropins were not measured due to the patient's age group, as no additional clinical decisions were anticipated based on those results. Secondary hypothyroidism was diagnosed postoperatively. Moving forward, it is important to emphasize certain clinical signs and symptoms for diagnosing CD. The combination of low bone mineral density (Likelihood Ratio [LR] +21.33), central obesity (LR +3.10), and arterial hypertension (LR + 2.29) [9] has a higher positive LR than some symptoms considered “characteristic,” such as reddish-purple striae, plethora, proximal muscle weakness, and unexplained bruising [2, 10]. It is essential to give relevance to the signs the patient may present, emphasizing signs that have been proven to have an increased odds ratio (OR) such as osteoporosis (OR 3.8), myopathies (OR 6.0), metabolic syndrome (OR 2.7) and adrenal adenoma (OR 2.4) [9‐11]. The simultaneous development and worsening of these conditions should raise suspicion for underlying issues. Understanding the evolving nature of CD signs highlights the importance of vigilance during medical examinations, prioritizing the diagnostic focus, and enabling prompt initiation of treatment. Recognizing the overlap of certain clinical features in CS is fundamental to achieving a timely diagnosis. Learning Points CS diagnosis is challenging due to the absence of pathognomonic signs and symptoms and the overlap of features present in many pathologies, such as metabolic syndrome. Early detection of CS is crucial, given its association with high morbidity and mortality resulting from chronic exposure to glucocorticoids. Recognizing the combination of low bone mineral density, obesity, hypertension, and diabetes as valuable clinical indicators is key in identifying CS. Interdisciplinary collaboration is essential to achieve a comprehensive diagnostic approach. Acknowledgments We extend our gratitude to Pontificia Universidad Javeriana in Bogotá for providing essential resources and facilities that contributed to the successful completion of this case report. Special acknowledgment is reserved for the anonymous reviewers, whose insightful feedback significantly enhanced the quality of this manuscript during the peer-review process. Their contributions are sincerely appreciated. Contributors All authors made individual contributions to authorship. A.B.O. was involved in the diagnosis and management of this patient. M.A.G., J.M.H., and A.B.O. were involved in manuscript drafting and editing. All authors reviewed and approved the final draft. Funding This research received no public or commercial funding. Disclosures The authors declare that they have no conflicts of interest related to the current study. Informed Patient Consent for Publication Signed informed consent could not be obtained from the patient or a proxy but has been approved by the treating institution. Data Availability Statement Restrictions apply to the availability of some or all data generated or analyzed during this study to preserve patient confidentiality or because they were used under license. The corresponding author will on request detail the restrictions and any conditions under which access to some data may be provided. References 1 Hakami OA , Ahmed S , Karavitaki N . Epidemiology and mortality of Cushing's syndrome . Best Pract Res Clin Endocrinol Metab . 2021 ; 35 ( 1 😞 101521 . Google Scholar Crossref PubMed WorldCat 2 Nieman LK , Biller BMK , Findling JW , et al. The diagnosis of Cushing's syndrome: an endocrine society clinical practice guideline . J Clin Endocrinol Metab . 2008 ; 93 ( 5 😞 1526 ‐ 1540 . Google Scholar Crossref PubMed WorldCat 3 Gutiérrez Restrepo J , Latorre Sierra G , Campuzano Maya G . Síndrome de cushing . Med Lab . 2009 ; 15 : 411 ‐ 430 . Google Scholar WorldCat 4 Petersenn S , Newell-Price J , Findling JW , et al. High variability in baseline urinary free cortisol values in patients with Cushing's disease . Clin Endocrinol (Oxf) . 2014 ; 80 ( 2 😞 261 ‐ 269 . Google Scholar Crossref PubMed WorldCat 5 Lila AR , Sarathi V , Jagtap VS , Bandgar T , Menon P , Shah NS . Cushing's syndrome: stepwise approach to diagnosis . Indian J Endocrinol Metab . 2011 ; 15 ( Suppl4 😞 S317 ‐ S321 . Google Scholar PubMed WorldCat 6 Dogra P , Vijayashankar NP . Dexamethasone suppression test. In: StatPearls StatPearls Publishing; 2024 . Accessed January 29, 2024. http://www.ncbi.nlm.nih.gov/books/NBK542317/ 7 Müller OA , Dörr HG , Hagen B , Stalla GK , von Werder K . Corticotropin releasing factor (CRF)-stimulation test in normal controls and patients with disturbances of the hypothalamo-pituitary-adrenal axis . Klin Wochenschr . 1982 ; 60 ( 24 😞 1485 ‐ 1491 . Google Scholar Crossref PubMed WorldCat 8 Nieman LK , Biller BMK , Findling JW , et al. Treatment of Cushing's syndrome: an endocrine society clinical practice guideline . J Clin Endocrinol Metab . 2015 ; 100 ( 8 😞 2807 ‐ 2831 . Google Scholar Crossref PubMed WorldCat 9 Aron DC . Cushing's syndrome: why is diagnosis so difficult? Rev Endocr Metab Disord . 2010 ; 11 ( 2 😞 105 ‐ 116 . Google Scholar Crossref PubMed WorldCat 10 Braun LT , Vogel F , Zopp S , et al. Whom should we screen for cushing syndrome? the Endocrine Society practice guideline recommendations 2008 revisited . J Clin Endocrinol Metab . 2022 ; 107 ( 9 😞 e3723 ‐ e3730 . Google Scholar Crossref PubMed WorldCat 11 Schneider HJ , Dimopoulou C , Stalla GK , Reincke M , Schopohl J . Discriminatory value of signs and symptoms in Cushing's syndrome revisited: what has changed in 30 years? Clin Endocrinol (Oxf) . 2013 ; 78 ( 1 😞 153 ‐ 154 . Google Scholar Crossref PubMed WorldCat Abbreviations ACTH adrenocorticotropic hormone CD Cushing disease CRH corticotropin-releasing hormone CS Cushing syndrome HDDST high-dose dexamethasone suppression test HPA hypothalamic-pituitary-adrenal LDDST low-dose dexamethasone suppression test LNSC late-night salivary cortisol MRI magnetic resonance imaging OR odds ratio RV reference value UFC urinary free cortisol © The Author(s) 2024. Published by Oxford University Press on behalf of the Endocrine Society. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. From https://academic.oup.com/jcemcr/article/2/4/luae048/7643486?login=false

Abstract In Cushing syndrome (CS), prolonged exposure to high cortisol levels results in a wide range of devastating effects causing multisystem morbidity. Despite the efficacy of treatment leading to disease remission and clinical improvement, hypercortisolism-induced complications may persist. Since glucocorticoids use the epigenetic machinery as a mechanism of action to modulate gene expression, the persistence of some comorbidities may be mediated by hypercortisolism-induced long-lasting epigenetic changes. Additionally, glucocorticoids influence microRNA expression, which is an important epigenetic regulator as it modulates gene expression without changing the DNA sequence. Evidence suggests that chronically elevated glucocorticoid levels may induce aberrant microRNA expression which may impact several cellular processes resulting in cardiometabolic disorders. The present article reviews the evidence on epigenetic changes induced by (long-term) glucocorticoid exposure. Key aspects of some glucocorticoid-target genes and their implications in the context of CS are described. Lastly, the effects of epigenetic drugs influencing glucocorticoid effects are discussed for their ability to be potentially used as adjunctive therapy in CS. epigenetic, glucocorticoids, Cushing syndrome Issue Section: Mini-review In Cushing syndrome (CS), adrenocorticotropic hormone (ACTH) hypersecretion by a pituitary adenoma or an ectopic source, or autonomous cortisol hypersecretion by an adrenal tumor, induces chronic endogenous hypercortisolism with loss of the cortisol circadian rhythm (1). CS is more prevalent in women than men and frequently occurs in the fourth to sixth decades of life (2). Glucocorticoids (GC) have extensive physiological actions and regulate up to 20% of the expressed genome, mainly related to the immune system, metabolic homeostasis, and cognition. Therefore, the prolonged exposure to high cortisol levels results in a wide range of devastating effects, including major changes in body composition (obesity, muscle atrophy, osteoporosis), neuropsychiatric disturbances (impaired cognition, depression, sleep disturbances), the metabolic syndrome (obesity, hypertension, insulin resistance, and dyslipidemia), hypercoagulability, and immune suppression (3, 4). The consequences of hypercortisolism lead to compromised quality of life and increased mortality rate (5). The mortality rate in patients with CS is 4 times higher than the healthy control population (6). Risk factors such as obesity, diabetes, and hypertension contribute to the increased risk of myocardial infarction, stroke, and cardiac insufficiency. As a result, cardiovascular disease is the leading cause of the premature death in CS (5). Infectious disease is also an important cause of death in CS (5). Therefore, prompt treatment to control hypercortisolism is imperative to prevent complications and an increased mortality rate. Despite the efficacy of treatment leading to disease remission, the clinical burden of CS improves, but does not completely revert, in every patient (7). Indeed, obesity, neuropsychiatric disturbances, hypertension, diabetes, and osteoporosis persist in a substantial number of biochemically cured patients. For instance, in a study involving 118 CS patients in remission for about 7.8 years (median), resolution of comorbidities such as diabetes occurred in only 36% of cases, hypertension in 23% of cases, and depression in 52% of the cases (8). It has been proposed that epigenetic changes as a consequence of hypercortisolism is a mechanism of the persistence of some comorbidities (9-12). Epigenetics is a reversible process that modifies gene expression without any alterations in DNA sequence; frequently it is mediated by histone modification and DNA methylation together with microRNAs (13-15). GCs use the epigenetic machinery as a mechanism of action to regulate gene expression in physiological circumstances, such as metabolic actions and stress response. Its networks involve DNA and histone modifying enzymes, such as DNA methyltransferases (DNMTs), histone acetyltransferases (HATs), and histone deacetylases (HDACs) (16). (Fig. 1) The DNA methylation process catalyzed by DNMTs is usually associated with downregulation of gene expression (17). Histone modifications catalyzed by HAT enzymes induce gene transcription, while those by HDAC enzymes induce transcriptional repression (17). Drugs interfering with these enzymes (so-called epigenetic drugs) may affect the GC genomic actions confirming the interaction between GC and the epigenetic system (18, 19). Furthermore, GC can modulate HDAC and DNMT expression and activity (16, 19, 20). Based on these data it might be speculated that in CS, epigenetic modifications induced by long-term GC exposure plays a role in the development of the disease-specific morbidity (9, 10). Figure 1. Open in new tabDownload slide Glucocorticoid (GC) and its epigenetic machinery. GC through its receptor interacts with DNA and histone modifying enzymes, such as DNA methyltransferases (DNMTs), histone acetyl transferases (HATs), and histone deacetylases (HDAC) to modulate gene expression. In this review we provide an overview of epigenetic aspects of GC action in physiological conditions and in the context of CS. We start with a detailed characterization of how GC, using the epigenetic system, can change chromatin structure in order to activate or silence gene expression. (Fig. 2) Subsequently, we describe the role of epigenetic mechanisms in the regulation of expression of several GC-target genes related to CS. Finally, we present the current evidence of epigenetic changes caused by the long-term of GC exposure and the potential use of epidrugs influencing GC actions. Figure 2. Open in new tabDownload slide Epigenetic mechanisms of the glucocorticoid action to regulate gene expression. The GR is located in cytoplasm in a multi-protein complex; after GC binding, GR dissociates from the multi-protein complex, crosses the nuclear membrane, dimerizes, and binds to the GRE of the target gene. One of the mechanisms of action of GC is through the recruitment of co-regulators together with epigenetic enzymes, such as HAT, to change the chromatin structure, resulting in activation of gene transcription. Also, GR decreases gene expression by tethering other transcriptional factors and recruiting HDAC2, causing histone deacetylation, which leads to a repressed chromatin. GC can cause hypomethylation through downregulation in the expression of DNMT1. Abbreviations: Ac, acetylation; DNMT1, DNA methyltransferase 1; GC, glucocorticoid; GR, glucocorticoid receptor; GRE, glucocorticoid responsive elements; HAT, histone acetyltransferase; HDAC, histone deacetylases; Me: methylation. Search Strategy A search of the PubMed database was conducted using the advanced search builder tool for articles in the English language on the following terms “glucocorticoids,” “glucocorticoid receptor,” “Cushing,” “hypercortisolism,” “epigenetic,” “DNA methylation,” “histone deacetylase,” “histone acetyltransferase,” “microRNA” “fkbp5,” “clock genes,” and “POMC.” Moreover, references were identified directly from the articles included in this manuscript. The articles were selected by the authors after being carefully analyzed regarding their importance and impact. Epigenetic Aspects of Genomic Action of Glucocorticoids GCs regulate gene expression positively or negatively. GC-responsive genes include genes encoding for proteins associated with inflammation, metabolic processes, blood pressure and fluid homeostasis, apoptosis, cell cycle progression, circadian rhythm, and intracellular signaling (21). The GC actions are cell type–specific (22). For instance, in an in vitro study, the comparison of GC-expressed genes between 2 cell lines, corticotroph (AtT20) and mammary (3134) cell lines, showed a different set of GC-regulated genes, revealing the cell type–specific nature of GC effects (23). GC function depends on the accessibility of glucocorticoid receptor (GR)-binding sites in the DNA of the target tissue, which in turn is mostly established during cell differentiation. Therefore, different chromatin organization explains the distinct GR-binding sites among different tissues (22, 24, 25). The chromatin accessibility is determined by histone modifications such as acetylation, methylation, phosphorylation, and/or DNA methylation, processes that are both dynamic and reversible (26). Furthermore, gene expression is regulated in a GC-concentration-dependent manner which is tissue-specific. Only a few genes can be upregulated or downregulated at low concentrations of GC. For example, a dose of dexamethasone (Dex) as low as 0.5 nM selectively activated PER1 (period 1, transcription factor related to circadian rhythm) expression in lung cancer (A549) cells (21, 27). Additionally, continuous GC exposure or pulsed GC (cortisol fluctuation during circadian rhythm) may cause different responses with respect to gene expression (26, 28). For example, constant treatment with corticosterone induced higher levels of PER1 clock gene mRNA expression compared with pulsatile treatment, as demonstrated in an in vitro study using 3134 cell line (28). The time course for gene expression in response to Dex is fast, with repression occurring slightly slower compared to activation. Half of activated and repressed genes are detected within, respectively, about 40 minutes and 53 minutes following Dex exposure (21). In short, the transcriptional output in response to GC depends on cell type, as well as on the duration and intensity of GC exposure (21, 24, 26, 27). GCs act as a transcriptional regulatory factor resulting in activating or repressing the expression of genes. The GC exerts its function through binding to corticosteroid receptors, specifically, the mineralocorticoid receptor and the GR, members of the nuclear receptor superfamily (29, 30). Glucocorticoid Receptor The GR is located in the cytoplasm in a chaperone complex which includes heat-shock proteins (70 and 90) and immunophilins (such as FK506 binding protein [FKBP5]). Cortisol diffuses across the cell membrane and binds with high affinity to the GR. The activated GR bound to GC dissociates of the multi-protein complex and is transferred to the nucleus, where it ultimately regulates gene expression (26, 31). GR is a transcription factor encoded by nuclear receptor subfamily 3, group C member 1 (NR3C1) gene, located in chromosome 5, and consisting of 9 exons. It is composed of 3 major functional domains, namely a DNA binding domain (DBD), the C-terminal ligand-binding domain (LBD) and the N-terminal domain (NTB). The LBD recognizes and joins the GC. NTB contains an activation function-1 (AF1) which connects with co-regulators and the members of the general transcription machinery to activate target genes. The DBD comprises 2 zinc fingers motifs that are able to identify and bind to glucocorticoid responsive elements (GREs) (32, 33). GRα is the most expressed and functionally active GR. GRβ is another isoform which is the result of an alternative splicing in exon 9 of the GR transcript. The difference between the 2 isoforms is the distinct ligand-binding domain in GRβ. This variance prevents the GRβ from binding to GC. In fact, the GRβ counteracts GRα function by interfering with its binding to a GRE in the target gene, and GRβ expression is associated with GC resistance (32). In addition, GRβ has its own transcriptional activity which is independent and distinct from GRα (34). Another splice variant of human GR, GRγ, is associated with GC resistance in lung cell carcinoma and childhood acute lymphoblastic leukemia (33, 35). There is an additional amino acid (arginine) in the DBD of the GRγ that reduces, by about half, the capacity to activate or suppress the transcription of the target gene, as compared with GRα (32). One study identified GRγ in a small series of corticotroph adenomas (36). Glucocorticoid Mechanism of Action The GR-GC complex induces or represses gene expression directly by binding to DNA, indirectly by tethering other transcription factors or yet in a composite manner that consists in binding DNA in association with binding to other co-regulators (35, 37). The GR has the ability to reorganize the chromatin structure to become more or less accessible to the transcriptional machinery. In the classical mechanism of direct induction of gene expression, the GR dimerizes and binds to a GRE in DNA. The receptor recruits co-regulators, such as CREB binding protein, which has intrinsic histone acetyltransferase (HAT) activity that modifies the chromatin structure from an inactive to an active state. This model, called transactivation, upregulates the expression of some genes related to glucose, protein, and fat metabolism. Gene repression, on the other hand, is accomplished by GR binding to a negative GRE (nGRE) leading to the formation of a chromatin remodeling complex composed by co-repressor factors, such as NCOR1 and SMRT, and histone deacetylases (HDACs), that ultimately turn chromatin less accessible and suppress gene transcription. The gene repression through direct binding events occurs less frequently when compared to gene induction (25, 35, 38). Another mechanism of GC action is through binding to other transcription factors (tethering). In case of switching off inflammatory genes, GR binds to transcriptional co-activator molecules, such as CREB binding protein with intrinsic HAT activity, and subsequently recruits HDAC2 to reverse histone acetylation, thus resulting in a suppression of the activated inflammatory gene (39). In the same model, GC interacts with other cofactors, such as the STAT family, to induce chromatin modifications resulting in increased gene expression (26). Furthermore, the transcriptional dynamics of some genes follow a composite manner. In this model, GR, in conjunction with binding to GRE, also interacts with cofactors in order to enhance or reduce gene expression (35). GCs can also modulate gene expression by influencing the transcription of epigenetic modifiers. An experimental study demonstrated that GC mediated the upregulation of HDAC2 in rats exposed to chronic stress, which in turn decreased the transcription of histone methyltransferase (Ehmt2) that ultimately upregulated the expression of Nedd4. Nedd4 is a ubiquitin ligase, expression of which has been related to cognitive impairment (40). Additionally, GC was found to interact with another epigenetic eraser, namely JMJD3, a histone demethylase, suppressing its transcription in endothelial cells treated with TNFα that led to decreased expression of other genes related to the blood-brain barrier (41). GCs have the ability to induce (de)methylation changes in DNA, ultimately affecting gene expression. The DNA methylation process triggered by GC involves the family of DNA methyltransferases (DNMT) and ten-eleven translocation (TET) protein (20, 42-44). The DNMT, DNMT1, DNMT3A, and DNMT3B are able to transfer a methyl group to a cytosine residue in DNA, forming 5-methylcytosine (5mC), which negatively impacts gene expression. In contrast, TET protein chemically modifies the 5mC to form 5-hydroxymethylcytosine (5hmC), which ultimately leads to unmethylated cytosine, positively influencing gene expression (45). Glucocorticoids mainly induce loss of methylation events rather than gain of methylation across the genome (11, 46). The DNA demethylation process can be either active or passive. The active mechanism is linked to the upregulation of TET enzyme expression that follows GC treatment, which was described in retinal and osteocyte cell line model studies (42, 43). The passive demethylation event involves the downregulation (Fig. 2) or dysfunction of DNMT1. DNMT1 is responsible for maintaining the methylation process in dividing cells (45). In case of GC exposure, GC can cause hypomethylation through downregulation in the expression of DNMT1, a process described in the AtT20 corticotroph tumor cell model, or through GC hindering DNMT activity, particularly DNMT1, as demonstrated in the retinal cell (RPE) line (20, 42, 44). Glucocorticoid-Induced Epigenetic Changes There are several molecular mechanisms connecting GR activation and epigenetic modifications ultimately affecting gene expression (Fig. 2). As described above, GC uses epigenetic machinery, such as DNA and histone modifying enzymes, to restructure the chromatin in order to induce or silence gene transcription (16, 47). In an in vitro study using murine AtT20 corticotroph tumor and neuronal cell lines, after chronic GC exposure followed by a recovery period in the absence of GC, the cells retained an “epigenetic memory” with persistence of loss of methylation content in FKBP5 gene but with no increased gene expression at baseline. The functionality of this “epigenetic memory” only became evident in a second exposure to GC, when the cells responded sharply with a more robust expression of FKBP5 gene compared to the cells without previous exposure to GC (44). Another in vitro study, using a human fetal hippocampal cell line, confirmed long-lasting DNA methylation changes induced by GC. The cells were treated for 10 days with dexamethasone, during the proliferative and cell differentiation phases of the cell line, followed by 20 days without any treatment. The second exposure to GC resulted in an enhanced gene expression of a subset of GC-target genes (48). Additionally, using an animal model subjected to chronic stress, a distinct gene expression profile was demonstrated in response to acute GC challenge compared to those without chronic stress history. The proposed mechanism was that chronic stress resulted in GC-induced enduring epigenetic changes in target genes, altering the responsiveness to a subsequent GC exposure (49). In general, it seems that the majority of differential methylation regions (DMRs) induced by GC are loss of methylation rather than gain of methylation. In an experimental study, an association between hypomethylation and GC exposure was demonstrated in mice previously exposed to high levels of GC. Further analysis demonstrated that the genes linked with DMR were mostly related to metabolism, the immune system, and neurodevelopment (11). Human studies have also shown that excess of cortisol can induce modifications in DNA methylation. DNA methylation data obtained from whole blood samples from patients with chronic obstructive pulmonary disease (COPD) treated with GC revealed DMR at specific CpG dinucleotides across the genome. These DMR were confirmed by pyrosequencing and annotated to genes, such as SCNN1A, encoding the α subunit of the epithelial sodium channel, GPR97, encoding G protein coupled receptor 97, and LRP3, encoding low-density lipoprotein receptor-related protein 3 (50). Furthermore, it has been proposed that the negative impact of chronic GC exposure on the immune system, which increases the risk of opportunistically infections, may be epigenetically mediated (51). In a clinical study, using whole blood samples, an analysis of genome-wide DNA methylation was performed on patients before and after exposure to GC (51). Long-term GC exposure disrupts, through a persistent modification of the cytosine methylation pattern, the mTORC1 pathway which affects CD4+ T cell biology (51). Taken together, these data clearly show the interplay between GC signaling and methylation and histone modifications processes suggesting that GC interferes in the epigenetic landscape modulating gene expression. It is possible that most of these GC-induced epigenetic events are dynamic and temporary, while others may persist leading to long-lasting disorders. Further research to provide insight into what makes some events reversible is warranted. Epigenetic Changes as a Consequence of Long-Term Glucocorticoid Exposure in Cushing Syndrome The comorbidities associated with CS are associated with increased mortality mainly due to cardiovascular events (52). GC-induced comorbidities in CS may be at least in part epigenetically mediated. Previous study using whole blood methylation profile demonstrated that specific hypomethylated CpG sites induced by GC were associated with Cushing comorbidities, such as hypertension and osteoporosis (46). The study identified a methylator predictor of GC excess which could be used as a biomarker to monitor GC status (46). The long-term exposure to high cortisol levels may be crucial for the persistence of some morbidities in CS through epigenetic changes. Hypercortisolism-induced persistent changes in visceral adipose tissue gene expression through epigenetic modifications was investigated in a translational study (12). This study combined data from patients with active CS and data from an animal model of CS in active and remitted phase. Interestingly, the study demonstrated long-lasting changes in the transcriptome of adipose tissue that were associated with histone modifications induced by GC. Therefore, these epigenetic fingerprints observed even after the resolution of hypercortisolism may elucidate the mechanism of persistent modifications in gene expression in the visceral adipose tissue (12). With regard to the persistence of GC-induced DMR, a genome-wide DNA methylation analysis showed a lower average of DNA methylation in patients in remission of CS compared to controls. Interestingly, the most common biologically relevant affected genes were retinoic acid receptors, thyroid hormone receptors, or hormone/nuclear receptors, important genes related to intracellular pathways and regulators of gene expression (9). In summary, this large body of evidence supports the concept that prolonged GC exposure modulates the epigenetic landscape across the genome by inducing DMR and histone modifications. Some epigenetic modifications are persistent, and this may partially explain the incomplete reversibility of some of CS features following clinical remission. Glucocorticoid-Target Genes in Cushing Syndrome A detailed identification and characterization of GC-target genes may shed light in the understanding of the pathophysiology and treatment response in patients with CS. For instance, the GC regulation of pro-opiomelanocortin (POMC) expression as part of the physiologic GC negative feedback may be impaired in Cushing disease (CD), which is an important mechanism for the maintenance of high GC levels (53). Another example is the interaction between GC and clock genes, which may interfere in the loss of the GC circadian rhythm and may contribute to metabolic disorders in CS (54). Furthermore, the suppressive action of GC on drug targets, such as the somatostatin receptor (subtype 2), may influence the efficacy of first-generation somatostatin receptor ligands in normalizing cortisol levels in CD (55). Here we describe how GCs using epigenetic machinery influence the expression of important target genes and their implications in CS. FKBP5 FK506 binding protein (FKBP5) plays an important role in the regulation of hypothalamic-pituitary-adrenal (HPA) system (56). As part of the GC negative feedback loop, GC binds to hypothalamic and pituitary GR. In the cytoplasm, GR is bound to a multi-protein complex including FKBP5. FKBP5 modulates GR action by decreasing GR binding affinity to GC and by preventing GR translocation from cytoplasm to nucleus (57, 58). In other words, an increase of FKBP5 expression is inversely correlated with GR activity and results in GC resistance leading to an impaired negative feedback regulation in the HPA axis (59). FKBP5 is a GC-responsive gene; its upregulation by GC is part of an intracellular negative short-feedback loop (60). The mechanism by which GC regulates FKBP5 expression was shown to include inhibition of DNA methylation (44). In a model for CS, mice treated with corticosterone for 4 weeks had a reduced level of DNA methylation of FKBP5 in DNA extracted from whole blood, which was strongly correlated in a negative manner with GC concentration. Interestingly, a negative correlation was also observed between the degree of FKBP5 gene methylation measured at 4 weeks of GC exposure and the percentage of mice visceral fat (61). Accordingly, previous studies have provided compelling evidence of decreased methylation in the FKBP5 gene in patients with active CS compared to healthy control (10, 46). Even in patients with CS in remission, previous data have suggested a small decrease in FKBP5 methylation levels compared to healthy controls (9, 10). In an in vitro study, it was demonstrated that, by decreasing DNMT1 expression, GC is able to reduce FKBP5 methylation levels and, therefore, increase its expression (44). Likewise, FKBP5 mRNA is also sensitive to GC exposure. A time-dependent increase in blood FKBP5 mRNA after single-dose prednisone administration has been demonstrated in healthy humans (62). Accordingly, patients with ACTH-dependent CS had higher blood FKBP5 mRNA levels compared with healthy controls, and after a successful surgery, FKBP5 mRNA returned to baseline levels (63). Furthermore, in another study, blood FKBP5 mRNA was inversely correlated with FKBP5 promoter methylation and positively correlated with 24-hour urine free cortisol (UFC) levels in patients with CS (46). Taken together, this fine-tuning of FKBP5 DNA methylation and mRNA according to the level of GC suggests that FKBP5 can be used as a biomarker to infer the magnitude of GC exposure. POMC and Corticotropin-Releasing Hormone The partial resistance of the corticotroph adenoma to GC negative feedback is a hallmark of CD. Indeed, the lack of this inhibitory effect constitutes a method to diagnose CD, that is, with the dexamethasone suppression test. One of the mechanisms related to the insensitivity to GC can be attributed to GR mutations which are, however, rarely found in corticotrophinomas (64). Another mechanism that was uncovered in corticotroph adenomas is an overexpression of the HSP90 chaperone resulting in reduced affinity of GR to its ligand and consequently GR resistance (53, 65). In addition, the loss of protein expression of either Brg1, ATPase component of the SWI/SNF chromatin remodeling complex, or HDAC2 has been linked to GC resistance in about 50% of some adenomas (66). The trans-repression process on POMC transcription achieved by GC involves both the histone deacetylation enzyme and Brg1. One mechanism of corticotropin-releasing hormone (CRH)-induced POMC expression is through an orphan nuclear receptor (NR) related to NGFI-B (Nur77). NGFI-B binds to the NurRE sequence in the promoter region of POMC gene and recruits a co-activator to mediate its transcription. In a tethering mechanism, the GR directly interacts with NGFI-B to form a trans-repression complex, which contains the GR itself, Brg1, the nuclear receptor, and HDAC2; the latter being essential to block the gene expression through chromatin remodeling process (53, 66). In CD, hypercortisolism exerts a negative feedback at CRH secretion from the hypothalamus (67). The mechanism involved in GR-induced suppression of CRH expression is through direct binding to a nGRE in the promoter region of CRH gene and subsequent recruitment of repressor complexes. In a rat hypothalamic cell line, it was demonstrated that Dex-induced CRH repression occurs through coordinated actions of corepressors involving Methyl-CpG-binding protein 2 (MeCP2), HDAC1, and DNA methyltransferase 3B (DNMT3B). Possibly, GR bound to nGRE recruits DNMT3B to the promoter in order to methylate a specific region, subsequently binding MeCP2 on these methylated sites followed by the recruitment of chromatin modify corepressor HDAC1, ultimately resulting in CRH suppression. Another possibility is that 2 independent complexes, one consisting of GR with DNMT3 for the methylation and the other the MeCP2, bound to methylated region, interact with HDAC1 to induce repression (68). Clock Genes The clock system and the HPA axis are interconnected regulatory systems. Cortisol circadian rhythm is modulated by the interaction between a central pacemaker, located in the hypothalamic suprachiasmatic nuclei, and the HPA axis (69). At the molecular level, mediators of the clock system and cortisol also communicate with each other, both acting as transcription factors of many genes to influence cellular functions. In CS, the impact of chronic GC exposure on clock genes expression was recently evaluated using peripheral blood samples from patients with active disease compared with healthy subjects. The circadian rhythm of peripheral clock gene expression (CLOCK, BMAL, PER1-3, and CRY1) was abolished as a result of hypercortisolism, and that may contribute to metabolic disorders observed in Cushing patients (70). Another study, which investigated persistent changes induced by hypercortisolism in visceral adipose tissue, found that the expression of clock genes, such as PER1, remained altered in association with persistent epigenetic changes in both H3K4me3 and H3K27ac induced by hypercortisolism even after the resolution of hypercortisolism (12). This suggests that chronic exposure to GC may induce sustained epigenetic changes that can influence clock genes expression. Nevertheless, further studies are warranted to better elucidate how long-term exposure to GC impacts clock genes expression using the epigenetic machinery. Glucocorticoid Effects on MicroRNAs Along with histone modification and DNA methylation, microRNAs (miRNAs) have emerged as an epigenetic mechanism capable of impacting gene expression without changing DNA sequence (15). Interestingly, miRNA expression itself is also under the influence of epigenetic modifications through promoter methylation like any other protein-encoding genes (71). MicroRNAs are small (about 20-25 nucleotides in length) non-coding RNAs that are important in transcriptional silencing of messenger RNA (mRNA). By partially pairing with mRNA, miRNAs can either induce mRNA degradation or inhibit mRNA translation to protein. MiRNAs regulate the translation of about 50% of the transcriptome, allowing them to play an important role in a wide range of biological functions, such as cell differentiation, proliferation, metabolism, and apoptosis under normal physiological and pathological situations. Some miRNAs can be classified as oncogenes or tumor suppressing genes, and aberrant expression of miRNAs may be implicated in tumor pathogenesis (71-73). Insight into the regulation of miRNA expression is, therefore, crucial for a better understanding of tumor development and other human diseases, including cardiac, metabolic, and neurological disorders (73, 74). There are different regulatory mechanisms involved in miRNA expression, including transcriptional factors such as GR-GC. GC may modulate miRNA expression through direct binding to GRE in the promoter region of the host gene, as observed in hemopoietic tumor cells (75). In addition to transcriptional activation, in vascular smooth muscle cells, Dex treatment induces downregulation of DNMT1 and DNMT3a protein levels and reduces the methylation of miRNA-29c promoter, resulting in an increased expression of miRNA-29c (76). Interestingly, it was demonstrated that the increased expression of miRNA-29 family (miRNA-29a, -29b, and -29c) associates with metabolic dysfunction, such as obesity and insulin resistance, which pertains to CS (77, 78). With regard to metabolic dysfunction, miRNA-379 expression was shown to be upregulated by GC and its overexpression in the liver resulted in elevated levels of serum triglycerides associated with very low-density lipoprotein (VLDL) fraction in mice (79). In obese patients, the level of hepatic miRNA-379 expression was higher compared to nonobese patients and positively correlated with serum cortisol and triglycerides (79). Hence, GC-responsive miRNA may be, at least in part, a mediator to GC-driven metabolic conditions in CS. In pathological conditions, such as seen in CS, prolonged exposure to an elevated cortisol level results in a wide range of comorbidities. It can be hypothesized that the chronic and excessive glucocorticoid levels may induce an aberrant miRNA expression that might impact several cellular processes related to bone and cardiometabolic disorders. A recent study addressed the impact of hypercortisolism on bone miRNA of patients with active CD compared to patients with nonfunctional pituitary adenomas. Significant changes in bone miRNA expression levels were observed, suggesting that the disruption of miRNA may be partially responsible for reduced bone formation and osteoblastogenesis (80). Similarly, altered expression levels of selected miRNAs related to endothelial biology in patients with CS may point to a contribution to a high incidence of cardiovascular disorders in Cushing patients (81). Therefore, dysregulated miRNAs as a consequence of high cortisol levels may underpin the development and progression of comorbidities related to CS. To the best of our knowledge, it is currently not clear whether miRNA dysregulation persists after resolution of hypercortisolism, thus contributing to the persistence of some comorbidities. This hypothesis needs to be further investigated. MicroRNA can also be used as a diagnostic tool in CS. A study was performed to identify circulating miRNA as a biomarker to differentiate patients with CS from patients with suspected CS who had failed diagnostic tests (the control group) (82). It was observed that miRNA182-5p was differentially expressed in the CS cohort compared to the control group; therefore, it may be used as a biomarker (82). However, a large cohort is necessary to validate this finding (82). In corticotroph tumors, downregulation of miRNA 16-1 expression was observed relative to normal pituitary tissue (83). In contrast, the plasma level of miRNA16-5p was found to be significantly higher in CD compared to ectopic Cushing (EAS) and healthy controls (84). This finding suggests that miRNA16-5p may be a biomarker capable to differentiate the 2 forms of ACTH-dependent Cushing (84). Epidrugs and Glucocorticoid Action in Cushing's Syndrome The interest in understanding the epigenetic mechanism of GC action in the context of CS is based on reversibility of epi-marks, such as DNA methylation and histone modifications, using epidrugs (85, 86). The biological characteristics of epigenetic drugs and their target have been extensively explored. Their effectiveness as antitumor drugs have been tested on corticotroph tumors using in vitro studies (87-89). However, a limited number of studies have explored the role of epidrugs as a therapeutic tool in reversing the genomic action of GC in CS, particularly in comorbidities induced by hypercortisolism (90, 91). The use of histone deacetylase inhibitors (HDACi) may reduce the genomic action of GC (90-92). It has been demonstrated that the use of the HDAC inhibitor valproic acid increases the acetylation level of GR, consequently attenuating the genomic action of GC. In an experimental Cushing model in rats, the use of valproic acid decreased expression of genes related to lipogenesis, gluconeogenesis, and ion regulators in the kidney that ultimately reduces hepatic steatosis, hyperglycemia, and hypertension in ACTH-infused rats (90, 91). More studies evaluating the effects of epidrugs influencing the GC actions are warranted to further elucidate the underlying mechanisms and to explore potential treatment modalities to reverse long-lasting consequences of chronic corticoid exposure. Conclusions In physiologic conditions, GC are secreted in pulses following a circadian rhythm pattern, as opposed to a constant, chronic, and high GC exposure in CS. This pathological pattern may account for numerous devastating effects observed in CS (7). Yet, the expressed genome in response to chronic GC exposure may potentially be abnormal, leading to dysregulation in clock genes, among other effects. GC levels may return to a normal circadian pattern in response to a successful treatment, but with incomplete reversibility of some CS features, which may in part be explained by epigenetic changes. The epigenetic machinery is used by GC to induce dynamic changes in chromatin to modulate gene expression. (Fig. 2) It seems that most of chromatin modifications are reversible, but some may persist resulting in long-term epigenetic changes. (Table 1) Table 1. Evidence of interaction between glucocorticoid and epigenetic machinery Epigenetic changes/epigenetic enzymes Action Histone acetylation (HAT) Glucocorticoid receptors (GR) recruit co-regulators, such as CREB binding protein (CBP), which has intrinsic histone acetyltransferase (HAT) activity that modifies the chromatin structure from an inactive to an active state (25, 33, 35). Histone deacetylation (HDAC) GR recruit histone deacetylases (HDACs) to turn chromatin less accessible and suppress gene transcription (25, 35). The trans-repression process on POMC transcription achieved by glucocorticoids (GC) involves the histone deacetylation enzyme (HDAC2). GC mediates the upregulation of HDAC2 in rats exposed to chronic stress (40). Histone demethylase (JMJD3) GC suppress transcription of JMJD3 in endothelial cells treated with TNFα (41). Histone modifications Using ChIP-seq, a study in mice treated for 5 weeks with corticosterone showed higher levels of histone modifications (H3K4me3, H3K27ac) compared to control mice. In mice after a 10-week washout period, persistence of this epigenetic fingerprint was observed, which was associated with long-lasting changes in gene expression (12). DNA methylation (DNMT3B) and histone deacetylation (HDAC1) GC mediates CRH downregulation through DNMT3B to the promoter in order to methylate a specific region and recruitment of chromatin modify corepressor HDAC (68). DNA hypomethylation GC induces downregulation of DNMT1 in AtT20 (mouse corticotroph adenoma cell line) (20). GC induces upregulation of TET enzyme expression which was described in retinal and osteocyte cell line model (42, 43). An experimental study in mice previously exposed to high levels of GC showed differentially methylated regions (DMR) induced by GC treatment, of which the majority was loss of the methylation (11). Reduced DNA methylation in FKBP5 gene was found in patients in active disease and also in remission state of Cushing syndrome (CS) as compared to a healthy control group (10). A genome-wide DNA methylation analysis showed a lower average of DNA methylation in patients in remission of CS compared to controls (9). A study using whole blood methylation profile demonstrated an association between cortisol excess and DNA hypomethylation in patients with CS (46). Open in new tab Further studies are needed to elucidate how chronic exposure to GC leads to incomplete reversibility of CS morbidities via sustained modulation of the epigenetic machinery and possibly other mechanisms. Subsequent identification of therapeutic targets may offer new perspective for treatments, for example, with epidrugs, aiming to reverse hypercortisolism-related comorbidities. Funding The authors received no financial support for this manuscript. Disclosures T.P., R.A.F., and L.J.H. have nothing to declare. Data Availability Data sharing is not applicable to this article, as no datasets were generated or analyzed during the current study. From https://academic.oup.com/jcem/advance-article/doi/10.1210/clinem/dgae151/7633538?searchresult=1

