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MaryO

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  1. Key takeaways: Crinecerfont was granted FDA breakthrough therapy designation for the treatment of congenital adrenal hyperplasia. The medication met primary and secondary endpoints in a pair of phase 3 trials. The FDA granted breakthrough therapy designation for an oral non-glucocorticoid medication for the treatment of congenital adrenal hyperplasia, according to an industry press release. Crinecerfont (Neurocrine Biosciences) is a selective corticotropin-releasing factor type 1 receptor antagonist under development to lower excess adrenal androgens for people with congenital adrenal hyperplasia due to 21-hyroxylase deficiency. The medication met its primary and secondary endpoints in two phase 3 CAHtalyst trials, one assessing use of crinecerfont by children and the other by adults. In the pediatric trial, children and adolescents receiving crinecerfont had a decrease in serum androstenedione from baseline to 4 weeks. Participants receiving the medication also had a greater reduction in daily glucocorticoid at 28 weeks than placebo. As Healio previously reported, in the adult trial, crinecerfont was associated with a greater reduction in daily glucocorticoid while maintaining androgen control compared with placebo. The most common adverse events in the pediatric study were headache, fever, vomiting, upper respiratory tract infection and nasopharyngitis. Among adults, the most common adverse events were fatigue, headache and COVID-19 infection. No serious adverse events related to crinecerfont were reported. Breakthrough therapy is the latest designation granted to crinecerfont by the FDA. The medication was previously granted fast track and rare pediatric disease designations. "We are very pleased that the FDA granted breakthrough therapy designation for crinecerfont, thus recognizing both the seriousness of congenital adrenal hyperplasia and the significant unmet need currently faced by patients and families living with this condition,” Eiry W. Roberts, MD, Chief Medical Officer for Neurocrine Biosciences, said in a press release. "The outstanding safety and efficacy results from the phase 3 CAHtalyst studies in pediatric and adult patients suggest that crinecerfont has the potential to represent a substantial improvement over current standard of care in congenital adrenal hyperplasia by controlling androgen levels and allowing for reduced steroid doses. We remain on track to submit the new drug application in 2024." From https://www.healio.com/news/endocrinology/20231206/fda-grants-breakthrough-therapy-designation-for-oral-congenital-adrenal-hyperplasia-drug?utm_source=selligent&utm_medium=email&utm_campaign=news&fbclid=IwAR2WXDd3ajhKG0s2h0XD9ZQAstUkSotJYl1KLicH3gmxEPF6hvg6sZu2dCU
  2. In this study, we will investigate the possible side effects of psoriasis patients using long-term topical corticosteroids (TCS) such as adrenal insufficiency, Cushing’s Syndrome (CS) and osteoporosis and determine how these side effects develop. Forty-nine patients were included in the study. The patients were divided into two groups based on the potency of the topical steroid they took and the patients’ ACTH, cortisol and bone densitometer values were evaluated. There was no significant difference between the two groups regarding the development of surrenal insufficiency, CS and osteoporosis. One patient in group 1 and 4 patients in group 2 were evaluated as iatrogenic CS. ACTH stimulation tests of these patients in group 2 showed consistent results with adrenal insufficiency, while no adrenal insufficiency was detected in the patient in Group 1. Patients who used more than 50g of superpotent topical steroids per week compared to patients who used 50g of superpotent topical steroids per week. It was identified that patients who used more than 50g of superpotent topical steroids had significantly lower cortisol levels, with a negatively significant correlation between cortisol level and the amount of topical steroid use ( < .01).Osteoporosis was detected in 3 patients in group 1 and 8 patients in Group 2. Because of the low number of patients between two groups, statistical analysis could not be performed to determine the risk factors. Our study is the first study that we know of that investigated these three side effects. We have shown that the development of CS, adrenal insufficiency and osteoporosis in patients who use topical steroids for a long time depends on the weekly TCS dosage and the risk increases when it exceeds the threshold of 50 grams per week. therefore, our recommendation would be to avoid long-term use of superpotent steroids and to choose from the medium-potent group if it is to be used. ABOUT THE CONTRIBUTORS Betul Erdem Department of Dermatology, Van Training and Research Hospital, Van, Turkey. Muzeyyen Gonul Department of Dermatology, Ministry of Health, Ankara Etlik City Hospital, Ankara, Turkey. Ilknur Ozturk Unsal Department of Endocrine and Metabolic Disease, Ministry of Health, Ankara Etlik City Hospital, Ankara, Turkey. Seyda Ozdemir Sahingoz Department of Biochemistry, Ministry of Health, Ankara Etlik City Hospital, Ankara, Turkey. From https://www.physiciansweekly.com/evaluation-of-psoriasis-patients-with-long-term-topical-corticosteroids-for-their-risk-of-developing-adrenal-insufficiency-cushings-syndrome-and-osteoporosis/
  3. Answered by Dr. Howard E. Lewine M.D. Chief Medical Editor, Harvard Health Publishing · 40 years of experience · USA Cushing’s syndrome refers to an excess amount of cortisol in the body. This happens most commonly when a person needs to take a high dose of a corticosteroid like prednisone for an extended period of time. Much less commonly, a hormonal problem arising from either the pituitary gland in the brain or the adrenal gland in the abdomen leads to excess cortisol production. Because these situations can be corrected, life expectancy will likely not be directly related to the Cushing’s syndrome itself. Answered by Dr. Shobha S Reddy MBBS, Masters in Diabetology, General Practitioner & Diabetologist · 15 years of experience · India Cushing's syndrome is a disorder in which cortisol hormone (the stress hormone that helps the body in stress) levels in the blood are excess (maybe due to endogenous or exogenous causes). This hormone helps in maintaining blood pressure, blood sugar, reduce inflammation. This hormone is secreted by the adrenal glands in our body. Complications of Cushing's syndrome include Hypertension, DM, infection, Bone fracture, mood swings, memory loss. If left untreated then life expectancy would be around 5 years. Answered by Dr. Sharath Chandra MBBS Spl in ENT, Head Neck Surgery from AIIM · 8 years of experience · India Cushing's syndrome is the condition where the adrenal glands in our body produce excessive cortisol hormones. Symptoms like 1) weight loss. 2)purple striae. 3)Acne, fatigue. Life expectancy in various studies indicates the mean survival would not be more than 4. 5 years in untreated Cushing's syndrome. Answered by Dr. Mohan P. Abraham M.D., FAAFP (Family Physician) · 40 years of experience · USA The life expectancy is very normal when treated, but uncontrolled it may be 4 - 5 years. Disclaimer: This is for information purpose only, and should not be considered as a substitute for medical expertise. These are opinions from an external panel of individual doctors, and not to be considered as opinion of Microsoft. Please seek professional help regarding any health conditions or concerns. From https://www.msn.com/en-au/health/medical/advice-from-harvard-health-publishing-and-3-more-doctors-what-is-the-life-expectancy-of-someone-with-cushing-s-syndrome/ar-AA1m2Fdw
  4. Abstract Background There is an increasing number of cases of aldosterone- and cortisol-producing adenomas (A/CPAs) reported in the context of primary aldosteronism (PA). Most of these patients have PA complicated with subclinical Cushing's syndrome; cases of apparent Cushing's syndrome (CS) complicated with aldosteronism are less reported. However, Co-secretory tumors were present in the right adrenal gland, a cortisol-secreting adenoma and an aldosterone-producing nodule (APN) were present in the left adrenal gland, and aldosterone-producing micronodules (APMs) were present in both adrenal glands, which has not been reported. Here, we report such a case, offering profound insight into the diversity of clinical and pathological features of this disease. Case presentation The case was a 45-year-old female from the adrenal disease diagnosis and treatment centre in West China Hospital of Sichuan University. The patient presented with hypertension, moon-shaped face, central obesity, fat accumulation on the back of the neck, disappearance of cortisol circadian rhythm, ACTH < 5 ng/L, failed elevated cortisol inhibition by dexamethasone, orthostatic aldosterone/renin activity > 30 (ng/dL)/(ng/mL/h), and plasma aldosterone concentration > 10 ng/dL after saline infusion testing. Based on the above, she was diagnosed with non-ACTH-dependent CS complicated with PA. Adrenal vein sampling showed no lateralization for cortisol and aldosterone secretion in the bilateral adrenal glands. The left adrenocortical adenoma was removed by robot-assisted laparoscopic resection. However, hypertension, fatigue and weight gain were not alleviated after surgery; additionally, purple striae appeared in the lower abdomen, groin area and inner thigh, accompanied by systemic joint pain. One month later, the right adrenocortical adenoma was also removed. CYP11B1 were expressed in the bilateral adrenocortical adenomas, and CYP11B2 was also expressed in the right adrenocortical adenomas. APN existed in the left adrenal gland and APMs in the adrenal cortex adjacent to bilateral adrenocortical adenomas. After another surgery, her serum cortisol and plasma aldosterone returned to normal ranges, except for slightly higher ACTH. Conclusions This case suggests that it is necessary to assess the presence of PA, even in CS with apparent symptoms. As patients with CS and PA may have more complicated adrenal lesions, more data are required for diagnosis. Peer Review reports Background Because both adrenal Cushing's syndrome and primary aldosteronism (PA) can manifest as adrenocortical adenomas, it is difficult to distinguish between them on the sole basis of adrenal computed tomography (CT). There may also be multiple adenomas with different functions in the same adrenal gland [1], which also leads to the difficulty in the interpretation of adrenal vein blood collection results. With the increased reports on cases of PA complicated with subclinical Cushing's syndrome in clinical practice, increasing attention is being given to the screening of PA complicated with subclinical Cushing's syndrome. However, PA screening may be ignored in the diagnosis and treatment of adrenal Cushing's syndrome. Although it has been reported that PA with a diameter > 2 cm may be complicated with aldosterone- and cortisol-producing adenomas (A/CPAs) [2], cases of apparent Cushing's syndrome complicated with PA are less well known. Recently, Y. Fushimi et al. [3] reported a case of apparent Cushing's syndrome complicated with PA. The cortisol-producing enzyme cytochrome P450 (CYP) 11B1 was diffusely expressed in the adenoma, but based on staining, the aldosterone synthase CYP11B2 was significantly expressed in the adjacent adrenal cortex. This finding indicated that aldosterone-producing micronodules (APMs) in the adjacent adrenal cortex may be the pathological basis of PA. Here, a case of bilateral co-secretory lesions presenting with coexisting Cushing syndrome and primary aldosteronism detected by AVS and confirmed by immunohistochemical analysis after surgical resection is reported. Moreover, APMs were found in the adrenal cortex adjacent to bilateral adrenocortical adenomas; an aldosterone-producing nodule was detected adjacent to the unilateral adenoma. Case presentation A 45-year-old female patient was admitted to the adrenal disease diagnosis and treatment centre in West China Hospital of Sichuan University due to "increased blood pressure, weight gain for one year and facial oedema for half a year". After nifedipine controlled-release tablets 30 mg daily and terazosin 2 mg daily were applied, the blood pressure of this patient was still as high as 179/113 mmHg. She had no family history of endocrine disease or malignant tumour. Her body mass index (BMI) was 25.6 kg/m2 at admission, with a moon-shaped face, fat accumulation on the back of the neck and thin skin. Hormonal, glucose, renal function, lipid, and blood electrolyte tests were completed, and the physiological rhythm of cortisol had disappeared. Aldosterone-renin-angiotensin system (RAAS) results showed a significant decrease in renin activity and a significantly higher aldosterone/renin ratio (ARR) (as provided in Table 1). Dynamic testing for hormones was conducted, and the results were as follows: (i) in terms of the saline infusion test (SIT) in supine position, the before and after aldosterone level was 17.03 ng/dL and 15.45 ng/dL, respectively; (ii) in terms of the captopril challenge test (CCT), the before and after aldosterone level was 18.49 ng/dl and 15.25 ng/mL, respectively, with an inhibition rate of 17.52%; (iii) in terms of the standard low-dose dexamethasone suppression test, the before and after serum cortisol level was 467.9 nmol/L and 786.3 nmol/L, respectively; the before and after 24-h urine free cortisol (24-h UFC) level was 332.3 µg/24 and 480.4 µg/24, respectively. An enhanced CT scan revealed adenoma lesions in both adrenal glands (Fig. 1a and b). Bone mineral density measurement with dual-energy X-ray absorptiometry indicated osteoporosis. Chest CT showed old fractures of the 9th rib on the left side and the 2nd rib on the right side. Table 1 Peripheral blood laboratory data for this case Full size table Fig. 1 Adrenal CT of the patient: A nodule with a size of approximately 1.6 × 1.5 cm was found in the left adrenal gland, and a nodule with a size of approximately 2.2 × 1.8 cm was found in the right adrenal gland. Irregular mild to moderate enhancement was on enhanced CT, and the surrounding fat gap was clear Full size image Based on the above clinical features, the patient was diagnosed with "non-ACTH-dependent Cushing's syndrome complicated with PA". To assess lateralization, adrenal vein sampling (AVS) stimulated by ACTH was performed after obtaining informed consent. The results showed no lateralization of cortisol and aldosterone secretion (Table 2). Table 2 Results of AVS Full size table After communicating with the patient, the left adrenocortical adenoma was first removed by robot-assisted laparoscopic resection; the thickened adrenal cortex near the left adrenocortical adenoma was also resected during the surgery. The pathological report revealed adrenocortical adenoma, the Weiss score was 1, and immunohistochemistry showed weak CYP11B1 expression in the adenoma and positive CYP11B2 expression in an adjacent nodule. Hypertension was not alleviated after surgery. One month later, purple lines appeared on both sides of the lower abdomen, groin area and inner thigh, accompanied by weight gain, apparent systemic joint pain and fatigue in both lower limbs. The patient was readmitted to the hospital, and examination revealed orthostatic ALD at 11.99 ng/dL, PRA at 0.08 ng/mL/h, angiotensin II at 39.38 ng/L (reference range: 55.3–115.3 ng/L) and ARR at 149.88 (ng/dL)/(ng/mL/h). In addition, ACTH was 2.37 ng/L, serum cortisol was 352.30–353.50–283.90 nmol/L at 8 h-16 h-24 h, 24-h UFC was 112.8 µg, and serum cortisol was 342.10 nmol/L in the morning after the 1 mg dexamethasone suppression test. Enhanced CT of the kidneys and adrenal glands showed no solid nodules or masses in the left adrenal gland, though a nodule with a size of approximately 2.2*1.8 cm was detected in the right adrenal gland. Enhanced CT showed irregular mild to moderate enhancement. Therefore, the diagnosis was still "non-ACTH-dependent Cushing's syndrome complicated with PA". Subsequently, the right adrenocortical adenoma and the thickened adrenal cortex near the right adrenocortical adenoma were removed by robot-assisted laparoscopic resection. The pathological report indicated adrenocortical adenoma, and immunohistochemistry showed diffuse homogeneous expression of CYP11B1 and CYP11B2. Antibodies against CYP11B1 (MABS502) and CYP11B1 (MABS1251) were purchased from the Millipore Corporation. There were APMs in the adrenal cortex adjacent to the bilateral cortical adenomas. The fluorescence staining image of the left cortical adenoma is shown in Fig. 2. The immunohistochemistry image of the left adrenal gland is given in Fig. 3 and that of the right adrenal gland in Fig. 4. The immunofluorescence method used in this study was indirect immunofluorescence double staining procedure. Paraffin-embedded human adrenal tissues were prepared using heat-induced epitope retrieval after deparaffinization. Tissue sections were blocked with 5% goat serum in PBS, pH 7.4, containing 0.5% SDS, for 1 h. The slides were incubated with individual primary antibodies at 4℃ overnight, followed by incubation with Alexa Fluor 488-, and Alexa Fluor 647-conjugated secondary antibodies specific to the species of the primary antibodies with DAPI for immunofluorescence staining. Antibodies used included anti-CYP11B1 (Millipore, Cat. No. MABS502, 1:100), anti-CYP11B2(Millipore, Cat. No. MABS1251, 1:100), Alexa Fluor 488-conjugated anti-rat IgG secondary antibody (CYP11B1; Green) and Alexa Fluor 647-conjugated anti-mouse IgG secondary antibody (CYP11B2; Red). Nuclei were stained with DAPI. Fig. 2 Routine hematoxylin and eosin (H&E) staining and immunofluorescence of the left adrenocortical adenoma (green represents expression of CYP11B1 and red that of CYP11B2). This adrenocortical adenoma and the surrounding