Abstract Disclosure: C.M. Godar: None. E.B. Noble: None. N.O. Vietor: None. T.S. Knee: None. Background: Cushing’s syndrome may rarely present as an emergency known as Florid Cushing’s Syndrome. Patients can exhibit severe hyperglycemia, hypertension, hypokalemia, infections, and hypercoagulability. Cushing’s syndrome is a rare disease, and the constellation of clinical features can be overlooked if clinicians are not aware of the manifestations of hypercortisolism. We present the case of a patient with Cushing’s syndrome that went unrecognized with life-threatening sequelae. Case presentation: A 52-year-old woman with well-controlled type 2 diabetes and hypertension was admitted to the hospital for severe left lower extremity cellulitis. Prior to hospitalization she had noted rapid weight gain, fatigue, weakness, mental clouding, and moodiness. She was admitted for antibiotics and surgical debridement. The infection persisted despite broad spectrum antibiotics, multiple surgical debridements, and skin grafting. She became bacteremic, and extremity amputation was considered. She additionally developed hypertensive emergency, refractory hypokalemia, and hyperglycemia to 396 mg/dL. Exam was notable for facial plethora, supraclavicular fullness, dorsocervical fat pad, and violaceous abdominal striae. Cushing’s Syndrome was suspected, and labs revealed a significantly elevated random serum cortisol of 60.5mcg/dL (Ref 6.2-19.4), significantly elevated 24H urine cortisol of 2157mcg/24H (Ref 0-50), and ACTH elevated to 81.8pg/mL (Ref 7.2-63.3) that confirmed Cushing’s Disease. MRI sella and octreotide scans did not localize a lesion. Inpatient therapy included multiple antihypertensive agents, insulin drip, aggressive potassium repletion, and initiation of ketoconazole to reduce cortisol levels. Ketoconazole was maximally dosed and she underwent surgical exploration and removal of a small pituitary microadenoma. Following surgery, she developed transient adrenal insufficiency requiring hydrocortisone and she no longer required antihypertensives, insulin, or potassium therapy. Follow up 7 years later has revealed no recurrence of Cushing’s Disease. Discussion: Cushing’s Syndrome may present with a variety of clinical features and rarely may present as a medical emergency. Delay in diagnosis can lead to Florid Cushing’s Syndrome which carries high risk for morbidity and mortality. This case illustrates the need for clinician awareness of the features of Cushing’s Syndrome: hypertension, hyperglycemia, rapid weight gain, cushingoid exam features, hypokalemia, hirsutism, virilization, infection, and/or hypercoagulable state. Severe hypercortisolism was responsible for this patient’s refractory infection, and if not controlled, she likely would have endured a lower extremity amputation. Rapid detection with elevated random serum and/or urine cortisol and treatment with a cortisol-lowering agent is critical and lifesaving. Presentation: Thursday, June 15, 2023 Issue Section: Neuroendocrinology and Pituitary PDF This content is only available as a PDF.

Highlights Aim to identify independent risk factors for postoperative delirium after pituitary adenoma surgery. Select matched subjects by Propensity Score Matching to reduce potential biases caused by variables. Enhance preoperative communication to minimize the occurrence of delirium, for patients at high risk of postoperative delirium. Minimize surgery duration and general anesthesia, optimize perioperative sedation regimen. Reducing unnecessary or excessive protective physical restraints. Abstract Objectives The primary aim of this study is to explore the factors associated with delirium incidence in postoperative patients who have undergone endoscopic transsphenoidal approach surgery for pituitary adenoma. Methods The study population included patients admitted to Tianjin Huanhu Hospital's Skull Base Endoscopy Center from January to December 2022, selected through a retrospective cohort study design. The presence of perioperative delirium was evaluated using the 4 'A's Test (4AT) scale, and the final diagnosis of delirium was determined by clinicians. Statistical analysis included Propensity Score Matching (PSM), χ2 Test, and Binary Logistic Regression. Results A total of 213 patients were included in this study, and the incidence of delirium was found to be 29.58 % (63/213). Among them, 126 patients were selected using PSM (delirium:non-delirium = 1:1), ensuring age, gender, and pathology were matched. According to the results of univariate analysis conducted on multiple variables, The binary logistic regression indicated that a history of alcoholism (OR = 6.89, [1.60–29.68], P = 0.010), preoperative optic nerve compression symptoms (OR = 4.30, [1.46–12.65], P = 0.008), operation time ≥3 h (OR = 5.50, [2.01–15.06], P = 0.001), benzodiazepines for sedation (OR = 3.94, [1.40–11.13], P = 0.010), sleep disorder (OR = 3.86, [1.40–10.66], P = 0.009), and physical restraint (OR = 4.53, [1.64–12.53], P = 0.004) as independent risk factors for postoperative delirium following pituitary adenoma surgery. Conclusions For pituitary adenoma patients with a history of alcoholism and presenting symptoms of optic nerve compression, as well as an operation time ≥3 h, enhancing communication between healthcare providers and patients, improving perioperative sleep quality, and reducing physical restraint may help decrease the incidence of postoperative delirium. Introduction In clinical practice, patients admitted to the intensive care unit (ICU) during the postoperative period after endoscopic transsphenoidal tumorectomy of pituitary adenoma often experience episodes of delirium. According to a recent retrospective analysis conducted at a single center, the incidence of postoperative delirium among these patients was found to be 10.34 % (n = 360) [1]. Delirium is a common complication following neurosurgery, characterized by acute distraction, confusion in thinking, sleep disorders, and cognitive decline. The incidence of delirium in admitted patients after neurosurgery has been reported to be 19 %, with a range of 12 % to 26 % depending on clinical features and the methods used for delirium assessment [2], [3], [4]. The incidence of postoperative delirium varied across different types of neurosurgical diseases, as reported in a meta-analysis [2]. Specifically, the incidences were 8.0 % for patients with neurological tumors, 20 % for those undergoing functional neurosurgery, 24.0 % for microvascular decompression patients, 19.0 % for traumatic brain injury patients, 42.0 % for neurovascular patients, and 17.0 % for the mixed population undergoing neurosurgery procedures. Furthermore, the incidence rates of delirium in intensive care units (ICUs), general wards, or both combined were found to be 24.0 %, 17 %, and 18 %, respectively. The aforementioned issue not only leads to prolonged hospital stays and increased healthcare costs, but also exerts a significant impact on patient consciousness and cognitive function. Therefore, early and accurate identification of delirium in post-neurosurgical patients is crucial. However, due to frequent co-occurrence with primary brain injury, related complications can also lead to cognitive impairment or even decreased levels of consciousness, posing challenges for timely and precise identification of delirium. Currently, the primary focus lies in the prevention of delirium within the neurosurgical ICU setting. Early identification and comprehensive pre-surgical assessment are positively significant measures for preventing postoperative delirium occurrence [5], [6]. In this study, a retrospective cohort design was employed to collect pertinent data and statistically analyze the incidence of delirium, as well as its associated influencing factors, among patients admitted to the neurosurgical ICU for pituitary adenoma treatment. And now it is reported as follows. Section snippets Patient selection A retrospective cohort study design was employed to select 213 pituitary adenomas admitted to the Skull Base and Endoscopy Center of Tianjin Huanhu Hospital between January 2022 and December 2022 as the subjects for investigation, with a review of their medical records. The mean age was (50.03 ± 15.72) years, ranging from 20–79 years old (Fig. 1). Informed consent was obtained from all patients or their families, ensuring compliance with the requirements stated in the Declaration of Helsinki. Inclusion criteria a. Propensity score matching The present study enrolled a total of 213 patients with pituitary tumors, among whom 63 exhibited symptoms related to delirium while the remaining 150 did not. Consequently, the incidence rate of delirium was determined to be 29.58 % in this cohort of patients admitted to the intensive care unit following pituitary tumor surgery. The univariate analysis revealed no significant differences in age (≥65y old, 23.8 % vs. 23.3 %, P = 0.940) and gender (male, 49.2 % vs. 56.7 %, P = 0.318) between the Background of perioperative delirium in transsphenoidal endoscopic pituitary adenoma surgery The pituitary gland is situated within the sella turcica and comprises two distinct components. The anterior pituitary, known as the adenohypophysis, functions as an endocrine organ responsible for secreting growth hormone, prolactin, adrenocorticotropic hormone, thyrotropin, follicle-stimulating hormone and luteinizing hormone. On the other hand, the posterior pituitary, referred to as the neurohypophysis, serves as a direct extension of the hypothalamus and acts as a storage site for Conclusions To enhance the evaluation of postoperative patients at risk of delirium, it is anticipated that optimizing doctor-nurse-patient communication and minimizing unnecessary and indiscriminate protective measures will mitigate the incidence of delirium following pituitary tumor surgery. This study is a single-center prospective study conducted at our institution, which has several inherent limitations. A large-scale multicenter prospective study is anticipated to further investigate the associated Limitations There are multiple factors that influence the occurrence of delirium following neurosurgery. This retrospective study solely focused on analyzing and comparing general patient data, medical history, and potential perioperative factors contributing to delirium, without considering any other known or unknown variables in this analysis. The pituitary gland functions as a neuroendocrine organ involved in the regulation of neuroendocrine processes. Changes in hormone levels following surgery for Funding All authors affirm that this study was conducted without any fund support from external organizations. CRediT authorship contribution statement Shusheng Zhang: Writing – original draft, Methodology, Investigation, Formal analysis, Data curation, Conceptualization. Yanan Chen: Writing – original draft, Investigation, Data curation. Xiudong Wang: Validation, Supervision, Project administration, Methodology, Conceptualization. Jun Liu: Software, Formal analysis, Data curation. Yueda Chen: Validation, Supervision, Methodology, Investigation. Guobin Zhang: Writing – review & editing, Validation, Supervision, Methodology, Conceptualization. Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. References (21) G. Sousa et al. Postoperative delirium in patients with history of alcohol abuse Rev Esp Anestesiol Reanim (2017) M. Terzaghi et al. Sleep disorders and acute nocturnal delirium in the elderly: a comorbidity not to be overlooked Eur J Intern Med (2014) S. Lee et al. Opioid and benzodiazepine use in the emergency department and the recognition of delirium within the first 24 hours of hospitalization J Psychosom Res (2022) A.J. Slooter et al. Delirium in critically ill patients Handb Clin Neurol (2017) E. Wang et al. Effect of perioperative benzodiazepine use on intraoperative awareness and postoperative delirium: a systematic review and meta-analysis of randomized controlled trials and observational studies Br J Anaesth (2023) E. Rollo et al. Physical restraint precipitates delirium in stroke patients J Neurol Sci (2021) H. Chen et al. The incidence and predictors of postoperative delirium after brain tumor resection in adults: A cross-sectional survey World Neurosurg (2020) J. Liu et al. Identifying hormones and other perioperative risk factors for postoperative delirium after endoscope-assisted transsphenoidal pituitary adenoma resection: A retrospective, matched cohort study Brain Behav (2023) P.R. Kappen et al. Delirium in neurosurgery: a systematic review and meta-analysis Neurosurg Rev (2022) J. Wang et al. Risk factors for the incidence of delirium in cerebrovascular patients in a Neurosurgery Intensive Care Unit: A prospective study J Clin Nurs (2018) There are more references available in the full text version of this article. From https://www.sciencedirect.com/science/article/abs/pii/S0967586824001279

Abstract Avascular necrosis (AVN), also called osteonecrosis, stems from blood supply interruption to the bone and is often idiopathic. It has risk factors like trauma, alcohol, and corticosteroids. AVN in the talus (AVNT) is less common than in the femoral head. Most cases of talar osteonecrosis are associated with trauma, while a smaller proportion is linked to systemic conditions such as sickle cell disease or prolonged prednisone use. Glucocorticoids are a key nontraumatic cause. We report a middle-aged woman with Cushing’s syndrome symptoms, such as hypertension and moon face, since her youth. A few years ago, she experienced pain and swelling in her ankle, which was diagnosed as atraumatic AVNT and treated with hindfoot fusion. Years later, she was diagnosed with Cushing’s disease caused by an adrenocorticotropic hormone (ACTH)-producing pituitary adenoma in laboratory tests and imaging, which was resected in 2020. She experienced significant weight loss, and her Cushing’s syndrome symptoms were relieved after tumor resection. Mechanisms behind AVN in hypercortisolism involve fat cell hypertrophy, fat embolization, osteocyte apoptosis, and glucocorticoid-induced hypertension. Traditional X-rays may miss early AVN changes; MRI is preferred for early detection. Although there are some cases of femoral AVN caused by endogenous hypercortisolism in the literature, as far as we know, this is the first case of AVNT due to Cushing’s disease. AVNT treatment includes conservative approaches, hindfoot fusion, and core decompression. Cushing’s disease is a rare cause of AVNT, and a multidisciplinary approach aids in the rapid diagnosis of elusive symptoms. Introduction Avascular necrosis (AVN), also known as osteonecrosis, is a condition arising from the temporary interruption or permanent cessation of blood supply to a bone, leading to tissue necrosis or its demise. While AVN is frequently idiopathic, certain established risk factors are known including trauma, alcohol abuse, and the use of exogenous corticosteroids [1]. While not as prevalent as in the femoral head, AVN of the talus (AVNT) in the ankle presents a painful and disabling issue for patients and poses a challenging dilemma for orthopedic surgeons [2]. About 75% of cases of talar osteonecrosis stem from traumatic injuries, while approximately 25% of nontraumatic instances are typically associated with systemic conditions such as sickle cell disease or prolonged use of prednisone, which impede blood flow. [3] The use of glucocorticoids is one of the most important non-traumatic causes of AVN. Nevertheless, there are some case reports where AVN in the femoral head is reported as a manifestation of endogenous hypercortisolism, particularly associated with Cushing's syndrome [4-12]. In this article, we describe the case of a middle-aged woman who was diagnosed with idiopathic severe progressive AVNT for two years. She had retrogradely diagnosed masked symptoms of Cushing’s disease since her youth, but the diagnosis was confirmed after undergoing ankle arthrodesis. Case Presentation A 43-year-old woman visited our office in June 2018 with a complaint of severe pain and swelling in her left ankle, which had persisted for the past two years. She had hypertension since her youth and blurry vision since 2013 but had no other significant medical or family history. She was also diagnosed with major depressive disorder (MDD) in 2015 when she lost her husband. She had no history of smoking, alcohol consumption, or addiction. She had not experienced any significant trauma during this period and sought consultations from various specialties, including neurology, psychology, internal medicine, nephrology, rheumatology, and orthopedics. She had received a platelet-rich plasma (PRP) injection in the ankle, but it did not improve her symptoms. Despite undergoing various diagnostic workups, no precise diagnosis had been established. Back in 2013, she remembers suddenly experiencing blurry vision in her right eye. This condition underwent a misdiagnosis, which almost led to a loss of vision. She had been struggling with her eye problems until her last visit, during which she received intravitreal bevacizumab injections. Additionally, she previously had iron deficiency anemia, which was treated with ferrous sulfate before 2018. In our first visit, during the physical examination, the pain was localized in the ankle mortise with some posterolateral pain along the course of the peroneal tendons posterior to the fibula. Based on the physical examination and available ankle radiographs, we diagnosed AVNT. The patient subsequently underwent ankle arthroscopy through the standard anterior portals, the joint was cleaned, the synovium was shaved, and a small incision was conducted for peroneal assessment; this procedure revealed a subchondral collapse and extensive necrosis in the talus. Following the procedure, she experienced a partial improvement in her symptoms. However, six months later, she returned with a recurrence of symptoms (Figure 1). Upon further inquiry, she mentioned that her symptoms had recurred a month ago when she was dancing at a family party. Radiographs showed a stress fracture in her fibula and extensive AVNT. This diagnosis was confirmed through a CT scan, MRI, and bone scan (Figure 2). Figure 1: Ankle X-ray six months after arthroscopy Pain had reduced for four months, then pain increased with activity and disabled her after a night of dancing. Subchondral fracture and fibular stress fracture are evident (A and B, respectively). Figure 2: MRI, CT scan, and technetium-99m (Tc-99m) bone scan Coronal MRI confirmed avascular necrosis of the talar dome with subchondral fracture (A and B, respectively). CT scan (C) and Tc-99 bone scan (D) images also revealed the pathologies. In the second visit after arthroscopy, upon confirmation of a fibular stress fracture and significant subchondral collapse, and following a discussion of the next available options with the patient, the second procedure was performed as an ankle arthrodesis with an anterior approach. A 6 cm longitudinal incision was made anteriorly, and through the plane between the tibialis anterior and extensor hallucis longus, the ankle joint was accessed. Joint preparation was done with an osteotome, ensuring a bleeding surface on both sides. Then, manual compression with provisional pin fixation in the corrective position was performed. The fusion was planned at less than 5 degrees of valgus, 10 degrees of external rotation, and approximately 10 degrees of plantar flexion, suitable for the high-heeled shoes that she was using in her daily living activities. After confirming fluoroscopy in two planes, final 6.5 mm cannulated cancellous screws were used, and fixation was augmented with an anterior molded 4.5 mm narrow dynamic compression plate (DCP), according to our previously published anterior ankle fusion technique [13]. The foot was placed in a splint for 10 days, after which stitches were removed, and a cast was applied for four weeks. Then, walking with gradual, as-tolerated weight-bearing was initiated (Figure 3). Three months after surgery, she was pain-free, and by the sixth month, she could walk without any boot or brace, only using high-heeled shoes. Figure 3: Post-operative radiographies Six months after the ankle surgery, a huge osteonecrosis and fibular stress fracture were managed with an acceptable, painless ankle fusion (not solid) despite the remaining necrosis (A and B, respectively). In 2024, four years after the tumor resection, complete healing of talus necrosis and solid fusion were achieved (C and D, respectively). In 2020, two years after her ankle surgery, she was referred to an endocrinologist due to excessive weight gain and hirsutism. The biochemical assessment revealed the following: cortisol (8 AM) (chemiluminescence immunoassay (CLIA)) was 96 µg/dl (normal range: 4.82 - 19.5 µg/dl), and it was 22.1 µg/dl after overnight dexamethasone (normal range: < 1.8 µg/dl). Adrenocorticotropic hormone (ACTH) (CLIA) was 44.4 pg/ml (normal range: 7.2-63.3 pg/ml), and cortisol measured 5.7 µg/dl after the 48-hour low-dose dexamethasone suppression test (normal < 5 µg/dl). The results, along with symptoms (Table 1), are documented in the laboratory tests (Table 2). She was diagnosed with Cushing’s syndrome, which was subsequently confirmed as Cushing's disease due to an ACTH-producing pituitary adenoma observed in the MRI and Brain CT (Figure 4). Sign/symptom Severity Weight Gain Severe Hirsutism Severe Hypertension Severe Easy bruising Severe Depression Severe Moon face Moderate (masked with makeup) Lethargy Moderate Headache Moderate Peripheral edema _ Buffalo hump _ Myopathy _ Acne _ Purple striae _ Table 1: Cushing's disease symptoms and signs The hyphens in the table indicate that the patient does not have those symptoms or signs. Laboratory test Result Reference range Cortisol (8 AM) (CLIA) 96 µg/dl 4.82-19.5 µg/dl Cortisol (8 AM) (after overnight dexamethasone) (CLIA) 22.1 µg/dl <1.8 µg/dl ACTH (CLIA) 44.4 pg/ml 7.2-63.3 pg/ml Cortisol after 48 hours of LDDST (CLIA) 5.7 µg/dl < 5 µg/dl Table 2: Laboratory tests CLIA: chemiluminescence immunoassay; ACTH: adrenocorticotropic hormone; LDDST: low-dose dexamethasone suppression test Figure 4: Brain MRI Finally, a pituitary adenoma was diagnosed using a Brain MRI as the cause of Cushing’s disease symptoms (A and B). Finally, she underwent a tumor resection and had a dramatic response after treatment (30 kg weight loss). She revealed that she had Cushing’s syndrome symptoms since she was young. These symptoms included a puffy face, which she covered with makeup, high blood pressure, and hirsutism. In January 2024, four years after her brain surgery, during our last visit, her symptoms had significantly improved. She reported no problems with her ankle, and talus necrosis was completely healed, with a solid fusion achieved in radiographs (Figure 3). Discussion As far as we are aware, this case presentation represents the first instance of AVNT attributed to Cushing’s disease in the existing literature. Nevertheless, some individuals with endogenous Cushing's syndrome have been reported to experience AVN of the femoral head [4-12]. Cushing's syndrome is an uncommon endocrine condition marked by manifestations of hypercortisolism. The predominant cause is often an adenoma in the anterior pituitary gland that produces ACTH, referred to as Cushing's disease [14]. The presentation of Cushing's syndrome can vary significantly in both adults and children, influenced by the extent and duration of hypercortisolemia. However, the typical signs and symptoms of Cushing's syndrome are widely known [15]. Although some individuals may perceive these alterations as normal and physiological, the disease can go unnoticed for an extended period, as in our case, in which it remained undiagnosed for more than 20 years. However, it is known that steroid use is a significant contributing factor to the occurrence of bone osteonecrosis, accounting for up to 40% of non-traumatic instances of AVN [16]. The mechanisms leading to AVN due to either endogenous hypercortisolism or excess exogenous glucocorticoids are not completely understood. There are just some hypotheses that suggest that the hypertrophy of fat cells, embolization of fat, and osteocytes' apoptosis result in impaired blood flow in the bone, ultimately causing ischemic tissue necrosis [17]. An alternative proposed theory suggests that elevated levels of glucocorticoids may cause insulin resistance and subsequently contribute to AVN [18]. Traditional X-rays often fail to detect the initial changes of AVN (as observed in our case). MRI stands as the preferred method for identifying AVN in its early phases, providing an opportunity for timely therapeutic interventions [19,20]. In an analysis of 321 cases of AVNT, the predominant treatment modalities included conservative therapies (n = 104), hindfoot fusion (n = 62), and core decompression (n = 85) [21]. These approaches reflect the primary methods employed in contemporary clinical practice for addressing AVNT. After all, we confirmed the AVNT diagnosis using MRI and bone scan and managed it with hindfoot fusion. Subsequently, the underlying issue, endogenous hypercortisolism due to an ACTH-producing pituitary adenoma, was identified and treated through resection of the tumor (Figure 5). Figure 5: Case report timeline * Avascular necrosis in the talus Conclusions Cushing’s syndrome is a rare endocrine disorder characterized by excessive cortisol levels, commonly caused by an ACTH-producing adenoma in the pituitary gland, known as Cushing’s disease. Cushing’s disease may be one of the rare causes of AVNT. To the best of our knowledge, this is the first instance of AVNT due to Cushing’s disease described in the literature. Since atraumatic AVNT is rare in itself, a multidisciplinary approach can lead us to a more rapid and proper diagnosis, as each symptom may be masked or considered rare within its subspecialty field. References Chang CC, Greenspan A, Gershwin ME: Osteonecrosis: current perspectives on pathogenesis and treatment. Semin Arthritis Rheum. 1993, 23:47-69. 10.1016/s0049-0172(05)80026-5 Zhang H, Fletcher AN, Scott DJ, Nunley J: Avascular osteonecrosis of the talus: current treatment strategies. Foot Ankle Int. 2022, 43:291-302. 10.1177/10711007211051013 Parekh SG, Kadakia RJ: Avascular necrosis of the talus. J Am Acad Orthop Surg. 2021, 29:e267-78. 10.5435/JAAOS-D-20-00418 Belmahi N, Boujraf S, Larwanou MM, El Ouahabi H: Avascular necrosis of the femoral head: an exceptional complication of Cushing's disease. 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Objective Postoperative data on Cushing’s disease (CD) are equivocal in the literature. These discrepancies may be attributed to different series with different criteria for remission and variable follow-up durations. Additional data from experienced centers may address these discrepancies. In this study, we present the results obtained from 96 endoscopic transsphenoidal surgeries (ETSSs) for CD conducted in a well-experienced center. Methods Pre- and postoperative data of 96 ETSS in 87 patients with CD were included. All cases were handled by the same neurosurgical team between 2014 and 2022. We obtained data on remission status 3−6 months postoperatively (medium-term) and during the latest follow-up (long-term). Additionally, magnetic resonance imaging (MRI) and pathology results were obtained for each case. Results The mean follow-up duration was 39.5±3.2 months. Medium and long-term remission rates were 77% and 82%, respectively. When only first-time operations were considered, the medium- and long-term remission rates were 78% and 82%, respectively. The recurrence rate in this series was 2.5%. Patients who showed remission between 3−6 months had higher longterm remission rates than did those without initial remission. Tumors >2 cm and extended tumor invasion of the cavernous sinus (Knosp 4) were associated with lower postoperative remission rates. Conclusion Adenoma size and the presence/absence of cavernous sinus invasion on preopera-tive MRI may predict long-term postoperative remission. A tumor size of 2 cm may be a supporting criterion for predicting remission in Knosp 4 tumors. Further studies with larger patient populations are necessary to support this finding. Key Words: Complete remission · Neuroendoscopy · Pituitary-dependant Cushing syndrome · Treatment outcome. Go to : INTRODUCTION Cushing’s disease (CD) is characterized by excessive secretion of adrenocorticotropic hormone (ACTH) by a corticotropic adenoma in the pituitary gland. In patients with CD whose hypercortisolism is inadequately corrected, morbidity and mortality can increase by up to 4.8 times due to Cushingrelated complications such as osteoporosis, hypertension, dyslipidemia, insulin resistance, and hypercoagulability [11,18]. Endoscopic transsphenoidal surgery (ETSS), the first-line treatment for CD [7], is performed to decrease complications while achieving remission and long-term disease control. Previous studies on CD have reported varying remission rates between 45% and 95% and recurrence rates ranging from 3−66% [2,4,9,16,21,30]. This wide range of differences can be primarily attributed to differences in surgical experience among centers: centers with higher surgical experience have fewer postoperative complications and higher remission rates [4,6]. However, despite initial remission, patients with CD may eventually experience recurrence. The mean recurrence rate at the 5-10-year follow-up is 23% for microadenomas and 33% for macroadenomas [19,23,30]. Since the postoperative rates in the literature are variable, additional data from experienced centers may be necessary to resolve these discrepancies. In this study, we present the medium- and long-term follow-up data from 96 operations for CD that were conducted in a center with a high level of experience for ETSS. Go to : MATERIALS AND METHODS The study was conducted in accordance with the Declaration of Helsinki (as revised in 2013). The study was approved by the Ethics Committee of Basaksehir Cam and Sakura City Hospital (No. 2022185). Informed consent was obtained from all patients. The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. This retrospective study included pre and postoperative data of 96 ETSS performed in 87 patients with CD (Fig. 1). CD was diagnosed based on unsuppressed cortisol levels (>1.8 µg/dL) following the 1-mg dexamethasone suppression test, high levels of urinary free cortisol, or late night salivary cortisol and plasma ACTH levels >20 pg/mL [28]. Between 2014 and 2022, all surgeries were conducted by the experienced neurosurgical team (Ö.G., O.T., B.E., E.A.) responsible for endoscopic transsphenoidal procedures at the Pituitary Research Center. The surgeries were performed under perioperative glucocorticoid coverage. Fig. 1. Number of operations and patients included in the study. Size, cavernous sinus invasion, sellar and suprasellar infiltration of adenoma on preoperative magnetic resonance imaging (MRI) scans, residual tumor on postoperative MRI scans, postoperative complications, pathology results, remission and recurrence status, and additional postoperative management were evaluated in addition to patients’ demographic data. For follow-up assessments, data obtained 3−6 months postoperatively and during the latest follow-up were included. Three different classifications obtained during radiologic evaluation using MRI were used for pituitary adenomas : 1) maximum size of tumor (MST) : 0−5 mm (group 1), 6−10 mm (group 2), 11−20 mm (group 3), and >20 mm (group 4); 2) Knosp classification : for evaluation of cavernous sinus invasion [22]; and 3) modified Hardy classification : for evaluation of sellar and suprasellar infiltrations [20,39]. In cases of CD without a lesion or with a lesion <6 mm on MRI, confirmation of the central origin and lateralization was provided by inferior petrosal sinus sampling (IPSS) with corticotropin-releasing hormone stimulation [25,26,29]. Under neuronavigation guidance, pure ETSS surgical interventions were performed for all patients by a single surgical team using the Medtronic StealthStation™ S7 and S8 systems (Medtronic, Minneapolis, MN, USA) together with 4-mm 0°, 30°, and 45° rigid optical instruments and an endoscope. A nasal decongestant spray was administered 1 hour before the operation. The sphenoid ostium was detected from both nostrils, and a bi-nostril approach was used by resecting the posterior nasal septum. After sphenoidectomy, the standard sellar approach was used for lesions in the sellar region. The details of these surgical procedures are described in previous study [14]. Selective adenectomy with ETSS was performed for preoperatively localized and visible tumors, whereas hemihypophysectomy was performed for non-lesional cases. In cases with cavernous sinus-invading tumors, particularly Knops 3-4, the defect which was created by the tumor on the medial wall of anterior cavernous sinus was identified and, it was expanded for resection of the tumor tissue within the cavernous sinus. If a defect was not visible, blunt-ended hook-shaped dissectors were used to create a defect on the medial wall, allowing access for the tumor to enter the cavernous sinus. Hematoxylin and Eosin (H&E) and immunohistochemistry staining were performed for the specimens obtained during ETSS. Adenomas showing positive immunohistological staining for ACTH were diagnosed histologically as corticotropinomas. CD was considered to be in remission when the cases showed basal cortisol levels <5 µg/dL or suppressed cortisol levels (≤1.8 µg/dL) following the 1-mg dexamethasone suppression test, 3-6 months postoperation, and during the latest follow-up. The study protocol was approved by the ethics committee of our institution. Data were statistically analyzed using the SPSS 15.0 package (IBM Corp., Armonk, NY, USA). The chi-square test was used for categorical variables. Sample distribution was evaluated with the Kolmogorov-Smirnov test. Continuous independent variables with a normal distribution were compared using the Student’s t-test. Continuous variables with non-normal distributions were compared using the Mann-Whitney U test. p<0.05 was considered statistically significant. A Kaplan-Meier survival analysis was conducted to determine probability and time to recurrence in cases with initial remission. Go to : RESULTS Demographic data A total of 96 ETSS were performed for 87 patients with CD. Of the 87 patients, 68 (79%) were female, and 19 (21%) were male. The mean patient age was 42.2±12.9 years, and the mean duration of follow-up was 39.5±3.2 months. Of the 96 surgeries, 79 (82%) were performed for the first time, six (6%) were performed for residual tumors, and 11 (12%) were performed following a recurrence of the disease. Eight of the 17 patients who underwent reoperations had undergone their first operation at another center. Preoperative imaging Table 1 shows the maximum tumor size on preoperative pituitary MRI before each surgical procedure. Preoperative IPSS for lateralization was performed in 42 operations (44%), all of which were first-time cases. Knosp classification based on preoperative pituitary MRI and the modified Hardy classification is presented in Table 1. Table 1. Preoperative pituitary magnetic resonance imaging scans Number of tumors (n=96) Maximum tumor size  Group 1, 0−5 mm 41 (42.7)  Group 2, 6−10 mm 24 (25.0)  Group 3, 11−20 mm 20 (20.8)  Group 4, >20 mm 11 (11.5) Knosp classification  Grade 0 52 (54.2)  Grade 1 22 (22.9)  Grade 2 6 (6.3)  Grade 3 8 (8.3)  Grade 4 8 (8.3) Modified Hardy classification  0   A 41 (42.8)   B -   C -   D -   E -  1   A 14 (14.6)   B -   C -   D -   E 4 (4.2)  2   A 5 (5.2)   B -   C -   D -   E 5 (5.2)  3   A 1 (1.0)   B 2 (2.1)   C -   D -   E 1 (1.0)  4   A 1 (1.0)   B -   C -   D 1 (1.0)   E 3 (3.1)  NA 18 (18.8) Values are presented as number (%). Invasion : 0, sella normal; 1, sella focally expanded and tumor ≤10 mm; 2, sella enlarged and tumor ≥10 mm; 3, localized perforation of the sellar floor; 4, diffuse destruction of the sellar floor. Suprasellar extension : A, no suprasellar extension; B, anterior recesses of the third ventricle obliterated; C, floor of the third ventricle grossly displaced with parasellar extension; D, intracranial (intradural) : anterior, middle or middle fossa; E, into/beneath the cavernous sinus (extradural). NA : not available Postoperative results Remission was achieved between the 3rd and 6th months in 74 (77%) of the 96 operations, and long-term remission in 79 operations (82%). Among all 96 operations, eight (8%) concluded with a residual tumor. Regarding only first-time operations, five (6%) of the 79 concluded with a postoperative residual tumor. Of the 79 first-time operations, there were 62 cases (78%) of remission between 3 and 6 months. Two (2.5%) of these 79 operations involved recurrence during follow-up, while 60 (97%) showed sustained remission. Those with sustained remission had a median disease-free survival time of 31 months (interquartile range, 14-64) during long-term followup, two cases with recurrence had their recurrence 49 and 54 months after their operation. Survival analysis of cases with remisson and recurrence is presented in Fig. 2. CD persisted after 17 (21.5%) of the 79 first operations. Fig. 2. Survival analysis after the first operation in cases with remission at 3-6 months. Dashed line represents cases with recurrence and, straight line represents cases with sustained remission during long-term follow-up. Ten (13%) of the 79 cases underwent reoperation; two were due to recurrence, and eight due to disease persistence. In five cases (29%), the patients were initially unresponsive but showed remission later during the long-term follow-up. Remission was achieved with stereotactic radiosurgery (STRS) and medical treatment in one of these cases, with only STRS in two and only medical treatment in two cases. At the latest follow-up visit, the total number of cases showing remission after the first operation was 65 (82%). Additional details regarding the results of the first operations are provided in Fig. 3. Fig. 3. Results of the cases who had operation for the first time. Of the 18 reoperations, the results for one case were excluded since the patient was operated at another center. After the reoperation (n=17), the medium and long-term remission rates were 71% (n=12) and 77% (n=13), respectively. The 3-6-month remission rate did not differ significantly between first-time and reoperations (p=0.5). Residual tumors were present in three cases (18%) after reoperation. Of the early non-responders, one case showed remission after STRS, and none of the responders showed recurrence during long-term follow-up. Additional details regarding the results of reoperations are provided in Fig. 4. Fig. 4. Results of the reoperations in our center. Remission rates based on tumor size are presented in Table 2. The initial remission rates of the tumors in MST group 4 were significantly lower than those in the other MST groups (MST 1 vs. 4, p=0.01; MST 2 vs. 4, p=0.001; and MST 3 vs. 4, p=0.006). Comparisons of the other MST groups showed no significant differences. When adenomas were stratified using the 10-mm cut-off, the remission rates did not differ significantly (remission rate, 81% for adenomas <10 mm and 68% for adenomas ≥10 mm; p=0.2). Postoperative residual tumors were observed in five of the 11 tumors (46%) >2 cm (MST group 4) and in one tumor in each of MST groups 1-3 (2%, 4%, and 5%, respectively, p<0.001). Reoperation rate was 17% (n=7) for adenomas ≤5 mm, 18% (n=10) for adenomas ≥6 mm (p=0.9), and 27% (n=3) for adenomas >20 mm (among all grades, p=0.3). Table 2. Comparison of remission rates in preoperative pituitary magnetic resonance imaging scans 3−6-month remission Long-term remission Maximum tumor size  Group 1, 0−5 mm (n=41) 31 (75.6) 33 (80.5)  Group 2, 6−10 mm (n=24) 22 (91.7) 22 (91.7)  Group 3, 10−20 mm (n=20) 17 (85.0) 17 (85.0)  Group 4, >20 mm (n=11) 4 (36.4) 7 (63.6)  p-value 0.003* 0.200 Knops classification  0 (n=52) 41 (78.8) 44 (84.6)  1 (n=22) 21 (95.5) 21 (95.5)  2 (n=6) 4 (66.7) 3 (50.0)  3 (n=8) 7 (87.5) 7 (87.5)  4 (n=8) 1 (12.5) 4 (50.0)  p-value <0.001* 0.010* Modified Hardy classification  0   A (n=41) 32 (78.0) 34 (82.9)  1   A (n=14) 12 (85.7) 12 (85.7)  2   E (n=4) 3 (75.0) 3 (75.0)   A (n=5) 5 (100.0) 5 (100.0)  3   E (n=5) 2 (40.0) 2 (40.0)   A (n=1) 1 (100.0) 1 (100.0)   B (n=2) 2 (100.0) 2 (100.0)  4   E (n=1) 0 (0.0) 0 (0.0)   A (n=1) 1 (100.0) 1 (100.0)   D (n=1) 0 (0.0) 0 (0.0)   E (n=3) 1 (33.3) 3 (100.0)  p-value 0.10 0.06 Pathology result  Corticotropinoma (+) (n=71) 58 (81.7) 60 (84.5)  Corticotropinoma (-) (n=25) 16 (64.0) 19 (76.0)  p-value 0.07 0.30 Values are presented as number (%). Invasion : 0, sella normal; 1, sella focally expanded and tumor ≤10 mm; 2, sella enlarged and tumor ≥10 mm; 3, localized perforation of the sellar floor; 4, diffuse destruction of the sellar floor. Suprasellar extension : A, no suprasellar extension; B, anterior recesses of the third ventricle obliterated; D, intracranial (intradural) with anterior, middle, or middle fossa; E, into/beneath the cavernous sinus (extradural). * Statistically significant p-value Remission rates based on Knosp and Hardy classifications are presented in Table 2, respectively. The medium-term remission rates in Knosp group 4 were significantly lower than the rates in the other groups (Knosp 0 vs. 4, p<0.001; Knosp 1 vs. 4, p<0.001; Knosp 2 vs. 4, p=0.04; and Knosp 3 vs. 4, p=0.003). Additionally, the medium-term remission rate of tumors in Knosp group 2 was lower than that in Knosp group 1 (p=0.04). However, remission rates did not differ significantly among the other groups. Comparing invasive (Knosp 3 and 4) and noninvasive (Knosp 0, 1, and 2) tumors, remission rates within 3-6 months were 50% and 83% in the invasive and noninvasive groups, respectively. We further stratified cases with tumor size ≥20 mm (n=11) using Knosp classification; one case (9%) was Knosp 0, one case (9%) was Knosp 1, two cases (18%) were Knosp 3, and seven cases (64%) were Knosp 4 tumors. For ≥20 mm, all cases with Knosp 0, 1, and 3 tumors achieved remission within 3-6 months postoperatively, while none of the cases with Knosp 4 tumors had remission (p=0.01). All the cases with Knosp 0, 1, and 3 tumors sustained remission, and three cases with Knosp 4 tumor later achieved long-term remission (p=0.3). Of the cases that achieved long-term remission, two underwent STRS, and one had medical therapy with additional STRS. Of the 96 tissue specimens obtained during ETSS, 71 (74%) stained positive for ACTH and were histologically identified as corticotropic adenomas, while 25 (26%) were negative. Remission rates based on the pathology results are compared in Table 2. Of the lesions with conclusive findings on MRI (≥6 mm lesions), 89% (n=49) were pathologically confirmed as corticotropinomas, whereas 54% (n=22) of those with inconclusive MRI f indings were pathologically conf irmed (p<0.001). Among the lesions that showed negative results for both conclusive MRI findings (≤5 mm) and pathologic confirmation (negative for a corticotropinoma) (n=19), 12 (63%) showed remission at 3-6 months and 14 (74%) showed remission during long-term follow-up. During the exploration of the cavernous sinus in one patient (1%), postoperative lateral gaze paralysis of the eye developed due to right abducens nerve palsy. The patient was treated with anti-inflammatory doses of steroids, and the symptom completely resolved within 1 month. In three other patients (3%), severe epistaxis was observed in the postoperative period, 1 to 3 weeks after surgery. Nasal packing was applied for 3 days. Additionally, three patients (3%) experienced postoperative rhinorrhea. To address this issue, a reconstruction of the skull base was performed using fat tissue harvested from the leg, fascia lata graft, and tissue adhesive material. These patients were monitored with a lumbar drain for 1 week. Among the patients who developed rhinorrhea, one patient also developed meningitis and received intravenous antibiotic therapy for about 3 weeks and, the situation compeletly resolved during follow-up. The postoperative complications are summarized in Table 3. Comparison of various characteristics of the cases with and without medium and long-term remission are presented in Table 3, respectively. Table 3. Comparison of cases with and without remission, postoperative complications 3−6-month remission Long-term remission Number of cases (n=96) Remission (+) (n=74) Remission (-) (n=22) p-value Remission (+) (n=79) Remission (-) (n=17) p-value Operation 0.500 0.08  First time 62 (83.8) 17 (77.3) 66 (83.5) 13 (76.5)  Re-operation 12 (16.2) 5 (22.7) 13 (16.5) 4 (23.5) Tumor characteristics 0.003* 0.20  MST   Grade 1 31 (42.0) 10 (45.0) 33 (41.8) 8 (47.1)   Grade 2 22 (30.0) 2 (9.0) 22 (27.8) 2 (11.8)   Grade 3 17 (23.0) 3 (14.0) 17 (21.5) 3 (17.6)   Grade 4 4 (5.0) 7 (32.0) 7 (8.9) 4 (23.5)  Knosp classification <0.001* 0.01*   0 41 (56.0) 11 (50.0) 44 (55.5) 9 (53.0)   1 21 (28.0) 1 (4.5) 21 (26.5) 2 (12.0)   2 4 (5.0) 2 (9.0) 3 (4.0) 1 (6.0)   3 7 (10.0) 1 (4.5) 7 (9.0) 1 (6.0)   4 1 (1.0) 7 (32.0) 4 (5.0) 4 (23.0)  Hardy classification 0.09 0.06   0A 32 (43.2) 9 (41.0) 34 (43.0) 7 (41.0)   1A 12 (16.2) 2 (9.0) 12 (15.0) 2 (12.0)   1E 3 (4.0) 1 (4.5) 3 (4.0) 1 (6.0)   2A 5 (6.7) 0 (0.0) 5 (6.0) 0 (0.0)   2E 2 (2.7) 3 (14.0) 2 (3.0) 3 (17.0)   3A 1 (1.4) 0 (0.0) 1 (1.0) 0 (0.0)   3B 2 (2.7) 0 (0.0) 2 (3.0) 0 (0.0)   3E 0 (0.0) 1 (4.5) 0 (0.0) 1 (6.0)   4A 1 (1.4) 0 (0.0) 1 (1.0) 0 (0.0)   4D 0 (0.0) 1 (4.5) 0 (0.0) 1 (6.0)   4E 1 (1.4) 2 (9.0) 3 (4.0) 0 (0.0)   NA 15 (20.3) 3 (13.5) 16 (20.0) 2 (12.0) Postoperative  Complication 0.900 0.30   (+) 10 (13.5) 3 (13.6) 12 (15.2) 1 (5.9)   (-) 64 (86.5) 19 (86.4) 67 (84.8) 16 (94.1)  Pathologic diagnosis 0.070 0.30   Corticotropinoma 58 (78.4) 13 (59.1) 60 (75.9) 11 (64.7)   Negative 16 (21.6) 9 (40.9) 19 (24.1) 6 (35.3)  Remission during long-term F/U <0.001*   (+) 72 (97.3) 7 (31.8)   (-) 2 (2.7) 15 (68.2)  Residual tumor 0.001*   (+) 3 (3.8) 5 (29.4)   (-) 76 (96.2) 12 (70.6)  Remission during long-term F/U <0.001*   (+) 72 (91.1) 2 (11.8)   (-) 7 (8.9) 15 (88.2) Postoperative complication  DI-temporary 4 (4.2)  DI-permanent 4 (4.2)  Meningitis 1 (1.0)  CSF leak 3 (3.1)  Epistaxis 3 (3.1)  Cranial nerve palsy, transient 1 (1.0) Hypopituitarism 4 (4.2)  Hypocortisolism 2 (2.1)  Hypothyroidisim 2 (2.1) Values are presented as number (%). *Statistically significant p-values. MST : maximum size of tumor, NA : not available, F/U : follow up, DI : diabetes insipidus, CSF : cerebrospinal fluid Go to : DISCUSSION This study reported an overall postoperative 3-6 month remission rate of 77% and a long-term remission rate of 82% after 3 years of follow-up. The initial and long-term remission rates after first operations were 78% and 82%, respectively, with a recurrence rate of 2.5% over a follow-up period of 3-3.5 years. Additionally, our findings revealed that tumor size >2 cm and extended tumor invasion of the cavernous sinus (Knosp 4) might be associated with lower postoperative remission rates. Patients who showed remission within 3-6 months showed higher rates of long-term remission than those in patients without initial remission. Pituitary surgery is the first-line treatment modality for CD. ETSS is a safe and less invasive method for treating pituitary adenomas; therefore, it has been increasingly preferred in CD [5,15]. However, the postsurgical outcomes in patients with CD have shown variable remission and recurrence rates [2,4,9,16,17,21,30]. These discrepancies may be attributable to differences in population and number of cases involved in the studies, tumor characteristics, criteria for remission and recurrence used by the centers, laboratory parameters, time of evaluation and followup durations, surgical and imaging techniques used by different centers, and neurosurgical expertise. In this study, we present the medium- and long-term postoperative results of 96 ETSS procedures performed in 87 patients. The medium-term results (obtained 3-6 months postoperation) were preferred to immediate results since a subset of cases may show delayed remission, and immediate postoperative results could be misleading in almost 6% of cases [37]. The overall medium-term remission rate was 77%, consistent with the results published by Serban et al. [34], who reported an overall remission rate of 77% 2 months postoperation. A larger series of 1106 cases reported an immediate remission rate of 72.5% within 7 days postoperation; however, this rate decreased to 67% after delayed remission rates and recurrences 56 months postoperation were considered [12]. The long-term remission rate obtained over a median period of 3 years was 82% in our series. The increased long-term remission rate was attributed to reoperations, additional medical therapies, and the use of STRS in those who did not show remission initially. Of the 96 procedures, 79 were performed for the first time. The medium-term remission rate after first operations was 78%. Recent studies have reported remission rates of 74-82% after first operations [12,34]. The recurrence rates reported previously varied between 3% and 66% [5,12,34]. However, the duration of follow-up differed among the studies. Dai et al. [12] and Brady et al. [5] reported recurrence rates of 12% and 3%, respectively, after a follow-up period of 2 years. In contrast, Serban et al. [34] reported a recurrence rate of 17% after a longer followup duration of 6 years. In this series, after a median follow-up period of 3 years, the overall recurrence rate was 2.5%. Residual tumors were observed in five cases (6%), and the reoperation rate after the first operation was 13%. Including the eight patients admitted for reoperation after having undergone their first surgery in another center, 17 cases involved reoperations in our center. Of these cases, 71% (n=12) showed remission between 3-6 months postoperation, while none showed recurrence; thus, the long-term remission rate was 77%. Residual tumors were detected in three cases (18%), and the disease persisted in four (24%) of these 17 reoperated cases. Previous studies have reported remission rates of 22-75% after repeated surgery in CD [5,12,34,38]. Although the success rates after reoperations were lower than those in first-time operations in some studies [38], the remission rates after the first and reoperations did not differ significantly in our study. Tumor size has been reported to contribute to the success of transsphenoidal surgery [12,34], with microadenomas showing a higher success rate after surgery [5,12,34]. Our remission rates for micro- and macroadenomas were similar to those reported by Dai et al. [12] : 81% for adenomas <10 mm and 68% for adenomas ≥10 mm. However, the statistical significance of our study differed from that in the series presented by Dai et al. [12] (p=0.2 vs. p=0.002). This may be due to the large difference in the number of cases included in the two studies and the differences in size scales for tumors ≥10 mm. In our series, when the tumors were stratified further by the tumor size, the medium-term remission rate further decreased to 36% for tumors ≥20 mm in size, although the remission rates for other groups <20 mm were all above 75% (p=0.003). Sharifi et al. [35] classified pituitary MRI scans in CD showing a tumor size <6 mm as “inconclusive” because incidentalomas are frequent among tumors in this size range, and this size is not indicative of CD. Previously published series reported that the rate of inconclusive MRI scans in CD was 36-64%, and the remission rates varied between 50% and 71% for those with an inconclusive MRI scan [10,24,27,32,33]. In our series, 54% of the preoperative MRI scans were inconclusive. In the series presented by Sharifi et al. [35] and some other series [8,12,32,36], no significant difference was observed between the remission rates of CD cases with and without a conclusive MRI.This finding is controversial since other studies showed decreased remission rates with preoperative inconclusive MRIs [13,40]. Similar to the results reported by Sharifi et al. [35], we did not find a statistically significant difference between the remission rates of tumors <6 mm and those between 6-20 mm. However, a significant difference was observed between tumors <6 mm and those ≥20 mm. Residual tumors were more frequent after operating tumors >20 mm compared to those <20 mm, but the number of reoperations did not differ among the groups. Additionally, tumors >20 mm were primarily Knosp 4 (64%), probably contributing to lower remission rates in this group. Interestingly, two Knosp 3 cases had postoperative remission within 3-6 months without additional intervention. In these two cases, the surgical team explored the cavernous sinus and could resect the tumor. However, complete excision was not feasible with Knosp 4 tumors, where there is a complete encasement of the intracavernous internal carotid artery. Thus, a tumor size of 20 mm may be supportive data in predicting non-remission in the presence of complete cavernous sinus infiltration. Cavernous sinus invasion, determined by the Knosp classification, and sellar invasion and/or suprasellar extension, determined by the Hardy-Wilson classification, indicate the radiologic status of local invasion in cases of pituitary tumors [20,22,39]. Invasion to surrounding structures and tissues may be a limiting factor for postoperative remission of pituitary tumors. In the series presented by Dai et al. [12], remission rates of corticotropinomas with Knosp grade 4 (definitive cavernous sinus invasion) dropped to 53% from a remission rate of 77% for corticotropinomas with less likely or no cavernous sinus invasion (p<0.001). Similarly, our results showed that both medium- and long-term remission rates for Knosp grade 4 tumors decreased to 13% and 50%, respectively, and were lower than the remission rates in other grades (p<0.001 and p=0.01, respectively). While remission rates in Knosp group 3 were not inferior to noninvasive tumors, remission rates in Knosp group 4 were lower than all the other groups. In this regard, the extent of invasion may be more determinative. In contrast, in our series, the modified Hardy classification did not show a significant effect on postoperative remission rates in medium- and long-term follow-up assessments. Araujo-Castro et al. [3] had previously shown that for pituitary adenomas, the Hardy-Wilson classification lacked utility in predicting postoperative remission compared to the Knosp classification. The difference in the utility of these classifications for predicting postoperative remission may be due to differences in accessing tissues during surgery. In the present series, 74% (n=71) of tissues were histologically proven to be corticotropinomas, while 26% (n=25) did not show histologic confirmation. Medium- and long-term remission rates did not differ between histologically proven and unproven CD cases (medium-term remission rates, 82% vs. 64%, p=0.07; long-term remission rates, 85% vs. 76%, p=0.3). A conclusive finding of an adenoma on MRI increased the rate of histologic proof of corticotropinoma in our series, implying that adenomas showing a ≥6-mm lesion on MRI more frequently stained positive for ACTH. In previous studies 12-53% of CD did not have postoperative pathologic identification and the rate increased in those with a preoperative inconclusive MRI [25,31,38]. However, this did not have a significant influence on our remission rates. The remission rates did not decrease even for CD cases that were not conclusively detected on MRI and could not be histologically proven. On the other hand, in previous studies, ACTH positivity was higher, and the lack of proof for a corticotropinoma decreased the remission rates [1,12,31,32,34]. The higher remission rates despite reduced localization with MRI and/or lower rates of histologic confirmation in our series may be explained by the successful preoperative IPSS lateralization, surgical exploration, and hemi-hypophysectomy procedure. Furthermore, tumor tissues might have been aspirated along with blood and other materials through the suction tube, potentially resulting in less histological confirmation despite postoperative remission of CD. Additionally, tumor tissues might have been aspirated along with blood and other materials through the suction tube, potentially resulting in less histological confirmation despite postoperative remission of CD. The total rate of complications in this series was 20%, and the most frequent complication was diabetes insipidus (DI; 8%, both permanent and temporary). The incidence of hypopituitarism was relatively lower (4%), mainly involving hypocortisolism and hypothyroidism. Recent studies have shown postoperative DI rates of 25-66% and hypothyroidism rates of 11-23% [5,34]. Although our neurosurgical team was experienced in conducting pituitary surgeries, other factors may have resulted in these differences. Since not all the cases were postoperatively followed in our center, with some patients lost to follow-up, there may be missing data. Comparing cases with and without remission in the medium term, cases of remission frequently involved adenomas >20 mm and less cavernous sinus invasion. Additionally, cases that achieved medium-term remission showed long-term remission more frequently. In the long term, those showing remission had less cavernous sinus invasion and residual tumors compared to those without remission. Therefore, we may conclude that a tumor size of 20 mm may predict medium-term remission, while the absence of/less cavernous sinus invasion, early remission, and absence of residual tumor may predict long-term remission. This study had limitations. First, the retrospective nature of the study and the limited number of cases, which was inevitable due to the low incidence of CD, may have distorted our results. Although the same neurosurgical team operated on all patients, they were followed up pre and postoperatively at different endocrinology centers, causing difficulty in obtaining the full postoperative data of certain cases. Lastly, some patients recently underwent ETSS; thus, they had a shorter follow-up period. However, we intend to present the longer-term outcomes of all patients in a separate study. Although ETSS is the first-line treatment for CD, previous studies on the use of ETSS for CD have reported a wide range of remission and recurrence rates, which can be primarily attributed to differences in the surgical experience levels among centers. This trend highlights the need for additional data from experienced centers to resolve the discrepancies in the existing data. Therefore, we present medium- and long-term follow-up data from 96 operations for CD conducted in a center with a high level of experience for ETSS. We believe our study makes a significant contribution to the literature because the findings reconfirm the usefulness of ETSS for the treatment of CD and highlight the importance of the size of the adenoma and presence/absence of cavernous sinus invasion on preoperative MRI in predicting long-term remission postoperatively. Go to : CONCLUSION ETSS is a safe and effective method for the treatment of CD. Some characteristics of adenomas, such as size, cavernous sinus invasion, and postoperative residue, may predict long-term remission. A tumor size of 2 cm may be a supporting criterion for predicting remission status in the presence of complete cavernous sinus infiltration. Further studies with larger patient populations are necessary to support this finding. Go to : Notes Conflicts of interest No potential conflicts of interest relevant to this study exist. Informed consent Informed consent was obtained from all individual participants included in this study. Author contributions Conceptualization : BE, MB, EH; Data curation : EA, OH, DT, MM; Formal analysis : LŞP, DAB, DT, İÇ; Funding acquisition : OT, ÖG, DAB; Methodology : LŞP, İÇ, MM, ÖG; Project administration : BE, SÇ, EH; Visualization : EA, OT, OH; Writing - original draft : BE, MB, SÇ; Writing - review & editing : BE, EH Data sharing None Preprint None Go to : Acknowledgements This manuscript was edited by a certified English Proofreading Service (Editage). Go to : References 1. Acebes JJ, Martino J, Masuet C, Montanya E, Soler J : Early post-operative ACTH and cortisol as predictors of remission in Cushing’s disease. Acta Neurochir (Wien) 149 : 471-477; discussion 477-479, 2007 2. Aranda G, Enseñat J, Mora M, Puig-Domingo M, Martínez de Osaba MJ, Casals G, et al : Long-term remission and recurrence rate in a cohort of Cushing’s disease: the need for long-term follow-up. Pituitary 18 : 142-149, 2015 3. 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Nieman LK, Biller BM, Findling JW, Newell-Price J, Savage MO, Stewart PM, et al : The diagnosis of Cushing’s syndrome: an Endocrine Society clinical practice guideline. J Clin Endocrinol Metab 93 : 1526-1540, 2008 29. Oldfield EH, Doppman JL, Nieman LK, Chrousos GP, Miller DL, Katz DA, et al : Petrosal sinus sampling with and without corticotropin-releasing hormone for the differential diagnosis of Cushing’s syndrome. N Engl J Med 325 : 897-905, 1991 30. Petersenn S, Beckers A, Ferone D, van der Lely A, Bollerslev J, Boscaro M, et al : Therapy of endocrine disease: outcomes in patients with Cushing’s disease undergoing transsphenoidal surgery: systematic review assessing criteria used to define remission and recurrence. Eur J Endocrinol 172 : R227-R239, 2015 31. Prevedello DM, Pouratian N, Sherman J, Jane JA Jr, Vance ML, Lopes MB, et al : Management of Cushing’s disease: outcome in patients with microadenoma detected on pituitary magnetic resonance imaging. J Neurosurg 109 : 751-759, 2008 32. Salenave S, Gatta B, Pecheur S, San-Galli F, Visot A, Lasjaunias P, et al : Pituitary magnetic resonance imaging findings do not influence surgical outcome in adrenocorticotropin-secreting microadenomas. J Clin Endocrinol Metab 89 : 3371-3376, 2004 33. Semple PL, Laws ER Jr : Complications in a contemporary series of patients who underwent transsphenoidal surgery for Cushing’s disease. J Neurosurg 91 : 175-179, 1999 34. Serban AL, Del Sindaco G, Sala E, Carosi G, Indirli R, Rodari G, et al : Determinants of outcome of transsphenoidal surgery for Cushing disease in a single-centre series. J Endocrinol Invest 43 : 631-639, 2020 35. Sharifi G, Amin AA, Sabahi M, Echeverry NB, Dilmaghani NA, Mousavinejad SA, et al : MRI-negative Cushing’s disease: management strategy and outcomes in 15 cases utilizing a pure endoscopic endonasal approach. BMC Endocr Disord 22 : 154, 2022 36. 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World Neurosurg 77 : 525-532, 2012 From https://jkns.or.kr/journal/view.php?doi=10.3340/jkns.2023.0100

YOU’RE INVITED! Webinar on Dr. Theodore Friedman’s update on medical treatment for Cushing’s disease In this informative webinar, Dr. Friedman will discuss What medicines to use to treat Cushing’s disease Side effects and timing of the medicines The use of ketoconazole for a medication trial before surgery Longer-term treatment for Cushing’s How to determine when a patient should go to surgery Sunday • March 31 • 6 PM PDT here to join the meeting or https://us02web.zoom.us/j/4209687343?pwd=amw4UzJLRDhBRXk1cS9ITU02V1pEQT09&omn=88672684111 OR +16699006833,,4209687343#,,,,*111116# US (San Jose) OR Join on Facebook Live https://www.facebook.com/goodhormonehealth Slides will be available on the day of the talk here. There will be plenty of time for questions using the chat button. For more information, email us at mail@goodhormonehealth.com

Cushing’s syndrome (CS) secondary to adrenocorticotropic hormone (ACTH) producing tumours is a severe condition with a challenging diagnosis. Ectopic ACTH-secretion often involves neuroendocrine tumours (NET) in the respiratory tract. ACTH-secreting small intestine neuro-endocrine tumours (siNET) are extremely rare entities barely reported in literature. This review is illustrated by the case of a 75-year old woman with fulminant ectopic CS caused by a ACTH-secreting metastatic siNET. Severe hypokalemia, fluid retention and refractory hypertension were the presenting symptoms. Basal and dynamic laboratory studies were diagnostic for ACTH-dependent CS. Extensive imaging studies of the pituitary and thorax-abdomen areas were normal, while [68Ga]Ga-DOTATATE PET-CT revealed increased small intestine uptake in the left iliac fossa. The hypercortisolism was well controlled with somatostatin analogues, after which a debulking resection of the tumour was performed. Pathological investigation confirmed a well-differentiated NET with sporadic ACTH immunostaining and post-operative treatment with somatostatin analogues was continued with favourable disease control. © Acta Gastro-Enterologica Belgica. ABOUT THE CONTRIBUTORS B alliet, c severi, t veekmans, j cuypers, h topal, c m deroose, t roskams, m bex, j dekervel B Alliet Department of Gastroenterology, UZ Leuven, Leuven, Belgium. C Severi Department of Gastroenterology, ZOL, Genk, Belgium. T Veekmans Department of Pathology, UZ Leuven, Leuven, Belgium. J Cuypers Department of Endocrinology, AZ Turnhout, Turnhout, Belgium. H Topal Department of Abdominal Surgery, UZ Leuven, Leuven, Belgium. C M Deroose Department of Nuclear Medicine, UZ Leuven, Leuven, Belgium. T Roskams Department of Pathology, UZ Leuven, Leuven, Belgium. M Bex Department of Endocrinology, UZ Leuven, Leuven, Belgium. J Dekervel Department of Gastroenterology – Digestive Oncology, UZ Leuven, Leuven, Belgium. From https://www.physiciansweekly.com/fulminant-ectopic-cushings-syndrome-caused-by-metastatic-small-intestine-neuroendocrine-tumour-a-case-report-and-review-of-the-literature/