cortex was cut into three parts. A and C show the overall appearance of the resected portion, with a nodule adjacent to the adenoma. B shows a neoplastic lesion formed by clear cells (aldosterone-producing cell) within nodules, lacking a fibrous envelope. C clearly shows the weak and diffuse expression of CYP11B1 in adrenocortical adenoma and CYP11B2 expression in a nodule in the cortex adjacent to the adenoma. D shows local enlargement of the aldosterone-producing nodule and three aldosterone-producing micronodules adjacent to it Full size image Fig. 3 Resected adrenocortical adenoma and part of the adrenal cortex on the left side. A shows expression of Aldosterone-producing micronodule CYP11B2 in the cortex adjacent to the adenoma. B shows an aldosterone-producing nodule with a diameter of approximately 2 mm. C shows weak positive expression of CYP11B1 in the adenoma and D negative expression of CYP11B1 in the aldosterone-producing nodule Full size image Fig. 4 Resected adrenocortical adenoma and part of the adrenal cortex on the right side. A and B show several Aldosterone-producing micronodules (positive expression of CYP11B2) in the cortex adjacent to the adenoma. C shows diffuse expression of CYP11B1 in the adenoma. D shows diffuse expression of CYP11B2 in the adenoma Full size image The Cushing's syndrome in this patient disappeared after surgery, and glucocorticoids were discontinued after 15 months according to medical advice. Follow-up was conducted for half a year after drug discontinuance, and the patient had no fatigue or dizziness; she was satisfied with the outcomes. Her systolic and diastolic blood pressure remained at 100–120 mmHg and 70–80 mmHg, respectively. During the most recent re-examination, the following results were obtained: (1) orthostatic ALD of 19.1 ng/dL and orthostatic renin concentration of 12.59 µIU/mL, with an aldosterone/renin ratio (ARR) of 1.52; (2) PTC at 8 AM of 247 nmol/L, ACTH of 93.55 ng/L and 24-h UFC of 26.8 µg; (3) parathyroid hormone of 3.86 pmol/L; (4) 25-OH-VitD of 119.5 nmol/L; (5) serum creatinine of 60 µmol/L; (6) serum sodium of 140.4 nmol/L, serum potassium of 3.87 mmol/L and serum calcium of 2.27 mmol/L. Discussion and conclusions Adrenal Cushing's syndrome is caused by excessive autonomic secretion of cortisol induced by adrenal cortical tumours or adrenal cortical hyperplasia; primary aldosteronism (PA) is caused by excessive autonomic secretion of aldosterone induced by adrenal cortical tumours or adrenal cortical hyperplasia. More adverse symptoms occur if aldosterone and cortisol-producing adenomas are present. Specifically, (1) it is more difficult to control hypertension; (2) the incidence of major adverse cardiovascular and cerebrovascular events would increase [4]; (3) glucose intolerance and other metabolic complications would be aggravated [5, 6]; (4) patients would be prone towards osteoporosis [7, 8]; (5) adrenal vein sampling results may be misinterpreted [9]; and (6) adrenal insufficiency may occur after surgery. Therefore, it is of great clinical significance to avoid missed diagnosis of A/CPAs. Despite many reports on A/CPAs, the majority of these patients may have subclinical Cushing's syndrome (SCS), and cases of apparent Cushing's syndrome complicated with PA are rarely reported. In the present case, the clinical manifestation of Cushing's syndrome were more apparent, and it would be appropriate to call it cortisol-aldosterone cosecretoma. Naoyoshi Onoda et al. [10] reported a case of Cushing's syndrome caused by a left adrenocortical adenoma (30 mm in diameter) and PA caused by a right adrenocortical adenoma (20 mm in diameter), and Fushimi et al. [3] reported a case of right A/CPA (25 mm*22 mm in size). Interestingly, in the present report, the patient had bilateral A/CPAs, and the clinical manifestations of Cushing's syndrome became more apparent after unilateral resection was performed. Similar to the above two cases, APMs were found in the adrenal cortex adjacent to the A/CPAs, but aldosterone-producing nodules were found near the cortisol-producing adenoma on the left side. The biochemical phenotype of APM-inducing autonomic aldosterone secretion has not been clarified. APMs can also be found in the adrenal tissue of 30% of individuals with normal blood pressure [11] and surrounding areas of APA [12, 13]. APMs do not express CYP11B1 or CYP17A1, which are necessary for the generation of cortisol [12, 14]. In our patient, the aldosterone-producing nodule in the left adrenal gland may have developed from APM. More than one-third of APMs carry known mutations in CACNA1D and ATP1A1, promoting the generation of aldosterone [14, 15]. Unfortunately, we did not perform whole-exome sequencing on the DNA of the peripheral blood and adenoma tissues of this patient. Due to the existence of APMs adjacent to the adenoma, it remains unclear whether there is a risk of the relapse of PA in these cases after resection of adrenal the adenoma. Therefore, it was necessary to conduct medical follow-up for this patient. Remi Goupil et al. performed AVS on 8 patients with cortisol-producing adenoma (CPA), and the results showed that cortisol on the CPA side was higher than that on the contralateral side (median, 6.7 times [range: 2.4–27.2]); P = 0.012]) [16]. There was no significant difference in bilateral cortisol and aldosterone concentrations after AVS in this patient, which is consistent with bilateral A/CPA. Although immunohistochemical results revealed weak expression of CYP11B1 for the first time, expression of cortisol in bilateral adrenal venous blood samples increased significantly after ACTH stimulation. Hence, cortisol was over-synthesized on both sides, and bilateral A/CPAs was definitively diagnosed. In summary, this case highlights the need for A/CPA screening. The complicated pathological features of these cases impose challenges to our understanding of this disease. Due to the presence of APMs in the adrenal cortex near bilateral adrenocortical adenomas, more clinical data are required to identify whether the disease might relapse after simple resection of the adenoma in these patients. Therefore, further medical follow-up of these patient is needed. Availability of data and materials Not applicable. Abbreviations CS: Cushing's syndrome PA: Primary aldosteronism ACTH: Adrenocorticotropic hormone UFC: Urinary free cortisol AVS: Adrenal vein sampling A/CPA: Aldosterone-and cortisol producing adenoma APN: Aldosterone-producing nodules APM: Aldosterone-producing micronodule CYP: Cytochrome P450 CT: Computed tomography PAC: Plasma aldosterone concentration PRA: Plasma renin activity ARR: Aldosterone /renin ratio References Stenman A, Shabo I, Ramström A, Zedenius J, Juhlin CC: Synchronous aldosterone- and cortisol-producing adrenocortical adenomas diagnosed using CYP11B immunohistochemistry. SAGE open medical case reports. 2019, 7:2050313x19883770. Hiraishi K, Yoshimoto T, Tsuchiya K, Minami I, Doi M, Izumiyama H, Sasano H, Hirata YJ. Clinicopathological features of primary aldosteronism associated with subclinical Cushing’s syndrome. Endocr J. 2011;58(7):543–51. Article CAS PubMed Google Scholar Fushimi Y, Tatsumi F, Sanada J, Shimoda M, Kamei S, Nakanishi S, Kaku K, Mune T, Kaneto H. 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Article CAS PubMed Google Scholar Download references Acknowledgements Not applicable Funding This study was supported by the Discipline Excellence Development 1.3.5 Project of West China Hospital, Sichuan University (No. ZYGD18022). Author information Authors and Affiliations Department of Endocrinology and Metabolism, Adrenal Center, West China Hospital of Sichuan University, Chengdu, 610041, Sichuan, China Hongjiao Gao, Yan Ren, Tao Chen & Haoming Tian Department of Endocrinology and Metabolism, The Third Affiliated Hospital of Zunyi Medical University (The First People’s Hospital of Zunyi), Zunyi, Guizhou, China Hongjiao Gao Institute of Clinical Pathology, West China Hospital of Sichuan University, Chengdu, Sichuan, China Li Li & Fei Chen Contributions HG, TC researched data and/or wrote the manuscript. LL, FC contributed to immumohistochemical staining. HT, TC, YR contributed to discussion. All authors have read and approved the manuscript. Corresponding authors Correspondence to Tao Chen or Haoming Tian. Ethics declarations Ethics approval and consent to participate Not applicable. Consent for publication Written informed consent was obtained from the patient for publication of this Case report and any accompanying images. A copy of the written consent is available for review by the Editor of this journal. Competing interests We do not have any potential conflicts of interest relevant to this article. Additional information Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Rights and permissions Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Reprints and Permissions Cite this article Gao, H., Li, L., Chen, F. et al. Bilateral co-secretory lesions presenting with coexisting Cushing syndrome and primary aldosteronism: a case report. BMC Endocr Disord 23, 263 (2023). https://doi.org/10.1186/s12902-023-01454-8 Download citation Received08 April 2022 Accepted24 August 2023 Published29 November 2023 DOIhttps://doi.org/10.1186/s12902-023-01454-8 Share this article Anyone you share the following link with will be able to read this content: Get shareable link Provided by the Springer Nature SharedIt content-sharing initiative Keywords Cushing’s syndrome Primary aldosteronism Adrenal vein sampling Immunohistochemistry Aldosterone-producing cell cluster Download PDF Sections Figures References Abstract Background Case presentation Discussion and conclusions Availability of data and materials Abbreviations References Acknowledgements Funding Author information Ethics declarations Additional information Rights and permissions About this article From https://bmcendocrdisord.biomedcentral.com/articles/10.1186/s12902-023-01454-8
  5. 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
  6. 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. 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  7. 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
  8. 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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Cortical thickness abnormalities in long-term remitted Cushing’s disease. Transl Psychiatry. 2020;10(1):293. 55. Wang X, Zhou T, Wang P, Zhang L, Feng S, Meng X, et al. Dysregulation of resting-state functional connectivity in patients with Cushing’s disease. Neuroradiology. 2019;61(8):911–20. © 2023 The Author(s). Published by S. Karger AG, Basel Open Access License / Drug Dosage / Disclaimer This article is licensed under the Creative Commons Attribution-NonCommercial 4.0 International License (CC BY-NC). Usage and distribution for commercial purposes requires written permission. Drug Dosage: The authors and the publisher have exerted every effort to ensure that drug selection and dosage set forth in this text are in accord with current recommendations and practice at the time of publication. 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. This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License . From https://karger.com/nen/article/doi/10.1159/000534789/869375/Reversibility-of-Impaired-Large-Scale-Functional
  9. 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
  10. 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
  11. 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
  12. Abstract Paraneoplastic syndromes are rare and diverse conditions caused by either an abnormal chemical signaling molecule produced by tumor cells or a body’s immune response against the tumor itself. These syndromes can manifest in a variable, multisystemic and often nonspecific manner posing a diagnostic challenge. We report the case of an 81-year-old woman who exhibited severe hypokalemia, metabolic alkalosis, and worsening hyperglycemia. The investigation was consistent with adrenocorticotropin (ACTH)-dependent Cushing’s syndrome and, eventually, the patient was diagnosed with stage IV primary small-cell lung cancer (SCLC). SCLC is known to be associated with paraneoplastic syndromes, including Cushing’s syndrome caused by ectopic adrenocorticotropin (ACTH) secretion. Despite being associated with very poor outcomes, managing these syndromes can be challenging and may hold prognostic significance. Introduction Adrenocorticotropin (ACTH)-dependent Cushing’s syndrome (CS) is caused by excessive ACTH production by corticotroph (Cushing’s disease (CD)) or nonpituitary (ectopic) tumors, leading to excessive cortisol production. Ectopic ACTH syndrome (EAS) is a rare condition, accounting for 10 to 20% of all cases of ACTH-dependent CS and 5 to 10% of all types of CS [1]. The normal glucocorticoid-induced suppression of ACTH is reduced in ACTH-dependent CS, especially with ectopic ACTH production. Studies show that a wide variety of neoplasms, usually carcinomas rather than sarcomas or lymphomas, have been associated with EAS. Most cases are caused by neuroendocrine tumors of the lung, pancreas, or thymus, in which the hypercortisolism state is not apparent clinically, resulting, all too often, in delayed diagnosis [2,3]. Current diagnostic tests for EAS aim to confirm high cortisol levels, the absence of a cortisol circadian rhythm, as well as the reduced response to negative feedback from glucocorticoid administration, and imaging to identify the site of ACTH production. Prompt diagnosis and management are crucial in EAS, highlighting the importance of physician awareness and early recognition of this syndrome. Treatment options depend on the underlying tumor. Surgical removal is often the primary approach, followed by radiation therapy or chemotherapy. Additionally, medications to control cortisol levels may be necessary to manage the various comorbid conditions associated with CS, such as cardiovascular disease, diabetes, electrolyte imbalances, infections and thrombotic risk [4,5]. Case Presentation We report the case of an 81-year-old woman with a fully active performance status (ECOG 0) and a medical history of diabetes, hypertension, dyslipidemia, and depressive disorder. She was admitted to an internal medicine ward due to an acute hydroelectrolytic disorder, including metabolic alkalosis, severe hypokalemia (2 mmol/L), hypochloremia (85 mmol/L), hypocalcemia (0.95 mmol/L), hypophosphatemia (1.4 mg/dL), hypomagnesemia (0.9 mg/dL), and hyperlactatemia (5.8 mmol/L), after she reportedly self-medicated herself with higher doses of metformin (four to five pills a day) due to high blood glucose levels. The patient presented with asthenia, nausea, vomiting, and diarrhea for three days and reported uncontrolled blood glucose levels for the last eight days. The physical examination was unremarkable, without any altered mental status or signs of infection. Arterial blood gas samples showed metabolic alkalemia (pH 7.59) and hyperlactatemia, associated with severe hypokalemia, normal bicarbonate (27 mmol/L), and mildly elevated glycemia and ketonemia (232 mg/dL and 1.7 mmol/L, respectively). Lab tests confirmed the serum potassium levels as well as the other aforementioned electrolyte disturbances. Kidney function and hepatic enzymes were normal. Considering the possible relationship between the electrolyte disorder and the gastrointestinal presentation, the patient was given intravenous (IV) fluids and received potassium and magnesium replacement therapy. Despite receiving 200 milliequivalents (mEq) of IV potassium chloride and 4 grams of magnesium sulfate, in the first 48 hours, the ion deficits persisted. Given the persistent electrolyte derangement, the patient was admitted to the Internal Medicine ward for etiological investigation and monitoring of ionic correction. The initial period was remarkable for refractory hypokalemia and uncontrolled diabetes under respective therapeutic measures, including 80 to 130 mEq of IV potassium chloride and progressive titration of spironolactone to 200 mg a day. Laboratory investigation revealed high parathormone levels (PTHi 167 pg/mL; reference range: 10-65 pg/mL), vitamin D deficiency (3.3 ng/mL; reference range >20 ng/mL) and apparent ACTH-dependent hypercortisolism (serum cortisol 80.20 ug/dL; ACTH 445 pg/mL), as well as high urinary potassium and glucose concentrations (190 mEq/24 h and 21161 mg/24 h). A dexamethasone suppression test was performed twice (standard low and high dose) without any changes in cortisol levels, leading to the suspicion of a CS caused by abnormally high ACTH production. Cranioencephalic computed tomography (CT) and magnetic resonance imaging (MRI) were performed, excluding the presence of pituitary anomalies. A follow-up whole-body CT scan was performed, revealing a suspicious pulmonary mass in the left lower lobe, associated with ipsilateral hilar lymphadenopathy and hepatic and adrenal gland lesions suggestive of secondary involvement. An endobronchial ultrasound bronchoscopy and biopsy were performed, documenting anatomopathological findings of small-cell lung carcinoma with a Ki67 expression of 100% (Figures 1-3). Figure 1: Pulmonary mass (SCLC) in the left lower lobe with ipsilateral hilar lymphadenopathy and pleural effusion. SCLC: small-cell lung cancer. Figure 2: Secondary involvement of the liver with hypodense multilobar hepatic lesions (arterial phase). Figure 3: Bilateral suprarenal lesions suggestive of secondary involvement. The patient was referred to oncology, and chemotherapy was deferred, considering the infectious risk associated with hypercortisolism. The patient started metyrapone 500 mg every eight hours, resulting in a reduction in cortisol levels and control of hypokalemia. Later on, a fluorodeoxyglucose-positron emission tomography (FDG-PET) scan was performed, confirming disseminated disease with additional bone involvement. Unfortunately, despite endocrinological