Highlights Phaeochromocytoma with ectopic ACTH secretion. Its clinical presentation is varied, and diagnosis is challenging. Ectopic ACTH secretion from a phaeochromocytoma can rapidly progress to severe Cushing’s syndrome. Removal of the primary tumour often leads to full recovery. Abstract Introduction The occurrence of hypercortisolism resulting from adrenocorticotropic hormone (ACTH)-secreting pheochromocytoma is exceedingly uncommon, with limited documented instances thus far. Presentation of case We present a case of ectopic ACTH-secreting pheochromocytoma in a patient who suffered from severe metabolic disorders. Our clinical case outlines the diagnostic history, preoperative correction of the patient's metabolic disturbances and surgical strategy for management of a rare ectopic ACTH producing pheochromocytoma. Discussion Ectopic adrenocorticotropic hormone-secreting pheochromocytoma displays multifaceted clinical features and requires prompt diagnosis and multidisciplinary management in order to overcome the related severe clinical derangements. Conclusion The combination of biochemical and hormonal testing and imaging procedures is mandatory for the diagnosis of ectopic ACTH secretion, and in the presence of an adrenal mass, the possibility of an ACTH-secreting pheochromocytoma should be taken into account. Keywords Hypokalemia Adrenal gland Pheochromocytoma Ectopic cushing's syndrome Cushing's syndrome 1. Introduction Neuroendocrine tumors such as Pheochromocytoma and paraganglioma (PPGL) are an uncommon occurrence. The prevalence of PPGL has been estimated to be between (2–8)/1 million, with a population rate of 1:2500–1:6500 [1], and it is associated with symptoms such as headache, irregular heartbeats, profuse sweating, high blood pressure, nausea, vomiting, nervousness, irritability, and a sense of imminent mortality [2]. Hypercortisolism is also a rare disorder with an incidence of 5/1 million, <10 % of patients with hypercortisolism are caused by ectopic secretion of ACTH [3], and these are most commonly seen in APUD tumors such as small cell bronchopulmonary carcinoma, pancreatic islet carcinoma, medullary thyroid carcinoma, pheochromocytoma, and melanoma [4]. Tumors that secrete both ACTH and catecholamines are much rarer. Here, we present a case of ectopic ACTH-secreting pheochromocytoma with severe metabolic disorders. The case report is compliant with SCARE Guidelines [5]. 2. Case report The patient is a 46-year-old male who presented to our hospital with recurrent symptoms of pheochromocytoma. He reported that he experienced unexplained symptoms such as panic attacks, headache, sweating, nausea, vomiting, and a feeling of imminent death, which could be alleviated by rest. His blood pressure was around 160–220/110–120 mmHg, and he was taking oral antihypertensive drugs regularly, with poor control of his blood pressure. The patient was admitted with a body temperature of 36.7 °C, heart rate of 130 beats/min, respiratory rate of 20 cycles per minute, blood pressure of 138/88 mmHg, height of 175 cm, weight of 67 kg, Body Mass Index (BMI): 21.88, normal physical examination, emaciated body type, thin subcutaneous fat, self-reported weight loss of 20 kg within 10 months, and history of diabetes mellitus of >1 year. Laboratory tests showed that the blood potassium levels were within the normal range, while the blood sugar and beta-hydroxybutyrate levels were elevated (Table 1). Hormonal analysis showed plasma levels of free catecholamine and its metabolites were much higher than normal, in addition to a severe excess of cortisol secretion with circadian rhythm disorders and elevated serum ACTH (Table 2). Small dose dexamethasone suppression test (1 mg) yielded cortisol levels of over 1750 nmol/L (negative: no decrease in blood cortisol), thus confirming the presence of ACTH-dependent hypercortisolism. The results of electrocardiogram, chest computerized tomography (CT), cardiac ultrasound and thyroid ultrasound showed no obvious abnormality. Enhanced CT of the adrenal glands (Fig. 1) revealed the presence of a right adrenal tumor measuring approximately 5.3 ∗ 4.7 cm. Despite undergoing cranial MRI, no pituitary lesion was detected, thereby ruling out the possibility of Cushing's disease. The patient was further considered for possible ectopic ACTH syndrome and suspected ectopic ACTH-secreting pheochromocytoma. Table 1. Laboratory test results. Laboratory test Result Reference value Unit White blood cell 17.03 3.5–9.5 109/L Red blood cell 5.06 3.8–5.1 1012/L Hemoglobin 147 115–150 g/L Platelets 206 125–350 109/L Glucose 12.13 3.9–6.1 mmol/L β-Hydroxybutyric acid 8.680 0–0.30 mmol/L Creatinine 55.30 40–105 umol/L Calcium 2.47 2.2–2.7 mmol/L Phosphate 1.26 0.85–1.51 mmol/L Potassium 3.66 3.5–5.5 mmol/L Sodium 147.1 137–147 mmol/L Table 2. The patient's adrenal hormone results Empty Cell Preoperative Postoperative Reference value Unit Norepinephrine, free 11,900 118 217–1109 pg/ml Adrenaline, free 3940 <24 <95 pg/ml Dopamine 207 <18 <20 pg/ml Methoxy norepinephrine 4130 87.80 <145 pg/ml Methoxy adrenaline 1850 <12 <62 pg/ml Adrenocorticotropic hormone (8:00) 544 10.60 7.2–63.3 pg/ml Cortisol (8:00) >1750 246.00 166–507 nmol/L Adrenocorticotropic hormone (16:00) 647 33.50 – pg/ml Cortisol (16:00) >1750 536.00 73.8–291 nmol/L Adrenocorticotropic hormone (00:00) 566 – – pg/ml Cortisol (00:00) >1750 – nmol/L Renin 2.82 3.10 2.4–32.8 pg/ml Aldosterone 81.51 73.56 16–160 pg/ml Aldosterone/renin concentration ratio 28.90 23.73 0–25 Download : Download high-res image (184KB) Download : Download full-size image Fig. 1. Adrenal CT showed a 53 ∗ 47 mm mass in the right adrenal gland. In response to the patient's pheochromocytoma symptoms and improve preoperative preparation, we used α-blocker (Phenoxybenzamine 20 mg q8h) to lower blood pressure and increase blood volume, antihypertensive medication (nifedipine 30 mg q12h, olmesartan tablets 20 mg q12h) to assist in lowering blood pressure, and β-blocker (metoprolol 47.5 mg q12h) to control the heart rate. On the 4th day in hospital, the patient was lethargic and had weak limbs. Urgent blood workup showed severe hypokalemia (2.85 mmol/L) as well as hyperglycemia (10.26 mmol/L). Patient was transferred to intensive care to correct intractable hypokalemia and diabetic ketoacidosis. After the patient was transferred to ICU, a deep vein cannulation was performed with intravenous potassium chloride supplementation, and the patient's blood potassium was maintained at normal levels prior to surgery through a large amount of potassium supplementation (Fig. 2A). For diabetic ketoacidosis, insulin administration, rehydration, ketone elimination and other treatments were given and the amount of access was recorded, and it was found that the patient was polyuric, with the highest urine volume of 21,800 ml in a single day (Fig. 2B), and the amount of urine did not decrease by taking oral desmopressin tablets 0.1 mg bid. Download : Download high-res image (255KB) Download : Download full-size image Fig. 2. Changes in blood potassium and urine volume during the patient's hospitalization. A: Blood potassium level. B: Daily urine vlume. Eventually, the patient underwent laproscopic right adrenal tumor resection. Intraoperative changes in blood pressure and heart rate are shown in Fig. 3. On day 1 after surgery, the morning (8:00) ACTH level was 10.60 pg/ml, antihypertensive medications were discontinued, and his blood pressure was 100–120/60–90 mmHg. The patient's daily urine output and blood glucose gradually returned to normal levels after surgery. Pathology (Fig. 4😞 Adrenal pheochromocytoma with ACTH immunopositive staining, cellular heterogeneity was unremarkable, nuclear schizophrenic images were rare, no pericytes, choroidal invasion and necrosis were seen. The patient was discharged from the clinic in a satisfactory condition with adrenal insufficiency compensated by daily intake of Prednisone Acetate Tablets (20 mg), discontinued 6 months after surgery. No signs of recurrence were noted upon frequent follow-up examinations. Download : Download high-res image (295KB) Download : Download full-size image Fig. 3. Changes in patient's intraoperative blood pressure and heart rate. Download : Download high-res image (313KB) Download : Download full-size image Fig. 4. Immunohistochemistry. A: hematoxylin and eosin staining B: ACTH. 3. Discussion We share the management of a patient with ectopic ACTH-secreting pheochromocytoma with severe metabolic disturbances, where, in addition to the rare etiology, perioperative management of the clinical complications of catecholamines and hypercortisolism is very challenging [6]. Patients suffering from ectopic ACTH syndrome caused by pheochromocytoma commonly exhibit severe Cushing's syndrome (CS), significant diabetes mellitus, hypertension, and hypokalemia [7]. Additionally, a retrospective study revealed that the majority of patients presented with Cushing's syndrome [8], whereas another report indicated that only 30 % of patients presented with typical Cushing's syndrome, but weight loss was frequently observed [9]. Our patient's recent weight loss may be attributed to the body's hypermetabolic condition caused by catecholamines. Recent reports claim that catecholamines directly reduce subcutaneous and visceral fat [10]. Rapid onset of cortisolism appears to be a feature of ACTH-secreting pheochromocytomas, because of the rapid onset of severe hypercortisolism, and our patient did not exhibit typical Cushing's symptoms [8]. Despite the absence of typical Cushing-like symptoms, this patient displayed persistent hypokalemia, a prevalent metabolic manifestation of Cushing's syndrome, particularly in ectopic ACTH syndrome, where hypokalemia is observed in 74 %–95 % of patients, in contrast to 10 % of patients with Cushing's disease [11]. Glucocorticoids have the ability to interact with aldosterone receptors, resulting in specific aldosterone-like reactions, while ectopic ACTH syndrome typically generates a higher amount of cortisol compared to Cushing's disease, ultimately causing more pronounced hypokalemia [7]. The perioperative management of patients with ACTH-secreting pheochromocytomas poses a significant challenge due to severe hypokalemia, and our patient's potassium levels remained within the normal range through extensive central venous potassium supplementation, without the need for cortisol secretion inhibition medications. The severity of hypertension in patients with ACTH-secreting pheochromocytomas seems to surpass that of patients with pheochromocytomas alone [12]. Hypercortisolism amplifies catecholamine-induced hypertension [13]. In the case of hypertension in patients with pheochromocytomas, alpha-blockers are favored for reducing blood pressure and enlarging blood volume, while for individuals whose blood pressure is not adequately managed with alpha-blockers alone, a combination of medications is recommended. Proper preoperative readiness for expanding the volume is crucial for a successful surgical procedure. Patients with ACTH-secreting pheochromocytoma have a greater prevalence and intensity of diabetes mellitus compared to those with pheochromocytoma alone [14], and our patient displayed a combination of severe diabetes mellitus and ketoacidosis. Insulin exhibits swift action and adaptable dosage, effectively averting hypoglycemia and effectively addressing hyperglycemia, rendering it the preferred medication for regulating blood glucose levels in individuals with ectopic CS [6]. Managing the water-electrolyte balance in this patient proved to be an arduous task, and the diabetes insipidus may have been one of the complications, with a maximum urine output of 21,800 ml in a single day (Fig. 2), and we hold the belief that the patient's diabetes insipidus is caused by a range of factors, such as hypokalemia, hypercortisolism, and severe diabetes mellitus. Indeed, hypokalemia may cause renal impairment, which reduces the ability to concentrate urine and lack of response to antidiuretic hormone (ADH), leading to nephrogenic diabetes insipidus [15]. Cortisol increases renal plasma flow and glomerular filtration rate, and also inhibits the secretion of antidiuretic hormone, leading to neurogenic diabetes insipidus [16]. For hypercortisolism, surgery to target the cause is the first-line treatment, and surgical removal of primary tumor may lead to 40 % radical treatment and 80 % complete remission of ectopic ACTH syndrome [17]. 4. Conclusion Preoperative diagnosis and management of pheochromocytoma, an extremely rare cause of ectopic ACTH syndrome, is challenging. Proper preoperative recognition of complications of both hypercortisolism and catecholamines excess is the key to prevent the morbidity and mortality of an ACTH-producing pheochromocytoma. If diagnosed successfully and managed intensively, they are curable. Consent Written informed consent was obtained from the patient for publication of this case report and accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal on request. Ethical approval Shandong Provincial Hospital Affiliated to Shandong First Medical University does not require ethical approval for publication of case reports. Signed consent from the patient has been received. Funding No funding was received for this research. Author contribution Shangjian Li: study concept or design, data collection, data analysis or interpretation, writing the paper Xudong Guo: study concept or design, data collection, data analysis or interpretation, writing the paper Hanbo Wang: study concept or design, data analysis or interpretation Ni Suo: study concept or design, data analysis or interpretation Xiuqing Mi: study concept,data collection Shaobo Jiang: study concept or design, data analysis or interpretation, writing the paper Guarantor Shangjian Li Xudong Guo Shaobo Jiang Conflict of interest statement All authors declare no conflict of interest. Acknowledgements None. References [1] A. Jain, R. Baracco, G. Kapur Pheochromocytoma and paraganglioma-an update on diagnosis, evaluation, and management Pediatr. Nephrol., 35 (2020), pp. 581-594 View article CrossRefView in ScopusGoogle Scholar [2] F.A. Farrugia, A. Charalampopoulos Pheochromocytoma Endocr. 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Abstract Context Patients with Cushing’s disease (CD) face challenges living with and receiving appropriate care for this rare, chronic condition. Even with successful treatment, many patients experience ongoing symptoms and impaired quality of life (QoL). Different perspectives and expectations between patients and healthcare providers (HCPs) may also impair well-being. Objective To examine differences in perspectives on living with CD between patients and HCPs, and to compare care goals and unmet needs. Design Memorial Sloan Kettering Pituitary Center established an annual pituitary symposium for pituitary patients and HCPs. Through anonymous pre-program surveys distributed at the 2020 and 2022 symposia, patients and HCPs answered questions related to their own sense, or perception of their patients’ sense, of hope, choice, and loneliness in the context of living with CD. Participants From 655 participants over two educational events, 46 patients with CD and 116 HCPs were included. Median age of both groups was 51 years. 78.3% of the patients were female vs. 53.0% of the HCPs. Results More patients than HCPs reported they had no choices in their treatment (21.7% vs. 0.9%, P < 0.001). More patients reported feeling alone living with CD than HCPs’ perception of such (60.9% vs. 45.5%, P = 0.08). The most common personal care goal concern for patients was ‘QoL/mental health,’ vs. ‘medical therapies/tumor control’ for HCPs. The most common CD unmet need reported by patients was ‘education/awareness’ vs. ‘medical therapies/tumor control’ for HCPs. Conclusions CD patients experience long term symptoms and impaired QoL which may in part be due to a perception of lack of effective treatment options and little hope for improvement. Communicating experiences and care goals may improve long term outcomes for CD patients. Introduction Patients with rare diseases face challenges receiving appropriate care. Cushing’s disease (CD), a condition associated with excess endogenous glucocorticoids due to an ACTH-secreting pituitary tumor, is a rare disease, occurring in 0.7 to 2.4 per million per year [1]. Patients with CD are at high risk for metabolic, cardiovascular, and psychiatric disease, in addition to long-term symptom burden and impaired quality of life (QoL), despite adequate treatment [1,2,3]. A critical aspect of effective patient care is communication and mutual understanding between healthcare provider (HCP) and patient. Patients with pituitary tumors experience significant anxiety associated with their diagnosis, in large part due to difficulties interacting with healthcare systems and limited communication of information [4]. Many pituitary patients express concern regarding the complexity of their care, and satisfaction improves with the delivery of more information by the HCP [4]. Patients with pituitary tumors, and CD specifically, require multidisciplinary care which necessitates effective communication in order to provide the best possible outcomes [5]. Similar to acromegaly patients [6], CD patients’ long-term well-being may be adversely affected by different perspectives and expectations between patients and HCPs, especially after treatment [7]. While HCPs primarily use biochemical data to define successful treatment, patients rely more on their symptoms and ability to regain normal functioning [7]. Despite achieving biochemical remission, CD patient perception of having persistent disease negatively impacts QoL [8]. In addition, 67.5% of Cushing’s syndrome patients report receiving insufficient information from their HCPs regarding the recovery experience after surgery despite the fact that all HCPs report providing this information [9]. Improved communication between HCPs and CD patients is vital to optimizing patients’ QoL and long term outcomes. Recently there has been a growing emphasis on the use of internet-based platforms for healthcare delivery and education [10]. With the goals of offering HCP and patient education and assessing pituitary patients’ needs, since 2019 the pituitary center at Memorial Sloan Kettering (MSK) has offered annual virtual educational programs for pituitary patients, caregivers, HCPs, and members of the pharmaceutical industry. For the current study, we gathered deidentified information from 2020 to 2022 MSK program participants on CD patients’ and HCPs’ attitudes about CD, related to their sense of hope, choice, and loneliness, through anonymous pre-program surveys. Our specific aims were to: (1) Assess differences in perspectives between patients’ and HCPs’ responses in the pre-program survey; (2) Compare patients’ and HCPs’ perceived care goals and unmet needs. Methods Educational program enrollment The MSK program was offered to patients with any type of pituitary tumor as well as HCPs, family members, caregivers, and members of industry. The role of the registrant as a patient, caregiver/family member, HCP, and/or member of industry was determined for all registrants of the virtual programs. Any patient with a pituitary tumor treated at our center and outside institutions, inclusive of patients at all points along their treatment journey, were invited to register for the virtual education program. HCPs, including endocrinologists, neurosurgeons, otolaryngologists, radiation oncologists, neurologists, ophthalmologists, neuro-oncologists, family medicine and internal medicine physicians, physicians in training and other allied health professionals who treat and manage patients with pituitary diseases were also invited to register. Invitations were sent through email to neuroendocrine experts and endocrinologists, patient support groups on social media, direct messaging to patients with pituitary tumors by their treating physicians and via patient databases, advertisements through endocrine societies, brochure/postcard mailing, and Eventbrite, a virtual platform for live events. Study participants Registrants from MSK virtual programs held on December 5, 2020, (n = 328) and April 9, 2022, (n = 327) were included in the pool of subjects, among which the qualifying participants were determined. Of the 655 total registrants from the 2020 and 2022 programs, 320 (48.9%) were patients or caregivers and 309 (47.2%) were HCPs (Fig. 1). Of the 147 providers (88 in 2020 and 59 in 2022) that attended and filled out a pre-program survey 31 were excluded from our analysis. Eight filled out surveys in both 2020 and 2022, 4 were members of industry, 3 did not fill out any responses, and 1 was not in the healthcare field. In addition, 12 providers had at least three fields missing in the survey and 3 had filled out two surveys for the same year, so they were also excluded. A total of 116 providers (72 from 2020 to 44 from 2022) were included in the analysis. Fig. 1 Enrollment flowchart Full size image Among the 320 pituitary patients who attended the programs (157 from 2020 to 163 from 2022), 53 identified as ‘patients with Cushing’s’ and submitted surveys (34 participants from 2020 to 19 from 2022). Seven patients were excluded from the 2022 surveys as they had also filled out surveys in 2020, leaving a final group of 46 patients who were included in the analysis. Virtual education programs For each program, there was a single day of live interactive programming, meaning that all participants attended at the same time. The programs were recorded and made available for several weeks as enduring material for registrants on an online website. After joint sessions in the morning, both programs consisted of two tracks in the afternoon: the ‘provider/clinical track’ and the ‘patient/caregiver track’. During the programs, an ongoing chat reeled through the virtual program which allowed patients to continually ask questions. Faculty experts answered these questions in written responses directly within the chat and/or in spoken responses during one of the live broadcasted Q&A sessions. Additionally, the programs both included panel discussions answering patient questions and moderated patient discussions with invited patient speakers. Study procedures Through anonymous pre-program surveys distributed at the 2020 and 2022 symposia, patients and HCPs answered questions related to their own sense, or perception of their patients’ sense, of hope, choice, and loneliness in the context of living with CD. This survey was developed by a multidisciplinary team and has been reported previously [11]. Demographic and clinical information was also assessed including year of diagnosis, prior treatments, and current medications (for patients) and specialty and practice type (for providers), as shown in Tables 1 and 2. Multiple-choice questions assessing patients’ attitudes toward their disease included possible answers of ‘I have no hope for improvement,’ ‘I have some hope for improvement,’ and ‘I have a lot of hope for improvement;’ and ‘I have no choice in my treatment,’ ‘I have some choices in my treatment,’ and ‘I have many choices in my treatment.’ Patients were also asked to respond ‘TRUE’ or ‘FALSE’ to the following statements: ‘I feel alone living with my Cushing’s,’ ‘Hearing the journeys of other patients helps me better understand my own,’ and ‘I feel anxious about my Cushing’s diagnosis.’ Table 1 Patient demographic data Full size table Table 2 Provider demographic data Full size table Multiple-choice questions assessing providers’ attitudes about their patients' Cushing’s included possible answers of ‘I have no hope for their improvement,’ ‘I have some hope for their improvement,’ and ‘I have a lot of hope for their improvement;’ and ‘my patients have no choice in their treatment,’ ‘my patients have some choices in their treatment,’ and ‘my patients have many choices in their treatment.’ Providers were also asked to respond ‘TRUE’ or ‘FALSE’ to the following statements: ‘my patients feel alone living with their Cushing’s,’ ‘Hearing the journeys of other patients helps will help my patients better understand their own,’ and ‘my patients feel anxious about their Cushing’s diagnosis.’ Additionally, patients were surveyed on care goals and unmet needs related to their treatment. Specifically, patients were asked, ‘What are the healthcare outcomes/goals that matter to you the most?’ and ‘What do you think are unmet needs for the diagnosis or treatment of your condition?’ The first question was intended to refer to the patient specifically, while the second question was meant to examine how the condition is treated in general. Survey responses were submitted as free text and subsequently grouped by the authors (AH and EBG) into nine different categories: (a) Quality of life (QoL)/Mental Health; (b) Medical Therapies/Tumor Control; (c) Education/Awareness; (d) Communications/Multidisciplinary Care; (e) Insurance/Access; (f) Fertility; (g) Controlling Comorbidities; (h) Support System and (i) none. Responses could receive multiple designations if applicable. AH coded the free text themes independently, then EBG reviewed each answer and corresponding grouping to confirm accuracy. If there was disagreement or confusion, coding from our prior work [11] was reviewed. HCPs were also surveyed on care goals and unmet needs related to their patient’s treatment. Providers were asked, ‘What are the healthcare outcomes/goals that matter to you the most?’ and ‘what do you think are unmet needs for the diagnosis or treatment of your patient’s condition?’ The first question was intended to refer to the provider and their goals related to Cushing’s, while the second question was meant to examine how the condition is treated in general. Survey responses were submitted as free text and subsequently grouped by the authors (AH and EBG) into nine different categories: (a) Quality of life (QoL)/Mental Health; (b) Medical Therapies/Tumor Control; (c) Education/Awareness; (d) Communications/Multidisciplinary Care; (e) Insurance/Access; (f) Fertility; (g) Controlling Comorbidities; (h) Support System and (i) none. Responses could receive multiple designations if applicable. Statistical analysis Descriptive statistics were presented as counts and percentages for categorical variables and as medians and interquartile range (IQR) for continuous variables. The Chi-square test or Fisher’s exact test was used to compare gender and survey responses between the CD patient group and the HCP group. All statistical tests were two-tailed, and a P-value of < 0.05 was considered statistically significant. SAS Software® (version 9.4; SAS Institute Inc., Cary, NC) was used for all analyses. Results Between the 2020 and 2022 events, there was combined representation from 25 different countries. A map and a full list of the countries is shown in Fig. 2. Fig. 2 Map of registrant locations. Locations (listed alphabetically): Argentina, Australia, Belgium, Brazil, Canada, Chile, China, Greece, Hong Kong, India, Israel, Jamaica, Latvia, Malaysia, Netherlands, New, Zealand, Oman, Peru, Philippines, Qatar, Romania, Saudi Arabia, Singapore, UK, US Full size image From a total of 655 participants over two educational events, 46 patients with CD and 116 HCP caring for CD patients were included in the analysis. The demographic data of the patients and HCPs are outlined in Tables 1 and 2, respectively. Median age of the patients and HCPs was 51 years. 78.3% of the CD group was female vs. 53.0% of the HCP group (P = 0.003). CD patients ranged from newly diagnosed to being diagnosed 33 years prior. The HCPs who filled out the pre-program surveys were in practice for a mean duration of 18.5 years, with a range from 1 to 54 years. As shown in Table 1, CD patients had a mean duration of suspected active disease prior to diagnosis of 5.26 years, as defined by onset of CD symptoms until diagnosis, and a mean duration of disease since diagnosis of 5.9 years. 42 (91%) had undergone surgical treatment of their Cushing’s. For those who underwent surgery, the mean number of surgeries was 1.17 (range 0–4). 20% had received pituitary radiation. Overall, 31% of patients were on medical therapy for Cushing’s. Metyrapone was the most used CD therapy (in 11%), followed by ketoconazole (in 9%). Of those requiring pituitary hormone replacement, 34.8% had one pituitary hormone deficiency and 21.7% had multiple hormone deficiencies. Thyroid hormone replacement (37%) and adrenal replacement (30%) were the most common. As shown in Table 2, the majority of the HCPs were endocrinologists (72%) followed by neurosurgeons (9%) and nurses (8%). There was a total of 9 different specialties represented by the provider group. 16% of the providers worked in private practice, 16% were hospital based, and 16% worked in ‘unspecified clinical care.’ 38% of the providers practice type was ‘unspecified.’ Based on the pre-program survey responses, we identified different attitudes between patients and HCPs in several domains. Table 3 depicts pre-program survey responses from CD patients and HCPs assessing their attitudes about CD. 21.7% of patients reported they had no choices in their treatment, compared to 0.9% of HCPs (P < 0.001). Almost all HCPs (99.1%) reported that CD patients had least some choice in their management. In addition, less than half (45.7%) of patients reported they had a lot of hope for improvement whereas 71.3% of HCPs had a lot of hope for their patients’ improvement. Surprisingly, fewer CD patients reported feeling anxious about their diagnosis compared to HCPs’ perceived patient anxiety (65.2% vs 94.6%, P < 0.001). However, more patients tended to feel more alone living with CD than HCPs’ perception of such (60.9% vs. 45.5%, P = 0.08). Both CD patients and HCPs agreed that hearing the journeys of other CD patients would help patients better understand their own disease (97.8% vs 100%). Table 3 Patient and provider attitudes by pre-program survey Full size table CD patients and HCPs were also surveyed on their personal care goals and unmet needs, results of which are shown in Figs. 3A, B and 4A, B. The most common personal care goal concern for patients was ‘QoL/mental health’ which was reported by 70%, followed by ‘controlling comorbidities’ (39%) and ‘medical therapies/tumor control’ (24%). HCPs prioritized the same three care goals as patients but ‘medical therapies/tumor control’ was the most common (44%). ‘Controlling comorbidities’ and ‘QoL/mental health’ were the second and third most often HCP reported care goals (31 and 22% respectively). ‘Education/awareness’ was the most common perceived CD unmet need by patients (59%). HCPs reported both ‘medical therapies/tumor control’ and ‘education/awareness’ to be the most common unmet needs (35 and 26%, respectively). Examples of patient and provider responses, and how they were coded, are shown in Supplemental Table 1. Fig. 3 A Care goals according to participants with Cushing’s who completed pre-program survey. This pie graph represents the free-text survey response from patients regarding their personal care goals as categorized by topic. B Care goals according to providers who completed pre-program survey. This pie graph represents the free-text survey response from providers regarding their personal care goals as categorized by topic Full size image Fig. 4 A Unmet needs for the field of Cushing’s disease according to participants with Cushing’s who completed pre-program survey. This pie graph represents the free-text survey response from patients regarding unmet needs in Cushing’s as categorized by topic. B Unmet needs for the field of Cushing’s disease according to providers who completed pre-program survey. This pie graph represents the free-text survey response from providers regarding unmet needs in Cushing’s as categorized by topic Full size image Discussion This study examined the differences between patients and HCP-reported perceptions of living with CD. We identified several differences in disease outlook between CD patients and HCPs. We found that more patients than HCPs reported they had no choices in their treatment. Furthermore, less than half of patients reported they had a lot of hope for improvement whereas most (71.3%) of HCPs had a lot of hope for their patients’ improvement. Interestingly, fewer CD patients reported feeling anxious about their diagnosis compared to HCPs’ perceived patient anxiety, although a higher percentage of patients reported feeling alone living with CD compared to the HCPs’ perception of patient loneliness. We also identified HCP and patient differences in reported personal care goals and perceived unmet needs in the field. The most common personal care goal concern for patients was ‘QoL/mental health,’ whereas it was ‘medical therapies/tumor control’ for HCPs. ‘Education/awareness’ was the most commonly perceived unmet need by patients, whereas it was ‘medical therapies/tumor control’ for HCPs. Our findings support prior work demonstrating a discrepancy between patients and HCPs regarding the need for improved multidisciplinary care [12]. 43% of patients listed ‘communication/multidisciplinary care’ as an unmet need in the field, compared to 3% of providers. Pituitary centers of excellence provide expert multidisciplinary care in the neuroendocrine, neurosurgical, and radiation oncology domains, but often lack expertise in mental and physical health domains salient for CD patients, who suffer from depression, anxiety, myopathy and joint pain. In order to offer comprehensive care, psychiatrists, psychologists, social workers, pain medicine experts, physical therapists, and nutritionists with expertise in CD should be included in the pituitary center multidisciplinary team [13]. Our findings suggest that pituitary centers of excellence should take into account the most important personal care goal reported by CD patients, which is Qol/mental health, and provide expert treatment in this domain. It is not surprising that Qol/mental health is the personal care goal most reported by CD patients. Prior assessment of acromegaly patients demonstrated the same finding: QoL/mental health was the most common personal care goal concern [11]. While surgical [14] and medical [15,16,17,18] treatment of Cushing’s improves QoL, QoL has been shown to remain impaired over time after treatment [19]. Several factors may contribute to long-term Qol impairments, including the presence of persistent disease, imperfect treatment modalities which themselves may be associated with burden and adverse side effects, and persistent comorbidities including depression, anxiety, fatigue, and overweight. Perception of disease status may also play a role in QoL. In surgically remitted CD patients, there may be discordance between biochemical remission and perceived disease status [8]. Specifically, this study found that of those with self-identified persistence of disease, 65% were in fact biochemically remitted. This group had lower QoL scores than the concordant group who self-identified as in remission with biochemical evidence of remission. CD patients’ outlook on their condition, including their perception of choices and hope for change, has not been previously well described, despite the fact that these perceptions likely inform long term Qol. Patient outlook may be a modifiable target that if addressed, could improve long term patient well-being and outcomes. Aside from continuing progress in the development of new therapies for CD patients which can offer patients more objective choices in their treatment, other modalities should be considered. Prior work has shown that virtual educational programs improve acromegaly patients’ hope for improvement, perception of having choices in their treatment, and sense of loneliness [11]. Educational programs have also been shown to result in improved physical activity and sleep, and reduced pain levels in CS patients [20]. More work is needed to develop effective education programming tailored for CD patients to provide the appropriate support that these patients need. Difference in HCP and patient disease perceptions may also play a role in Cushing’s patients’ quality of life and outcomes. Among a cohort of patients who underwent surgical resection for Cushing’s, 32.4% reported not receiving information from their doctors about the recovery experience, despite the fact that all physicians surveyed reported giving information about the recovery process [9]. Furthermore, 16.1% of patients in this cohort reported that not enough medical professionals were familiar with the symptoms of Cushing’s. Recovery time was also reported to be longer by patients than providers [9]. Similarly, discordance was found between acromegaly patients and HCPs regarding reported severity of symptoms, with patients more frequently reporting symptoms as severe compared to HCPs, and many patients reporting symptoms which were not reported by HCPs [6]. Improving communication between HCP and patients may positively affect CD patient outlook and QoL. We identified a similar disparity between CD patients and HCP regarding care goals and unmet needs. 70% of patients surveyed considered QoL/mental health to be a top care goal, but only 22% of provider shared this goal. 59% of patients reported education/awareness as an unmet need, compared to 26% of HCPs. These findings support data shown by Acre et al. in which Cushing’s patients report a lack of symptom recognition by their providers [9]. HCPs should be aware that their patients may have different treatment priorities. Our finding that more HCPs reported patient anxiety living with CD compared to patients themselves needs further exploration. This could reflect inadequate communication between HCP and patient, or skewed HCP perceptions of CD. This, and other findings in our study should be viewed in light of the small cohort, and as such, needs confirmation in larger cohorts and more in-depth symptom assessments. Additional limitations of our study include lack of paired patient-HCP responses as the HCPs included were not providing care for this specific CD cohort. Since this was a pituitary educational forum, likely most or all patients who identified as having Cushing’s had CD. However, our survey did not specify the type of surgery patients underwent or the etiology of their Cushing’s. Additionally, we used multidisciplinary team agreed upon measures and not validated assessments. Further work should consider validating a tool to assess patient-provider discordances. Our findings may also be confounded by selection bias, given that the patients participating in our virtual education programs are more likely to be under the care of experts in the field and may not represent the attitudes of all patients living with CD. Finally, the included HCPs were representatives from a range of specialties with different levels of experience taking care of patients with CD which may also affect their responses. Our findings highlight the importance of understanding CD patients’ outlook and perspective in their condition, and that they may differ from their HCP. More than half of CD patients did not have a lot of hope for improvement and reported feeling alone, and many patients felt they had no choices in their treatment. QOL/mental health was the most commonly reported care goal for patients, which was not the case for HCPs. Comprehensive multidisciplinary care for CD patients should include mental health professionals with expertise in CD. Regular open communication between HCPs and CD patients will help bridge perception differences and facilitate personalized care, which will ultimately improve long-term outcomes for CD patients. Data availability The data that support the findings of this study are available from the authors upon request. References Newell-Price J, Bertagna X, Grossman AB, Nieman LK (2006) Cushing’s syndrome. 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Endocrine 53:199–209. https://doi.org/10.1007/s12020-015-0737-0 Article CAS PubMed Google Scholar Download references Acknowledgements The authors would like to thank the HCP and patient participants who attended the events, the MSK faculty, invited speakers, Leslie Edwin of Cushing’s Support and Research Foundation, Amy Edouard and the MSK CME team, and Recordati Rare Diseases, Inc., Amryt Pharma (previously Chiasma, Inc.), Crinetics, Sparrow Pharmaceuticals, Corcept Therapeutics, and Xeris Biopharma (previously Strongbridge Biopharma) for providing educational grants for these educational activities. Funding This research was funded in part through the NIH/NCI Cancer Center Support Grant P30 CA008748. Author information Authors and Affiliations Division of Endocrinology, Department of Medicine, Weill Cornell Medicine, New York, NY, USA Amanda Halstrom Department of Epidemiology and Biostatistics, Memorial Sloan Kettering Cancer Center, New York, NY, USA I.-Hsin Lin Multidisciplinary Pituitary & Skull Base Tumor Center, Memorial Sloan Kettering Cancer Center, New York, NY, USA Andrew Lin, Marc Cohen, Viviane Tabar & Eliza B. Geer Department of Neurology, Memorial Sloan Kettering Cancer Center, New York, NY, USA Andrew Lin Department of Neurosurgery, Memorial Sloan Kettering Cancer Center, New York, NY, USA Andrew Lin, Marc Cohen & Eliza B. Geer Head and Neck Service, Department of Surgery, Memorial Sloan Kettering Cancer Center, New York, NY, USA Marc Cohen & Viviane Tabar Department of Medicine, Memorial Sloan Kettering Cancer Center, New York, NY, USA Eliza B. Geer Contributions A.H. and E.B.G. wrote the manuscript text and prepared the figures. All authors reviewed the manuscript. Corresponding author Correspondence to Eliza B. Geer. Ethics declarations Competing interests The authors declare no competing interests. Ethical approval As an educational quality initiative project using de-identified data, it was determined that our project did not constitute human subjects research and thus did not require IRB oversight. Additional information Publisher's Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Supplementary Information Below is the link to the electronic supplementary material. Supplementary file1 (DOCX 15 kb) Rights and permissions Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. Reprints and permissions From https://link.springer.com/article/10.1007/s11102-024-01381-4

Abstract Pituitary surgery, a critical intervention for various pituitary disorders, has sparked ongoing debates regarding the preference between endoscopic and microscopic transsphenoidal approaches. This systematic review delves into the outcomes associated with these techniques, taking into account the recent advancements in neurosurgery. The minimally invasive nature of endoscopy, providing improved visualization and reduced morbidity, stands in contrast to the well-established track record of the conventional microscopic method. Examining outcomes for disorders such as Cushing's disease and acromegaly, the review synthesizes evidence from Denmark, Bulgaria, and China. Noteworthy advantages of endoscopy encompass higher resection rates, shorter surgery durations, and fewer complications, endorsing its effectiveness in pituitary surgery. While emphasizing the necessity for prospective trials, the review concludes that endoscopic approaches consistently showcase favorable outcomes, influencing the ongoing discourse on the optimal surgical strategies for pituitary disorders. Introduction & Background Pituitary surgery is a critical intervention for various pituitary disorders, and the choice between endoscopic and microscopic transsphenoidal approaches has been a subject of ongoing debate within the medical community. This systematic review aims to explore and analyze the outcomes associated with endoscopic and microscopic transsphenoidal pituitary surgery. As advancements in surgical techniques continue to shape the field of neurosurgery, understanding the comparative effectiveness of these two approaches becomes imperative. The endoscopic approach, characterized by its minimally invasive nature, has gained popularity for pituitary surgery in recent years [1]. Proponents argue that it provides enhanced visualization, improved maneuverability, and reduced patient morbidity. On the other hand, traditional microscopic transsphenoidal surgery has been the conventional method for decades, known for its familiarity among surgeons and established track record [2]. Several studies have investigated the outcomes of these approaches in treating pituitary disorders, including but not limited to Cushing's disease, pituitary adenomas, and other tumors. For instance, a systematic review and meta-analysis by Chen et al. compared endoscopic and microscopic transsphenoidal surgery specifically for Cushing's disease, shedding light on the effectiveness of these approaches in managing this specific condition [3]. Moreover, Møller et al. reported promising results for endoscopic pituitary surgery based on the experiences of experienced microscopic pituitary surgeons, indicating a potential shift towards the adoption of the endoscopic technique [1]. Guo et al. conducted a meta-analysis comparing the effectiveness of microscopic and endoscopic surgery for treating pituitary disorders, contributing valuable insights into the overall efficacy of these approaches [4]. This review aims to contribute to the ongoing discourse on pituitary surgery by providing a comprehensive analysis of the outcomes associated with endoscopic versus microscopic transsphenoidal approaches. By synthesizing the existing evidence, we strive to offer valuable insights that can guide both clinicians and researchers in making informed decisions regarding the optimal surgical approach for pituitary disorders. Review Materials and methods This systematic review strictly adheres to the established Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines, employing a comprehensive approach to investigate the outcomes of endoscopic versus microscopic transsphenoidal pituitary surgery. The subsequent sections delineate the criteria for study inclusion, the search strategy utilized, and the methodology employed for data synthesis. Search Strategy We conducted a meticulous search across prominent electronic databases, including PubMed, Embase, and the Cochrane Library, to identify pertinent articles. Our search strategy comprised a combination of Medical Subject Headings (MeSH) terms and keywords related to pituitary surgery, encompassing both endoscopic and microscopic approaches. Boolean operators (AND, OR) were strategically employed to refine the search and identify studies meeting our predetermined inclusion criteria. The search string used for PubMed was ("Outcomes" OR "Treatment Outcome" OR "Surgical Outcome") AND ("Endoscopic Transsphenoidal Pituitary Surgery" OR "Endoscopic Pituitary Surgery" OR "Endoscopic Hypophysectomy") AND ("Microscopic Transsphenoidal Pituitary Surgery" OR "Microscopic Pituitary Surgery" OR "Microscopic Hypophysectomy" OR "Endoscopy"[Mesh] OR "Endoscopy, Surgical"[Mesh] OR "Transsphenoidal Hypophysectomy"[Mesh] OR "Microsurgery"[Mesh] OR "Microscopic Hypophysectomy"[Mesh]). Eligibility Criteria Stringent inclusion criteria were predefined to ensure the selection of high-quality and relevant studies. The included studies focused on investigating the outcomes of endoscopic versus microscopic transsphenoidal pituitary surgery. Only articles published in peer-reviewed journals within the timeframe from the inception of relevant databases until October 2023 were considered. Exclusion criteria encompassed studies on other interventions, those lacking sufficient data on surgical outcomes, and studies solely involving animal cells. Additionally, only studies in the English language with full-text availability were included, and gray literature was not considered eligible. Data Extraction and Synthesis Two independent reviewers meticulously screened titles and abstracts to identify potentially eligible studies. Subsequently, full-text articles were retrieved and evaluated for adherence to inclusion criteria. Discrepancies between reviewers were resolved through discussion and consultation with a third reviewer. Relevant data, including study design, patient characteristics, interventions, and surgical outcomes, were systematically extracted using a predefined data extraction form. Data Analysis A narrative synthesis approach was employed to summarize findings from included studies due to anticipated heterogeneity in study designs and outcome measures. Key themes and patterns related to the outcomes of endoscopic versus microscopic transsphenoidal pituitary surgery were identified and presented. Results Study Selection Process Following four database searches, 97 articles were initially identified. After eliminating eight duplicates, the titles and abstracts of the remaining 89 publications were evaluated. Subsequently, 17 potential studies underwent eligibility verification through a thorough examination of their full texts. Ultimately, three articles satisfied the inclusion criteria. No additional studies meeting the eligibility criteria were found during the examination of references in the selected articles. The entire process is visually depicted in the PRISMA flowchart (Figure 1). Figure 1: PRISMA flow diagram of the selection of studies for inclusion in the systematic review. PRISMA: Preferred Reporting Items for Systematic Reviews and Meta-Analyses Characteristics of Selected Studies Overall, three papers met the inclusion criteria. Two studies were randomized controlled trials (RCTs), one each from Bulgaria and China. One study was an observational study from Denmark. The main findings and characteristics of the included studies are mentioned in the following tables (Table 1 and Table 2). Author Year Country Study type Sample size No. of participants in the endoscopic group No. of participants in the microscopic group Main findings Møller et al. [1] 2020 Denmark Observational study 240 45 195 The study comparing endoscopic and microscopic transsphenoidal pituitary surgery revealed that the endoscopic technique exhibited advantages, achieving a higher rate of gross total resection (39% vs. 22%) and shorter surgery duration (86 minutes vs. 106 minutes). Complications within 30 days were lower with the endoscope (17% vs. 27%), and grade II complications or higher were significantly reduced (4% vs. 20%) compared to the microscopic approach. Pituitary function outcomes favored the endoscope, with fewer new deficiencies in the HPA axis (3% vs. 34%) and TSH-dependent deficiencies (15% vs. 38%). The HPG axis also showed better normalization in the endoscopic group (32% vs. 19%). Visual field impairment and postoperative improvement did not significantly differ between the two techniques. Overall, the findings suggest that endoscopic transsphenoidal pituitary surgery may offer superior outcomes compared to the microscopic approach, particularly in terms of resection rates and complication profiles. Vassilyeva et al. [5] 2023 Bulgaria RCT 83 43 40 The study compared endoscopic and microscopic transsphenoidal pituitary surgery in acromegaly patients, revealing comparable demographic profiles between the groups. Endoscopic surgery demonstrated advantages with shorter anesthesia and surgery times, as well as a reduced postoperative hospital stay. Complete tumor removal was more frequent with endoscopic adenomectomy, while microscopic surgery showed a higher rate of sub-total removal. Both techniques led to a tendency for somatic improvement, with more pronounced visual function improvement in the endoscopic group. Complications, such as liquorrhea and endocrine disorders, were generally low, with endoscopic surgery showing mainly mild complications. Disease remission rates were similar between the groups at various follow-up intervals. In conclusion, while both techniques proved effective in achieving remission, endoscopic surgery exhibited favorable outcomes in terms of efficiency and some aspects of complication profiles. Zhang et al. [6] 2021 China RCT 46 23 23 Endoscopic transsphenoidal pituitary surgery for the treatment of Cushing's disease showed comparable efficacy to microscopic transseptal pituitary surgery but with the added benefits of shorter operative time, reduced estimated blood loss, shorter hospital stays, and fewer complications. Table 1: Summary of the studies included in this systematic review. RCT: randomized controlled trial; HPA: hypothalamic-pituitary-adrenal; TSH: thyroid-stimulating hormone; HPG: hypothalamic-pituitary-gonadal Technique Møller et al. [1] Vassilyeva et al. [5] Zhang et al. [6] Male-to-female ratio (endoscopic) 25:20 17:26 13:10 Male-to-female ratio (microscopic) 107:88 16:24 12:11 Mean age in years (endoscopic) 61 43.26 55.6 Mean age in years (microscopic) 58 44.12 53.2 Functional tumors (endoscopic) 15 All functional All functional Non-functional tumors (endoscopic) 29 - - Functional tumors (microscopic) 69 All functional All functional Non-functional tumors (microscopic) 115 - - Microadenoma size (mm) (endoscopic) - 4 19 Macroadenoma size (mm) (endoscopic) - 39 4 Microadenoma size (mm) (microscopic) - 3 18 Macroadenoma size (mm) (microscopic) - 37 5 Mean operative time (min) (endoscopic) 86 142 108 Mean operative time (min) (microscopic) 106 176 174 Mean hospital stay (days) (endoscopic) - 5 2.8 Mean hospital stay (days) (microscopic) - 7 5.1 Postoperative complications (endoscopic) 2 15 3 Postoperative complications (microscopic) 39 10 8 Table 2: Summary of demographics, tumor characteristics, and postoperative outcomes of the studies included in this systematic review. The quality assessment of the selected studies was done using the Newcastle-Ottawa Quality Assessment Scale. All three studies included in this study turned out to be of high quality with a rating of 9/9 stars (Table 3). Author Selection Comparability Outcome Total stars Møller et al. [1] ★★★★ ★★ ★★★ ★★★★★★★★★ Vassilyeva et al. [5] ★★★★ ★★ ★★★ ★★★★★★★★★ Zhang et al. [6] ★★★★ ★★ ★★★ ★★★★★★★★★ Table 3: Quality assessment of the included studies using the Newcastle-Ottawa Quality Assessment Scale. Discussion This systematic review thoroughly assesses the effectiveness and results of endoscopic transsphenoidal pituitary surgery in comparison to microscopic transsphenoidal surgery, with a specific focus on pituitary adenomas, including Cushing's disease and acromegaly. The results contribute significant insights into the evolving landscape of pituitary surgery, highlighting the benefits and limitations of both surgical techniques. The selected studies offer valuable insights into the comparative outcomes. Møller et al.'s observational study in Denmark suggests that endoscopic surgery exhibits superior outcomes with higher gross total resection rates, shorter surgery duration, and fewer complications [1]. Vassilyeva et al.'s RCT in Bulgaria, focusing on acromegaly patients, indicates endoscopic advantages such as shorter anesthesia and surgery times, reduced postoperative stay, and comparable remission rates [5]. Zhang et al.'s RCT in China, specifically for Cushing's disease, suggests comparable efficacy with added benefits favoring endoscopy [6]. The endoscopic approach has been advocated for its panoramic visualization and superior mobility, which are crucial in resecting tumors while preserving normal structures [7,8]. Studies have shown a higher remission rate in endoscopic procedures for endocrine-active tumors, like growth hormone or adrenocorticotropic hormone (ACTH)-secreting adenomas, compared to the microscopic approach [9,10]. Patient comfort and recovery play a significant role in evaluating surgical methods. The endoscopic technique, by avoiding submucosal excision of nasal tissues, typically results in less postoperative pain and rhinological dysfunction. Studies, including ours, have reported shorter operative times and hospital stays with endoscopic surgery, attributed to fewer intraoperative and postoperative complications and a reduced need for wound management [11-13]. Safety is paramount to any surgical intervention. The endoscopic method has shown a decrease in common complications such as cerebrospinal fluid (CSF) leak, pituitary hormone dysfunction, and diabetes insipidus. Additionally, the endoscopic procedure exhibited fewer complications, which could be attributed to the enhanced visualization allowing for more precise tumor excision and preservation of vital structures [14-16]. In the context of acromegaly patients, the endoscopic technique has demonstrated increased radicality in tumor removal. Our review aligns with these findings, showing a higher rate of total tumor resection in endoscopic patients compared to those undergoing microscopic surgery. This improved outcome is likely due to better illumination and a wider angle of vision provided by endoscopic operations [5,17]. The endoscopic technique has shown a statistically significant improvement in visual function post surgery compared to the microscopic method. However, the frequency of certain postoperative complications, such as intraoperative liquorrhea, was higher in microscopic surgery. These differences underline the importance of the surgical technique in influencing the outcomes and complications of pituitary surgery [5,18]. Despite these findings, it is important to recognize the limitations inherent in these studies. Factors such as tumor size, density, and localization significantly influence surgical outcomes and procedure times. Additionally, the retrospective nature of many studies introduces potential biases, underscoring the need for more prospective, randomized trials for a comprehensive understanding of the long-term outcomes of these techniques. Conclusions This systematic review comparing endoscopic and microscopic transsphenoidal pituitary surgery outcomes indicates consistent evidence favoring the endoscopic approach. Notable studies from Denmark, Bulgaria, and China reveal superior results with endoscopic surgery, demonstrating higher resection rates, shorter surgery duration, and fewer complications. Endoscopy's benefits extend to patient comfort, as evidenced by shorter operative times and hospital stays. Safety considerations also support endoscopy, showing a decrease in common complications such as CSF leaks and hormonal dysfunction. Despite these strengths, the review underscores the need for prospective, randomized trials to address limitations and provide a comprehensive understanding of long-term outcomes. References Møller MW, Andersen MS, Glintborg D, Pedersen CB, Halle B, Kristensen BW, Poulsen FR: Endoscopic vs. microscopic transsphenoidal pituitary surgery: a single centre study. Sci Rep. 2020, 10:21942. 10.1038/s41598-020-78823-z Gao Y, Zhong C, Wang Y, et al.: Endoscopic versus microscopic transsphenoidal pituitary adenoma surgery: a meta-analysis. World J Surg Oncol. 2014, 12:94. 10.1186/1477-7819-12-94 Chen J, Liu H, Man S, et al.: Endoscopic vs. microscopic transsphenoidal surgery for the treatment of pituitary adenoma: a meta-analysis. Front Surg. 2022, 8:806855. 10.3389/fsurg.2021.806855 Guo S, Wang Z, Kang X, Xin W, Li X: A meta-analysis of endoscopic vs. microscopic transsphenoidal surgery for non-functioning and functioning pituitary adenomas: comparisons of efficacy and safety. Front Neurol. 2021, 12:614382. 10.3389/fneur.2021.614382 Vassilyeva N, Mena N, Kirov K, Diatlova E: Comparative effectiveness of endoscopic and microscopic adenoma removal in acromegaly. Front Endocrinol (Lausanne). 2023, 14:1128345. 10.3389/fendo.2023.1128345 Zhang T, Zhang B, Yuan L, Song Y, Wang F: Superiority of endoscopic transsphenoidal pituitary surgery to microscopic transseptal pituitary surgery for treatment of Cushing's disease. Rev Assoc Med Bras (1992). 2021, 67:1687-91. 10.1590/1806-9282.20210732 Yadav Y, Sachdev S, Parihar V, Namdev H, Bhatele P: Endoscopic endonasal trans-sphenoid surgery of pituitary adenoma. 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World J Otorhinolaryngol Head Neck Surg. 2020, 6:84-93. 10.1016/j.wjorl.2020.01.005 Qiao N: Endocrine outcomes of endoscopic versus transcranial resection of craniopharyngiomas: a system review and meta-analysis. Clin Neurol Neurosurg. 2018, 169:107-15. 10.1016/j.clineuro.2018.04.009 Nie D, Fang Q, Wong W, Gui S, Zhao P, Li C, Zhang Y: The effect of endoscopic transsphenoidal somatotroph tumors resection on pituitary hormones: systematic review and meta-analysis. World J Surg Oncol. 2023, 21:71. 10.1186/s12957-023-02958-2 Butenschoen VM, Schwendinger N, von Werder A, Bette S, Wienke M, Meyer B, Gempt J: Visual acuity and its postoperative outcome after transsphenoidal adenoma resection. Neurosurg Rev. 2021, 44:2245-51. 10.1007/s10143-020-01408-x From https://www.cureus.com/articles/213241-navigating-the-surgical-landscape-a-comprehensive-analysis-of-endoscopic-vs-microscopic-transsphenoidal-pituitary-surgery-outcomes#!/

Abstract We investigated the impact of metformin on ACTH secretion and tumorigenesis in pituitary corticotroph tumors. The mouse pituitary tumor AtT20 cell line was treated with varying concentrations of metformin. Cell viability was assessed using the CCK-8 assay, ACTH secretion was measured using an ELISA kit, changes in the cell cycle were analyzed using flow cytometry, and the expression of related proteins was evaluated using western blotting. RNA sequencing was performed on metformin-treated cells. Additionally, an in vivo BALB/c nude xenograft tumor model was established in nude mice, and immunohistochemical staining was conducted for further verification. Following metformin treatment, cell proliferation was inhibited, ACTH secretion decreased, and G1/S phase arrest occurred. Analysis of differentially expressed genes revealed cancer-related pathways, including the MAPK pathway. Western blotting confirmed a decrease in phosphorylated ERK1/2 and phosphorylated JNK. Combining metformin with the ERK1/2 inhibitor Ulixertinib resulted in a stronger inhibitory effect on cell proliferation and POMC (Precursors of ACTH) expression. In vivo studies confirmed that metformin inhibited tumor growth and reduced ACTH secretion. In conclusion, metformin inhibits tumor progression and ACTH secretion, potentially through suppression of the MAPK pathway in AtT20 cell lines. These findings suggest metformin as a potential drug for the treatment of Cushing's disease. Introduction Pituitary neuroendocrine tumors (PitNETs) are common intracranial tumors with an incidence of 1/1000, and pituitary corticotroph tumors (corticotroph PitNETs) account for approximately 15% of all PitNETs. Most corticotroph PitNETs are functional tumors with clinical manifestations of Cushing's disease characterized by central obesity, hypertension, diabetes mellitus, and psychosis (Cui et al., 2021). The increased cortisol due to the overproduction of adrenocorticotropic hormone (ACTH) significantly reduces the overall quality of survival and life expectancy of patients (Sharma et al., 2015; Barbot et al., 2018). Currently, treatment of corticotroph PitNETs mainly relies on surgery resection, pharmacologic therapy or radiotherapy may be considered for patients with residual tumors or those who are unable to undergo surgery. While several agents, such as cabergoline and pasireotide, are clinically approved, the effect is unsatisfactory, and potentially serious side effects exist. Therefore, there is an urgent need to develop novel therapeutic drugs for corticotroph PitNETs. Metformin is a biguanide hypoglycemic agent for the treatment of type 2 diabetes. In addition to its hypoglycemic effect, numerous studies identified the therapeutic role of metformin in the prevention and treatment of various tumors including small cell lung cancer, colorectal cancer, breast cancer, ovarian cancer, and neuroendocrine tumors (Lu et al., 2022; Kamarudin et al., 2019; Wang et al., 2019; Thakur et al., 2019), making metformin a promising adjuvant drug in the therapy of cancers. Besides, it has been reported that metformin improves metabolic and clinical outcomes in patients treated with glucocorticoids. However, to date, limited studies explore the potential anti-cancer effect of metformin in corticotroph PitNETs. Recent studies report the use of metformin for blood glucose and body weight control in patients with Cushing's disease (Ceccato et al., 2015), while the role of metformin on ACTH secretion and tumor growth in corticotroph PitNETs remains to be elucidated. In the current study, we investigated the effect of metformin in corticotroph PitNETs and performed RNA-sequencing to identify the potential mechanisms of metformin. We found that metformin inhibited cell proliferation and ACTH secretion of AtT20 cells in a dose-dependent manner. Besides, metformin induced cell cycle arrest via decreased ERK1/2 phosphorylation and increased P38 phosphorylation. Our results revealed that metformin is a potential drug for corticotroph PitNET therapy. Section snippets Cell culture The ACTH-secreting mouse pituitary tumor cell line AtT-20 was purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA). Cells were cultured in F-12K medium (ATCC; Catalog No. 30-2004), supplemented with 15% fetal bovine serum (FBS; Gibco), and 2.5% horse serum (Gibco) as suggested. AtT20 cells were cultured in a humidified incubator at 37 °C in 5% CO2. Reagents and drugs Metformin and Ulixertinib were purchased from MedChemExpress (MCE), Metformin was dissolved in sterile H2O and prepared as a Results Metformin inhibits cell proliferation and ACTH secretion, and leads to cell cycle arrest in AtT20 cells. We used CCK-8 assay to detect the cell viability of AtT20 cells after treatment with different concentrations of metformin at 24 h, 48 h, and 72 h. The results showed that metformin significantly inhibited the proliferation of AtT20 cells in a dose-dependent manner (Fig. 1A). Similarly, prolonged (6 days) treatment of AtT20 cells with a lower concentration (400 μM) of metformin also inhibited Discussion Metformin, acting by binding to PEN2 and initiating the subsequent AMPK signaling pathway in lysosomes, is the most commonly used oral hypoglycemic agent (Hundal et al., 2000; Ma et al., 2022). Previous reports demonstrated metformin as a potential anti-tumor agent in cancer therapy (Evans et al., 2005). Metformin, either alone or in combination with other drugs, has been shown to reduce cancer risk in a variety of tumors including pituitary neuroendocrine tumors (PitNETs) (Thakur et al., 2019; Conclusion Our study demonstrated that metformin suppressed cell proliferation and decreased ACTH secretion in AtT20 cells via the MAPK pathway. Our results revealed that metformin is a potential anti-tumor drug for the therapy of corticotroph PitNETs, which deserves further study. Funding This study was supported by the National Natural Science Foundation of China (82072804, 82071559). CRediT authorship contribution statement Yingxuan Sun: Conceptualization, Formal analysis, Investigation, Writing – original draft, Writing – review & editing. Jianhua Cheng: Data curation, Formal analysis, Visualization, Writing – original draft, Writing – review & editing. Ding Nie: Formal analysis, Writing – review & editing. Qiuyue Fang: Data curation, Formal analysis, Writing – review & editing. Chuzhong Li: Conceptualization, Supervision, Writing – original draft, Writing – review & editing, Funding acquisition. Yazhuo Zhang: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgement We thank Mr. Hua Gao (Cell Biology Laboratory, Beijing Neurosurgical Institute, China) for support with the techniques. References (30) K. Jin et al. Metformin suppresses growth and adrenocorticotrophic hormone secretion in mouse pituitary corticotroph tumor AtT20 cells Mol. Cell. Endocrinol. (2018) R. Krysiak et al. The effect of metformin on prolactin levels in patients with drug-induced hyperprolactinemia Eur. J. Intern. Med. (2016) X. Liu et al. Combination treatment with bromocriptine and metformin in patients with bromocriptine-resistant prolactinomas: pilot study World neurosurgery (2018) J. Sinnett-Smith et al. Metformin inhibition of mTORC1 activation, DNA synthesis and proliferation in pancreatic cancer cells: dependence on glucose concentration and role of AMPK Biochem. Biophys. Res. Commun. (2013) C.R. Triggle et al. Metformin: is it a drug for all reasons and diseases? Metab., Clin. Exp. (2022) J.C. Wang et al. Metformin inhibits metastatic breast cancer progression and improves chemosensitivity by inducing vessel normalization via PDGF-B downregulation J. Exp. Clin. Cancer Res. : CR (2019) J. An et al. Metformin inhibits proliferation and growth hormone secretion of GH3 pituitary adenoma cells Oncotarget (2017) M. Barbot et al. Diabetes mellitus secondary to Cushing's disease Front. Endocrinol. (2018) F. Ceccato et al. Clinical use of pasireotide for Cushing's disease in adults Therapeut. Clin. Risk Manag. (2015) M. Cejuela et al. Metformin and breast cancer: where are we now? Int. J. Mol. Sci. (2022) From https://www.sciencedirect.com/science/article/abs/pii/S0303720723002915