stabilization, the patient's condition worsened, and she ended up dying one month after the diagnosis. Discussion When this patient was admitted, it was assumed that the metabolic alkalosis and various electrolyte disturbances were related to the gastrointestinal presentation and hyperlactatemia secondary to metformin overdose. However, the unusual persistence and refractory hypokalaemia raised some concerns that an alternative etiology might be involved and incited subsequent testing. The high cortisol levels were unexpected given the subclinical presentation, which seems to be more frequent in cases of EAS. In fact, because of this, the true incidence of EAS is unknown and probably underdiagnosed since patients often have subclinical presentations and do not exhibit catabolic features. Since the patient wasn’t on any steroid medication, the association between the high cortisol and ACTH levels, non-responsive to the dexamethasone suppression test, along with the absence of a pituitary lesion, raised suspicion of a probable EAS, which was later confirmed by the body CT scan and endobronchial ultrasound (EBUS). EAS is a rare disease with a poor prognosis. It reportedly occurs in 3.2 to 6% of neuroendocrine neoplasms, and the tumor often originates in the lung, thyroid, stomach, and pancreas. Locoregional and/or distant metastasis can be seen at the time of diagnosis in 15% of typical carcinoids and about half of atypical carcinoids with visible primaries [6,7]. The presence of a typical CS presentation, with or without electrolyte abnormalities, should raise suspicion and serum levels of both ACTH and cortisol should be assessed to determine if they are elevated and to distinguish between an ACTH-dependent (pituitary or nonpituitary ACTH-secreting tumor) and an independent mechanism (e.g., from an adrenal source). The diagnosis of CS is established when at least two different first-line tests are unequivocally abnormal and cannot be explained by any other conditions that cause physiologic hypercortisolism. Additional evaluation is performed to rule out a pituitary origin (with brain MRI) and to assess for a possible ectopic ACTH-secreting tumor. In the aforementioned case, the production of ACTH was caused by primary neuroendocrine SCLC. The recommended approach to EAS involves the initial normalization of serum cortisol levels and the treatment of related comorbidities before performing a complete diagnostic evaluation and addressing the underlying cause [5-7]. This approach seems to improve survival and prevent complications such as sepsis following a combined steroid-induced immunosuppression and chemotherapy-induced agranulocytosis [6,7]. Direct therapies vary according to the tumor, but surgery is usually the first line of treatment (transsphenoidal surgery in cases of CD or tumor resection in cases of non-metastatic EAS). However, our patient presented with stage IV SCLC with EAS, in which chemotherapy remains the first-line treatment. SCLC patients with EAS have a poorer prognosis than those without EAS, with a life expectancy of only three to six months. This makes early diagnosis more important [2,7], as controlling the high cortisol levels and then administering systemic chemotherapy may achieve longer survival [8]. Apart from systemic chemotherapy, ketoconazole (widely accepted but highly toxic), metyrapone, mitotane (adrenocortical suppressant drug with significant side effects), and mifepristone (glucocorticoid antagonist, mainly used for the treatment of hyperglycemia in CS) can be used to reduce circulating glucocorticoids. Moreover, thromboprophylaxis and Pneumocystis jirovecii pneumonia prophylaxis should be started. Because ketoconazole may increase the risk of chemotherapy toxicity by inhibiting cytochrome P450 3A4, metyrapone has been reported to be a better choice [5,7]. Nonetheless, administration of chemotherapy in the setting of a hypercortisolism-induced immunosuppressive state, cancerous background and metabolic disorders featuring electrolyte disturbance and hyperglycemia, aggravate the condition and can be life-threatening. Thus, a palliative approach can sometimes be reasonable. Conclusions The diagnosis of CS is a three-step process that includes its suspicion based on the patient's laboratory and semiologic findings, the documentation of hypercortisolism, and the identification of its cause, which can be either ACTH-dependent or independent. The ectopic secretion of ACTH (EAS) by nonpituitary tumors is a relatively rare cause of CS and often presents as paraneoplastic syndromes, adding therapeutic and prognostic concerns. This case, in particular, highlights the importance of seeking alternative explanations for common electrolyte disturbances, particularly when they don't resolve promptly. Clinicians should be aware of EAS and its frequent subclinical presentation in order to initiate the diagnostic workup as soon as suspicion arises. References Hayes AR, Grossman AB: The ectopic adrenocorticotropic hormone syndrome: rarely easy, always challenging. Endocrinol Metab Clin North Am. 2018, 47:409-25. 10.1016/j.ecl.2018.01.005 Ilias I, Torpy DJ, Pacak K, Mullen N, Wesley RA, Nieman LK: Cushing's syndrome due to ectopic corticotropin secretion: twenty years' experience at the National Institutes of Health. J Clin Endocrinol Metab. 2005, 90:4955-62. 10.1210/jc.2004-2527 Lacroix A, Feelders RA, Stratakis CA, Nieman LK: Cushing’s syndrome. Lancet. 2015, 29:913-27. 10.1016/S0140-6736(14)61375-1 Nieman LK: Molecular derangements and the diagnosis of ACTH-dependent Cushing's syndrome. Endocr Rev. 2022, 43:852-77. 10.1210/endrev/bnab046 Nieman LK, Biller BM, Findling JW, Murad MH, Newell-Price J, Savage MO, Tabarin A: Treatment of Cushing's syndrome: an endocrine society clinical practice guideline. J Clin Endocrinol Metab. 2015, 100:2807-31. 10.1210/jc.2015-1818 Bostan H, Duger H, Akhanli P, et al.: Cushing's syndrome due to adrenocorticotropic hormone-secreting metastatic neuroendocrine tumor of unknown primary origin: a case report and literature review. Hormones (Athens). 2022, 21:147-54. 10.1007/s42000-021-00316-z Richa CG, Saad KJ, Halabi GH, Gharios EM, Nasr FL, Merheb MT: Case-series of paraneoplastic Cushing syndrome in small-cell lung cancer. Endocrinol Diabetes Metab Case Rep. 2018, 2018:4. 10.1530/EDM-18-0004 Zhang HY, Zhao J: Ectopic Cushing syndrome in small cell lung cancer: a case report and literature review. Thorac Cancer. 2017, 8:114-7. 10.1111/1759-7714.12403 From https://www.cureus.com/articles/198133-adrenocorticotropin-dependent-ectopic-cushings-syndrome-a-case-report#!/
  13. Background: Cushing’s disease (CD) poses significant challenges in its treatment due to the lack of reliable biomarkers for predicting tumor localization or postoperative clinical outcomes. Sphingosine-1-phosphate (S1P) has been shown to increase cortisol biosynthesis and is regulated by adrenocorticotropic hormone (ACTH). Methods: We employed bilateral inferior petrosal sinus sampling (BIPSS), which is considered the gold standard for diagnosing pituitary sources of CD, to obtain blood samples and explore the clinical predictive value of the S1P concentration ratio in determining tumor laterality and postoperative remission. We evaluated 50 samples from 25 patients who underwent BIPSS to measure S1P levels in the inferior petrosal sinuses bilaterally. Results: Serum S1P levels in patients with CD were significantly higher on the adenoma side of the inferior petrosal sinus than on the nonadenoma side (397.7 ± 15.4 vs. 261.9 ± 14.88; P < 0.05). The accuracy of diagnosing tumor laterality with the interpetrosal S1P and ACTH ratios and the combination of the two was 64%, 56% and 73%, respectively. The receiver operating characteristic curve analysis revealed that the combination of interpetrosal S1P and ACTH ratios, as a predictor of tumor laterality, exhibited a sensitivity of 81.82% and a specificity of 75%, with an area under the curve value of 84.09%. Moreover, we observed that a high interpetrosal S1P ratio was associated with nonremission after surgery. Correlation analyses demonstrated that the interpetrosal S1P ratio was associated with preoperative follicle-stimulating hormone (FSH), luteinizing hormone (LH), and postoperative ACTH 8 am levels (P < 0.05). Conclusion: Our study demonstrated a significant association between the interpetrosal S1P ratio and tumor laterality, as well as postoperative remission in CD, suggesting that the interpetrosal S1P ratio could serve as a valuable biomarker in clinical practice. 1 Introduction Cushing’s disease (CD), also known as adrenocorticotropic hormone (ACTH)-secreting pituitary adenoma, arises from the pituitary corticotroph cells and induces endogenous hypercortisolism by stimulating the adrenal glands to produce excessive amount of cortisol (1). Patients with CD typically exhibit symptoms of hypercortisolism, such as hypertension, diabetes, purplish skin striae, mental disturbances, hyposexuality, hirsutism, menstrual disorders, acne, fatigue, obesity, and osteoporosis (1). The overall mortality of patients with CD is twice that of the general population, and if left untreated, hypercortisolism resulting from CD increases this rate to approximately four times the expected value (2–4). Transsphenoidal surgery continues to be the primary treatment for CD (5). However, previous studies reported variable remission rates, ranging from 45% to 95% (6–8). Long-term follow-up data have revealed recurrence in 3–66% of patients who had initially achieved complete remission (9, 10). The rate of surgical remission in CD can be influenced by various factors, including the size and location of the tumor, expertise of the neurosurgeon, and criteria used for assessing remission (11). Preoperative clinical variables, such as age, gender, disease duration, and severity of clinical signs and symptoms, cannot reliably identify patients at a higher risk of nonremission (12, 13). Therefore, predicting postsurgical remission in CD remains a challenging goal. Accumulating evidence has shown that sphingosine-1-phosphate (S1P), an intracellular pleiotropic bioactive sphingolipid metabolite synthesized by sphingosine kinase 1 (SPHK1), plays a pivotal role in diverse endocrine disorders (14–16). Overexpression of SPHK1 promotes the progression of multiple neuroendocrine tumors (17, 18). ACTH can rapidly activate sphingolipid metabolism, causing an increase in S1P secretion in the adrenal cortex (19). Furthermore, the activation of S1P signaling in H295R cells, a human adrenocortical tumor cell line, has been suggested to induce increased transcription of hormone-sensitive lipase and steroidogenic acute regulatory protein, ultimately elevating cortisol production (20). Recently, surgical removal of ACTH-secreting adenoma has been reported to cause a decline in sphingomyelin levels (21). However, whether they have a similar role in the pituitary gland remains to be investigated. Bilateral inferior petrosal sinus sampling (BIPSS) is a highly effective procedure for diagnosing pituitary sources of ACTH in CD (22, 23). Contemporaneous differences in ACTH concentration during venous sampling between the two sides of the adenoma can predict the location of the adenoma within the pituitary (on the side of the gland with a microadenoma) and may guide surgical treatment in cases with inconclusive magnetic resonance imaging findings. Previous studies demonstrated that an ACTH gradient of ≥1.4 between the inferior petrosal sinuses can indicate microadenoma lateralization in patients with CD (24–26). However, the correct lateralization only occurs in 57–68% of all cases (27–29). Therefore, we analyzed the clinical behavior of a well-characterized cohort of patients with CD who underwent BIPSS before surgery. We measured the difference in the concentration of S1P in bilateral petrosal sinus blood samples and explored the clinical predictive value of the S1P concentration ratio in determining tumor laterality and postoperative remission. 2 Materials and methods 2.1 Patients and study design This study was conducted at a tertiary center, involving a cohort of 25 patients diagnosed with CD who had undergone BIPSS and surgery, with a minimum follow-up duration of 2 years. Comprehensive chart reviews were conducted to collect data on demographics, clinical characteristics, pituitary imaging findings, tumor pathology, and biochemical tests. The criteria used for diagnosing CD encompassed the presence of characteristic signs and symptoms of hypercortisolism, along with biochemical evaluation of two urinary free cortisol measurements exceeding the normal range for the respective assay, serum cortisol level >1.8 μg/dL (50 nmol/L) after an overnight 1-mg dexamethasone suppression test, and two late-night salivary cortisol measurements exceeding the normal range for the respective assay (30). A diagnosis of Cushing’s syndrome was established if the patient had positive test results for at least two of the three aforementioned tests. Adrenal insufficiency was diagnosed if patients exhibited symptoms or signs of adrenal insufficiency or if serum cortisol levels were ≤3 μg/dL, even in the absence of clinical signs or symptoms. Remission was defined as normalization of the levels of 24-h urinary free cortisol, late-night salivary cortisol, and overnight 1-mg dexamethasone suppression test in patients without concurrent central adrenal insufficiency after surgery (31). 2.2 Patients and tissue/serum samples Surgical specimens of CD-affected tissues were collected from Xiangya Hospital, Central South University. Three normal pituitary tissues were obtained from cadaveric organ donors without any history of endocrine disease (Central South University). A total of 25 CD tissue samples were obtained for immunohistochemistry analysis. This study was conducted in compliance with the Helsinki Declaration and was ethically approved by the Xiangya Hospital Ethics Committee, Xiangya Hospital (Changsha, China). Tumor samples and corresponding clinical materials were obtained with written consent from all patients. 2.3 BIPSS After obtaining informed consent, BIPSS was performed using standard techniques described in previous studies (32, 33). Briefly, the patient’s head was immobilized to ensure midline positioning and prevent any potential bias towards asymmetric pituitary drainage by the petrosal sinuses. After placing peripheral catheters and cannulating both inferior petrosal sinuses, blood samples were collected at baseline and at 3, 5, 10, and 15 min following intravenous administration of DDAVP, which stimulates pituitary production of ACTH. Additional samples for experimental purposes were collected immediately following the 15-min sample collection to avoid interference with the patient’s diagnostic study. 2.4 Measurement of baseline plasma S1P concentration Blood samples were obtained from both petrosal sinuses and were centrifuged to remove cellular components. Samples that exhibited hemolysis or coagulation were excluded from the study. Plasma samples were stored at −80°C. The S1P levels in plasma were analyzed using a S1P competitive ELISA kit (Echelon Biosciences, Salt Lake City, UT) according to the manufacturer’s instructions (34). 2.5 Immunofluorescence staining The pituitary tissues were post-fixed and dehydrated with alcohol as follows: 70% for 24 h, 80% for 3 h, 90% for 4 h, 95% for 3 h, and finally in absolute alcohol for 2 h. Tissue slices with a 5-μm thickness were cut using a microtome (Thermo Fisher Scientific), blocked with 3% BSA, and then treated with primary antibodies to SPHK1 (CST, #3297) and ACTH (Proteintech, CL488-66358). Subsequently, the tissue slides were incubated with Alexa Fluor 488-conjugated anti-rabbit (Invitrogen, A21206, 1:200) or Alexa Fluor 555-conjugated anti-rabbit (Invitrogen, A21428, 1:200) secondary antibodies. Specimens were visualized and imaged using a fluorescence microscope. 2.6 Statistical analysis The Mann–Whitney U test was used to assess the clinical–molecular associations in adenoma samples, whereas the chi-square test was used to compare categorical data. The Kruskal–Wallis analysis and ANOVA were conducted for multiple comparisons. Statistical analyses were conducted using SPSS v20 and GraphPad Prism version 7. All results were presented in graphs and tables as median ± interquartile range. The distribution of each parameter was presented as the minimum–maximum range. Parametric or nonparametric statistical tests were applied, as appropriate, after testing for normality. The receiver operating characteristic curve was used to determine the cut-off value for predicting tumor laterality. Pearson correlation analyses was used to examine the correlations between variables. Proportions were expressed as percentages, and significance was defined as P < 0.05. 3 Results 3.1 Clinical characteristics of remission and nonremission in patients with CD This study included 25 patients with CD who underwent BIPSS before surgery (Figure 1). Among them, 12 patients had microadenomas, whereas the remaining 13 had inconclusive magnetic resonance imaging findings; clinicopathological data are summarized in Supplementary Table 1. Table 1 displays the demographics of patients who achieved remission (n = 16) and those who did not (n = 9). No significant differences were observed in terms of sex, age at diagnosis, or radiological variables between patients who achieved and those who did not achieve remission (P > 0.05). Patients who achieved remission exhibited a higher prevalence of emotional lability (P < 0.05). However, no significant differences were observed in other parameters (P > 0.05). Figure 1 Figure 1 Flowchart of the screening process employed to select eligible participants for the study. Table 1 Table 1 Baseline clinical features of patients with pituitary tumors secreting adrenocorticotropin. Several recent studies have established morning cortisol level measured on postoperative day 1 (POD1) as a predictive biomarker for long-term remission of CD (35, 36). For biochemical features, patients who did not achieve remission exhibited higher serum cortisol (19.16 ± 5.55 vs. 5.95 ± 1.42; P = 0.014) and median serum (8 am) ACTH (10.26 ± 8.24 vs. 5.15 ± 3.68; P = 0.042) levels on POD1. No significant differences were observed in the preoperative baseline 4 pm serum cortisol levels, preoperative baseline 0 am serum cortisol levels, preoperative 8 pm ACTH levels, 4 pm ACTH levels, and 0 am ACTH levels (P > 0.05) (Table 2). In addition preoperative FT3, FT4, TSH, GH, FSH, LH, and PRL levels were comparable in patients with and without remission. Table 2 Table 2 Baseline clinical and biochemical features of patients with pituitary tumors secreting adrenocorticotropin. 