Here, we report the first adult case of pancreatic yolk sac tumor with ectopic adrenocorticotropic hormone (ACTH) syndrome. The patient was a 27-year-old woman presenting with abdominal distension, Cushingoid features, and hyperpigmentation. Endogenous Cushing’s syndrome was biochemically confirmed. The ACTH level was in the normal range, which raised the suspicion of ACTH precursor-dependent disease. Elevated ACTH precursors were detected, supporting the diagnosis of ectopic ACTH syndrome. Functional imaging followed by tissue sampling revealed a pancreatic yolk sac tumor. The final diagnosis was Cushing’s syndrome due to a yolk sac tumor. The patient received a steroidogenesis inhibitor and subsequent bilateral adrenalectomy for control of hypercortisolism. Her yolk sac tumor was treated with chemotherapy and targeted therapy. Cushing’s syndrome secondary to a yolk sac tumor is extremely rare. This case illustrated the utility of ACTH precursor measurement in confirming an ACTH-related pathology and distinguishing an ectopic from a pituitary source for Cushing’s syndrome. Introduction Ectopic adrenocorticotrophic hormone (ACTH) syndrome, also termed paraneoplastic Cushing’s syndrome, can be caused by the secretion of ACTH and/or ACTH precursors from ectopic tumors. The tumors concerned secrete ACTH precursors, including unprocessed proopiomelanocortin (POMC) and POMC-derived peptides, owing to the altered post-translational processing of POMC (1). These tumors are associated with intense hypercortisolism and various complications, such as hypertension, hyperglycemia, osteoporosis, infection risks, and thrombotic tendencies (2). Distinguishing ectopic from pituitary-dependent Cushing’s syndrome is often challenging. The two conditions are classically distinguished by their variable responses to dynamic endocrine tests, including the high-dose dexamethasone suppression test, the corticotrophin-releasing-factor (CRF) test, and the desmopressin test (3). Pituitary imaging may sometimes provide a diagnosis if a pituitary macroadenoma is identified at this juncture. The gold standard for diagnosing pituitary Cushing’s is a positive inferior petrosal sinus sampling (IPSS) result. The measurement of ACTH precursors is reported to have diagnostic value in this scenario (4). The most common source of ectopic ACTH is intrathoracic tumors, including bronchial carcinoid and small cell lung cancers. Other possible sources include gut neuroendocrine tumors and medullary thyroid cancer. Recognizing the potential causes of ectopic ACTH syndrome is essential as this provides guidance in locating the causative tumor and allows tumor-directed therapies. A yolk sac tumor as a cause of ectopic ACTH syndrome has only been reported in a 2-year-old child but not in adults (5). Here, we present a case of a 27-year-old Chinese woman who had Cushing’s syndrome due to ectopic ACTH precursor production from a pancreatic yolk sac tumor. Case description A 27-year-old Chinese woman, who had unremarkable past health and family history, presented with right upper quadrant abdominal pain and nausea in early 2020. Abdominal ultrasonography was unrevealing. A few months later, she developed Cushingoid features and oligomenorrhea. At presentation, her blood pressure was 160/95 mmHg, body weight was 65.6 kg, and body mass index was 23.2 kg/m2. She had a moon face, hirsutism, proximal myopathy, bruising, thinning of the skin, and acne. She also had hyperpigmentation on the nails and knuckles of both hands (Figure 1). Figure 1 Figure 1. Cushingoid features at presentation include moon face, acne, thin skin, and easy bruising. Hyperpigmentation on the nails and knuckles was also noted. Diagnostic assessments Her 9 am and 9 pm cortisol were both >1,700 nmol/L. Her 24-h urine-free cortisol was beyond the upper measurable limit at >1,500 nmol/L. Her serum cortisol was 759 nmol/L after a 1 mg overnight-dexamethasone suppression test, confirming endogenous Cushing’s syndrome. The morning ACTH was 35 pg/mL (upper limit of normal is 46 pg/mL). After excluding a high dose-hook effect, her blood sample was concomitantly sent for ACTH measurement using two different platforms to eliminate possible interference, which might cause a falsely low ACTH reading. ACTH was 19 pg/mL (upper limit of normal is 46 pg/mL) using an IMMULITE 2000 XPI, Siemens Healthineers, Erlangen, Germany, and 17 pg/mL (reference range: 7–63 pg/mL) using a Cobas e-801, Roche Diagnostics, Indianapolis, IN, United States, therefore verifying the ACTH measurement. In view of this being ACTH-dependent Cushing’s syndrome, a high-dose-dexamethasone suppression test (HDDST) was performed, and her cortisol was not suppressed at 890 nmol/L, with ACTH 42 pg/mL. The serum cortisol day profile showed a mean cortisol level of >1,700 nmol/L (i.e., higher than the upper measurable limit of the assay) and an ACTH of 17 pg/mL. A CRF test using 100 μg of corticorelin showed less than a 50% rise in ACTH and no rise in cortisol levels (Supplementary Table S1). She suffered from multiple complications of hypercortisolism, including thoracic vertebral collapse with back pain, diabetes mellitus (HbA1c 6.7% and fasting glucose 7.6 mmol/L), and hypokalemic hypertension, with a lowest potassium level of 2.3 mmol/L. The rapid onset of intense hypercortisolism and refractory hypokalemia, as well as the responses in the HDDST and CRF tests raised the suspicion of ectopic ACTH syndrome. Tumor markers were measured. Alpha-fetoprotein (AFP) was markedly raised at 33,357 ng/mL (reference range: <9 ng/mL). Beta-human chorionic gonadotropin (beta-hCG) was not elevated. Carcinoembryonic antigen (CEA) was 4.0 ng/mL (reference range: <3 ng/mL) and CA 19–9 was 57 U/mL (reference range: <37 U/mL). The marked hyperpigmentation in the context of normal ACTH levels pointed to the presence of an underlying tumor producing circulating ACTH precursors. Hence, magnetic resonance imaging (MRI) of the pituitary gland was not performed at this juncture. ACTH precursors were measured using a specialized immunoenzymatic assay (IEMA) employing in-house monoclonal antibodies against the ACTH region and the gamma MSH region. Both monoclonal antibodies have to bind to these regions in POMC and pro-ACTH to create a signal. The patient had a level of 4,855 pmol/L (upper limit of normal is 40 pmol/L) (6). This supported Cushing’s syndrome from an ectopic source secondary to an excess in ACTH precursors. Localization studies were arranged to identify the source of ectopic ACTH precursors. Computed tomography (CT) of the thorax did not show any significant intrathoracic lesion but incidentally revealed a pancreatic mass. Dedicated CT of the abdomen confirmed the presence of a 7.9 × 5.6 cm lobulated mass in the pancreatic body; the adrenal glands were unremarkable. 18-FDG and 68Ga-DOTATATE dual-tracer positron-emission tomography-computed tomography (PET-CT) showed that the pancreatic mass was moderately FDG-avid and non-avid for DOTATATE (Supplementary Figure S1). Multiple FDG-avid nodal metastases were also present, including left supraclavicular fossa lymph nodes. Fine needle aspiration of the left supraclavicular fossa lymph node yielded tumor cells featuring occasional conspicuous nucleoli, granular coarse chromatin, irregular nuclei, and a high nuclear-to-cytoplasmic ratio. Mitotic figures were infrequent. On immunostaining, the tumor cells were positive for cytokeratin 7 and negative for cytokeratin 20. Focal expression of CDX-2, chromogranin, and synaptophysin was noted. They were negative for TTF-1, GCDPF, Gata 3, Pax-8, CD56, ACTH, inhibin, and S-100 protein. Further immunostaining was performed in view of highly elevated AFP. The tumor cells expressed AFP, Sall4, and MNF-116. They were negative for c-kit, calretinin, Melan A and SF-1. Placental ALP (PLAP) was weak and equivocal. The features were in keeping with a yolk sac tumor. Therapeutic intervention and outcome The patient had significant hypokalemic hypertension requiring losartan 100 mg daily, spironolactone 100 mg daily, and a potassium supplement of 129 mmol/day. Co-trimoxazole was given for prophylaxis against Pneumocystis jirovecii pneumonia. Metyrapone was started and up-titrated to 1 gram three times per day. However, in view of persistent hypercortisolism, with urinary free cortisol persistently above the upper measurable limit of the assay, bilateral adrenalectomy was performed. The tumor was mainly in the periadrenal soft tissue, with vascular invasion. The tumor formed cords, nests, and ill-defined lumen (Figure 2). The tumor cells were polygonal and contained pale to eosinophilic cytoplasm and pleomorphic nuclei, some with large nucleoli. Mitosis was present while tumor necrosis was not obvious. The stroma was composed of vascular fibrous tissue, with minimal inflammatory reaction. Immunohistochemical study showed that the tumor was positive for cytokeratin 7, MNF-116, AFP, and glypican-3, and also positive for Sall4 and HNF1β. The tumor cells were negative for cytokeratin 20, PLAP, CD30, negative for neuroendocrine markers including S100 protein, synaptophysin, chromogranin, and also negative for Melan-A, inhibin, and ACTH. Histochemical study for Periodic acid–Schiff–diastase (PAS/D) showed no cytoplasmic zymogen granules like those of acinar cell tumor. The features were compatible with yolk sac tumor. She was put on glucocorticoid and mineralocorticoid replacements post-operatively. Figure 2 Figure 2. Histology and immunohistochemical staining pattern of tumor specimen. (A) HE stain x 40 showing tumor cells in the soft tissue and peritoneum. (B) HE × 400 showing that the tumor forms cords, nests, and ill-formed lumen in the vascular stroma. The tumor cells are polygonal with pale cytoplasm and pleomorphic nuclei. (C) PAS/D stain showing no cytoplasmic zymogen granules. (D) Tumor is diffusely positive for cytokeratin 7. (E) Tumor is positive for AFP. (F) Tumor is positive for glypican-3. (G) Tumor is diffusely positive for HNF1β. (H) Tumor is diffusely positive for SALL4. Regarding her oncological management, she received multiple lines of chemotherapy, but the response was poor. Due to limited access to the ACTH precursor assay, serial measurement was unavailable. Treatment response was monitored by repeated imaging and monitoring of AFP. Figure 3 shows a timeline indicating the key events of the disease, showing the trends of the AFP and cortisol levels. Apart from (i) bleomycin, etoposide, and platinum, she was sequentially treated with (ii) etoposide, ifosfamide with cisplatin, and (iii) palliative gemcitabine with oxaliplatin. Next-generation sequencing showed a BRAF V600E mutation, for which (iv) dabrafenib and trametinib were given. Unfortunately, the disease progressed, and the patient succumbed approximately one year after the disease was diagnosed. Figure 3 Figure 3. Timeline with serial cortisol and alpha-fetoprotein levels from diagnosis to patient death. Discussion This case demonstrates the diagnostic value of ACTH precursor measurement in the diagnosis of ectopic Cushing’s syndrome. ACTH precursors are raised in all ectopic tumors responsible for Cushing’s syndrome and could be useful in distinguishing ectopic from pituitary Cushing’s syndrome (4). Moreover, Cushing’s syndrome due to a yolk sac tumor has been reported only once in a pediatric case, and this is the first adult case reported in the literature (5). POMC is sequentially cleaved in the anterior pituitary into pro-ACTH and then into ACTH, which is released into the circulation and binds to ACTH receptors in the adrenal cortex, leading to glucocorticoid synthesis (5, 7). Due to incomplete processing, ACTH precursors are found in normal subjects at a concentration of 5–40 pmol/L (6). Pituitary tumors are traditionally well-differentiated and can also relatively efficiently process ACTH precursors. However, this processing is less efficient in ectopic tumors that cause Cushing’s syndrome (8). Some less differentiated pituitary macroadenomas can secrete ACTH precursors into the circulation; however, these tumors are diagnosed by imaging and so do not, in general, cause problems with differential diagnosis (9). Measurement of ACTH precursors by immunoradiometric assay (IRMA) was first described by Crosby et al. (10). The assay utilized monoclonal antibodies specific for ACTH and the other binding gamma-MSH. The assay only detects peptides expressing both epitopes and therefore measures POMC and pro-ACTH. The assay does not cross-react with other POMC-derived peptides such as beta-lipotropin, ACTH, and N-POMC. Oliver et al. demonstrated that, compared to the pituitary adenomas in Cushing’s disease, all ectopic tumors responsible for Cushing’s syndrome in their study produce excessive POMC and pro-ACTH (4). The excessive production of ACTH precursors may reflect neoplasm-induced modification and amplification of POMC production. It is suggested that POMC binds to and activates the ACTH receptor because it contains the ACTH amino-acid sequence, or it is cleaved to ACTH in the adrenal glands to cause hypercortisolism (5) (Figure 4). Moreover, cleavage of POMC may produce peptides that exert mitogenic actions on adrenal cells and lead to adrenocortical growth. Outside the adrenal tissue, excessive ACTH precursors in Cushing’s syndrome caused by ectopic tumors can lead to marked hyperpigmentation. Both hypercortisolism and hyperpigmentation were observed in the reported case. Figure 4 Figure 4. Postulated pathological mechanism of ectopic ACTH precursors. In patients with ACTH-dependent Cushing’s syndrome, ectopic tumors should be distinguished from pituitary tumors. The HDDST, at a cut-off of 50% cortisol suppression, gives a sensitivity of 81% and a specificity of 67% for pituitary dependent Cushing’s syndrome (11). The CRF test provides 82% sensitivity and 75% specificity for pituitary disease (8). IPSS is the gold standard in distinguishing pituitary from ectopic tumors in Cushing’s syndrome. Utilization of CRF-stimulated IPSS provides 93% sensitivity and 100% specificity for pituitary disease. It also allows correct lateralization in 78% of patients with pituitary tumors. However, it is only available in specialized centers. In a retrospective cohort, the ACTH precursor level distinguished well between Cushing’s disease and ectopic ACTH syndrome (4). With a cut-off of 100 pmol/L, the test achieved 100% sensitivity and specificity for ectopic ACTH syndrome. More recently, this assay has been used to diagnose patients with occult ectopic ACTH syndrome, with ACTH precursors above 36 pmol/L (8). Unfortunately, the immunoassay for ACTH precursor measurement utilizes in-house monoclonal antibodies, which are not widely available. Cross-reactivity of POMC in commercially available ACTH assays ranges from 1.6% to 4.7% (12). In cases of ectopic tumors causing Cushing’s syndrome with markedly raised ACTH-precursors and intense hypercortisolism, the cross-reactivity would give significantly high ‘ACTH’ measurements to suggest an ACTH-related pathology. The degree of cross-reactivity, which is variable, should ideally be provided by the assay manufacturer as it affects result interpretation. Lower levels of ACTH precursor production might not be detected, especially by assays with low precursor cross-reactivity. Clinical vigilance is crucial in reaching the correct diagnosis. In patients with marked hypercortisolism and a normal ACTH concentration, like in this case, the measurement of ACTH precursors would allow the accurate diagnosis of Cushing’s syndrome caused by ACTH precursors. Ectopic tumors causing Cushing’s syndrome are associated with more intense hypercortisolism than Cushing’s disease (11). However, due to variable cross-reactivity, commercial ACTH assays might not accurately detect the excessive ACTH precursors responsible for the clinical syndrome. For this reason, ACTH measurements in these two conditions can significantly overlap and may not differentiate between ectopic and pituitary diseases (4). On the other hand, the more specific POMC assay described in 1996, which does not cross-react with pro-ACTH, has a low sensitivity of 80% for ectopic Cushing’s syndrome and is not now available (13). Hence, the ACTH precursor assay used in this reported case, which detects POMC and pro-ACTH, appears to provide the best diagnostic accuracy from the available literature. Serial measurement of ACTH precursors may play a role in monitoring the treatment response in an ACTH precursor secreting tumor. In the case of ectopic ACTH secretion, the corticotropic axis is slowed down and ACTH is almost exclusively of paraneoplastic origin. Immunotherapy is known to alter the functioning of the hypothalamic–pituitary corticotropic axis; however, its effect on ectopic secretions is not known. More data is required before the role of ACTH precursor measurement for disease monitoring in these scenarios can be ascertained. The incidence of endogenous Cushing’s syndrome is reported to be 2 to 4 per million people per year (14). Ectopic sources of Cushing’s syndrome are responsible for 9 to 18% of these cases. Typical sources of these ectopic tumors include bronchial carcinoid tumors, small-cell lung cancer, and gut neuroendocrine tumors. Notably, germ cell tumors, including teratomas, ovarian epithelial tumors, and ovarian endometrial tumors, are also possible ectopic sources of Cushing’s syndrome. The histological diagnosis of germ cell tumor in a non-genital site is challenging, especially for the poorly differentiated, or with somatic differentiation. Immunostaining, chromosomal, or genetic study are very important in confirming the diagnosis. AFP elevation in our case limited the differential diagnoses to germ cell tumors/yolk sac tumors, hepatocellular carcinoma, and rare pancreatic tumors. The specimen was biopsied from the retroperitoneum, and the morphology was a dominant trabecular pattern or a hepatoid pattern. It showed diffuse positive immunostaining for cytokeratin, AFP, and glypican-3. It was also diffusely and strongly positive for HNF1β and SALL4, supporting the diagnosis of yolk sac tumor. Both HNF1β and SALL4, being related with the expression of genes associated with stem cells or progenitor cells, are used as sensitive and specific markers for germ cell tumors/yolk sac tumors (15, 16). Staining related to pancreatic acinar cell carcinoma and neuroendocrine tumor were performed. PAS/D staining showed a lack of zymogen granules. A lack of nuclear β-catenin positivity was shown. Staining for neuroendocrine markers, including chromogranin and synaptophysin, was negative. Bcl-10 and trypsin were not available in the local setting. Cushing’s syndrome due to a yolk sac tumor was reported only once, in a 2-year-old child (5). The abdominal yolk sac tumor was resistant to cisplatin, with rapid disease progression, and the patient succumbed 1.5 years after initial presentation. Yolk sac tumor in the pancreas is also rare, with only 4 cases reported so far. The first case was reported in a 57-year-old woman with an incidentally detected abdominal mass (17). The tumor stained positive for AFP, PLAP, and CEA. The second case was a 70-year-old asymptomatic woman with histology showing a group of tumor cells with features of a yolk sac tumor, and another group showing features of pancreatic ductal adenocarcinoma with mucin production, suggesting a yolk sac tumor derived from pancreatic ductal adenocarcinoma (18). The tumor showed partial positivity for AFP, Sall4, glypican-3, and cytokeratin 7, as found in our case, while MNF-116 and PLAP staining results were not described. The third was in a 33-year-old man with a solitary pancreatic head mass with obstructive jaundice (19). The patient had undergone Whipple’s procedure followed by cisplatin-based chemotherapy, resulting in at least 5 years of disease remission. The latest reported case was in a 32-year-old man presenting with abdominal pain (20). Notably, initial imaging showed diffuse enlargement of the pancreas and increased FDG uptake without a distinct mass. Reassessment imaging 11 months later showed a 13 cm pancreatic mass. The initial imaging findings suggested initial intraductal growth of the tumor, as reported in some subtypes of pancreatic carcinoma. None of the reported cases of adult pancreatic yolk sac tumors were associated with abnormal hormone secretion. We reported the first adult case of pancreatic yolk sac tumor with ectopic ACTH syndrome. The case represents an overlap of two rarities. It demonstrates that pancreatic yolk sac tumor is a possible cause of ectopic ACTH syndrome. Conclusion ACTH precursor measurement helps to distinguish ectopic ACTH syndrome from Cushing’s disease. The test has superior diagnostic performance and is less invasive than IPSS. Nonetheless, the limited availability of the assay may restrict its broader use in patient management. We describe the first adult case of pancreatic yolk sac tumor with ACTH precursor secretion resulting in Cushing’s syndrome. This adds to the list of origins of ectopic ACTH syndrome in adults. Data availability statement The original contributions presented in the study are included in the article/Supplementary material, further inquiries can be directed to the corresponding author. Ethics statement Written informed consent was obtained from the individual to publish any potentially identifiable images or data in this article. Author contributions JC wrote the manuscript. JC, CW, WC, AW, KW, and PT researched the data. WC, AL, EL, YW, KT, KL, and CL critically reviewed and edited the manuscript. DL initiated and conceptualized this case report and is the guarantor of this work. All authors contributed to the article and approved the submitted version. Funding The author(s) declare that no financial support was received for the research, authorship, and/or publication of this article. Conflict of interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Publisher’s note All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher. Supplementary material The Supplementary material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fmed.2023.1246796/full#supplementary-material References 1. Stewar, PM, Gibson, S, Crosby, SR, Pennt, R, Holder, R, Ferry, D, et al. ACTH precursors characterize the ectopic ACTH syndrome. Clin Endocrinol. (1994) 40:199–204. doi: 10.1111/j.1365-2265.1994.tb02468.x PubMed Abstract | CrossRef Full Text | Google Scholar 2. Young, J, Haissaguerre, M, Viera-Pinto, O, Chabre, O, Baudin, E, and Tabarin, A. 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Edited by: Alessandro Vanoli, University of Pavia, Italy Reviewed by: Petar Brlek, St. Catherine Specialty Hospital, Croatia Wafa Alaya, Hospital University Tahar Sfar, Tunisia Copyright © 2023 Chang, Woo, Chow, White, Wong, Tsui, Lee, Leung, Woo, Tan, Lam, Lee and Lui. 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: David Tak Wai Lui, dtwlui@hku.hk 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. 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Lydia, a 28-year-old Florida resident, wife, and mother of two, first noticed a drastic increase in her weight around Easter of 2022 in a family photo. She was shocked by how different she looked despite not making any drastic changes to her diet. “While those I loved would say ‘you look beautiful’, to me I looked like a completely different person,” recalled Lydia. When Lydia asked her mother, Jeanne, if she had noticed her weight gain, her mother observed that some days Lydia’s face looked swollen. They both recognized that this was not normal, and decided, like many pituitary patients, to make an appointment with a primary care provider. “I remember her saying to me ‘something is wrong with me’ and ‘something is not right’”, recalled Jeanne. Lydia’s weight gain was most noticeable in her face and around her abdomen. “She was exercising all the time and trying to watch what she ate and cut down on sugars,” said Jeanne. “But she kept putting on more weight. We knew something was not right.” Lydia scheduled the first of what would be many doctor appointments hoping for answers. Her primary care provider recognized that her rapid weight gain was abnormal and ordered standard blood work. When that blood work came back normal, her doctor referred her to an endocrinologist and to her OBGYN. In addition to her weight gain, Lydia had begun developing other symptoms including excessive sweating day and night, severe acne, hair loss, hair gain on her face, insomnia, thin skin, and brittle nails. “The worst symptom was the constant feeling of fight or flight,” recalled Lydia. “I always felt on edge and was letting things bother me.” Lydia would later learn that this feeling was caused by the drastic increase of cortisol in her body. When Lydia first met with her OBGYN to address her weight gain and the overall feeling that something was wrong with her body, her concerns were quickly dismissed. “He told me ‘You’re almost 30 and you’ve had two kids, no wonder you feel the way that you do,’” said Lydia. “He blew me off and told me that I needed more diet and exercise. He didn’t order other tests.” Figure illustrates the drastic physical changes and symptoms caused by a pituitary tumor and Cushing’s disease. (Medical illustration by Mark Schornak, MS, CMI) A couple of months later, Lydia went to see an endocrinologist. Despite watching her calories and exercising almost every day of the week, she had gained more weight and felt more miserable. When her labs came back, Lydia’s cortisol levels were so high that the endocrinologist thought there had been a lab error. A 24-hour urine test confirmed that Lydia’s cortisol levels were off the charts. “I was in full panic mode at this point,” said Lydia. Lydia could not get back in to see her endocrinologist in a timely manner, so she ended up back at her primary care provider’s office. Her primary care provider suggested that it could be a tumor on her adrenal glands and that it was probably not in her brain since she was not experiencing headaches. A CT scan of the adrenal glands came back clean. “I remember telling my primary care doctor ‘I just don’t feel normal’”, recalls Lydia. “His response was ‘everyone’s normal is different’ and I told him ‘I’m not normal for me.’” At this point, Lydia was desperate for answers. “All these doctors were telling me it could be in my head or because I was almost 30,” said Lydia. “I kept getting shut down. I told friends and family that there was something seriously wrong with me and no one was believing me.” Finally, a friend sent Lydia information on another endocrinologist in Florida. “He was the first doctor to care about me,” said Lydia. “He said, ‘I’m so sorry you’ve been treated like this. Everyone you have seen before me is an idiot.’” More specific bloodwork and an MRI confirmed that Lydia had a macroadenoma, a benign tumor in the pituitary gland, and Cushing’s disease. After the diagnosis, Lydia was told that she would need to have the tumor removed. “He told me, ‘Find where you want to go and I’ll refer you,’” said Lydia. Lydia and her mother Jeanne began searching online for the right pituitary tumor surgeon. “Once I realized how serious it was, we started researching different doctors,” recalled Jeanne. Both Lydia and Jeanne spent time researching different doctors, but could not find a doctor that had experience treating Cushing’s disease. “We researched all kinds of surgeons to find the best one,” said Jeanne. “Then we found Dr. Oyesiku. He understood Cushing’s disease. That was important to me.” Jeanne is the one who found world-renowned pituitary tumor surgeon, Dr. Nelson Oyesiku. “I called him and said, ‘I have a 28-year-old daughter with a pituitary tumor and Cushing’s disease and I need you to operate on her,’” said Jeanne. Dr. Oyesiku has performed over 4,000 pituitary tumor operations and is currently the Chair of the Department of Neurosurgery at UNC Health. “Cushing’s is a rare disease so not many surgeons have a lot of experience with the various technical nuances required to achieve a high likelihood of cure and reduce the incidence of re-operations and complications,” said Dr. Oyesiku. Since Lydia lives in Florida, her initial consultation with Dr. Oyesiku was over Zoom. “I Zoomed with another local neurosurgeon and I was going back and forth,” said Lydia. “Dr. Oyesiku told me that he looks at the whole picture and what the tumor is doing to you. He said that he wanted to get the tumor out and then cure the Cushing’s disease.” Jeanne was also with her daughter during the initial Zoom appointment with Dr. Oyesiku. “I couldn’t find anyone else that had that background knowledge for Cushing’s disease,” said Jeanne. Dr. Oyesiku ordered more labs. “He told me ‘I want to measure twice and cut once,’” recalled Lydia. “That phrase is something my dad always said growing up and that felt like fate. So that made my decision for me and made me want to see him.” After her initial consultation with Dr. Oyesiku, both Lydia and her mother felt confident that they had found the right surgeon. Lydia met with Dr.Oyesiku in December of 2022, then had her surgery on January 23, 2023. “I called UNC and made sure that I could go in with her and stay while she was recovering,” said Jeanne. “We had contacted a different hospital early on, and I would have had to drop her off and not see her until after her surgery and only during visiting hours.” Patient coordinator David Baker, who also played an important role in Lydia’s care, helped Lydia and Jeanne find a local hotel for them to stay in before surgery at a discounted rate. After surgery, UNC Health endocrinologist Dr. Atil Kargi spoke with Lydia and her mom to help them understand the severity of Cushing’s disease and the importance of monitoring Lydia closely. “Dr. Kargi and David Baker really helped us to truly understand Cushing’s disease,” said Jeanne. Jeanne was impressed with the level of patient care that Lydia experienced during her surgery at UNC Health. “Lydia had her own nurse that would text me or call me to let me know how things were progressing.” Jeanne said. Jeanne explained that the same nurse was with her daughter going into the surgery and when she woke up after the surgery. She was also able to stay with Lydia in the hospital while she recovered from the surgery. “UNC was such an uplifting place. All these residents, they all love what they’re doing,” said Jeanne. Lydia stayed in the hospital for six days so Dr. Oyesiku and the endocrine team could monitor her levels. “I was in the normal range, and then I started to tank,” said Lydia. “I had read that a lot of patients are sent home right after surgery. If they would have sent me, I would have been adrenally insufficient.” Lydia also expressed gratitude for ENT surgeon, Dr. Brian Thorp. “During my surgery, Dr. Thorp also repaired my deviated septum,” said Lydia. “Even after surgery when I was home miles away in Florida, he was always available to me. I appreciate Dr. Oyesiku and everyone at UNC,” said Lydia. “I can’t imagine going anywhere else for this. Dr. Oyesiku truly saved my life.” After her discharge, Jeanne drove Lydia back to Florida. “Dr. Oyesiku followed-up after surgery with the Cushing’s disease treatment,” said Jeanne. “Our local endocrinologist could not believe how fast she recovered.” Jeanne also noted that she was always able to get ahold of Dr. Oyesiku, Dr. Thorp, or David Baker to answer her questions. “You feel like you’re their only patient,” said Jeanne. “We are 8-9 hours away, and it didn’t feel like it.” After Lydia weaned off of her medication, she started to lose weight, her face changed, and her body started to feel “normal” again. “My biggest symptom that I am thankful went away was my literally going crazy feeling,” said Lydia. “I am very thankful that I was able to catch it early enough so that this awful disease didn’t leave me with any lifelong complications.” Lydia, like many pituitary tumor patients, still has lingering feelings of anxiety and frustration with the long road from initial symptoms to diagnosis. It takes the average pituitary tumor patient 5-8 years to be properly diagnosed. Lydia and her mother were extremely proactive and still spent 18 months looking for answers. Lydia went to her primary care provider, her OB, a second OB, and two endocrinologists before she had a proper diagnosis. “Cushing’s disease can mimic many other vastly common medical disorders and is often misdiagnosed or mistaken for something else such as diabetes, hypertension, obesity, infertility, depression, or autoimmune disorders,” said Dr. Oyesiku. “Making the diagnosis requires expert clinical acumen supported by sophisticated medical tests, and many of these tests have to be repeated to confirm the diagnosis.” Because Cushing’s disease is so rare, many of the providers that initially saw Lydia dismissed it. After her surgery, Lydia returned to her OB office in Florida for her annual exam and was seen by the OB that told her that her symptoms were “all in her head”. “I told him, ‘Remember that you blew me off? I had a brain tumor that caused Cushing’s disease,’” said Lydia. “He told me that in all his years practicing, he had never had a patient with an endocrine disorder caused by a pituitary tumor.” Lydia’s story and other pituitary tumor patient stories serve as a reminder that while Cushing’s disease is rare, it is worth ruling out when a patient complains of these symptoms. “Part of the problem is that people just do not have access to good doctors,” said Jeanne. “If we had not had that endocrinologist, I don’t know how much longer it would have taken. It makes me sad that other women and even men can have it for so long because they cannot figure out what is going on.” Both Jeanne and Lydia are thankful that the surgery was a success, but the symptoms and long road to a diagnosis left Lydia with a few emotional scars. “I’m fine and healthy on paper, but still battling the mental aspects and the toll it took on me,” said Lydia. “Sometimes I feel resentful because it took away a year and a half of my life. I feel very blessed to be on the other side of this disease, but I’m ready to not be a patient anymore.” From https://www.med.unc.edu/neurosurgery/i-dont-feel-normal-the-diagnosis-of-a-pituitary-tumor-cushings-disease/?fbclid=IwAR1I12ND084Ato5lloDalTEcIFycV5HpLiR7S1brNxr7Lux1BZ6g_vySHOA

ABSTRACT Objective To determine whether accurate inferior petrosal sinus sampling (IPSS) tumor lateralization is associated with improved clinical outcomes following the surgical treatment of Cushing’s disease. Methods The presented study was performed in accordance with PRISMA guidelines. Data regarding patient demographics, IPSS tumor lateralization, and postoperative endocrinologic outcomes were abstracted and pooled with random effects meta-analysis models. Additional meta-regression models were used to examine the association between the accuracy of IPSS tumor lateralization and postoperative outcomes (recurrence/persistence or remission/cure). Statistical analyses were performed using the Comprehensive Meta-Analysis software (significance of P<0.05). Results Seventeen eligible articles were identified, yielding data on 461 patients. Within average follow-up duration (∼59 months), the rate of correct IPSS tumor lateralization was 69% [95% Confidence Interval: 61%, 76%], and the rate of postoperative remission/cure was 78% [67%, 86%]. Preoperative IPSS tumor lateralization was concordant with MRI lateralization for 53% of patients [40%, 66%]. There was no significant association between the rate of correct IPSS tumor lateralization and postoperative remission/cure among study-level data (P=0.735). Additionally, there was no association among subgroup analyses for studies using stimulatory agents during IPSS (corticotropin-releasing hormone or desmopressin, P=0.635), nor among subgroup analyses for adult (P=0.363) and pediatric (P=0.931) patients. Conclusions Limited data suggest that the rate of correct IPSS tumor lateralization may not be positively associated with postoperative remission or cure in patients with Cushing’s disease. These findings bring into question the utility of IPSS tumor lateralization in the context of preoperative planning and surgical approach rather than confirming a pituitary source. From https://www.sciencedirect.com/science/article/abs/pii/S187887502301745X