3.2 Overexpression of SPHK1 and higher concentrations of serum S1P on the tumor side in patients with CD Prior studies have demonstrated that ACTH acutely activates SPHK1 to increase S1P concentrations (19). Upregulation of SPHK1 is associated with poor prognosis in endocrine-related cancer (17, 18, 21). To investigate the role of SPHK1 in CD, we performed a heatmap analysis of key genes involved in phospholipid metabolism and signaling pathways in CD adenomas and surrounding normal tissues using the GEO dataset (GEO208107). This analysis revealed the activation of crucial genes involved in phospholipid metabolism and signaling pathways in ACTH-secreting pituitary adenomas (Supplementary Figure 1). Subsequently, we compared the association between pituitary SPHK1 expression and proopiomelanocortin, corticotropin-releasing hormone, corticotropin releasing hormone receptor 1, and corticotropin releasing hormone receptor 2 in pituitary tumor tissues and identified a positive correlation between SPHK1 and ACTH tumor-related genes in the TNM plot database (Supplementary Figure 2). To investigate the potential role of SPHK1 in CD, we compared the expression values of SPHK1 in the normal pituitary tissues and those obtained from patients with CD in the remission/nonremission groups. Immunofluorescence staining (Figures 2A, B; Supplementary Figure 3) revealed an increased number of double-positive cells for SPHK1 and ACTH in CD-affected pituitary tissues than those in the normal pituitary tissues. Furthermore, the proportion of double-positive cells for SPHK1 and ACTH was significantly higher in the nonremission CD adenomas tissues than that in the remission CD adenomas. Furthermore, we investigated the concentration of S1P in bilateral petrosal sinus blood samples and observed that the concentration was significantly higher on the adenoma side than that on the nonadenoma side (397.7 ± 15.4 vs. 261.9 ± 14.88; P < 0.05, Figure 2C). Thus, these findings suggested a close association between S1P concentration and the development of ACTH-secreting tumor. Figure 2 Figure 2 (A) Representative images of immunofluorescence double staining for SPHK1 (green) and ACTH (pink) in normal pituitary glands and ACTH-secreting pituitary adenomas from the remission and nonremission groups (Normal: n = 3, ACTH pituitary adenoma: remission vs. nonremission: n = 16 vs. 9); scale bars: 100-μm upper and 50-μm lower. (B) Quantitative analysis; white arrows indicate double-positive cells for ACTH and SPHK1. (C) The concentration of S1P in the plasma obtained from the inferior petrosal sinus of the adenoma side and nonadenoma side. ***P < 0.001. Bar represents mean ± SD. 3.3 Combination of interpetrosal S1P and ACTH ratios improved the diagnostic performance for adenoma laterality The pathology of patients with CD was classified based on adenomatous tissue with ACTH-positive immunostaining into adenoma or nonadenoma sides. To evaluate the correlation between the interpetrosal S1P ratio lateralization and tumor location, we compared the accuracy of predicting tumor laterality using the interpetrosal S1P ratio (>1) and interpetrosal ACTH ratio (>1.4) (the interpetrosal ACTH ratio >1.4 is acknowledged for its positive role in predicting tumor laterality), as well as their combination. Our results indicated that using the interpetrosal S1P or ACTH ratios alone yielded accuracies of 64% and 56% respectively. Notably, the combination of both demonstrated a significantly improved accuracy of 73% (Figure 3A). Figure 3 Figure 3 (A) Bar graph illustrating the accuracy of predicting tumor laterality. (B) Receiver operating characteristic (ROC) curve analysis of interpetrosal ACTH ratio to predict tumor location. (C) ROC curve analysis of the interpetrosal S1P ratio to predict tumor location. (D) ROC curve analysis of the combination of the interpetrosal S1P and ACTH ratios to predict tumor location. Thereafter, the receiver operating characteristic analysis was performed to determine the role of predicting tumor laterality. In particular, the interpetrosal ACTH ratio with an AUC of 75.32% (95% CI: 60.06–97.46%, P < 0.05) and the interpetrosal S1P ratio demonstrated a clinically significant diagnostic accuracy for lateralization, with an AUC of 79.17% (95% CI: 44.40–85.84%, P < 0.05). Furthermore, combining the interpetrosal S1P and ACTH ratios generated an receiver operating characteristic curve with an AUC of 84.09% (95% CI: 52.3–96.77%, P < 0.05) for predicting lateralization with tumor location (cutoff value: interpetrosal S1P ratio ≥1.06, interpetrosal ACTH ratio ≥2.8, 81.82% sensitivity, and 75% specificity) (Figures 3B–D). 3.4 Interpetrosal S1P ratio serves as a predictive factor for early remission in CD To investigate whether the interpetrosal S1P ratio is associated with early postoperative remission in CD, we compared the baseline interpetrosal S1P ratio between patients with CD in the remission and nonremission groups. Interestingly, we observed that the nonremission group exhibited higher interpetrosal S1P ratios than those of the remission group (median, 1.28 ± 0.25 vs. 1.10 ± 0.09, P = 0.012) (Figure 4). Figure 4 Figure 4 Left picture: Scatter plot of bilateral S1P concentrations in the remission and nonremission groups; the slope represents the interpetrosal S1P ratio, blue dots represent the remission group, and red dots represent the nonremission group. Right picture: The interpetrosal S1P ratio in the remission and nonremission groups. *P < 0.05. Bar represents mean ± SD. To investigate potential factors affecting the interpetrosal S1P ratio, we compared the correlation between interpetrosal S1P ratio and various clinical indicators. This analysis revealed that the interpetrosal S1P ratio positively correlated with preoperative FSH and LH levels, as well as with postoperative 8 am ACTH levels. No significant difference was observed between the interpetrosal S1P ratio and other indicators (Supplementary Figure 4). 4 Discussion The use of BIPSS involves collection of samples from each inferior petrosal sinus simultaneously, enabling a direct comparison of ACTH concentrations between the left and right petrosal sinuses. BIPSS is used for two purposes: 1) to assist in the differential diagnosis of Cushing’s syndrome; and 2) to determine which side of the pituitary gland contains an adenoma in patients with CD. The interpetrosal ACTH ratio is also useful in determining the location/lateralization of pituitary microadenomas (24, 30, 37), thereby providing guidance to the neurosurgeon during surgery. To our knowledge, this is the first study to demonstrate that serum S1P levels in patients with CD are significantly higher on the adenoma side of the inferior petrosal sinus than on the nonadenoma side. The interpetrosal S1P ratio exhibited a positive significance in predicting tumor laterality, and the predictive performance was improved when S1P was combined with the interpetrosal ACTH ratio. Notably, the interpetrosal S1P ratio exhibited a positive significance in predicting remission after surgery. Furthermore, the interpetrosal S1P ratio demonstrated a positive and significant correlation with preoperative FSH and LH levels, as well as 8 am ACTH levels on POD1. ACTH is recognized for its role in controlling the expression of genes involved in steroid production and cortisol synthesis in the human adrenal cortex through sphingolipid metabolism (19). Specifically, ACTH rapidly stimulates SPHK1 activity, leading to an increased in S1P levels, which in turn, increases the expression of multiple steroidogenic proteins (20). Our study demonstrated that higher S1P concentrations were present on the tumor side than on the nontumor side in patients with CD, indicating that the regulatory relationship between ACTH and S1P also exists in ACTH-secreting pituitary adenomas. Several pieces of evidence have supported the potential relationship between S1P and the occurrence of CD. Interestingly, SPHK1 and S1P are known to be integral to the regulation of epidermal growth factor receptor (EGFR) (38), which is highly expressed in human corticotropinomas, where it triggers proopiomelanocortin (the precursor of ACTH) transcription and ACTH synthesis (39). Blocking EGFR activity with an EGFR inhibitor can attenuate corticotroph tumor cell proliferation (40). Furthermore, SPHK1 and proopiomelanocortin share a common transcriptional coactivator, P300 (41, 42). Notably, S1P also directly binds to and inhibits histone deacetylase 2, thereby regulating histone acetylation and gene expression (43). Notably, histone deacetylase 2 expression is deficient in ACTH-pituitary adenomas in CD, contributing to glucocorticoid insensitivity (44), which is a hallmark of CD and a feature associated with nonremission. These studies further demonstrated an association between high S1P ratio and nonremission of CD. Our study, for the first time, established an association between SPHK1/S1P and ACTH adenoma. Nevertheless, further experimental verification is required to confirm the existence of common pathways linking SPHK1 and ACTH. Thus, these findings indicated that the S1P ratio can, to some extent, reflect the differences in ACTH levels and may serve as a surrogate marker for detecting ACTH-secreting pituitary adenomas. BIPSS is a highly effective procedure for diagnosing pituitary sources of ACTH in CD and remains the gold standard diagnostic method. However, some findings indicated certain limitations associated with the use of the inferior petrosal sinus sampling (IPSS) method in predicting tumor lateralization. The possible causes of error include asymmetrical or underdeveloped petrosal sinus anatomy and placement of the catheter (27). The present study revealed a notable increase in the interpetrosal ACTH ratio among patients with accurate predictions of tumor laterality than among those with inaccurate predictions, although the positive predictive value remained low. These findings suggested that other mechanisms may exist that contribute to false-positive results. The limitations on lateralization highlighted the need for further research to understand the underlying mechanisms contributing to the accuracy of IPSS in predicting tumor lateralization. Further investigation is required to understand these potential mechanisms and improve the accuracy of IPSS in predicting tumor lateralization. We observed that the interpetrosal S1P ratio was slightly more effective than the ACTH ratio in predicting tumor laterality. However, combining both methods significantly improved the diagnostic sensitivity and specificity. These results have important implications for clinical practice as accurate tumor lateralization is essential for the correct management and treatment of pituitary adenomas. Overall, these findings highlighted the importance of using multiple measures in predicting tumor lateralization and suggested that combining measures may be more effective than relying on any single measure alone. Future research should investigate additional measures to improve the accuracy of tumor lateralization and optimize the use of existing measures for making clinical decisions. The initial treatment recommendation for CD is surgery. However, long-term surveillance is necessary because of the high recurrence rate (12). Therefore, identifying patients who are at a greater recurrence risk would be helpful in establishing an effective surveillance strategy. Our study revealed that the expression of SPHK1 in pituitary tissue was higher in postoperative nonremission group than in postoperative remission group. Moreover, patients in the nonremission group exhibited significantly higher interpetrosal S1P ratios than those of patients in the remission group. SPHK1 catalyzes the direct phosphorylation synthesis of S1P, and the S1P ratio can thus reflect the expression level of SPHK1 in ACTH tumors. Since S1P can increase the expression of multiple steroidogenic proteins, including steroidogenic acute regulatory protein, 18-kDa translocator protein, low-density lipoprotein receptor, and scavenger receptor class B type I (20), the interpetrosal S1P ratios may be indicative of disease prognosis. This finding is consistent with previous findings indicating the overexpression of SPHK1 is associated with poor prognosis in various neuroendocrine tumors, as factors associated with tumor proliferation, S1P and SPHK1, may play a key role in the proliferation and survival of ACTH pituitary adenomas. The high proportions of SPHK1/ACTH double-positive cells are likely associated with greater phenotypic severity, and CD tumors with this phenotype may have a poor prognosis. These findings hold clinically significance for predicting early postoperative remission in patients with CD. As aforementioned, the interpetrosal S1P ratios have been suggested as a useful diagnostic tool for determining adenoma lateralization in CD, which can also serve as a prognostic indicator for postoperative remission. Pearson correlation analysis indicated that ACTH 8 am on POD1 and FSH/LH levels were significantly associated with the interpetrosal S1P ratio, suggesting that these pituitary dysfunctions may have a role in the early remission of CD. However, the sample size in this study was relatively small, and further studies with larger sample sizes are needed to confirm these findings. Additionally, other factors affecting surgical outcomes, such as the experience of the surgeon, extent of surgical resection, and use of adjuvant therapy, should be considered when predicting postoperative remission in patients with CD. This study has some limitations. First, the study was retrospective in design, which limited the control of confounding factors. Additionally, because of the limited sample size, we did not specifically investigate cases where the ACTH ratio failed to accurately identify the correct tumor location. Finally, we did not explore the functional evidence of a common pathway between SPHK1 and ACTH. Despite these limitations, the study contributes to our understanding of the potential utility of the interpetrosal S1P ratio as a biomarker for CD and provides a basis for future research in this area. In conclusion, our study demonstrated a significant association between the interpetrosal S1P ratio and tumor laterality, as well as in early remission in CD. These findings suggested that the interpetrosal S1P ratio could serve as a useful biomarker in clinical practice. Moreover, targeting genes and drugs related to SPHK1/S1P could provide novel therapeutic strategies for treating CD. 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 The studies involving humans were approved by The Xiangya Hospital Ethics Committee, Xiangya Hospital (Changsha, China). The studies were conducted in accordance with the local legislation and institutional requirements. The participants provided their written informed consent to participate in this study. Author contributions HS: conceptualization, methodology, software, visualization, and investigation. CW and BH: software. YX: writing – review & editing. 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. Acknowledgments The authors gratefully acknowledge contributions from the GEO databases and TNMplot database (https://www.tnmplot.com/). Conflict of interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Publisher’s note All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher. Supplementary material The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fendo.2023.1238573/full#supplementary-material References 1. Tritos NA, Miller K. Diagnosis and management of pituitary adenomas: A review. JAMA (2023) 329(16):1386–98. doi: 10.1001/jama.2023.5444 PubMed Abstract | CrossRef Full Text | Google Scholar 2. 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Genes Dev (2006) 20:2871–86. doi: 10.1101/gad.1444606 PubMed Abstract | CrossRef Full Text | Google Scholar Keywords: ipss, sphingosine-1-phosphate, Cushing’s disease, remission, tumor laterality Citation: Sun H, Wu C, Hu B and Xiao Y (2023) Interpetrosal sphingosine-1-phosphate ratio predicting Cushing’s disease tumor laterality and remission after surgery. Front. Endocrinol. 14:1238573. doi: 10.3389/fendo.2023.1238573 Received: 12 June 2023; Accepted: 17 October 2023; Published: 31 October 2023. Edited by: Anton Luger, Medical University of Vienna, Austria Reviewed by: Guangwei Wang, Hunan University of Medicine, China Marie Helene Schernthaner-Reiter, Medical University of Vienna, Austria Copyright © 2023 Sun, Wu, Hu and Xiao. 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: Yuan Xiao, xiaoyuan2021@csu.edu.cn Disclaimer: All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article or claim that may be made by its manufacturer is not guaranteed or endorsed by the publisher. From https://www.frontiersin.org/articles/10.3389/fendo.2023.1238573/full