BACKGROUND Double pituitary adenomas are rare presentations of two distinct adenohypophyseal lesions seen in <1% of surgical cases. Increased rates of recurrence or persistence are reported in the resection of Cushing microadenomas and are attributed to the small tumor size and localization difficulties. The authors report a case of surgical treatment failure of Cushing disease because of the presence of a secondary pituitary adenoma. OBSERVATIONS A 32-year-old woman with a history of prolactin excess and pituitary lesion presented with oligomenorrhea, weight gain, facial fullness, and hirsutism. Urinary and nighttime salivary cortisol elevation were elevated. Magnetic resonance imaging confirmed a 4-mm3 pituitary lesion. Inferior petrosal sinus sampling was diagnostic for Cushing disease. Primary endoscopic endonasal transsphenoidal resection was performed to remove what was determined to be a lactotroph-secreting tumor on immunohistochemistry with persistent hypercortisolism. Repeat resection yielded a corticotroph-secreting tumor and postoperative hypoadrenalism followed by long-term normalization of the hypothalamic-pituitary-adrenal axis. LESSONS This case demonstrates the importance of multidisciplinary management and postoperative hormonal follow-up in patients with Cushing disease. Improved strategies for localization of the active tumor in double pituitary adenomas are essential for primary surgical success and resolution of endocrinopathies. Keywords: pituitary neuroendocrine tumor; PitNET; pituitary adenoma; Cushing disease; prolactinoma; transsphenoidal ABBREVIATIONS ACTH = adrenocorticotrophic hormone; BMI = body mass index; DHEA-S = dehydroepiandrosterone sulfate; FSH = follicle-stimulating hormone; GH = growth hormone; IHC = immunohistochemical; IPSS = inferior petrosal sinus sampling; LH = luteinizing hormone; MRI = magnetic resonance imaging; POD = postoperative day; T4 = thyroxine; TF = transcription factor; TSH = thyroid-stimulating hormone; UFC = urinary free cortisol Pituitary adenomas are adenohypophyseal tumors that can cause endocrinopathies, such as pituitary hormone hypersecretion or anterior hypopituitarism. Cell lineages are used to classify these tumors on the basis of immunohistochemical (IHC) staining of transcription factors, hormones, and other biomarkers.1 Pituitary adenomas differentiate from pluripotent stem cells along one of three lineage pathways, depending on the following active transcription factors (TFs): pituitary transcription factor 1 (PIT-1), T-box transcription factor (TPIT), or steroidogenic factor-1 (SF-1). Rarely, two or more discrete pituitary adenomas from different lineages are identified in patients; however, the etiology remains unclear.2 The incidence of multiple pituitary adenomas has been reported to be 1%–2% of all resected pituitary adenomas but is likely underestimated based on data from large autopsy series.1–4 Pluri-hormonal adenomas are also rare entities in which a single tumor contains multiple TF lineages with one or more hormonal excesses.1–3 Preoperative recognition of multiple or pluri-hormonal pituitary adenomas is rare, and most tumors are discovered incidentally upon autopsy, intraoperatively, or on histological analysis.2,3,5 In cases of multiple synchronous pituitary adenomas, only one hormone excess syndrome is most frequently evident on clinical presentation and endocrine workup. Silent pituitary tumors positive for prolactin on immunohistochemistry are the most prevalent additional, incidentally found tumor in cases of multiple pituitary adenomas.5 This is particularly true in Cushing disease.6,7 It is important to recognize the presence of multiple pituitary adenomas especially in the setting of hormonally active pituitary adenomas to provide optimal management for this subset of patients. Complete resection is curative for Cushing disease with the standard of care achieved through a transsphenoidal approach. Localization of the tumor presents a challenge because of suboptimal sensitivity of magnetic resonance imaging (MRI) in demonstrating microadenomas, the inconsistency of lateralization with inferior petrosal sinus sampling (IPSS), and delays in pathological analysis.1,8,9 Additionally, the presence of an additional pituitary adenoma can obscure the microtumor through its large size and mass effect and can act as a “decoy lesion” during MRI, IPSS, and resection.6 Consideration of multiple pituitary tumors is necessary for successful resection. In a patient with a biochemical picture of Cushing disease, the demonstration of an adenoma with negative adrenocorticotrophic hormone (ACTH) immunostaining and the absence of postoperative hypoadrenalism may indicate the existence of a double adenoma. Few cases have described a lack of remission of an endocrinopathy after transsphenoidal resection due to the presence of an additional adenoma,2,6,10 and even less so in the instance of the persistence of Cushing disease.6 We present a rare case of double pituitary adenomas in a patient presenting with Cushing disease who underwent two endoscopic endonasal transsphenoidal resections and immunostaining for prolactin and ACTH, respectively, with long-term normalization of her hypothalamic-pituitary-adrenal (HPA) axis. Illustrative Case History and Presentation A 32-year-old female, gravida 0 para 0, with a history of a pituitary lesion and hyperprolactinemia presented to our institution for the evaluation for Cushing disease. Ten years earlier, the patient had presented to a gynecologist with hirsutism, galactorrhea, and oligomenorrhea. Her endocrine workup was remarkable for an elevated prolactin at 33.8 ng/mL (2.3–23.3 ng/mL), while follicle-stimulating hormone (FSH), luteinizing hormone (LH), and thyroid-stimulating hormone (TSH) levels were normal. No ACTH or cortisol levels were available. MRI demonstrated a 5 × 6 × 5–mm T1-weighted isointense pituitary lesion protruding into the suprasellar cistern due to a small sella size. She was treated with bromocriptine 2.5 mg daily for 5 years, with normalization of her prolactin level. Subsequent MRI demonstrated a stable lesion size and T1 and T2 hyperintensity in the region of the known pituitary lesion, considered to be posttreatment cystic change with proteinaceous contents and blood. After the normalization of her prolactin levels, she continued to have oligomenorrhea and abnormal hair growth. Polycystic ovaries were not visualized on ultrasound. She was started on oral contraceptives and then switched to the etonorgestrel implant. A decade after initial presentation, she presented to endocrinology at our institution with 3 years of weight gain, hirsutism, and potential oligomenorrhea. Vital signs were stable (blood pressure: 122/86; heart rate: 72 beats/min), and facial fullness and striae on her bilateral breasts were appreciated on physical examination. She was taking isoniazid and pyridoxine for a recent diagnosis of latent tuberculosis and had discontinued bromocriptine 5 years earlier. Her weight was 66.3 kg and body mass index (BMI) was 23.9 kg/m2. She reported that her maternal uncle had a pituitary tumor. Laboratory analysis was positive for elevated urinary free cortisol (UFC) of 109 µg per 24 hours (2.5–45 µg/24 h; Table 1) and nighttime salivary cortisol of 142 ng/mL (<100 ng/dL) with high-normal prolactin of 22.8 ng/mL (2.3–23.3 ng/dL) and normal FSH, LH, TSH, and thyroxine (T4). Dehydroepiandrosterone sulfate (DHEA-S) was 128 µg/dL (98.8–340.0 µg/dL). Imaging demonstrated a 4 × 4 × 4–mm pituitary lesion with decreased T1-weighted and increased central T2-weighted signal intensity in the left lateral pituitary (Fig. 1A–C). Desmopressin (Ferring Pharmaceuticals DDAVP) stimulation increased a basal ACTH of 49.9 pg/mL to ACTH of 91.2 pg/mL, and cortisol increased from 13.7 µg/dL to 21.2 µg/dL, consistent with neoplastic hypercortisolism. IPSS was performed, which showed a right-sided, central-to-peripheral ACTH gradient (Table 2). The patient elected to undergo endoscopic endonasal resection with the initial target as the left-lateral pituitary mass to achieve a cure for Cushing disease. TABLE 1 Urinary free cortisol at baseline and 3, 5, and 7 months after the primary resection Variable Baseline 3 Mos 5 Mos 7 Mos on Osilodrostat Urinary free cortisol (4–50 µg/24 hrs) 109 134.2 125.4 40.3 Urinary creatinine (0.5–2.5 g/24 hrs) 0.995 1.17 1.42 1.11 Urinary vol (mL) 1950 2300 2100 2125 FIG. 1 Preoperative coronal precontrast (A) and postcontrast (B) T1-weighted magnetic resonance imaging (MRI) and T2-weighted MRI (C) demonstrated a 4-mm3 lesion (arrows) with decreased T1 and increased central T2 signal intensity in the left lateral pituitary. Two days after surgery, coronal precontrast (D) and postcontrast T1-weighted (E) and T2-weighted (F) MRI demonstrated the unchanged adenoma. TABLE 2 Preoperative inferior petrosal sinus sampling with corticorelin ovine triflutate 68 µg Time (mins) ACTH (pg/mL) Prolactin (ng/mL) Peripheral Petrosal Sinus ACTH Ratio Peripheral Petrosal Sinus Prolactin Ratio Rt Lt Rt Lt Rt Lt Rt Lt −5 50.6 225 1586 4.45 31.34 21 124 295 5.90 14.05 0 48.8 389 1376 7.97 28.20 22.2 185 198 8.33 8.92 3 69.8 4680 1333 67.05 19.1 22.1 396 32.5 17.92 1.47 5 80.9 4590 1623 56.74 20.06 22.1 436 32.2 19.73 1.46 10 112 4160 1660 37.14 14.82 20.2 367 42 17.90 2.05 ACTH or prolactin ratio = inferior petrosal sinus ACTH or prolactin/peripheral blood ACTH or prolactin. Primary Resection and Outcomes During the primary resection, abnormal tissue was immediately visible after a linear incision along the bottom of the dura, with an excellent plane of dissection. The inferomedial adenoma was distinct from the known left lateral lesion, and the resection was considered complete by the primary neurosurgeon. Subsequently, the left-sided adenoma was not pursued because of the historical prolactinoma diagnosis and an assumption that the newly discovered adenoma was the cause of ACTH hypersecretion. However, pathology of the inferomedial tumor was strongly and diffusely positive for prolactin (Fig. 2B), synaptophysin, and cytokeratin, with an Mindbomb Homolog-1 (MIB-1) proliferative index of 2.4%. ACTH, growth hormone (GH), FSH, LH, and TSH immunostaining were negative. TF immunohistochemistry was not available. On postoperative day (POD) 1, pituitary MRI was performed and demonstrated the unchanged 4-mm3 T1-weighted hypointense lesion with small central T2-weighted hyperintensity in the left lateral gland (Fig. 1D–F). Cortisol levels ranged from 9.7 to 76.2 µg/dL (4.8–19.5 µg/dL), and ACTH was 19.5 pg/mL (7.2–63.3 pg/mL) on POD 1. FIG. 2 Histological examination of surgical specimens from the inferomedial (A–C) and left lateral (D–F) lesions. The initial resection (hematoxylin and eosin [H&E], A) was strongly and diffusely positive for prolactin (B) with normal reticulin levels (C) indicating a lactotrophic pituitary adenoma. The second operation (H&E, D) was diagnostic for a corticotropic pituitary adenoma with diffusely positive adrenocorticotrophic hormone (ACTH) (E) and decreased reticulin (F). Original magnification ×100. Early reoperation was discussed with the patient based on the pathology and persistent hypercortisolism; however, she elected to pursue conservative management with close follow-up. Postoperative cortisol nadir was 4.8 µg/dL (4.8–19.5 µg/dL) on POD 2 during her 4-day hospital stay. DHEA-S was significantly decreased from baseline at 22.3 µg/dL (98.8–340.0 µg/dL) and a prolactin level of 3.4 ng/mL (2.3–23.3 ng/dL) was low-normal. No glucocorticoids were administered during her hospital course. There was no clinical evidence of vasopressin deficiency while she was an inpatient. Three months postoperatively, the patient reported insomnia, poor hair quality, fatigue, nocturnal sweating, and continued increasing weight gain with fat accumulation in the supraclavicular and dorsal cervical area. She had one spontaneous menstrual period despite the use of etonogestrel implant. UFC was increased at 134.2 µg/24 hours (4–50 µg/24 h; Table 1). The 8:00 am serum cortisol was 10.2 µg/dL (5.0–25.0 µg/dL). She was started on osilodrostat 2 mg twice daily for her persistent hypercortisolism, and she reported some clinical improvement; however, she had continued elevation in her late-night salivary cortisol levels up to 7.0 nmol/L. Other endocrine lab work was normal, with a prolactin of 13.5 ng/mL (2.8–23.3 ng/mL) and TSH of 3.67 µIU/mL (0.4–4.0 µIU/mL). Her weight had increased by 4.9 kg to 71.2 kg with a BMI of 25.3 kg/m2. Approximately 6 months postoperatively, she was amenable to a secondary resection targeting the remaining left lateral pituitary adenoma. Secondary Resection and Outcomes After obtaining adequate exposure for the secondary resection, the lesion in the left lateral aspect of the pituitary was targeted. The tumor was clearly identified and completely resected without intraoperative complication. IHC staining was diffusely positive for ACTH (Fig. 2E), synaptophysin, and cytokeratin with decreased reticulin and an MIB-1 index of 3.3%. Prolactin, GH, TSH, LH, and FSH immunostaining were negative. Postoperative cortisol monitoring demonstrated decreased levels, with a nadir of 2.0 µg/dL on POD 0. Levels of ACTH and DHEA-S were decreased at 4.4 pg/mL (7.2–63.3 pg/mL) and 13.3 µg/dL (98.8–340 µg/dL), respectively, on POD 1. Prolactin remained within the normal range at 8.2 ng/mL (2.8–23.3 ng/mL). The patient was started on intravenous hydrocortisone 50 mg every 8 hours for adrenal insufficiency. Postoperative symptoms of nausea, headache, and muscle weakness resolved with hydrocortisone administration. She was discharged on hydrocortisone 60 mg daily in divided doses for adrenal insufficiency and had no signs of vasopressin deficiency during her 2-day hospital course. By 3 months, the patient reported decreased fatigue, myalgia, and insomnia and improved overall well-being and physical appearance. She was weaned down to a total daily dose of 20 mg of hydrocortisone and had lost 5.2 kg. Her menstruation returned while having an etonogestrel implant. Rapid ACTH stimulation was abnormal, with decreased cortisol at 30 minutes of 4.1 µg/dL (7.2–63.3 pg/mL) demonstrating continued adrenal insufficiency. Follow-up MRI demonstrated miniscule remaining left pituitary adenoma (Fig. 3). Seven months after her second surgery, she was started on 50 µg levothyroxine for primary hypothyroidism in the setting of slightly elevated TSH of 4.1 µIU/mL (0.4–4.0 µIU/mL) and a low-normal T4 of 0.8 ng/dL (0.7–1.5 ng/dL). FIG. 3 Postoperative imaging 3 months after the second operation demonstrates near gross-total resection (yellow arrows: surgical cavity) of the left lateral pituitary adenoma on coronal precontrast (A) and postcontrast T1-weighted (B) and T2-weighted (C) MRI. Two years after the second resection, the patient lost 10.1 kg (weight, 61.1 kg; BMI, 21.76 kg/m2). Her ACTH stimulation test became normal, and hydrocortisone therapy was discontinued. At the 2-year time point, the patient and her husband successfully conceived a child. Patient Informed Consent The necessary patient informed consent was obtained in this study. Discussion Double or multiple pituitary adenomas are discovered in 0.37%–2.6% of resected pituitary lesions.3,4,6,11,12 A majority of multiple pituitary adenomas are not suspected before surgery with an inconclusive clinical presentation or endocrine laboratory workup.6 The presentation of multiple synchronous neoplasms is thought to be more common than having a single neoplasm with multiple lineages.1 Studies have shown that additional pituitary adenomas are seen at a rate of 1.6%–3.3% in Cushing disease in studies including both contiguous and noncontiguous double pituitary adenomas.6 Additional pituitary adenomas that are hormonally active make up 40% of resected double pituitary adenomas, with most staining for gonadotroph adenoma.13 Overall, the most common incidental pituitary adenoma is prolactinoma,6 which occurs most frequently with GH or ACTH adenomas.5 In very rare instances, Cushing cases can present with hyperprolactinemia and Cushing synchronously.6 Hormonal secretion and clinical presentation are variable, with the pathology most often attributed to only one component of double pituitary adenoma.3,14 The multiple-hit theory is the most common hypothesis for double pituitary adenoma etiology with coincidental monoclonal expansion of two or more lineages, which present with separate pseudo-capsules for each lesion.15 Observations On presenting with Cushing disease, the differential diagnosis before the initial operation considered that the known left lateral pituitary adenoma could be a mixed tumor with both prolactin and ACTH lineages. Therefore, it was the initial target of the resection until discovering the second adenoma intraoperatively. With two distinct adenomas, the inferomedial adenoma was presumed to be the source of the ACTH hypersecretion and was subsequently resected. The left lesion was thought to be a prolactinoma and hormonally inactive after historical dopaminergic therapy and thus was not pursued during the initial surgery. However, pathology confirmed that the opposite was true. Few cases have also involved incidental pituitary tumors that look like the hormonally active adenoma and encourage resection of it, leaving the primary pituitary adenoma behind.6,7 It has been reported that these “decoy lesions” can cause surgical failure and require secondary operations.6,7,10,16 Intraoperative localization and confirmation of the adenoma classification may have also been helpful during the case, including tissue-based ACTH antibody assay,9 plasma ACTH measurements with a immunochemiluminometric method,17 or intraoperative ultrasound.5,6 The inferomedial second tumor was not appreciated or reported throughout her serial MRI studies from 2010 to 2020. Interestingly, imaging did demonstrate the left pituitary adenoma that was medically treated as a prolactinoma, although it was later diagnosed as an ACTH-secreting lesion on IHC staining. Preoperative visualization of a pituitary adenoma in Cushing disease is reported to be limited, with a reported 50% incidence with negative MRI with standard 1.5 T.1,18,19 MRI technical refinements in magnet strength, slice thickness, or enhanced spin sequences have increased sensitivity, but one-third of patients with Cushing disease still have negative scans.20 Small prolactinomas, especially those near the cavernous sinus, are also notoriously difficult to visualize on MRI, although recent advances using co-registration of 11C-methionine positron emission tomography–computed tomography with MRI (Met-PET/MRICR) may prove useful.21 Difficulty with preoperative visualization complicates a diagnosis of multiple adenomas, with or without multiple endocrinopathies, and negatively affects surgical planning. In a single-institution retrospective review of MRI in all cases of double pituitary tumors, only one of eight patients (12.5%) over 16 years of age had a positive MRI for double pituitary tumors and was diagnosed preoperatively.2 The patient’s preoperative IPSS demonstrated a right central-to-peripheral gradient. This was incongruent with the MRI demonstrating the single left-sided tumor. While IPSS is useful in confirming Cushing disease, its sensitivity for lateralization has been reported at only 59%–71%.9 With this in mind and a known left-sided adenoma on MRI, exploration of the right side of the pituitary was not originally planned. Ultimately, the left-sided adenoma was the source of ACTH hypersecretion, which remains incongruent with preoperative IPSS. It has been suggested that multiple pituitary adenomas in Cushing disease could further decrease its accuracy.1,6 The patient’s initial historical prolactin levels (33.8 ng/dL) were lower than reported levels of 100–250 ng/dL for microadenoma and >250 ng/dL in cases of macroadenoma. Normally, in active single prolactinoma, prolactin secretion is correlated to size. We do not suspect that the presence of more than one pituitary adenoma would affect the level of prolactin hypersecretion.6 Slight elevations in prolactin can be attributed to causes such as pituitary stalk effect, medications, and physiological stimulation. During the 5 years of bromocriptine therapy, the effect on the inferomedial prolactinoma was unknown, as it was not appreciated on MRI. There are reports of prolactinomas being less responsive to dopaminergic agonist therapy in cases of double adenomas.14,22 Upon resection of the inferomedial prolactinoma during the initial operation, there was no further change in the patient’s prolactin levels, which could most likely be attributed to prior dopaminergic therapy. Unfortunately, the initial endocrine laboratory workup did not include levels of ACTH or cortisol. In addition to hyperprolactinemia, Cushing disease can also present with changes in menstruation. After the secondary resection and removal of the ACTH-secreting pituitary adenoma, the patient’s oligomenorrhea resolved and she achieved pregnancy. Retrospectively, it remains unclear if the prolactinoma was once truly active hormonally. Lessons The rare presence of two pituitary adenomas can complicate the diagnosis, medical and surgical management, and long-term outcomes for patients. A complete endocrine workup is essential when a pituitary adenoma is suspected and can help screen for pluri-hormonal and multiple pituitary adenomas. In our patient, it is unknown when the onset of hypercortisolism was with the limited initial hormonal workup. Currently, localizing and resecting the hormonally active adenoma in double or multiple pituitary adenomas remain a challenge, with limitations in preoperative imaging and intraoperative measures. After encountering the additional inferomedial lesion during surgery, resection of both adenomas during the initial surgery may have been prudent to ensure the resolution of Cushing disease. Although exploration for additional pituitary adenomas is not usually recommended, it could be considered in cases of multiple pituitary adenomas and uncertainty of the culprit of Cushing disease. The current characterization of pituitary tumors by the World Health Organization includes immunohistochemistry for both transcription factors and pituitary hormones, with clinical usefulness to be determined by future studies. Multiple lineages can occur mixed in a single pituitary adenoma or across different noncontiguous adenomas and can only be determined by TF immunostaining. The left ACTH-staining lesion in our patient had some shrinkage and MRI changes, which may have been a response to dopaminergic therapy. Full characterization of the tumor cell lineages in this case remains undetermined without staining for TFs. In conclusion, we report a rare case of Cushing disease concurrent with a prolactinoma leading to the need for repeat resection. This is one of the few reported cases of a double pituitary adenoma leading to a lack of biochemical remission of hypercortisolism after the initial surgery. Strategies for localization of the active tumor in double pituitary adenomas are essential for primary surgical success and the resolution of endocrinopathies. Author Contributions Conception and design: Zwagerman, Tavakoli, Shah, Findling. Acquisition of data: Zwagerman, Armstrong, Tavakoli, Shah, Ioachimescu, Findling. Analysis and interpretation of data: Zwagerman, Armstrong, Tavakoli, Shah, Coss, Ioachimescu, Findling. Drafting of the article: Zwagerman, Armstrong, Shah. Critically revising the article: Zwagerman, Armstrong, Tavakoli, Shah, Ioachimescu, Findling. Reviewed submitted version of the manuscript: Zwagerman, Armstrong, Tavakoli, Shah, Laing, Ioachimescu, Findling. Approved the final version of the manuscript on behalf of all authors: Zwagerman. Statistical analysis: Armstrong, Shah. Administrative/technical/material support: Zwagerman, Armstrong, Shah. Study supervision: Zwagerman, Tavakoli, Shah, Laing. References 1↑ Asa SL, Mete O, Perry A, Osamura RY. Overview of the 2022 WHO Classification of Pituitary Tumors. Endocr Pathol. 2022;33(1😞6–26. PubMed Search Google Scholar Export Citation 2↑ Roberts S, Borges MT, Lillehei KO, Kleinschmidt-DeMasters BK. Double separate versus contiguous pituitary adenomas: MRI features and endocrinological follow up. Pituitary. 2016;19(5😞472–481. PubMed Search Google Scholar Export Citation 3↑ Mete O, Alshaikh OM, Cintosun A, Ezzat S, Asa SL. Synchronous multiple pituitary neuroendocrine tumors of different cell lineages. Endocr Pathol. 2018;29(4😞332–338. PubMed Search Google Scholar Export Citation 4↑ Kontogeorgos G, Kovacs K, Horvath E, Scheithauer BW. Multiple adenomas of the human pituitary. A retrospective autopsy study with clinical implications. J Neurosurg. 1991;74(2😞243–247. PubMed Search Google Scholar Export Citation 5↑ Budan RM, Georgescu CE. Multiple pituitary adenomas: a systematic review. Front Endocrinol (Lausanne). 2016;7:1. 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PubMed Search Google Scholar Export Citation 19↑ Escourolle H, Abecassis JP, Bertagna X, et al. Comparison of computerized tomography and magnetic resonance imaging for the examination of the pituitary gland in patients with Cushing’s disease. Clin Endocrinol (Oxf). 1993;39(3😞307–313. PubMed Search Google Scholar Export Citation 20↑ Grober Y, Grober H, Wintermark M, Jane JA, Oldfield EH. Comparison of MRI techniques for detecting microadenomas in Cushing’s disease. J Neurosurg. 2018;128(4😞1051–1057. PubMed Search Google Scholar Export Citation 21↑ Bakker LEH, Verstegen MJT, Ghariq E, et al. Implementation of functional imaging using 11C-methionine PET-CT co-registered with MRI for advanced surgical planning and decision making in prolactinoma surgery. Pituitary. 2022;25(4😞587–601. PubMed Search Google Scholar Export Citation 22↑ Coiré CI, Smyth HS, Rosso D, Horvath E, Kovacs K. A double pituitary adenoma presenting as a prolactin-secreting tumor with partial response to medical therapy. Case report. Endocr Pathol. 2010;21(2😞135–138. PubMed Search Google Scholar Export Citation From https://thejns.org/caselessons/view/journals/j-neurosurg-case-lessons/6/22/article-CASE23485.xml

Abstract The occurrence of a second neoplasm possibly constitutes an adverse and uncommon complication after radiotherapy. The incidence of a second pituitary tumor in patients irradiated for adrenocorticotropic hormone secreting pituitary adenoma is rare. We report a case of a 40-year-old female with Cushing disease who underwent surgical management followed by radiotherapy. After 5 years of initial treatment, an increase in tumor size was evident at the same location, with a significant interval growth of the parasellar component of the lesion. Histology revealed an undifferentiated highly malignant sarcoma. In the span of next 2 years, the patient was followed with 2 repeat decompression surgeries and radiotherapy because of significant recurrent compressive symptoms by locally invasive malignant tumor. Despite the best efforts, the patient remained unresponsive to multiple treatment strategies (eg, surgical resections and radiotherapy) and succumbed to death. radiotherapy, second malignancy, Cushing disease Issue Section: Case Report Introduction Radiation therapy is a commonly used modality for primary or adjuvant treatment of pituitary adenoma. It is also used as an adjuvant therapy for Cushing disease with persistent or aggressive tumor growth or recurrent disease after surgery. The immediate sequelae of radiotherapy for pituitary tumors include nausea, fatigue, diminished taste and olfaction, and hair loss [1]. One frequent long-term side effect is hypopituitarism. The incidence rate of new-onset hypopituitarism after conventional radiotherapy is approximately 30% to 100% after a follow-up of 10 years, whereas after stereotactic radiosurgery or fractionated radiotherapy, the incidence is approximately 10% to 40% at 5 years [2]. The occurrence of a second neoplasm after cranial radiotherapy constitutes possibly one of the most adverse complications. Tumors such as meningioma, glioma, and sarcoma are the most frequently reported secondary neoplasms after pituitary irradiation [3]. The cumulative probability of a second brain tumor in patients irradiated for pituitary adenoma and craniopharyngioma is approximately 4% [4]. We report 1 such case with detailed clinical, histopathological, and radiological characteristics because of its rarity and associated high mortality of radiation-induced sarcoma. Case Presentation The patient first presented at 40 years of age with complaints of weight gain, new-onset diabetes mellitus, hypertension, and cushingoid features in 2014. She was diagnosed with Cushing disease (24-hour urinary cortisol 1384 mcg/24 hours [3819 nmol/24 hours; reference >2 upper limit of normal], low-dose dexamethasone suppression test serum cortisol 16.6 mcg/dL [457.9 nmol/L], ACTH 85 pg/mL [18.7 pmol/L; reference range, <46 pg/mL, <10 pmol/L]) caused by invasive adrenocorticotropic hormone-secreting giant adenoma. The initial imaging revealed a homogenously enhanced pituitary macroadenoma with a size of 42 × 37 × 35 mm with suprasellar extension and encasing both the internal carotid arteries with mass effect on optic chiasma and sellar erosion. The patient underwent tumor excision by endoscopic transsphenoidal transnasal approach. Partial excision of the tumor was achieved because of cavernous sinus invasion. Histopathology and immunohistochemical stains demonstrated a corticotrophin-secreting (ACTH-staining positive) pituitary adenoma with MIB labeling index of 1% to 2%. Because biochemical remission was not achieved (urinary cortisol 794 mcg/24 hours [2191 nmol/24 hours]; ACTH 66 pg/mL [14.5 pmol/L; reference range, <46 pg/mL, <10 pmol/L]), the patient was started on ketoconazole and was received fractionated radiotherapy with a dose of 5040 cGy in 28 fractions. Diagnostic Assessment For the next 5 years, at yearly follow-up, 400 mg ketoconazole was continued in view of insufficient control of ACTH secretion. During follow-up, the size of the tumor was stable at approximately 23 × 16 × 33 mm after radiotherapy with no significant clinical and biochemical changes. Five years after surgery and radiotherapy, the patient developed cerebrospinal fluid rhinorrhea; imaging revealed a cystic transformation of the suprasellar component and increase in the size of the tumor to 39 × 22 × 26 mm, which included visualization of a parasellar component of size 29 × 19 × 15 mm. The patient continued on ketoconazole. The patient was also advised to undergo hypofractionated radiotherapy but did not return for follow-up. Treatment In 2021, 1.5 years after the last visit, the patient developed severe headache, altered sensorium, ptosis, focal seizures, and left-sided hemiparesis. During this episode, the patient had an ACTH of 66 pg/mL (14.53 pmol/L; reference range, <46 pg/mL [<10 pmol/L]) and baseline cortisol of 25 mcg/dL (689 nmol/L; reference range, 4-18 mcg/dL [110-496 nmol/L]). Repeat imaging revealed a significant decrease in the suprasellar cystic component but an increase in the size of the parasellar component to 38 × 21 × 25 mm from 29 × 19 × 15 mm, which was isointense on T1 and T2 with heterogeneous enhancement. Significant brain stem compression and perilesional edema was also visible. The patient underwent urgent frontotemporal craniotomy and decompression of the tumor. On pathological examination, the tumor tissue was composed of small pleomorphic round cells arranged in sheets and cords separated by delicate fibrocollagenous stroma. Cells had a round to oval hyperchromatic nucleus with scanty cytoplasm. Areas of hemorrhage, necrosis, and a few apoptotic bodies were seen. The tumor tissue had very high mitotic activity of >10/10 hpf and MIB labeling index of 70%. Immunohistochemistry demonstrated positivity for vimentin, CD99, and TLE-1. Dot-like positivity was present for HMB 45, synaptophysin. INI-1 loss was present in some cells. Ten percent patchy positivity was present for p53. The tumor cells, however, consistently failed to express smooth muscle actin, CD34, Myf-4, epithelial membrane antigen, desmin, LCA, SADD4, CD138, and S-100 protein. ACTH and staining for other hormones was negative. Based on the immunological and histochemical patterns, a diagnosis of high-grade poorly differentiated malignant tumor with a probability of undifferentiated sarcoma was made. Because of the invasion of surrounding structures and surgical inaccessibility, repeat fractionated radiotherapy was given with a dose of 4500 cGy over 25 fractions at 1.8 Gy daily to the planned target volume via image-guided fractionated radiotherapy. During the next 1.5 years, patient improved clinically with no significant increase in the size of tumor (Fig. 1). The patient was gradually tapered from ketoconazole and developed hypopituitarism requiring levothyroxine and glucocorticoid replacement. There was a significant improvement in the power of the left side and ptosis. Figure 1. Open in new tabDownload slide Contrast-enhanced T1 magnetic resonance imaging dynamic pituitary scan (A, sagittal; B, axial; C, coronal sections) reveals postoperative changes with residual enhancing tumor in the right lateral sella cavity with extension into the right cavernous sinus and parasellar region encasing the cavernous and inferiorly extends through the foramen ovale below the skull base up to approximately 1.5 cm. Anteriorly, it extends up to the right orbital apex and posteriorly extends along the right dorsal surface of clivus. Outcome and Follow-up After 1.5 years of reradiation in 2022, the patient again developed palsies of the abducens, trigeminal, oculomotor, and trochlear cranial nerve on the right side and left-sided hemiparesis. A significant increase in tumor size to 50 × 54 × 45 mm with anterior, parasellar, and infratentorial extension was seen (Fig. 2). Again, repeat decompression surgery was done. Two months after surgery, there was no improvement in clinical features and repeat imaging suggested an increased size of the tumor by 30%, to approximately 86 × 68 × 75 mm. Nine years after initial presentation, the patient had an episode of aspiration pneumonia and died. Figure 2. Open in new tabDownload slide Contrast-enhanced T1 magnetic resonance imaging dynamic pituitary images (A, sagittal; B, axial; C, coronal sections) after 1.5 years of a second session of radiotherapy reveal a significant interval increase in size of heterogeneously enhancing irregular soft tissue in sellar cavity with extension into the right cavernous sinus and parasellar region when compared with previous imaging. Superiorly, it extends in the suprasellar region, causing mass effect on the optic chiasma with encasement of the right prechiasmatic optic nerve and right-sided optic chiasma. Inferiorly, the lesion extends into the sphenoid sinus. Posteriorly, there is interval increase in the lesion involving the clivus and extending into the prepontine and interpeduncular cistern. Anteriorly, mass has reached up to the right orbital apex optic nerve canal, which shows mild interval increase. Discussion Radiation-induced tumors were initially described by Cahan et al in 1948. They also described the prerequisites for a tumor to be classified as a radiation-induced sarcoma [5]. The modified Cahan criteria state that (1) the presence of nonmalignancy or malignancy of a different histological type before irradiation, (2) development of sarcoma within or adjacent to the area of the radiation beam, (3) a latent period of at least 3 years between irradiation and diagnosis of secondary tumor, and (4) histological diagnosis of sarcoma, can be classified as radiation-induced sarcoma [5]. Our patient fulfilled the criteria for a radiation-induced sarcoma with a highly malignant tumor on histopathology. Radiation-induced sarcomas after functional pituitary tumors, especially Cushing disease, are rarely reported. One of the case reports revealed a high-grade osteoblastic osteosarcoma 30 years after treatment for Cushing disease with transsphenoidal resection and external beam radiotherapy [6]. In our case, there was a lag period of approximately 5 years before the appearance of a second highly undifferentiated, malignant, histologically distinct tumor. The cellular origin of this relatively undifferentiated tumor cannot be determined with certainty. However, the interlacing sarcomatous and adenomatous components resulting from distinct positive immunohistochemistry may indicate that the sarcomatous component may be derived from the preexisting pituitary adenoma. A hormonally functional pituitary tumor is not itself expected to be associated with an increased risk of secondary malignancy, except in the case of GH-secreting tumors and those with a hereditary cancer syndrome. Although not proven, immunosuppression from hypercortisolism in Cushing disease has been proposed as a contributor to secondary tumor development [7]. Other mechanisms causing increased risk of secondary malignancy can be double-stranded DNA damage and genomic instability caused by ionizing radiation and germline mutations in tumor suppressor genes such as TP53 and Rb [7]. Radiation-induced intracranial tumors were studied in a multicenter, retrospective cohort of 4292 patients with pituitary adenoma or craniopharyngioma. Radiotherapy exposure was associated with an increased risk of a second brain tumor with a rate ratio of 2.18 (95% CI, 1.31-3.62, P < .0001). The cumulative probability of a second brain tumor was 4% for the irradiated patients and 2.1% for the controls at 20 years [7]. In another study including 426 patients irradiated for pituitary adenoma between 1962 and 1994, the cumulative risk of second brain tumors was 2.0% (CI, 0.9-4.4) at 10 years and 2.4% (95% CI, 1.2-5.0) at 20 years. The relative risk of a second brain tumor compared with the incidence in the normal population is 10.5 (95% CI, 4.3-16.7) [8]. The incidence of radiation-induced sarcomas has been estimated at 0.03% to 0.3% of patients who have undergone radiation therapy. The risk of radiation-induced sarcomas increases with field size and dose. In a systemic review and analysis of 180 cases of radiation-induced intracranial sarcomas, the average dose of radiation delivered was 51.4 ± 18.6 Gy and latent period of sarcoma onset was 12.4 ± 8.6 years. A total of 49 cases were developed after radiation treatment of pituitary adenomas (27.2%). The median overall survival time for all patients with sarcoma was 11 months, with a 5-year survival rate of 14.3% [9]. Our patient received approximately 50 Gy twice through fractionated radiotherapy, resulting in larger field size and significantly higher dose than one would expect with a modern stereotactic treatment. Such a high dose of radiation is indeed a risk factor for secondary malignancy. In our patient, in a period of 2 months, there was already >30% tumor growth after recent repeat decompression surgery. The risk of secondary malignancy is thought to be much lower with stereotactic radiosurgery than conventional external beam radiation therapy, with an estimated cumulative incidence of 0.045% over 10 years (95% CI, 0.00-0.34) [10]. However, long-term follow-up data for patients receiving stereotactic radiation therapy are shorter and thus definitive conclusions cannot be made at this stage. Our case highlights a rare but devastating long-term complication of pituitary tumor irradiation after Cushing disease. The limited response to various available treatment options defines the aggressive nature of radiation-induced malignancy. Learning Points The occurrence of a second neoplasm constitutes possibly one of the most adverse and rare complication after radiotherapy. The incidence of radiation-induced sarcomas has been estimated at 0.03% to 0.3% of patients, but cases after Cushing disease are rarely reported. Patients often present with advanced disease unresponsive to various treatment modalities because of aggressive clinical course. New modalities with stereotactic radiosurgery and proton beam therapy are to be reviewed closely for risk assessment of secondary tumor. Acknowledgments The authors acknowledge Dr. Ishani Mohapatra for her support with histopathology and interpretation. Contributors All authors made individual contributions to authorship. G.B., S.K.M., and V.A.R. were involved in diagnosis and management of the patient. G.B. was involved in the writing of this manuscript and submission. V.P.S. was responsible for patient surgeries. All authors reviewed and approved the final draft. Funding The authors received no financial support for the research, authorship, and/or publication of this article. Disclosures The authors have nothing to disclose. Informed Patient Consent for Publication Signed informed consent could not be obtained from the patient or a proxy but was approved by the treating institute. Data Availability Statement Data sharing is not applicable to this article as no data sets were generated or analyzed during the current study. © The Author(s) 2023. Published by Oxford University Press on behalf of the Endocrine Society. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. From https://academic.oup.com/jcemcr/article/1/6/luad119/7343968?login=false