  14. Abstract Background This single-center retrospective cohort study aimed to describe the findings and validity of Bilateral inferior petrosal sinus sampling (BIPSS) in the differential diagnosis of patients with ACTH-dependent Cushing’s syndrome (CS). Methods Eleven patients underwent BIPSS due to equivocal biochemical tests and imaging results. Blood samples were taken from the right inferior petrosal sinus (IPS), left IPS, and a peripheral vein before and after stimulation with desmopressin (DDAVP). ACTH and prolactin levels were measured. The diagnosis was based on the ACTH ratio between the IPS and the peripheral vein. Also, lateralization of pituitary adenoma in patients with Cushing’s disease (CD) was predicted. No significant complications were observed with BIPSS. Results Based on the pathology report, eight patients had CD, and three had ectopic ACTH syndrome (EAS). Unstimulated BIPSS resulted in a sensitivity of 87.5%, specificity of 100%, PPV of 100%, NPV of 75%, and accuracy of 91%. Stimulated BIPSS resulted in a sensitivity of 100%, specificity of 100%, PPV of 100%, NPV of 100%, and accuracy of 100%. However, pituitary magnetic resonance imaging (MRI) had a lower diagnostic accuracy (sensitivity:62.5%, specificity:33%, PPV:71%, NPV:25%, accuracy:54%). BIPSS accurately demonstrated pituitary adenoma lateralization in 75% of patients with CD. Conclusions This study suggests that BIPSS may be a reliable and low-complication technique in evaluating patients with ACTH-dependent CS who had inconclusive imaging and biochemical test results. The diagnostic accuracy is improved by DDAVP stimulation. Pituitary adenoma lateralization can be predicted with the aid of BIPSS. Peer Review reports Introduction All disorders with manifestations associated with glucocorticoid excess are called Cushing’s syndrome. Exogenous corticosteroids cause most CS cases, and endogenous CS cases are rare [1, 2]. The diagnosis of Cushing’s syndrome may be complicated, particularly in cases with ambiguous clinical findings, atypical presentations, and cyclic hypercortisolemia [3,4,5]. The initial laboratory tests for diagnosis of CS include 24-hour urinary free cortisol (UFC), late-night salivary cortisol, and low-dose dexamethasone suppression test (DST). These tests only represent hypercortisolemia [1, 2]. Once CS is diagnosed, further evaluations are needed to identify the etiology. The first step is to measure the plasma ACTH level. A low plasma ACTH level indicates ACTH-independent CS and a high level suggests ACTH-dependent CS. Normal ACTH can also occur in ACTH-dependent CS. Almost all cases of ACTH-dependent are due to pituitary adenoma (Cushing’s disease) or EAS [1, 2, 6]. Some ectopic sources include neuroendocrine tumors, bronchial carcinoma, and pancreatic carcinoma [7, 8]. Because of the high mortality in tumors associated with EAS, it is essential to differentiate CD from EAS. To distinguish CD from EAS, a high-dose dexamethasone suppression test (HDDST), corticotropin-releasing hormone (CRH), or DDAVP stimulation tests, or pituitary MRI is recommended [1, 2, 6, 9,10,11,12]. MRI can be equivocal in half of the patients, and only relatively large lesions (> 6 mm) detected on MRI reliably confirm the diagnosis of CD with biochemical confirmation and expected clinical symptoms [9]. Considering the relatively low sensitivity and specificity of non-invasive tests [13, 14] and the high complications of the surgery, it seems reasonable to use a test with high sensitivity and specificity and few complications before resection. BIPSS with CRH or DDAVP stimulation can be helpful for further evaluation [1, 2, 10, 15, 16]. The BIPSS procedure is the same in both stimulation methods. Due to its lower cost, availability, and comparable diagnostic accuracy, using DDAVP instead of CRH for BIPSS is an alternative [17, 18]. BIPSS has been reported to have high sensitivity and specificity and is a safe procedure when performed by experienced interventional radiologists [15, 16, 19, 20]. This case series describes the experience with BIPSS and examines the validity of BIPSS for differentiating CD from EAS in patients with ACTH-dependent CS who had ambiguous or equivocal results in non-invasive tests. Materials and methods Patients This retrospective cohort study included 11 patients with ACTH-dependent CS who underwent BIPSS between 2018 and 2020 in a tertiary care hospital. Data collection Well-trained nurses conducted anthropometric measurements, including height and weight. Standing height was measured with a portable stadiometer (rounded to the nearest 0.1 cm). Using a calibrated balance beam scale, this study measured weight in the upright position (rounded to the nearest 0.1 kg). Body mass index (BMI) was calculated by dividing weight (kg) by height squared (m2). Well-trained examiners measured blood pressure (systolic and diastolic) at the left arm in the sitting position after 5 min of rest using a calibrated mercury sphygmomanometer. The blood sample was taken, and fasting blood sugar (FBS), hemoglobin (Hb), potassium (K), and creatinine (Cr) were measured. All research was performed in accordance with the Declaration of Helsinki. Informed consent was obtained from all participants or their legal guardians. Biochemical tests and imaging Patients with signs and symptoms of CS underwent screening evaluations, and confirmatory tests were performed using serum cortisol and 24-hour UFC. After confirmation of CS, ACTH was measured using an immunoradiometric assay to categorize patients into ACTH-dependent or independent groups. ACTH test was performed with SIEMENS IMMULITE 2000 device with an analytical sensitivity of 5 pg/ml (1.1 pmol/l) and CV ∼7.5%. HDDST was conducted by administering 2 mg dexamethasone every 6 h for 48 h to all patients, and then serum cortisol and 24-hour UFC were rechecked. A pituitary MRI was performed with sagittal and coronal T1- and T2-weighted images before and after the gadolinium injection. BIPSS procedure After biochemical tests and imaging, an experienced interventional radiologist performed bilateral and simultaneous catheterization of the inferior petrosal sinuses. Venography was obtained to evaluate venous anatomy and catheter placement. The retrograde flow of contrast dye into the contralateral cavernous sinuses was used as a marker of adequate sampling. After the correct placement of catheters, blood samples were obtained from each of three ports (peripheral (P), left inferior petrosal sinus (IPS), and right IPS) at -15, -10, -5, and 0 min. The current study used DDAVP for stimulation. After peripheral injection of 10 micrograms of DDAVP, blood samples from these three sites were obtained at + 3, +5, + 10, and + 15 min. Three samples from these sites were also obtained to measure prolactin. Upon collection, BIPSS samples were placed in an ice-water bath. At the end of the procedure, samples were taken to the laboratory, where the plasma was separated and used for immediate measurement of ACTH. Specimens were refrigerated, centrifuged, frozen, and assayed within 24 h. After the samples were obtained, both femoral sheaths were removed, and manual compression was used to obtain hemostasis before transferring patients to the recovery room. The whole procedure took 1–2 h. Patients underwent strict bed rest for 4 h before discharge on the same day. All BIPSS were performed without significant complications, and only hematoma at the catheterization site was observed in some patients. BIPSS interpretation The ratio of IPS ACTH to peripheral ACTH level (IPS/P ACTH) for each side was calculated. Baseline sampling at minute 0 with IPS/P ≥ 2 or stimulated sampling at minute 3 with 1PS/P ≥ 3 is confirmatory for CD [1, 8]. Also, the IPS/P ratio was checked for prolactin level after DDAVP stimulation (stimulated IPS/P prolactin). A stimulated IPS/P prolactin ≥ 1.8 indicates successful catheterization, meaning the catheter is correctly placed in the IPS [21]. For further evaluation, the current study normalized the ACTH to the prolactin level by dividing stimulated IPS/P ACTH into stimulated IPS/P prolactin for each side. A normalized ACTH/prolactin IPS/P ratio ≥ 1.3 supports a pituitary ACTH source (Cushing’s disease), and a normalized ratio ≤ 0.7 an ectopic source (EAS) [22]. The values between 0.7 and 1.3 are equivocal. The inter-sinus ratio was defined as the ratio of the IPS/P ACTH level of one side with the higher level divided by the IPS/P ACTH level of the other side with the lower level, either before or after stimulation. An inter-sinus ratio ≥ 1.4 indicates lateralization to the side with a higher IPS/P ACTH level [23]. Statistical analysis This analysis used SPSS software version 18 (SPSS, Inc.) to perform analyses. Data were expressed as numbers and percentages. Continuous variables were presented as means (± SD). This study reported the median or range when the data did not follow a normal distribution. The Shapiro-Wilk test was used to test for normality. The nonparametric Mann-Whitney U Test was utilized to compare variables. The sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and accuracy of the tests were calculated based on standard statistical equations. Results Baseline characteristics and clinical manifestations This retrospective research studied 11 patients with ACTH-dependent CS, including eight females (72.7%) and three (27.3%) males. The median (Q1-Q3) age was 32.0 (22–45) years. The median (Q1-Q3) of BMI, systolic blood pressure (SBP), diastolic blood pressure (DBP), FBS, Hb, K, and Cr were 29.2 (24.8–33.3), 130.0 (125–140), 80.0 (80–95), 98.0 (88–103), 13.5 (12.4–13.9), 4.2 (3.9–4.5), and 1.0 (0.9–1.1), respectively. The demographic characteristics of patients are presented in Table 1. The Hb levels were not different in women and men (median 13.35 vs. 13.70, p-value = 0.776). In addition, no statistical difference between patients with a final diagnosis of CD and EAS was detected for Hb levels (Total: median 13.60 vs. 13.2, p-value > 0.05) (Women: median 13.5 vs. 13.2, p-value > 0.05) (Men: median 13.7 vs. 13.25, p-value > 0.05). Table 1 Demographic characteristics of the studied patients Full size table 90% of patients had at least one skin manifestation, such as striae, easy bruising, acne, hyperpigmentation, hirsutism, hair loss, edema, and hypertrichosis. Other symptoms were hypertension (HTN) (81%), reproductive dysfunction (81%), including infertility, oligomenorrhea, loss of libido, weight gain (72%), proximal muscle weakness (45%), and headache (27%) (Table 2). Table 2 Clinical manifestations of the studied patients Full size table Results of biochemical tests Biochemical tests results, including basal serum cortisol (median:26 mcg/dl, range:15-54.5 mcg/dl), basal 24-hour UFC (median:670 mcg/dl, range:422–1545 mcg/dl), ACTH (median:58.8 pg/ml, range:25–155 pg/ml), serum cortisol after HDDST (median:14.2 mcg/dl, range:2.63-36.0 mcg/dl), 24-hour UFC after HDDST (median:292 mcg/dl, range:29.5–581 mcg/dl) are presented in Table 3. According to the basal serum cortisol results, eight patients (Cases 1, 3, 5, 7, 8, 9, 10, and 11) had basal serum cortisol levels > 22 mcg/dl, which indicates hypercortisolemia. Other patients (Cases 2, 4, and 6) had basal serum cortisol in the normal range (5–25 mcg/dl) and were considered as false negative results of this test. Table 3 The results of biochemical tests in the studied patients Full size table All patients had elevated basal 24-hour UFC levels (422–1545 mcg/dl), indicative of hypercortisolemia (Table 3). There were six patients with elevated peripheral ACTH levels (> 58 pg/ml) (cases 5, 6, 8, 9, 10, and 11). Other patients had ACTH within the normal range (6–58 pg/ml) (cases 1, 2, 3, 4, 7) (Table 3). None of the patients showed suppression after 1 mg DST. After HDDST, cases 2, 3, 8, and 10 had more than 50% suppression of serum cortisol. In the other six patients, serum cortisol was not suppressed or suppressed by less than 50%. In one patient, serum cortisol levels were not measured (case 1) because the sample was not stored under standard test conditions. Also, eight patients had more than 50% 24-hour UFC suppression after HDDST (cases 1, 2, 3, 4, 6, 7, 9, and 10). In two patients, 24-hour UFC was suppressed less than 50% (cases 5 and 11), and in one patient (case 8), the 24-hour UFC sample was not tested due to the non-standard condition of the sample. BIPSS results BIPSS results before and after stimulation are shown in Table 4. The baseline value (sampling at minute 0) of IPS/P ACTH ≥ 2 confirms CD. According to this ratio, cases 1,3,4,5,6,7, and 8 were diagnosed as CD. The unilateral source for CD was confirmed in cases 1, 3, 7, and 8. BIPPS didn’t demonstrate lateralization in cases 4, 5, and 6. Table 4 Baseline and stimulated IPS/P ratio for ACTH and Prolactin in the studied patients Full size table The highest IPS/P ACTH ratio was 3 min after the DDAVP injection. A sampling at minute 3 with stimulated IPS/P ACTH ≥ 3 confirms CD. This ratio confirmed CD in cases 1–8 and showed a unilateral source for CD in cases 1, 2, 3, and 7. The ratio didn’t demonstrate lateralization in cases 4, 5, 6, and 8. The stimulated IPS/P prolactin was ≥ 1.8 in all cases. The variability in the IPS/P ACTH ratio in patients with CD is shown in Fig. 1. The peak of this ratio was 3 min after the DDAVP injection. In patients with EAS, there were no changes before or after the DDAVP stimulation. Fig. 1 Comparison of mean values of IPS/P ACTH in CD (Lt.) and EAS (Rt.). IPS; inferior petrosal sinus; P: peripheral; ACTH: adrenocorticotropic hormone; CD: Cushing’s disease; EAS: ectopic ACTH syndrome; Lt: left; Rt: right Full size image According to the Prolactin-normalized ACTH IPS/P ratios, eight patients (cases 1–8) were diagnosed as CD and three as EAS (cases 9–11). In cases 1, 2, 3, 7, and 8, unilateral sources of CD were confirmed, but in cases 4,5 and 6, bilateral sources were detected (Table 4). According to the inter-sinus ratio, BIPSS could lateralize the source of ACTH in all patients with CD. The inter-sinus ratio in patients with EAS could not lateralize any pituitary source for ACTH (Table 4). In five patients with CD and one with EAS, the highest peripheral ACTH level was observed 15 min after stimulation. Two patients with CD and one with EAS had the highest peripheral ACTH level 10 min after stimulation. Only one patient with CD and one with EAS had the highest peripheral ACTH level 5 min after stimulation. No patient had maximum peripheral ACTH levels in the first post-stimulation sample (minute 3). The larger numerator or smaller denominator produces a higher value in a ratio. In the samples obtained immediately after stimulation, the highest concentration of ACTH was in the IPS, and the lowest was in the peripheral blood. Therefore, as mentioned, the highest post-stimulation value of the IPS/P ACTH ratio was obtained at minute 3. MRI results MRI results showed pituitary adenoma in five patients, enhancement in one patient, pituitary mass and lesion in two patients, empty sella in two patients, and possible pituitary adenoma and adrenal mass in one patient (Table 5). Table 5 Final diagnosis, lateralization, MRI results, and management Full size table Immunohistochemistry (IHC) results According to the pathology report, eight patients were confirmed as CD (Table 5). The other two patients were EAS (one carcinoid tumor of the lung and one pheochromocytoma). One patient had no documented pathologic source of hypercortisolemia because the patient did not consent to surgery, and the diagnosis of EAS was made based on the results of biochemical tests. BIPSS vs. MRI results MRI results showed pituitary adenoma in five patients with CD. MRI and BIPSS showed the adenoma on a similar side in two of them. In the other three patients, MRI showed bilateral adenoma, but BIPSS lateralized the adenoma to one side. One of the other three patients had only left-sided enhancement but no overt adenoma on MRI, whereas BIPSS lateralized the adenoma to the right side. One patient had a low-signal pituitary mass on the right side on MRI, and BIPSS also lateralized to the right. Another patient with a history of transsphenoidal surgery (TSS), diagnosed as recurrent CD, had a partially empty sella. MRI was equivocal, but BIPSS lateralized to the left side. Among patients with EAS, one with an equivocal BIPSS result had an empty sella on MRI. Two other patients had pituitary lesions on MRI, but BIPSS results were equivocal. Comparison between BIPSS, MRI, and surgery Among patients with CD, the final diagnosis based on surgery in three patients was consistent with MRI and BIPSS results and lateralized the adenoma on the same side. In one patient, the surgery result was similar to the MRI findings and showed bilateral adenoma, but BIPSS showed adenoma on the left side. In the patient with equivocal MRI findings and a history of TSS, IHC could not identify ACTH +, although BIPSS lateralized to the left side. In three other patients, surgery results were concordant with BIPSS and lateralized the adenoma on the same side, although MRI showed discordant results. Validity of BIPSS Baseline IPS/P ACTH resulted in a sensitivity of 87.5%, specificity of 100%, PPV of 100%, NPV of 75%, and accuracy of 91%. Stimulation with DDAVP improved validity. Both stimulated IPS/P ACTH and normalized ACTH/prolactin IPS/P ratio resulted in a sensitivity of 100%, specificity of 100%, PPV of 100%, NPV of 100%, and accuracy of 100%. BIPSS, either unstimulated or stimulated, had higher validity than MRI, with a sensitivity of 62.5%, specificity of 33%, PPV of 71%, NPV of 25%, and accuracy of 54%. BIPSS accurately predicted pituitary adenoma lateralization in 75% of patients with CD. Discussion In this study, BIPSS before stimulation showed a sensitivity of 87.5% and a specificity of 100%. However, BIPSS after stimulation showed a sensitivity of 100% and specificity of 100%. It has been demonstrated that the sensitivity of BIPSS can vary from 88 to 100%, and its specificity from 67 to 100% in the diagnosis of CD [24]. Previous studies have reported sensitivity and specificity of more than 80% and 90% for BIPSS, and the combination of BIPSS with stimulation by CRH or DDAVP improves the sensitivity and specificity to more than 95 and 100%, respectively [15, 19, 25]. Chen et al. suggested the optimal IPS:P cutoff value of 1.4 before and 2.8 after stimulation [20]. Considering these cutoffs, the only patient in this study who was negative for CD before stimulation becomes positive, and the sensitivity before stimulation increases from 87.5 to 100%. The diagnostic accuracy after stimulation remains unchanged. Results of the current study showed that BIPSS is highly valued in final diagnosis, even without stimulation. In this investigation, the utilization of Prolactin-normalized ACTH IPS/P ratios exhibited a sensitivity and specificity of 100% for the CD diagnosis. This finding aligns with research conducted by Detomas et al., which reported a sensitivity of 96% and specificity of 100% for the normalized ACTH: Prolactin IPS/P ratio [26]. It seems that concurrently assessing prolactin levels may potentially enhance the diagnostic accuracy of BIPSS. However, the current literature is inconsistent. Some studies do not support the use of prolactin to diagnose CD [27]. In all patients, the IPS/P ACTH ratio at minute 15 did not show a considerable difference from this ratio at minute 0. Previous studies have shown that sampling at minute 15 is not helpful for diagnosis [1, 15, 20, 28]. Unlike the IPS/P ACTH ratio, six patients had the highest peripheral ACTH level at minute 15 after stimulation, but no patient had it at minute 3 after stimulation. However, more studies are needed to obtain more precise results, and this study’s sample size was limited. BIPSS accurately lateralized the adenoma in six patients with CD, but MRI was able to lateralize the adenoma in two patients correctly. BIPSS had higher validity than MRI in differentiating CD from EAS, both with and without stimulation. The current literature is controversial. Colao et al. reported that adenoma could be accurately localized in 65% of patients using IPSS [23]. However, Lefournier et al. showed that the diagnostic accuracy of IPSS in identifying the side of the pituitary adenoma was 57% [28]. Wind et al. showed that the PPV for IPSS to identify the tumor side correctly was 69%. Additionally, MRI was more accurate than IPSS in tumor lateralization [29]. Earlier studies have shown that MRI may show a pituitary lesion, and BIPSS indicates a pituitary adenoma. However, the lesion observed on the MRI is not related to the pituitary adenoma [1, 15, 19, 25, 28]. Also, MRI may show pituitary lesions, while BIPSS indicates EAS. In the current study, the concordance of IHC results with BIPSS and MRI findings was inconclusive, possibly due to the limited number of patients. However, there is disagreement about the role of pathological study in diagnosis [19, 28]. Eight patients had elevated basal serum cortisol levels in this study (Sensitivity:73%). Instead, all patients had hypercortisolemia according to basal 24-hour UFC results, and no false-negative results were observed (Sensitivity:100%). This study’s findings were consistent with previous studies regarding low sensitivity for basal serum cortisol and high sensitivity for 24-hour UFC as screening tests for hypercortisolemia [6, 30, 31]. After HDDST, basal serum cortisol suppression was observed in three patients with CD (cases 2, 3, and 😎 but not in the others with CD. Also, serum cortisol levels were suppressed after HDDST in a patient with EAS who had a lung carcinoid tumor. Arnaldi et al. showed that some carcinoid tumors might be sensitive to HDDST, and suppression of serum cortisol may be observed after this test [1, 32]. After HDDST, six patients with CD had suppressed 24-hour UFC, but one did not show more than 50% suppression. Two patients with EAS had more than 50% 24-hour UFC suppression. According to the final pathology report, the sensitivity of serum and urine cortisol level tests after HDDST was 43% and 86%, and the specificity was 67% and 33%, respectively. PPV in both was 75%, NPV was 33% and 50%, and accuracy was 50% and 70%, respectively, which shows that these preliminary tests cannot be a good guide for the final diagnosis and subsequent treatment planning. Previous studies showed that more than one biochemical test could improve the accuracy for differentiating between CD and EAS [1, 5, 6, 9, 31]. The current study confirms the importance of using more than one biochemical test for diagnosing hypercortisolemia and diagnosing CD from EAS. Detomas et al. reported that Hb levels were high in females with CS while they were low in males with CS. Furthermore, there were lower levels of Hb in EAS than in CD in females [33]. In the current study, the Hb levels were not different in women and men. Furthermore, no statistical difference was observed for Hb levels between patients with a final diagnosis of CD and EAS. Hb levels did not contribute to diagnosing ACTH-dependent CS in this analysis. There were some limitations in this study. First, the sample size was relatively small. Second, it was a retrospective study. Further studies could investigate the BIPSS in a larger sample size and determine the validity of this method in patients with CS. Conclusions The current study suggests that BIPSS can be a reliable and low-complication method in evaluating patients with ACTH-dependent CS who had equivocal results in imaging and biochemical tests, even before stimulation. Stimulation with DDAVP increases diagnostic accuracy. BIPSS can be used to predict the lateralization of the pituitary adenoma. Data Availability All data generated or analyzed during this study are included in this published article. Abbreviations BIPSS: Bilateral inferior petrosal sinus sampling ACTH: Adrenocorticotropic hormone CS: Cushing’s syndrome IPS: Inferior petrosal sinus DDAVP: Desmopressin CD: Cushing’s disease EAS: Ectopic ACTH syndrome MRI: Magnetic resonance imaging UFC: Urinary free cortisol DST: Dexamethasone suppression test HDDST: High-dose dexamethasone suppression test CRH: Corticotropin-releasing hormone BMI: Body mass index FBS: Fasting blood glucose Hb: Hemoglobin Cr: Creatinine PPV: Positive predictive value NPV: Negative predictive value SBP: Systolic blood pressure DBP: Diastolic blood pressure K: Potassium HTN: Hypertension IHC: Immunohistochemistry TSS: Transsphenoidal surgery References Arnaldi G, Angeli A, Atkinson A, Bertagna X, Cavagnini F, Chrousos G, et al. Diagnosis and Complications of Cushing’s syndrome: a consensus statement. J Clin Endocrinol Metabolism. 2003;88(12):5593–602. Article CAS Google Scholar Sharma ST, Nieman LK, Feelders RA. 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Author information Authors and Affiliations Endocrinology and Metabolism Research Center (EMRC), Vali-Asr Hospital, Tehran University of Medical Sciences, Tehran, Iran Mohammadali Tavakoli Ardakani, Soghra Rabizadeh, Amirhossein Yadegar, Fatemeh Mohammadi, Sahar Karimpour Reyhan, Reihane Qahremani, Alireza Esteghamati & Manouchehr Nakhjavani Advanced Diagnostic and Interventional Radiology Research Center (ADIR), Tehran University of Medical Sciences, Tehran, Iran Hossein Ghanaati Contributions MN and MTA and SR: Conception and design of the study. AY and FM and HG: Acquisition of data. MTA and AY and SR: Analysis and interpretation of data. FM and RQ and SK: Drafting the article. MN and AE and AY: Critical revision of the article. All authors read and approved the final manuscript. Corresponding author Correspondence to Manouchehr Nakhjavani. Ethics declarations Ethics approval and consent to participate This study was performed in line with the principles of the Declaration of Helsinki. Informed consent was obtained from all participants or their legal guardians. Approval was granted by the Research Ethics Committee of Tehran University of Medical Sciences (Approval number: IR.TUMS.MEDICINE.REC.1398.707). Consent for publication In order to publish this study, written informed consent was obtained from each participant. A copy of the written consent form is available for review by the journal editor. Competing interests The authors declare no competing interests. Additional information Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Rights and permissions Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Reprints and Permissions Cite this article Ardakani, M.T., Rabizadeh, S., Yadegar, A. et al. Bilateral inferior petrosal sinus sampling: validity, diagnostic accuracy in lateralization of pituitary microadenoma, and treatment in eleven patients with Cushing’s syndrome – a single-center retrospective cohort study. BMC Endocr Disord 23, 232 (2023). https://doi.org/10.1186/s12902-023-01495-z Download citation Received05 July 2023 Accepted19 October 2023 Published23 October 2023 DOIhttps://doi.org/10.1186/s12902-023-01495-z Share this article Anyone you share the following link with will be able to read this content: Get shareable link Provided by the Springer Nature SharedIt content-sharing initiative Keywords BIPSS Bilateral inferior petrosal sinus sampling Cushing’s Disease Cushing’s syndrome EAS From https://bmcendocrdisord.biomedcentral.com/articles/10.1186/s12902-023-01495-z
  15. Medically reviewed by Daniel More, MD You may notice that your face appears puffy or more round on certain days. This can happen as your weight and hormones fluctuate or when you experience allergies or a temporary illness. However, if the puffiness persists or if your facial swelling is severe, this may be a sign of moon face—a condition that causes your face to become rounder due to fluid buildup. Symptoms Moon face causes swelling in your face as a result of excess fluid buildup. You may notice extra puffiness in your cheeks, forehead, and chin. When your facial features enlarge, it creates a round shape that mimics the look of the moon—hence the name, "moon face." It’s important to pay attention to the way your face feels. Sometimes, moon face is mild and not easily noticeable. But other times, moon face can be painful or affect your breathing. Keep track of any pain and swelling you're experiencing. Before seeing a healthcare provider, it can also help to document the following: What pain you're feeling Where the pain is located When your swelling began What improves and worsens your pain and swelling Any other symptoms that accompany puffiness These notes can help your healthcare provider understand the severity of your symptoms and recommend appropriate treatment options. Causes A variety of factors can cause moon face—ranging from mild everyday reactions to more serious conditions. Infections Underlying infections and medical conditions can cause facial swelling and increase your risk of moon face. These include: Conjunctivitis, or “pink eye” Infection in your salivary glands (the glands that produce saliva) Sinusitis, or swelling of your sinuses Styes that cause swelling around your eye Tooth abscesses, or infections in your teeth that cause a pocket of pus Cellulitis, a type of bacterial skin infection Cushing's Syndrome Among the most common causes of moon face is Cushing's syndrome—a condition that occurs when your body makes too much cortisol, which is commonly referred to as the "stress hormone." One of the most common symptoms of Cushing's syndrome is moon face, but you might also experience darkening of the skin, weight gain, and muscle weakness. Corticosteroids If your body doesn’t produce enough cortisol, your healthcare provider may prescribe corticosteroids. These anti-inflammatory drugs can also help treat several conditions such as arthritis, severe allergies, multiple sclerosis, lupus, certain kinds of cancer, and other conditions related to your lungs, skin, eyes, blood, kidneys, thyroid, stomach, or intestines. One of the most common corticosteroids is Deltasone (prednisone). Excess amounts or long-term use of corticosteroids can cause moon face to occur. Medical Side Effects Besides corticosteroids, other types of medication and medical treatment can also cause moon face. Specifically, you may develop moon face as a reaction to a blood transfusion or a range of medications, such as Bayer (aspirin) and certain types of antibiotics. You can also experience moon face after head, nose, or jaw surgery. Weight Changes Both severe malnutrition (not eating enough to get the nutrients you need) and obesity may lead to moon face. Some people with malnutrition develop kwashiorkor—a condition that can lead to swelling of your arms, legs, and face. This can happen because not eating enough food or drinking enough water can cause low levels of fluid and force your body to retain excess salt, which can cause swelling. People with obesity may also be more likely to develop moon face. It's estimated that approximately three out of every four people with Cushing's syndrome experience obesity. When Cushing's syndrome causes excess weight on your body, you may also be at an increased risk of developing fat deposits in your face. Other Causes Other common causes of moon face include: Allergic reactions Burns or injuries to the face Angioedema—a condition that causes swelling under the skin due to an issue with your immune system functioning Myxedema, which is a severe form of hypothyroidism—a condition that occurs when your thyroid gland doesn't make enough thyroid hormone and causes symptoms like skin changes and weight gain Superior vena cava (SVC) syndrome—a condition that causes facial and neck swelling because your SVC (a type of vein in your body) becomes compressed and isn't able to drain or pump blood back to the heart How to Get Rid of Moon Face Because moon face is a symptom of other underlying health conditions, it’s best to consult a healthcare provider to understand what's causing your facial swelling and learn about treatment options. For example, if your moon face is the result of an injury, you might try using ice to reduce the swelling. In addition, propping your head up with extra pillows while you sleep may help improve fluid drainage and reduce swelling. But, if a condition like Cushing's syndrome is causing moon face, medication or surgery may help improve facial swelling. How to Prevent Moon Face There isn't one surefire way to prevent moon face—mostly because a variety of factors can cause symptoms to develop. If you are at risk or concerned about a particular cause of facial swelling, speak with your healthcare provider about your options. If you’re prescribed a corticosteroid, there are particular steps you can take to reduce your chances of developing moon face. When taking a prescribed corticosteroid like prednisone, pay close attention to your symptoms and let your healthcare provider know early if you are developing any symptoms of Cushing's syndrome, including moon face. The earlier they are able to recommend alternative treatment, the better your chances of preventing long-term swelling. When to Contact a Healthcare Provider It’s important to seek care from your provider if you have specific symptoms associated with moon face, including: Swelling that comes on suddenly, causes pain, or is severe Long-lasting swelling Signs of infection, including fever, redness, or tenderness What To Expect at Your Appointment If you seek medical care for moon face, your healthcare provider will likely begin your appointment by taking your medical history and performing a physical exam. They may also ask about: How long your face has been swollen and when it began Things that improve or worsen your symptoms What allergies you have Which medications you take Any recent facial injury, medical test, or surgery Additional symptoms you're experiencing Once they gather this information, your provider can order the necessary testing, understand the underlying cause of your symptoms, and offer treatment options for moon face. A Quick Review Moon face is a condition that occurs when fluid builds up under your skin and causes facial swelling. Several factors can cause moon face, like reactions to medication or surgery, allergies, infections, weight changes, and underlying health conditions. If you have symptoms of moon face or notice your face getting puffy without a clear reason, talk to your provider. They can help you pinpoint the underlying cause and recommend treatment. Adapted from https://www.yahoo.com/lifestyle/moon-face-180000163.html
  16. We have an opportunity for you to take part in a Cushing’s Disease, Type 2 Diabetes Study (M3_8994) for patients. Our project number for this study is M3_8994. Project Details: Web-assisted telephone interview (you must be by a computer with high-speed internet access while on the phone during the time of the interview) Interview is 60-minutes long 120 Dollar Reward Things to Note: We recommend using the web browsers Google Chrome or FireFox Study is open to patients Please do not share study links One participant per household only Want to share this opportunity? Let us know and we can provide a new link Please use a laptop/computer ONLY. No smartphones or tablets - Preliminary questions are mobile friendly! Save this email to reference if you have any questions about the study! If you have any problems, email tana.karamustafic@rarepatientvoice.com and reference the project number. If you are interested in this study, please click the link below to answer a few questions to see if you qualify. Study link: Start Here OR if the study hyperlink is not clickable above, please copy/paste this URL below. https://panel.rarepatientvoice.com/newdesign/site/rarepatientvoice/surveystart.php?surveyID=s4gl5f3v5tr8&panelMemberID=trfnbc7mvduh1gseff1h&invite=email Thanks as always for your participation! Please be aware that by entering this information you are not guaranteed that you will be selected to participate. As always, we do not share any of your contact information without your permission. We have recently updated our Privacy Policy. Please make sure you have read through it and agree to the terms and conditions before taking studies. Please contact us at research@rarepatientvoice.com if you have any questions.