Abstract Introduction: Chronic exposure to excessive endogenous cortisol leads to brain changes in Cushing’s disease (CD). However, it remains unclear how CD affects large-scale functional networks (FNs) and whether these effects are reversible after treatment. This study aimed to investigate functional network changes of CD patients and their reversibility in a longitudinal cohort. Methods: Active CD patients (N = 37) were treated by transsphenoidal pituitary surgery and reexamined 3 months later. FNs were computed from resting-state fMRI data of the CD patients and matched normal controls (NCs, N = 37). A pattern classifier was built on the FNs to distinguish active CD patients from controls and applied to FNs of the CD patients at the 3-month follow-up. Two subgroups of endocrine-remitted CD patients were identified according to their classification scores, referred to as image-based phenotypically (IBP) recovered and unrecovered CD patients, respectively. The informative FNs identified by the classification model were compared between NCs, active CD patients, and endocrine-remitted patients as well as between IBP recovered and unrecovered CD patients to explore their functional network reversibility. Results: All 37 CD patients reached endocrine remission after treatment. The classification model identified three informative FNs, including cerebellar network (CerebN), fronto-parietal network (FPN), and default mode network. Among them, CerebN and FPN partially recovered toward normal at 3 months after treatment. Moreover, the informative FNs were correlated with 24-h urinary-free cortisol and emotion scales in CD patients. Conclusion: These findings suggest that CD patients have aberrant FNs that are partially reversible toward normal after treatment. Journal Section: Research Article Keywords: Cushing’s disease, Reversibility, Functional networks, Cortisol, Emotion Introduction Cushing’s disease (CD) is characterized by chronic exposure to excessive endogenous glucocorticoid most commonly caused by an adrenocorticotropic hormone (ACTH) pituitary adenoma [1, 2]. The CD is accompanied by multiple physical manifestations such as hypertension and osteoporosis, as well as various neuropsychiatric symptoms including memory lapses, attention deficits, executive function decline, emotional dysfunction, visual-spatial disability, and language defects [3‒14]. These neuropsychiatric symptoms are indicative of the effects of CD on the brain anatomy and function. Therefore, CD provides a unique and naturalistic model for investigating both the effects of hypercortisolism on the human brain and the reversibility of these effects after resolution of hypercortisolism. Recent studies have documented brain structural and metabolic abnormalities in CD patients with a variety of neuroimaging techniques, including structural magnetic resonance imaging (sMRI) [11, 12, 15‒24], diffusion tensor imaging [10, 25‒27], proton magnetic resonance spectroscopy [21, 28‒30], positron emission topography [21, 31], and arterial spin labeling [32]. These studies have shown that brain structural and metabolic abnormalities in CD patients can be partially restored after resolution of hypercortisolism [16, 18, 20‒22, 24, 32‒34], typically after transsphenoidal pituitary surgery (TSS), a safe and effective first-line treatment with a high endocrine remission rate [35, 36]. Several functional magnetic resonance imaging (fMRI) studies have also documented brain functional abnormalities in CD patients [37‒42]. Particularly, aberrant functional connectivity between the anterior cingulate cortex and the limbic network, as well as the lateral occipital cortex and the default mode network (DMN) was observed in endocrine-remitted CD patients after TSS treatment in a cross-sectional resting-state fMRI (rs-fMRI) study [40]. However, the causal effects of hypercortisolism on brain functional connectivity cannot be well investigated in CD patients only through the cross-sectional study. Additionally, the large-scale functional networks (FNs) of CD patients were not well investigated through univariate analyses in previous studies, which only examined one or few FNs in CD patients independently [37‒42]. The present study aims to jointly investigate a number of whole-brain large-scale intrinsic FNs and their reversibility due to hypercortisolism in CD patients based on rs-fMRI with a longitudinal design through multivariate analysis. Particularly, intrinsic FNs altered by CD were identified using a multivariate pattern classification model optimized by selecting intrinsic FNs informative for distinguishing CD patients from matched normal controls (NCs). The changes in these informative FNs of endocrine-remitted CD patients after TSS treatment were quantified at the 3-month follow-up with the established pattern classification model. Furthermore, changes in clinical measures, including serum cortisol, 24-h urinary-free cortisol (24hUFC), ACTH, self-rating depression scale (SDS), and self-rating anxiety scale (SAS), were detected between active and endocrine-remitted CD patients using pseudo paired t tests. Finally, the association between aberrant FNs and clinical measures was investigated in CD patients. Materials and Methods Participants In this study, 50 CD patients undergoing TSS, and 38 NCs with no history of glucocorticoid treatment were recruited at the Department of Neurosurgery, Peking Union Medical College Hospital. All these participants were assessed for depression and anxiety measured by the SDS and SAS, respectively [43]. The inclusion criteria for NCs were no past or present heart history of disease, atherosclerosis, hyperlipidemia, diabetes, neurological/psychiatric disorders, and claustrophobia. The exclusion criteria for both CD patients and NCs were past or present brain trauma, other neurological disorders, history of radiotherapy, or contraindications to MRI. Besides the inclusion and exclusion criteria, the quality of the imaging data was controlled as follows. No participant had head motion exceeding 2.0 mm translation in any of the three directions or exceeding 2.0o maximum rotation around any of the axes during rs-fMRI scanning [44]. Additionally, no participant had root-mean-square value of maximum frame-wise displacement greater than 0.3 mm [45]. After quality control of the imaging data, 37 CD patients and 37 sex-, age-, and education level-matched NCs were included in the study. The diagnosis of active CD was confirmed by experienced endocrinologists along with dynamic enhanced pituitary MRI, low- and high-dose dexamethasone suppression tests, and/or inferior petrosal sinus sampling in accordance with the latest clinical practice guidelines [46]. The 37 active CD patients were treated with TSS rather than radiotherapy. All of the 37 CD patients reached endocrine remission after treatment, which was confirmed by their normal serum cortisol (<5 µg/dL within 7 days of surgery) [46]. These patients were asked to revisit the hospital for reexamination 3 months after surgery, and all of them had no recurrence at the 3-month follow-up. Serum cortisol, 24hUFC, and ACTH were measured by direct chemiluminescence immunoassays in CD patients before surgery and at the 3-month follow-up (Siemens Healthcare Diagnostics Inc., USA). This study was approved by the Medical Ethics Committee of Peking Union Medical College Hospital, and written informed consent was obtained from all participants after explaining to them the nature of the study. Imaging Data Acquisition The MRI data were scanned by using an 8-channel phase-array head coil with a 3.0-Tesla MR scanner (Discovery MR750, General Electric) for all participants, including NCs, active CD patients, and endocrine-remitted CD patients without recurrence at the 3-month follow-up. The rs-fMRI data were acquired axially by using a gradient echo-planar imaging sequence, and the scanning parameters were 200 whole-brain volumes, 36 transverse slices with a thickness of 4 mm, in-plane resolution = 3.75 × 3.75 mm2, field of view = 240 × 240 mm2, flip angle = 90°, repetition time = 2,000 ms, and echo time = 30 ms. The extra high-resolution sagittal 3D T1-weighted data were acquired by using a brain volume sequence, and the scanning parameters were 172 slices with a thickness of 1.0 mm, in-place matrix = 512 × 512, field of view = 256 × 256 mm2, voxel size = 0.5 × 0.5 × 1.0 mm3, flip angle = 12°, repetition time = 7.2 ms, echo time = 3.2 ms, and inversion time = 400 ms. Imaging Data Preprocessing The rs-fMRI data were preprocessed as follows: (1) discarding the first four volumes of the fMRI data; (2) correction for slice timing; (3) 3D rigid-body correction for head motion to the middle frame of the data; (4) global 4D intensity scaling of the fMRI data to yield a mean value of 10,000; (5) nonlinear registration of the fMRI data to the MNI template with the deformation field obtained from its co-registered T1-weighted data using DARTEL within statistical parametric mapping (SPM12) software, with a resampled resolution of 3×3×3 mm3; (6) spatial smoothing with a 6-mm full-width at half maximum Gaussian kernel; (7) motion artifacts removal from fMRI data with ICA-AROMA; (8) regressing out averaged signals of white matter, cerebrospinal fluid, and whole brain; (9) temporal band-pass filtering (0.009–0.08 Hz). The preprocessing procedures were performed by using SPM12 software (https://www.fil.ion.ucl.ac.uk/spm/software/spm12/). Identification of Informative FNs in Active CD Patients The flowchart for identifying informative FNs in active CD patients is shown in Figure 1. First, group information-guided independent component analysis was applied to rs-fMRI data of each participant from NCs, active CD patients, and endocrine-remitted CD patients at 3 months after treatment to extract subject-specific independent components (ICs), referred to as intrinsic FNs [47] (Fig. 1a). Specifically, group-level ICs were computed based on all participants from NC, active CD, and endocrine-remitted CD groups, by using the multivariate exploratory linear optimized decomposition into independent components (MELODIC) toolbox in FSL software (https://fsl.fmrib.ox.ac.uk/fsl/fslwiki/melodic). These group-level ICs were used as guidance information to compute subject-specific ICs of all individuals [47]. The number of ICs was empirically set to be 25, and therefore each individual was characterized by 25 FNs. Particularly, these FNs were restricted to gray matter in order to minimize the partial volume effects of cerebrospinal fluid and confounding effects on the estimated components, and to improve the sensitivity to the changes of blood-oxygen-level-dependent signals. Fig. 1. VIEW LARGEDOWNLOAD SLIDE Flowchart of the multivariate pattern classification method for distinguishing active CD patients from NCs, including data preparation (a), classification modeling (b), as well as identifying CD-associated ICs (c). CD, Cushing’s disease; active CD patients, CD patients before treatment; NCs, normal controls; rs-fMRI, resting-state functional magnetic resonance imaging; ICs, independent components; GIG-ICA, group information-guided ICA; SVM, support vector machine; LOOCV, leave-one-out cross-validation. Subsequently, a multivariate pattern classification method based on support vector machine (SVM) was applied to identify cross-sectional informative FNs, which were most discriminative in distinguishing active patients from NCs [48] (Fig. 1b). Specifically, sigmoid kernel SVM classifiers were built upon a subset of 25 FNs obtained via a forward selection technique to optimize the classification performance for differentiating active patients from NCs. The similarity between subjects in SVM classification was defined as the Riemannian distance of the subset of FNs on the Grassmann manifold [48, 49]. Initially, the forward component selection procedure built a classifier on each individual FN, and the performance of the classifier was estimated using leave-one-out cross-validation (LOOCV) so that each FN could be evaluated for its classification performance. The accuracy rate was chosen as the main metric for evaluating the classification performance. The FN with the best performance was selected to be included in the subsequent classification. Through combining the first selected FN and any one of the remaining FNs, classifiers were built upon all paired FNs which were evaluated based on the training data during the current outer round using an inner LOOCV procedure. The paired FNs with the best performance were selected to be included in the classification. The procedure was repeated to add more FNs in the classification one by one until a single classifier was built upon all available FNs. Accordingly, a subset of FNs with the best performance was deemed to be the final selected components in the classification, hereafter referred to as informative FNs. To avoid potential classification biases, a nested LOOCV procedure was applied to optimize the parameters of the sigmoid kernel SVM classifiers to improve the classification performance during the forward component selection procedure [48, 49]. Since different FNs might be selected in each training runs or each testing run during the nested LOOCV procedure, the informative FNs were selected as the best performing ones with higher frequency (selection frequency>0.5). Based on these informative FNs of 74 subjects (including 37 NCs and 37 active CD patients), the LOOCV classification model yielded 74 aggregated SVM classifiers with the nested LOOCV classifiers, respectively. Each aggregated classifier generated a classification score from its corresponding nested classifiers with a positive value indicating CD state and a negative value indicating healthy state. Finally, the classification performance was evaluated with metrics including classification accuracy, specificity, sensitivity, and the area under the receiver operating characteristic curve (AUROC) (Fig. 1c). Non-parametric permutation tests were adopted to examine the statistical significance of the classification performance. The classification rate for the null distribution was estimated by building sigmoid kernel SVM models upon cross-sectional informative FNs of all active CD patients and NCs with subject labels randomly permuted by using the LOOCV strategy. This procedure was repeated for 10,000 times. Finally, the null distribution of the classification rate based on permuted samples was obtained. Longitudinal Analyses of Informative FNs and Emotion Scales from Active to Endocrine-Remitted CD Patients To investigate the longitudinal functional connectivity changes, pseudo paired t tests between active and endocrine-remitted CD patients (10,000 permutations) were applied voxel-wisely to each of the informative FNs using statistical non-parametric mapping (SnPM) software (http://warwick.ac.uk/snpm). Brain regions with statistical significance within each informative FN were identified at a voxel-wise threshold of p < 0.01 and an extent threshold of 40 adjacent voxels (AlphaSim-corrected p < 0.01). Additionally, statistical analyses were performed to compare the IC’s z scores of FNs as well as emotion scales between any pair of NC, active CD, and endocrine-remitted CD groups to further examine the longitudinal brain functional connectivity changes. Particularly, a pseudo paired t test was applied to all IC’s z scores within each informative FN as well as SDS scores and SAS scores between active and endocrine-remitted CD patients (10,000 permutations). While a pseudo two-sample t test with age, sex, and years of school education as covariates was applied to all IC’s z scores within each informative FN as well as SDS scores and SAS scores between NCs and active CD patients and endocrine-remitted CD patients. Significant differences were determined at a false discovery rate (FDR) threshold of p < 0.05 after adjusting for multiple comparisons. Statistical Analyses of Informative FNs in Endocrine-Remitted CD Patients The established pattern classification model was applied to the FNs of the follow-up endocrine-remitted CD patients. Thus, each endocrine-remitted CD patient had a classification score that reflected the likelihood of the endocrine-remitted CD patient to be active CD or healthy state (a positive value indicating active CD state and a negative value indicating healthy state). Based on the follow-up classification scores, endocrine-remitted CD patients who were correctly classified as active CD patients before treatment were further stratified into two subgroups: subjects with negative classification scores, referred to as image-based phenotypically (IBP) recovered CD patients, and those with positive classification scores, referred to as IBP unrecovered CD patients. Additionally, statistical differences in the IC’s z scores within each of the informative FNs between the IBP recovered and unrecovered CD patients, were assessed to elucidate these endocrine-remitted CD patients’ brain recoveries in these informative FNs at 3 months after treatment. Specifically, a pseudo two-sample t test with age, sex, years of school education, and years of disease duration as covariates was applied to all IC’s z scores of each FN between IBP recovered and unrecovered CD patients, and significant differences were determined at an FDR threshold of p < 0.05 (10,000 permutations) after adjusting for multiple comparisons. Correlation Analyses between Informative FNs and Clinical Measures Correlation analyses were performed to investigate the relationship between informative FNs and clinical measures in all 37 CD patients. The clinical measures of interest were serum cortisol, 24hUFC, ACTH, SDS, and SAS. Specifically, the correlation between each clinical measure and the averaged IC’s z score of each informative FN of CD patients before treatment was computed using a general linear model with age, sex, years of school education, and years of disease duration as covariates. Significant correlations were determined at a threshold of p < 0.05 using FDR corrected for multiple comparisons. Additionally, the correlation between the changes of each clinical measure and the averaged IC’s z score of each informative FN for endocrine-remitted CD patients before and after treatment was computed by using a general linear model with age, sex, years of school education, years of disease duration, and this clinical measure before treatment as covariates. The change of the averaged IC’s z score of each informative FN for each CD patient was calculated as the value after treatment minus the value before treatment divided by the value before treatment, and the change of each clinical measure for each CD patient was calculated similarly. Significant correlations were identified at a threshold of p < 0.05 using FDR corrected for multiple comparisons. Results Demographics and Clinical Characteristics The demographic and clinical data, including age, sex, years of school education, hormones, and emotion scales, are summarized in Table 1. There were no significant differences in age, sex, and years of school education between NCs and CD patients before treatment or at the 3-month follow-up (p > 0.05). The hormone levels, including ACTH, 24hUFC, and serum cortisol, were significantly restored (lower to be precise) in endocrine-remitted CD patients at the 3-month follow-up compared to their pre-treatment levels (FDR-corrected p < 0.05). These CD patients reached endocrine remission confirmed by their normal serum cortisol (<5 µg/dL) within 7 days of surgery. The emotion scales, including SDS scores and SAS scores, were significantly improved (smaller to be precise) in endocrine-remitted CD patients at 3 months after treatment compared to their rating scales in active phase (FDR-corrected p < 0.05), and the SDS scores and SAS scores for these endocrine-remitted CD patients were comparable to those of NCs. There was also significant difference in SDS scores between endocrine-remitted CD patients and NCs (FDR-corrected p < 0.05), while no significant difference was found in SAS scores between endocrine-remitted CD patients and NCs (p = 0.70). These psychometric comparison results suggest that depressive symptoms were partially recovered in endocrine-remitted CD patients, while their anxiety symptoms were also not completely recovered. Table 1. Demographic and clinical data of the participants Characteristics NCs (N = 37) Active CDs (N = 37) Endocrine-remitted CDs (N = 37) p value Age, years 38.46±11.85 33.92±8.57 33.92±8.57 0.062a Sex (M/F) 10/27 8/29 8/29 0.83a Years of school education 12.84±3.53 13.27±3.11 13.27±3.11 0.55a ACTH, pg/mL - 75.70 (45.55, 103.25) 23 (10.33, 30.70) <0.01**b 24hUFC, μg/day - 582.34 (351.30, 991.56) 47.77 (14.41, 186.54) <0.01**b Serum cortisol, μg/dL - 26.58 (20.98, 31.84) 5.49 (1.75, 13.69) <0.01**b Depression (SDS) 38.72±7.45 53.99±9.20 45.54±10.24 <0.01**c <0.01**d Anxiety (SAS) 33.34±5.46 45.27±11.92 34.46±9.78 <0.01**c 0.70d Values for characteristics are presented as mean ± SD or median (25th percentiles, 75th percentiles) unless otherwise indicated. Group differences in age, years of school education, SDS, and SAS between NCs and CD patients before or at the 3-month follow-up were examined using pseudo two-sample t tests. Group differences in sex between NCs and the CD patients before treatment or at the 3-month follow-up were examined using a χ2 test. Group differences in ACTH, 24hUFC, serum cortisol, SDS, and SAS between CD patients before treatment and at the 3-month follow-up were examined using pseudo paired t tests. NCs, normal controls; CDs, patients with Cushing’s disease; ACTH, adrenocorticotropic hormone; 24hUFC, 24-h urinary-free cortisol; SDS, self-rating depression scale; SAS, self-rating anxiety scale; M, male; F, female; SD, standard deviations. **p < 0.01. aNCs versus active or endocrine-remitted CDs. bActive CDs versus endocrine-remitted CDs. cActive CDs versus NCs or endocrine-remitted CDs. dNCs versus endocrine-remitted CDs. Informative FNs in Active CD Patients Active CD patients were mostly different from the NCs in 3 out of 25 FNs (selection frequency>0.5), including cerebellar network (CerebN), fronto-parietal network (FPN), and DMN, as shown in Figure 2a and b. The classification models built upon these three informative FNs yielded an accuracy of 72% (sensitivity: 68%, specificity: 76%, AUROC: 0.81), as shown in Figure 2c. Non-parametric permutation tests demonstrated that the classification accuracy was promising and significant (p < 1.0e−04), as suggested by the histogram of permuted classification rates shown in Figure 2d. Particularly, 25 out of 37 (67%) CD patients were correctly classified as active CD patients before treatment. Fig. 2. VIEW LARGEDOWNLOAD SLIDE Three informative functional brain networks identified by the multivariate pattern classification method and the classification performance. a Three highly selected functional brain networks, including CerebN, FPN, and DMN, for differentiating active CD patients from NCs. b The frequency of the functional brain networks selected in the nested LOOCV experiments. c The receiver operating characteristic (ROC) curve (area under the ROC curve [AUROC] = 0.81) of the classification model built upon the selected most discriminative FNs. d The histogram of the classification rates of the permutation tests and the real classification rate. In panel (a), brain regions with significant functional connectivity were obtained by applying voxel-wise one-sample t tests to the IC’s z scores for each of the FNs across all active CD patients and NCs (p < 0.05, FWE corrected for multiple comparisons, and cluster size >400 voxels). CerebN, cerebellar network; FPN, fronto-parietal network; DMN, default mode network; CD, Cushing’s disease; Pres, CD patients before treatment (i.e., active CD patients); NCs, normal controls; FNs, functional networks; ICs, independent components; FWE, family-wise error; L, left; R, right. Changes in Informative FNs from Active to Endocrine-Remitted CD Patients Two out of the three informative FNs, i.e., CerebN and FPN other than DMN, exhibited significant functional connectivity changes in CD patients between active and endocrine-remitted states (Fig. 3a). Compared with their active state, the endocrine-remitted CD patients had significantly improved (increased to be precise) functional connectivity measured by IC’s z scores in both CerebN and FPN circuits at 3 months after treatment. These results indicate that the FNs of the endocrine-remitted CD patients partially recovered toward the NCs at 3 months after treatment (Fig. 3b). Fig. 3. VIEW LARGEDOWNLOAD SLIDE Two informative functional brain networks as well as emotion scales with significant longitudinal changes in CD patients before treatment and at the 3-month follow-up. a Brain regions with significant longitudinal changes in functional connectivity within circuits of CerebN and FPN for CD patients, identified using non-parametric permutation tests (AlphaSim-corrected p < 0.01). b, c Significantly different functional connectivity measured by IC’s z scores across voxels within circuits of CerebN and FPN as well as emotion scales measured by the self-rating depression scale (SDS) and self-rating anxiety scale (SAS) between any two of NCs, CD patients before the treatment (i.e., active CD patients), and endocrine-remitted CD patients at 3-month follow-up (FDR-corrected p < 0.05). A pseudo paired t test with age, sex, and years of school education as covariates was conducted to compare all IC’s z scores within each functional network as well as the SDS scores and SAS scores between CD patients before treatment and at the 3-month follow-up. While a pseudo two-sample t test with age, sex, and years of school education as covariates was conducted to compare IC’s z scores within each functional network as well as SDS scores and SAS scores between NCs and CD patients before treatment, and endocrine-remitted CD patients at 3-month follow-up. CerebN, cerebellar network; FPN, fronto-parietal network; CD, Cushing’s disease; Pres, CD patients before treatment; Posts, endocrine-remitted CD patients at 3-month follow-up; NCs, normal controls; ICs, independent components; FDR, false discovery rate. Changes in Informative FNs of Endocrine-Remitted CD Patients Among the endocrine-remitted CD patients who were correctly classified as active CD patient before treatment, 14 participants were classified as IBP-recovered patients, while 11 participants were classified as IBP-unrecovered patients. The IBP-recovered and -unrecovered CD patients were determined by using the established pattern classification model according to the opposite signs in their classification scores based on their follow-up rs-fMRI data at 3 months after treatment (Fig. 4b). The IBP recovered patients had better recovery of the impaired functional connectivity within the circuits of CerebN and FPN than the IBP-unrecovered patients, as shown in Figure 4a. Fig. 4. VIEW LARGEDOWNLOAD SLIDE Differences in functional connectivity measured by IC’s z scores across voxels within circuits of CerebN and FPN as well as classification scores between image-based phenotypically (IBP)-recovered and -unrecovered CD patients after treatment. In panel (a), statistical comparisons were performed using pseudo two-sample t tests with age, sex, years of school education, and years of disease duration as covariates (FDR-corrected p < 0.05). In panel (b), violin plots showed opposite signs in classification scores between IBP-recovered and -unrecovered CD patients. CerebN, cerebellar network; FPN, fronto-parietal network; CD, Cushing’s disease; CDs, patients with Cushing’s disease; ICs, independent components; FDR, false discovery rate. Relationship between Informative FNs and Clinical Measures Changes of 24hUFC for endocrine-remitted CD patients before and after treatment were negatively correlated with their changes of averaged IC’s z scores within the FPN circuits with statistical significance (r = −0.37, p = 0.020), as shown in Figure 5a. The emotion scales, including SDS and SAS, were significantly negatively correlated with the averaged IC’s z scores within the CerebN circuits in the active CD patients (r = −0.31, p < 0.042), as shown in Figure 5c and d. There was no significant correlation for other clinical measures. Fig. 5. VIEW LARGEDOWNLOAD SLIDE Correlations between clinical measures and averaged IC’s z scores of informative FNs in CD patients (FDR-corrected p < 0.05). a Scatter plot for the significantly negative correlation between changes in the averaged IC’s z scores of the FPN circuits and 24hUFC of these 37 endocrine-remitted CD patients. b Multi-slice view of the FPN circuits whose changes in the averaged z scores were significantly correlated with changes in 24hUFC for all 37 endocrine-remitted CD patients before and after treatment. c, d Scatter plots for the significantly negative correlations between the averaged IC’s z scores of the CerebN circuits, and the SDS scores and SAS scores in these 37 endocrine-remitted CD patients before treatment. In panel (a), the changes in the averaged IC’s z scores of the FPN circuits were adjusted by regressing out covariates including age, sex, years of school education, years of disease duration, and the pre-treatment 24hUFC. In panels (c) and (d), the averaged IC’s z scores of the CerebN circuits were adjusted by regressing out covariates including age, sex, years of school education, and years of disease duration. 24hUFC, 24-h urinary-free cortisol; SDS, self-rating depression scale; SAS, self-rating anxiety scale; FPN, fronto-parietal network; CerebN, cerebellar network; CD, Cushing’s disease; Pres, CD patients before treatment; Posts, endocrine-remitted CD patients at 3-month follow-up; ICs, independent components; FDR, false discovery rate. Discussion The present study investigated the large-scale FNs of CD patients before and after treatment based on longitudinal rs-fMRI data. To the best of our knowledge, this is the first study to characterize longitudinal large-scale functional brain network changes due to hypercortisolism in CD patients using multivariate analysis. Particularly, the active CD patients had aberrant functional connectivity within circuits of CerebN, FPN, and DMN, respectively. More importantly, the impaired functional connectivity within the circuits of the CerebN and FPN was partially recovered in the endocrine-remitted CD patients, respectively. The changes in 24hUFC of CD patients before and after treatment were correlated with their changes in the functional connectivity of the FPN circuits. In addition, the emotion scales, including SDS and SAS, were also correlated with the functional connectivity of the CerebN circuits in CD patients before treatment. Aberrant FNs in Active CD Patients The informative FNs identified by the multivariate method were able to distinguish active CD patients from NCs with an accuracy of 72% (sensitivity: 68%, specificity: 76%, AUROC: 0.81). The non-parametric permutation tests also suggested that the multivariate method performed well in differentiating active CD patients from NCs. The most frequently selected FNs (Fig. 2b), i.e., informative FNs, were CerebN, FPN, and DMN. The cross-sectional multivariate analyses have revealed that the active CD patients were mostly different from the NCs in the functional connectivity within 3 FNs out of 25 FNs, as shown in Figure 2a. The aforementioned cross-sectional results provided new insights into large-scale functional brain network abnormalities due to hypercortisolism in CD patients. Particularly, our study revealed that active CD patients had significantly disrupted functional connectivity within the cerebellum (Fig. 2a, 3b), and their emotional dysfunctions observed by the SDS and SAS were associated with the impaired functional connectivity within the cerebellum (Fig. 5c, d). Therefore, it was reasonable to speculate that the cognitive or emotional dysfunctions for active CD patients, documented in this study as well as numerous previous studies [3‒5, 7‒9, 11‒14, 50], might be closely related to the observed functional connectivity abnormalities in the cerebellum. Additionally, our study found that the functional connectivity within the FPN circuits was significantly reduced in active CD patients (Fig. 2a, 3b). It was postulated that cognitive impairments in active CD patients, reported in several early studies [6, 14, 51], might be associated with the observed functional connectivity abnormalities in the FPN circuits. While several recent studies reported that active CD patients had structural or metabolic abnormalities in two brain regions of the FPN, namely, the middle frontal gyrus and inferior parietal lobule [17, 21, 32] These local morphological or metabolic abnormalities might exacerbate the observed functional network (FPN) alterations in active CD patients. Moreover, our study found that the functional connectivity within the DMN circuits was vulnerable to the detrimental effects of hypercortisolism in active CD patients. Besides our finding, recent studies reported that active CD patients showed structural, metabolic, or spontaneous activity abnormalities in several brain regions of DMN, including the posterior cingulate cortex, precuneus, parahippocampal gyrus, ventral medial prefrontal cortex, superior frontal gyrus, inferior temporal gyrus, and lateral parietal cortex [21, 27, 30‒32, 38, 52] These local morphological, metabolic or activity abnormalities might exacerbate the newly discovered DMN impairments in active CD patients. Essentially, the functional, morphological, and metabolic abnormalities in regions of the DMN might be directly related to the adverse expressions of glucocorticoid receptor genes within these brain regions caused by excessive exposure to endogenous cortisol [53]. Reversible Impaired FNs in Endocrine-Remitted CD Patients after Treatment The longitudinal statistical analysis has revealed that the endocrine-remitted CD patients’ hormones, including ACTH, 24hUFC, and serum cortisol, maintained near-normal levels at 3 months after treatment, suggesting that these patients did not relapse according to the endocrine hormone levels (Table 1). Meanwhile, their functional connectivity within circuits of the FPN and CerebN was partially restored at the 3-month follow-up after resolution of hypercortisolism (Fig. 3b). Particularly, our combined longitudinal and cross-sectional study found that the functional connectivity within the FPN circuits in endocrine-remitted CD patients was partially restored after treatment. While a cross-sectional sMRI study reported that endocrine-remitted CD patients still had structural abnormalities in the FPN-related region, namely, the middle frontal gyrus [17]. Our study also found that the functional connectivity of the cerebellum in endocrine-remitted CD patients was partially restored after treatment (Fig. 3b). Besides this finding, two other cross-sectional sMRI studies reported that the structural abnormalities of the cerebellum in endocrine-remitted patients were present as well [16, 20]. Taken together, it was postulated that the reversibility of the observed functional connectivity impairments within circuits of the FPN and CerebN might be directly influenced by their local morphological abnormalities in endocrine-remitted CD patients. Moreover, our study uncovered that the IBP-recovered patients exhibited better recovery of the functional connectivity within circuits of the FPN and CerebN than the IBP-unrecovered ones, as shown in Figure 4a. This result demonstrated that different endocrine-remitted CD patients had different recovery levels for the impaired functional connectivity within circuits of these brain FNs. More importantly, our study further found that the recovered 24hUFC was associated with the improved functional connectivity within FPN circuits in endocrine-remitted CD patients at the 3-month follow-up after treatment (Fig. 5a). This finding indicated that chronic endogenous hypercortisolism in CD patients might be directly related to their FPN impairments. Strengths of This Study The combined longitudinal and cross-sectional analyses have confirmed that the brain functional network abnormalities in CD patients were partially reversible at 3 months after resolution of the hypercortisolism. Since the brain structural abnormalities in endocrine-remitted CD patients were not completely recovered [16], it merits further investigation how the brain structural and functional network recoveries couple with each other in a longitudinal design. The present study provided complementary information to existing neuroimaging studies of CD patients. The existing neuroimaging studies have reported that CD patients had brain volume loss in cortical and cerebellar regions, hippocampus, and amygdala, as well as enlarged ventricles. These structural abnormalities were partially recovered for endocrine-remitted CD patients after treatment [11, 15, 16, 18‒22, 24] or after resolution of the hypercortisolism [12, 18, 24]. CD patients also had reduced cortical thickness in many brain regions including superior frontal cortex, caudal middle frontal cortex, precentral gyrus, insula, precuneus, cuneus, caudal/rostral anterior cingulate gyrus, and posterior cingulate gyrus [17, 54]. In addition, disrupted white matter integrity was observed in CD patients throughout the brain including frontal lobe, temporal lobe, hippocampus, parahippocampal gyrus, cingulate cingulum, corpus callosum, uncinate fasciculus, and cerebellum [10, 25‒27]. Furthermore, metabolic abnormalities in CD patients have been reported in widely distributed brain regions [21, 28‒32], which could be almost completely restored after resolution of hypercortisolism. Besides aforementioned structural and metabolic abnormalities, functional abnormalities have also been reported in CD patients using fMRI [37‒42]. Particularly, abnormal functional activations in CD patients have been observed in the prefrontal cortex, superior/middle/inferior frontal gyrus, superior parietal lobule, superior/middle temporal gyrus, inferior occipital gyrus, rostral/dorsal anterior cingulate gyrus, anterior/middle/posterior hippocampus, amygdala, precuneus, cuneus, lingual gyrus, caudate body, pulvinar/lateral posterior nuclei of the thalamus, and substantia nigra using task fMRI [37‒39, 41]. Abnormal spontaneous functional activities measured by both the amplitude of low-frequency fluctuation and regional homogeneity for CD patients have been observed in the prefrontal cortex, occipital lobe, postcentral gyrus, posterior cingulate gyrus, precuneus, thalamus, and cerebellum [20]. The dysregulation of functional connectivity density of CD patients has been found primarily in the prefrontal cortex, lateral parietal cortex, anterior/posterior cingulate gyrus, and parahippocampal gyrus [55]. The abnormal functional connectivity for CD patients has also been observed between the prefrontal cortex and medial temporal lobe, ventromedial prefrontal cortex and posterior cingulate cortex, anterior cingulate gyrus and limbic network, and lateral occipital cortex and DMN using task fMRI or rs-fMRI [39, 40]. Limitations and Future Work This study has several limitations. First, the longitudinal sample size is not large enough due to the rarity of CD, which might lead to relatively low statistical power and potential biases. Second, our study mainly investigated the brain functional network reversibility of the CD. Studies of the CD’s structural reversibility may provide complementary information to the current study. Third, our study investigated the short-term (3 months) effects of hypercortisolism on large-scale functional brain networks in CD patients. Nevertheless, the long-term effects of hypercortisolism on large-scale functional brain networks remain unclear and merit further investigation. In future work, long-term follow-up data of the CD patients recruited in the current study will be collected to investigate the long-term dynamic changes of their impaired large-scale functional brain networks. Conclusion This is the first study to investigate large-scale functional brain networks and their reversibility in a longitudinal CD cohort by using multivariate analysis. The large-scale functional brain networks, including the CerebN, FPN, and DMN, were impaired due to elevated cortisol levels in active CD patients. More importantly, the impaired functional brain networks of these CD patients were partially restored when their hormone levels returned to normal at 3 months after treatment. The changes of the functional connectivity within the impaired FPN were correlated with changes of the 24hUFC in endocrine-remitted CD patients, while the functional connectivity within the impaired CerebN was closely associated with emotion dysfunctions in active CD patients. These findings suggest that pattern recognition techniques could help identify informative functional brain networks in CD patients, which may help open up novel avenues for their postoperative interventions and assessments after endocrine remission. Statement of Ethics This study confirmed to the Declaration of Helsinki and was approved by the Medical Ethics Committee of Peking Union Medical College Hospital (approval number S-424). Written informed consent was obtained from all participants. Conflict of Interest Statement All authors reported no financial interests or potential conflicts of interest. Funding Sources This study was supported in part by the China Postdoctoral Science Foundation (2020T130070, 2019M650567), and the Clinical Application Research of Capital Characteristic Fund from the Beijing Municipal Science and Technology Commission (Z151100004015099). Author Contributions Bing Xing, Feng Feng, and Yong Fan were involved in study conception and design. Bo Hou, Xiaopeng Guo, Yong Yao, and Ming Feng collected clinical and imaging data. Hewei Cheng, Lu Gao, and Rixing Jing performed data preparation and statistical analysis. Hewei Cheng, Lu Gao, Rixing Jing, Bing Xing, Feng Feng, and Yong Fan were involved in data interpretation. Hewei Cheng, Lu Gao, and Rixing Jing wrote the first draft of the manuscript. Hewei Cheng, Lu Gao, Rixing Jing, Bo Hou, Xiaopeng Guo, Zihao Wang, Ming Feng, Bing Xing, Feng Feng, and Yong Fan provided critical editing and revision of the manuscript for important intellectual content. All authors approved the final version of the manuscript. Additional Information Hewei Cheng and Lu Gao contributed equally to this work. Data Availability Statement All data generated or analyzed during this study are included in this article. Further inquiries can be directed to the corresponding author. References 1. Newell-Price J, Bertagna X, Grossman AB, Nieman LK. Cushing’s syndrome. Lancet. 2006;367(9522):1605–17. 2. 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However, in view of ongoing research, changes in government regulations, and the constant flow of information relating to drug therapy and drug reactions, the reader is urged to check the package insert for each drug for any changes in indications and dosage and for added warnings and precautions. This is particularly important when the recommended agent is a new and/or infrequently employed drug. Disclaimer: The statements, opinions and data contained in this publication are solely those of the individual authors and contributors and not of the publishers and the editor(s). The appearance of advertisements or/and product references in the publication is not a warranty, endorsement, or approval of the products or services advertised or of their effectiveness, quality or safety. The publisher and the editor(s) disclaim responsibility for any injury to persons or property resulting from any ideas, methods, instructions or products referred to in the content or advertisements. 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The following is a summary of “Diurnal Range and Intra-patient Variability of ACTH Is Restored With Remission in Cushing’s Disease,” published in the November 2023 issue of Endocrinology by Alvarez, et al. Distinguishing Cushing’s disease (CD) remission from other conditions using single adrenocorticotropic hormone (ACTH) measurements poses challenges. For a study, researchers sought to analyze changes in ACTH levels before and after transsphenoidal surgery (TSS) to identify trends confirming remission and establish ACTH cutoffs for targeted clinical trials. A retrospective analysis involved 253 CD patients undergoing TSS at a referral center from 2005 to 2019. Remission outcomes were assessed based on postoperative ACTH levels. Among 253 patients, 223 achieved remission post-TSS. The remission group exhibited higher ACTH variability at morning (AM) (P = .02) and evening (PM) (P < .001) time points compared to the nonremission group. Nonremission cases had a significantly narrower diurnal ACTH range (P < .0001). A ≥50% decrease in plasma ACTH from mean preoperative levels, especially in PM values, predicted remission. Absolute plasma ACTH concentration and the ratio of preoperative to postoperative values were associated with nonremission (adj P < .001 and .001, respectively). ACTH variability suppression was observed in CD, with remission linked to restored variability. A ≥50% decrease in plasma ACTH may predict CD remission post-TSS. The insights can guide clinicians in developing rational outcome measures for interventions targeting CD adenomas. Source: academic.oup.com/jcem/article-abstract/108/11/2812/7187942?redirectedFrom=fulltext

Dr. Theodore Friedman (the Wiz) will be giving a webinar on Optimal replacement for Hypopituitarism and Sheehan’s: Oxytocin, testosterone, growth hormone, stimulants and beyond Learn what most Endocrinologists don’t know about but will improve your quality of life Topics to be discussed include: • Oxytocin-the love hormone • Testosterone, not just for men • Stimulants to treat pituitary apathy • Growth hormone, not just for kids • Thyroid optimization • Cortisol, the right and wrong way to give • Learn about the common medicine you should never take if on growth hormone Wednesday • December 6th• 6 PM PST Via Zoom Click here to join the meeting or https://us02web.zoom.us/j/4209687343?pwd=amw4UzJLRDhBRXk1cS9ITU02V1pEQT09&omn=84521530646 OR +16699006833,,4209687343#,,,,*111116# Slides will be available before the webinar and recording after the meeting at slides or on Dr. Friedman’s YouTube channel OR Join on Facebook Live https://www.facebook.com/goodhormonehealth at 6 PM PST Meeting ID: 420 968 7343 Passcode: 111116 Your phone/computer will be muted on entry. There will be plenty of time for questions using the chat button. For more information, email us at mail@goodhormonehealth.com