  17. Objective: This study evaluated short- and long-term efficacy and safety of the second-generation somatostatin receptor ligand pasireotide alone or in combination with dopamine agonist cabergoline in patients with Cushing’s disease (CD). Study design: This is an open-label, multicenter, non-comparative, Phase II study comprising 35-week core phase and an optional extension phase. All patients started with pasireotide, and cabergoline was added if cortisol remained elevated. Eligible patients had active CD, with or without prior surgery, were pasireotide naïve at screening or had discontinued pasireotide for reasons other than safety. Primary endpoint was proportion of patients with a mean urinary free cortisol (mUFC) level not exceeding the upper limit of normal (ULN) at week 35 with missing data imputed using last available post-baseline assessments. Results: Of 68 patients enrolled, 26 (38.2%) received pasireotide monotherapy and 42 (61.8%) received pasireotide plus cabergoline during the core phase. Thirty-four patients (50.0%; 95% CI 37.6–62.4) achieved the primary endpoint, of whom 17 (50.0%) received pasireotide monotherapy and 17 (50.0%) received combination therapy. Proportion of patients with mUFC control remained stable during the extension phase up to week 99. Treatment with either mono or combination therapy provided sustained improvements in clinical symptoms of hypercortisolism up to week 99. Hyperglycemia and nausea (51.5% each), diarrhea (44.1%) and cholelithiasis (33.8%) were the most frequent adverse events. Conclusion: Addition of cabergoline in patients with persistently elevated mUFC on maximum tolerated doses of pasireotide is an effective and well-tolerated long-term strategy for enhancing control of hypercortisolism in some CD patients. Clinical trial registration: https://clinicaltrials.gov/ct2/show/NCT01915303, identifier NCT01915303. 1 Introduction Cushing’s disease (CD) is a rare condition arising from chronic overproduction of cortisol, secondary to an adrenocorticotropic hormone (ACTH)-secreting pituitary tumor (1). Untreated hypercortisolism results in substantial multisystem morbidity, impaired quality of life (QoL) and premature mortality (1–4). Pasireotide is a second-generation, multireceptor-targeted somatostatin receptor ligand (SRLs), with affinity for 4 of the 5 known somatostatin receptor subtypes (SSTRs) (5) and is approved for the treatment of patients with CD for whom surgery has failed or is not an option (6). Phase III trials of pasireotide monotherapy have shown sustained biochemical and clinical benefits up to 5 years (6–9). These benefits are also reflected in real-world evidence (10). Cabergoline, a potent dopamine agonist with high affinity for dopamine type 2 receptors (D2), is commonly used off-label for the treatment of CD (2). Small, retrospective, non-randomized studies have demonstrated long-term urinary free cortisol (UFC) control (24−;60 months) in 23−;40% of patients with CD, especially those with mild hypercortisolism (11–13). A meta-analysis of individual patient data from six observational studies (n=124) reported normalization of mean UFC (mUFC) levels in 34% of patients (14, 15). However, a short prospective study on cabergoline monotherapy showed a limited value in controlling UFC, possibly linked to short duration (16). As most corticotropinomas co­express SSTR5 and D2, combining pasireotide and cabergoline in a stepwise approach could potentially improve efficacy with achieving more rapid biochemical control (17), a premise supported by results from an 80-day pilot study of 17 patients with CD treated with cabergoline- pasireotide combination, and low-dose ketoconazole (in case of lack of complete control with the two-drug combination) (18). The current study aims to report the efficacy and safety of prolonged treatment with pasireotide alone or in combination with cabergoline from the largest prospective, multicentre study to date of a pituitary-targeting combination treatment regimen in patients with CD (NCT01915303). 2 Materials and methods 2.1 Patients Adults (≥18 years) with a confirmed diagnosis of CD or de novo CD, if they were not candidates for surgery or refused surgery were enrolled. Cushing’s disease was defined by a mean 24-hour (24h) UFC level greater than the upper limit of normal (ULN, 137.95 nmol/24h), calculated from three 24h samples collected within 2 weeks; a morning plasma ACTH level within or above the normal range; and a confirmed pituitary source of Cushing’s syndrome, determined by MRI confirmation of pituitary adenoma >6mm or inferior petrosal sinus sampling (IPSS) gradient >3 after CRH stimulation (or >2 if IPSS without CRH stimulation) for those patients with a tumor ≤6mm. For patients who had prior pituitary surgery, histopathology confirming an ACTH staining adenoma was considered confirmatory of CD. Key exclusion criteria included optic chiasm compression requiring surgery, poorly controlled diabetes (glycated hemoglobin [HbA1c] >8%) and having risk factors for torsades de pointes (for further details, see the Supplementary Appendix). 2.2 Study design This was a single-arm, open-label, multicenter, non-comparative, Phase II study. After 4 weeks of screening, patients were treated in a stepwise approach during the core phase. Patients received subcutaneous pasireotide 0.6 mg twice daily (bid) for 8 weeks. Patients with a mUFC level exceeding ULN after 8 weeks received pasireotide 0.9 mg bid for another 8 weeks. If mUFC level remained elevated with pasireotide 0.9 mg bid, oral cabergoline 0.5 mg once daily (qd) was added for 8 weeks and could be increased to 1.0 mg qd for another 8 weeks (Supplementary Figure S1). After 35 weeks of treatment in the core phase, patients could enter the extension phase of the trial. Addition or titration of cabergoline during the extension phase was at the discretion of investigators. Collection of extension data commenced from week 43, and patients continued their current study treatment up to study end (4 September 2019; date of last patient visit), week 257. Data beyond week 99 are not reported here because of small patient numbers. 2.3 End points and assessments The primary endpoint of the study was the proportion of patients with mUFC ≤ULN at week 35. Secondary endpoints (reported at 4-week intervals up to week 35 and 8-week intervals from week 43 to the date of the last patient visit) included changes from baseline in mUFC, plasma ACTH, serum cortisol, total cholesterol, and clinical signs (systolic/diastolic blood pressure, body mass index (BMI), weight, waist circumference, facial rubor, hirsutism, striae, supraclavicular and dorsal fat pads) and symptoms (CushingQoL). Treatment escape was defined as an increase in one UFC above the normal range during follow-up of complete responders (14). Cushing Quality of Life Questionnaire (CushingQoL) (19) scores were reported up to week 35 only. Details on the safety assessments are provided in the Supplementary Appendix. 2.4 Statistical analyses No formal hypothesis testing was performed because of the exploratory design of the study. Efficacy analyses were conducted on full analysis set, i.e., all patients to whom study treatment was assigned. Safety analyses were conducted on all patients who received ≥1 dose of pasireotide per day during the study. For patients with missing mUFC value at week 35, including those who discontinued, the last available assessment was carried forward. Details on the post hoc analyses and sample size estimation is provided in the Supplementary Appendix. Enrolled patients, who were observed for failed inclusion or exclusion criteria during the monitoring visits, were classified under protocol deviation. However, patients with no safety concerns were allowed to continue in the study and included in the full analysis set as intention to treat – assessing the study outcome, while some patients were excluded from the per protocol analysis. 3 Results 3.1 Study population A total of 68 patients were enrolled in the study. At baseline, 66 (97.1%) patients were pasireotide naïve, while 2 (2.9%) were treated with pasireotide previously with 4 weeks of washout period prior to screening (Table 1). Of 68 patients received treatment during the core phase, 26 (38.2%) received pasireotide monotherapy and 42 (61.8%) received combination therapy. Fifty-two (76.5%) patients completed the 35-week core phase while 16 (23.5%) discontinued (Figure 1). All 68 patients were included in the full analysis set based on the intention to treat (ITT) principle. One of the protocol deviations observed during the study, was inclusion of 3 patients with normal mUFC value at screening visit (baseline) and assigning a treatment. The deviation category for the 3 patients was ‘failed inclusion criteria’ with screening mUFC value ≤ULN (137.95 nmol/24h) or mUFC calculated using ❤️ UFC values or 2 out of 3 UFC values ≤ULN. One of these patients (baseline mUFC 37.37 nmol/24h ≤ULN) was discontinued from the study at Week 2 and due to lack of post-baseline mUFC assessment, was classified ‘non-responder’ at Week 35 assessment. The 2nd patient’s baseline mUFC value of 135.20 nmol/24h was close to ULN (137.95 nmol/24h) and was rescreened. Based on the rescreened mUFC value 306.5 nmol/24h, this patient was included in study, and the mUFC at Week 35 was 192.30 nmol/24h (non-responder at Week 35 assessment). For all study assessments, the scheduled screening visit’s first mUFC value (≤ULN) was used as baseline value. The 3rd patient (baseline mUFC value 131.77 nmol/24h) was discontinued from the study at Week 26 and was also observed for non-compliant schedule visit and medication dosages. The mUFC value recorded at Week 26 (88.95 nmol/24h) was ≤ULN and this last observation was carried forward to Week 35. Hence, the patient was classified ‘responder’, leaving one patient included in the study as responder as a protocol deviation. Table 1 Table 1 Patient demographics and baseline characteristics. Figure 1 Figure 1 Patient disposition. *If the study drugs were locally available at the end of the core phase, patients could switch over to the commercial supply and exit the extension phase. Only in countries where the drug was not locally available were patients given the option to enter the extension phase. Percentage for patients not entering the extension phase was calculated from the total number of patients enrolled in the study. Twenty-nine (42.6%) patients continued treatment in the extension phase; 10 (34.5%) received pasireotide monotherapy and 19 (65.5%) received combination therapy. Twelve (41.4%) patients completed the extension phase, while 17 (58.6%) discontinued treatment before study end, most commonly for unsatisfactory therapeutic effect (n=8). The most common reason for discontinuation was adverse events (AEs): 5 (17.2%) patients with pasireotide monotherapy and 2 (5.1%) patients with combination therapy. 3.2 Efficacy: biochemical response Overall, 34/68 (50.0%; 95% CI 37.6–62.4) patients achieved the primary endpoint, of whom 17 (50.0%) were receiving pasireotide monotherapy and 17 (50.0%) were receiving combination therapy. Patients with mild hypercortisolism (mUFC 1.0–<2.0 x ULN) at baseline were more likely to respond to both pasireotide monotherapy and combination therapy (n=15; 22.1%, Figure 2). Seven of 17 patients in the pasireotide monotherapy group met the primary endpoint based on their last available assessment prior to week 35. Even if the 3 patients who had mUFC ≤ULN at baseline were excluded from the primary analysis, 33/65 (50.7%; 95% CI 38.1–63.4) patients would have achieved the primary endpoint. The results are similar to the original analysis (34/68 (50.0%; 95% CI 37.6–62.4) based on the full analysis set. Figure 2 Figure 2 Patients achieving mUFC ≤ULN at week 35. †At baseline there were 23 patients with mild, 30 with moderate and 12 with severe hypercortisolism. mUFC, mean urine free cortisol; ULN, upper limit of normal. For the overall study population (n=68), mUFC rapidly decreased from 501.6 nmol/24h (3.6 x ULN; SD: 488.66 nmol/24h) to 242.1 nmol/24h (1.8 x ULN; SD: 203.47 nmol/24h) at week 4 and mUFC remained below baseline levels up to week 35 (184.8 nmol/24h; 1.3 x ULN; SD:140.13 nmol/24h). For patients who received pasireotide monotherapy (n=26), mUFC( ± SD) decreased from baseline (442.1± 557.13 nmol/24h [n=26]; 3.2 x ULN) to week 35 (136.6 ± 127.77 nmol/24h [n=14]; 1 x ULN) and at the end of the study (111.2 ± 40.39 nmol/24h [n=5]; 0.8 x ULN) using the last-observation-carried-forward (LOCF). For those who did not normalize on pasireotide monotherapy (n=42), mUFC ( ± SD) decreased from baseline, i.e., last observation before starting cabergoline (280.20 ± 129.03 nmol/24h [n=40]; 2.0 x ULN) to week 35 (206.6 ± 141.96 nmol/24h [n=31]; 1.5 x ULN) and at the end of the study (219.60 ± 83.78 nmol/24h [n=7]; 1.6 x ULN) using the LOCF. During the core phase, mean serum cortisol decreased from 738.6 nmol/L (1.3 x ULN) at baseline to 538.2 nmol/L (0.95 x ULN) and ACTH levels from 16.3 pmol/L (2.7 x ULN) to 11.0 pmol/L (1.8 x ULN) at week 35. During the extension phase, 25 patients had a mUFC assessment; of whom 12 (48%) had a mUFC ≤ULN at the end of the extension phase. During the extension phase, mUFC levels decreased slightly and fluctuated above and below the ULN up to the week 139 (Figure 3A), while mean serum cortisol remained below ULN (404 nmol/L; Figure 3B) and ACTH levels fluctuated from 8.2 pmol/L to 11.5 pmol/L) and remained above the ULN value (Figure 3C). Figure 3 Figure 3 Mean actual change over time in (A) mUFC (B) serum cortisol, and (C) ACTH. ACTH, adrenocorticotropic hormone; mUFC, mean urine free cortisol; ULN, upper limit of normal . Twenty-one of 38 (55%) patients achieved control with combination therapy at some point during the core or extension study, of whom 13 (62%) experienced escape (at least one UFC >ULN after previous control). The time to achieve control after starting cabergoline ranged from 14−;343 days. Notably, one patient received pasireotide 0.6 mg bid initially, dose increased to 0.9 mg bid at Week 17, followed by addition of cabergoline 0.5 mg od at Week 31. The patient achieved biochemical control (mUFC value of 120.15 nmol/24h) on the same day of the start of combination therapy. Clinically it is highly unlikely that biochemical control was achieved with single dose of cabergoline administration. Therefore, it could be considered that normalization was achieved while receiving pasireotide monotherapy. Also, the physician might have prescribed combination therapy before receiving the mUFC value of the (urinary) sample delivered on the morning of combination therapy initiation (while the patient was still on monotherapy). The patient continued combination therapy and maintained biochemical control up to Week 35 and beyond. Furthermore, at Week 59 the cabergoline dose was increased to 1.0 mg/day due to mUFC >ULN at previous visit (Week 51). The patient remained on pasireotide 0.9 mg bid/cabergoline 1.0 mg od combination therapy until the study end. The median time to escape after achieving control with the addition of cabergoline was 58 days (range 28−;344). 10/13patients regained biochemical control with combination therapy. No patients on pasireotide alone experienced escape, probably due to the short observation time. 3.3 Clinical signs and symptoms of CD Relative to baseline, pasireotide monotherapy was accompanied by reductions in median blood pressure, weight, BMI, waist circumference, and total cholesterol. Overall improvement in clinical measures persisted over time (Supplementary Table S1). Clinical improvements were also seen following the addition of cabergoline, particularly for hirsutism (Supplementary Figures S2, S3). Mean( ± SD) standardized CushingQoL score was 41.6(± 20.2) at baseline and increased to 47.6(± 20.8) at week 35 (Supplementary Table S2), indicating improvements in patients’ QoL (19). 3.4 Safety and tolerability Median duration of exposure to pasireotide was 35.0 weeks (range 0−;268), with a median dose of 1.53 mg/day (range 0.29−;1.80). Median duration of exposure to cabergoline was 16.9 weeks (range 1−;215), with a median dose of 0.50 mg/day (range 0.44−;0.97). All patients (N=68) reported at least one AE and 28/68 (41.2%) patients had a grade 3/4 AE (Table 2). The most common AEs (≥30%) were hyperglycemia and nausea (51.5% each), diarrhea (44.1%) and cholelithiasis (33.8%). Treatment-related AEs (TRAEs) were reported in 66/68 (97.1%) patients; the most frequent TRAEs (≥30%) were hyperglycemia and nausea (47.1% each), diarrhea (39.7%), and cholelithiasis (32.4%). Fourteen (20.6%) patients had ≥1 AE leading to discontinuation. Table 2 Table 2 Summary of adverse events (≥10%), overall and by treatment regimen. The most common AEs leading to discontinuation were increased gamma-glutamyl transferase (GGT) and hyperglycemia (two patients each, 2.9%). Twenty-three (33.8%) patients had ≥1 AE leading to dose adjustment or interruption. Details on special safety assessments such as hyperglycemia-related AEs, blood glucose, HbA1c, IGF-1 as well as hematological and biochemical abnormalities are presented in the Supplementary Appendix. Three (4.4%) patients died during the study, two (2.9%) during the core phase and one (1.5%) during the extension. All deaths were considered unrelated to study medication. The causes during the core phase were multi-organ dysfunction syndrome for one patient aged 79 years and unknown for the other aged 34 years. Uncontrolled hypertension was reported as the cause of death for the patient aged 47 during the extension phase. 4 Discussion The severe morbidity and increased mortality with uncontrolled CD highlight the importance of identifying an effective medical strategy. This study explored the potential of a synergistic benefit of the addition of cabergoline to pasireotide treatment in patients with CD. Complete normalization of cortisol production is required to reverse the risks of morbidity and mortality in patients with CD (1). Two small studies showed clinical improvement of normalized UFC when cabergoline and ketoconazole were combined (20, 21). Benefit has also been reported with triple therapy with pasireotide, cabergoline and ketoconazole (18) and triple therapy with ketoconazole, metyrapone and mitotane in severe CD (22). In the current study, 50% of patients achieved the primary endpoint of mUFC ≤ULN at week 35 and a similar proportion (48%) sustained biochemical control throughout the extension phase. Notably, combination treatment doubled the number of patients who attained mUFC ≤ULN from the core phase to the end of the extension phase. In particular, mUFC was rapidly reduced with treatment, i.e., in most patients within 2 months, while measures of patient-reported outcomes also improved including QoL. Twenty-three patients (33.8%) who completed the core phase did not enter the extension phase. This was because only patients from countries where a commercial supply was unavailable were given the option to enter the extension phase. This study confirms previous reports that patients with mild hypercortisolism at baseline were more likely to achieve mUFC control with pasireotide monotherapy than patients with moderate or severe hypercortisolism (6, 23). In addition, patients with moderate hypercortisolism at baseline were more likely to achieve mUFC control with the addition of cabergoline. This supports that a combination therapy can be effective for patients with a wider range of disease severity. Accordingly, in vitro data may indeed indicate synergism between SSTR and D2 that might increase therapeutic efficacy (24, 25). Improvements in clinical signs and symptoms with pasireotide monotherapy were consistent with published data (6, 10). In the core phase, an improvement of blood pressure and BMI was observed with pasireotide monotherapy and, to a lesser extent, with combination therapy which may related to the difference in duration of biochemical remission. The overall safety profile was consistent with that expected for pasireotide, with most AEs being mild/moderate (26, 27). There were no new safety signals identified with the addition of cabergoline. Common AEs including nausea, headache, dizziness, and fatigue are suggestive of steroid withdrawal symptoms associated with the decrease in UFC, although direct drug effects cannot fully be excluded. Adrenal insufficiency was not reported as side effect. Rates of hyperglycemia-related AEs (68%) were consistent with those in previous reports of pasireotide monotherapy (6, 10). FPG increased with pasireotide monotherapy during the first 8 weeks of treatment and stabilized for the remainder of the study, including following the addition of cabergoline. These data highlight the vital role of blood glucose monitoring in these patients. Both pasireotide and cabergoline are pituitary-targeted agents that act directly on the source of the disease via inhibition of ACTH release by the corticotroph tumor, which may be an advantage over steroid synthesis inhibitors. This study further confirms previous data reporting the benefits of pasireotide in combination with cabergoline in patients with CD (18). While not entirely elucidated, down-regulation of dopamine D2 receptors (D2R) expression, and post-receptor desensitization and/or tumor regrowth of corticotroph tumor cell were suggested as possible mechanisms for treatment escape (15). Moreover, different dopamine receptor patterns and/or D2R isoforms also influence the response and eventually the treatment escape. Treatment escape has been observed in some studies after long-term (7−;12 months) treatment with cabergoline (13), however it is possible that use of concomitant SRLs could potentially reduce the rate of escape. In this study, a total of 13 patients experienced treatment escape. However, 10 of these patients regained biochemical control. For 7 of these 10 patients, there was up titration of doses to a maximum of 1.8 mg/day of pasireotide and 1 mg/day of cabergolineAlthough pasireotide and cabergoline have shown long-term reduction in IGF-1 levels in patients with acromegaly (28, 29), there is little evidence for this effect in patients with CD (4, 30). One study (n=17) found significant decreases in IGF-1 after 28 days’ treatment with pasireotide that was independent of UFC reduction. One-third of patients had low IGF-1 (30). Our study showed that almost half of patients (47.6%) had IGF-1 levels either above ULN or below LLN prior to the addition of cabergoline, and IGF-1 levels decreased relative to the baseline, with majority of values within the normal range during the core and extension phases up to week 99. Baseline levels of IGF-1 may already be low because of the suppressive effect of excess cortisol on the somatotropic axis (31). Although clinicians have several therapeutic options at their disposal to treat hypercortisolemia associated with CD, the optimal treatment approach should be based on the individual clinical situation and the benefit–risk considerations for each patient. In this study, 13 patients had history of pituitary radiation, with a duration of at least 2.6 years (median 3.3 years) between the last radiation treatment and the observed response date. However, only 7/13 patients achieved the therapeutic target. Although there was a gap of > 2 years, we cannot exclude the role of radiation in normalizing UFC. Contrastingly, 6/13 patients treated with radiation did not achieve mUFC ≤ULN (responders) at Week 35. The impact of the adjuvant radiation therapy remains unclear. The strengths of this study are that this is the largest and longest prospective study with pituitary-directed pharmacotherapy, to date, evaluating the addition of cabergoline to pasireotide in patients with CD, and this stepwise approach reflects real-world clinical practice (18). The study is limited by the open-label design and the fact that it was not a head-to-head comparative study of pasireotide only versus pasireotide plus cabergoline. This may be of importance in interpreting patient-reported outcomes. Several patients continued treatment for almost 2 years; however, interpretation of long-term data should be made with caution because of the small patient numbers. Notably, the last available assessment was carried forward for patients with missing mUFC value at week 35 including those who discontinued and were considered for response analysis. It should also be noted that the definition of loss of response, also known as escape, used in this study (at least one UFC value >ULN after previously achieving UFC ≤ULN) may overestimate the rate of apparent escape as UFC values may have fluctuated about the ULN range or been marginally elevated. The definition of treatment escape differs across studies, and we have used a very stringent one in this study, requiring only a single high UFC to meet the classification as escape. Thus, it is likely that some loss of biochemical control interpreted as escape is actually fluctuation of cortisol around the upper limit of normal range. Other limitations include protocol deviations in including 3 patients with normal UFC at baseline (one patient was uncontrolled at rescreen, and one was discontinued at 2 weeks - both classified as non-responders), lack of data on impact of radiation therapy without study drug in patients who gained biochemical control with adjuvant radiation therapy, lack of pituitary magnetic resonance imaging to detect pituitary tumor changes, lack of data about effective cabergoline dose and absence of cardiac valve assessment for mild to moderate severity in the medium term. Both pasireotide and cabergoline can induce tumor shrinkage in CD (6, 9, 32–35) and it would be interesting to examine the combined effect on tumor size. This study used the subcutaneous formulation of pasireotide, whereas the most common usage currently is the long-acting formulation. Efficacy of long-acting pasireotide (36) seems higher compared to the subcutaneous formulation (7) and the effect of combination of long-acting pasireotide with cabergoline should be evaluated in future studies. No formal assessments were made for impulsive control disorders, which have been associated with dopamine agonists, including cabergoline (32, 33, 37, 38). The reason that several different terms were used for hyperglycemia-related AEs is that they were reported as per discretion of each investigator. No additional psychiatric AEs were reported, although they were not exhaustively searched. 5 Conclusions This is the first study demonstrating that pituitary-targeted combination treatment with pasireotide and cabergoline doubled the number of patients who attained mUFC ≤ULN. Both short- and long-term safety profile are consistent with known data for pasireotide and cabergoline. The low rate of discontinuation due to AEs suggests that pasireotide alone or as combination treatment is generally well-tolerated if appropriately monitored, even with prolonged treatment. The addition of cabergoline to pasireotide treatment in patients with persistently elevated mUFC could be an effective long-term strategy for enhancing the control of CD in a subset of patients, with close monitoring for possible escape. 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 The studies involving humans were approved by Hospital Britanico, Buenos Aires, Argentina; Ethische commissie University Hospitals Leuven, Leuven, Belgium; Universitair Ziekenhuis Gent, Gent, Belgium; Comite de Etica em Pesquisa Hospital Moinhos de Vento, Porto Alegre-RS, Brazil; Comitê de Ética em Pesquisa do Hospital de Clı́nicas, Universidade Federal do Paraná, Curitiba-PR, Brazil; Comissão de Ética para Análise de Projetos de Pesquisa, São Paulo - SP, Brazil; Ethics Committee for clinical trials, Sofia, Bulgaria; Comité Corporativo de Ética en Investigación, Bogotá DC, Colombia; Comite De Protection Des Personnes, Groupe Hospitalier Pellegrin - Bat, Bordeaux Cedex, France; Friedrich-Alexander Universitat Erlangen-Nurnberg, Medizinische Fakultat, Erlangen, Germany;National Ethics Committee, Cholargos, Athens, Greece; Ethics Committee for Clinical Pharmacology (ECCP), Budapest, Hungary; Institute Ethics Committee, New Delhi, India; Institutional Review Board (IRB) Ethics Committee Silver, Christian Medical College, Vellore, Tamil Nadu, India; Institute Ethics Committee, PGIMER, Chandigarh, India; Comitato Etico Dell’irccs Istituto Auxologico Italiano Di Milano, Milano, Italy; Comitato Etico Universita’ Federico Ii Di Napoli, Napoli, Italy; Jawatankuasa Etika & Penyelidikan Perubatan (Medical Research and Ethics Committee), d/a Institut Pengurusan Keshatan Jalan Rumah Sakit, Kuala Lumpur, Malaysia; Institutd Nacional De Neurologia Y Neurocirugia, Mexico City, Mexico; Clinica Bajio (CLINBA), Guanajuato, Mexico; Medische Ethische Toetsings Commissie, Rotterdam; Netherlands; CEIm Provincial de Málaga, Málaga, Spain; Istanbul University Cerrahpasa Medical Faculty, Istanbul, Turkey; WIRB, Puyallup, WA, USA; Research Integrity Office, Oregon Health & Science University Portland, OR USA. The studies were conducted in accordance with local legislations and institutional requirements. The participants provided their written informed consent to participate in this study. Author contributions All authors directly participated in the planning, execution, or analysis, and have had full control of complete primary data, and hold responsibility for data integrity and accuracy. All authors contributed to the article and approved the submitted version. Acknowledgments We thank Julie Brown, Mudskipper Business Ltd, and Manojkumar Patel and Sashi Kiran Goteti, Novartis Healthcare Private Limited, for medical editorial assistance with this manuscript. We would also like to thank all investigators, sub-investigators, study nurses and coordinators, and patients who have made this study possible. Conflict of interest HP and RM were Novartis employees and owned Novartis stocks. AMP was employed by Novartis and Recordati. AC is a Novartis employee and owns Novartis stocks. RF received research grants from Strongbridge and Corcept, consulting fee from Recordati, honoraria and financial support for meetings and/or travel from HRA Pharma and Recordati, and attended advisory boards for Recordati. MF has received research support to Oregon Health & Science University as a principal investigator from Recordati and Xeris Strongbridge and has performed occasional scientific consultancy for Recordati, HRA Pharma, Sparrow, and Xeris Strongbridge. PK attended advisory boards for Recordati. MB’s institution received consulting fee and attended advisory boards from Recordati. DG-D received research grants from Recordati Rare Disease and Bayer, consulting fee from Abbott-Lafrancol, Biotoscana, PTC lab, Glaxo/Helou, Recordati Rare Disease, and Bayer, honoraria from Valentech Pharma, Sanofi, and Bayer, travel grants from Recordati Rare Disease, advocacy groups and other leadership roles from Asociación Colombiana de Endocrinologia and Asociación Colombiana de Osteoporosis y Metabolismo, and other financial and non-financial interests include Asociacion Colombiana de Endocrinologia y Metabolismo, Hospital Universitario Fundación Santa Fé de Bogota, and Asociación Colombiana de Osteoporosis y Metabolismo. CB received research grants from Novartis and Recordati, and consulting and speaker fee from Novartis. BB served as the principal investigator for grants to Massachusetts General Hospital from Cortendo/Strongbridge Xeris, Millendo, and Novartis and has occasionally consulted for Cortendo/Strongbridge Xeris, HRA Pharma, Novartis Recordati, and Sparrow. RP and his institution received research grants and honoraria from Pfizer, Ipsen, Novartis, Merck Serono, IBSA Farmaceutici, Corcept, Shire, HRA Pharma, ICON, Covance, Neuroendocrine CAH, Camurus, Recordati, Janssen Cilag, and CMED Clinical Services, received consulting fee from Recordati Rare Disease, Organon Italia, Siunergos Pharma, Corcept, S&R Farmaceutici S.p.A., DAMOR Farmaceutici, and Pfizer, attended advisory boards from Crinetics Pharmaceuticals, Recordati Rare Disease, Pfizer, and HRA Pharma. The remaining author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The authors declare that this study received funding from Novartis Pharma AG. Novartis was involved in the study design, analysis, interpretation of data, and providing financial support for medical editorial assistance of this article. 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This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. *Correspondence: Richard A. Feelders, r.feelders@erasmusmc.nl †Present addresses: Alberto M. Pedroncelli, Chief Medical Office, Camurus AB, Lund, SwedenRicardo Maamari, Global Medical Affairs, Mayne Pharma, Raleigh, NC, United States ‡These authors have contributed equally to this work Disclaimer: All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article or claim that may be made by its manufacturer is not guaranteed or endorsed by the publisher. From https://www.frontiersin.org/articles/10.3389/fendo.2023.1165681/full
  18. Abstract Mifepristone and misoprostol are globally used medications that have become disparaged through the stigmatization of reproductive healthcare. Patients are hindered from receiving prompt treatment in clinical scenarios where misoprostol and mifepristone are the drugs of choice. It is no exaggeration to emphasize that in cases where reproductive healthcare is concerned. The aim of this paper is to discuss the different indications of mifepristone and to delineate where the discrepancy in accessibility arises. For this systematic review, we included publications citing clinical trials involving the use and efficacy of mifepristone published in English within the date range of 2000 to 2023. Five databases were searched to identify relevant sources. These databases are Google Scholar, MEDLINE with full text through EBSCO, and three National Center for Biotechnology Information (NCBI) databases (NCBI Bookshelf, PubMed, and PubMed Central). Twenty-three records were ultimately included in this review. Mifepristone has been shown to have therapeutic effects in the treatment of psychiatric disorders, such as major depressive disorder and psychotic depression. There was a significant decrease in depression and psychiatric rating symptoms for patients taking mifepristone versus placebo with no adverse events. Mifepristone has also been shown to improve treatment course in patients with Cushing’s disease (CD) who failed or are unable to undergo surgical treatment. In addition, mifepristone has been shown to be a successful treatment option for adenomyosis and leiomyomas. Patients had a statistically significant decrease in uterine volumes following mifepristone treatment, which aided in the alleviation of other symptoms, such as blood loss and pelvic discomfort. Mifepristone is a synthetic steroid that has immense potential to provide symptomatic relief in patients suffering from a wide array of complicated diseases. Historically, mifepristone has been proven to have an incredible safety profile. While further research is certainly needed, the politicization of its medical use for only one of its many indications has unfortunately led to the willful ignorance of its potential despite its evidence-based safety profile and efficacy. Introduction & Background Mifepristone is a synthetic steroid derived from norethindrone and therefore has antagonistic activity against progesterone and glucocorticoid receptors. Misoprostol is a synthetic prostaglandin E1 analog that works through the direct stimulation of prostaglandin E1 receptors. Recently, these medications have become disparaged due to their associations with the controversial medical procedure known as abortion. Abortions, however, have been so common that one out of four women will have had an abortion by the time they reach the age of 45 [1]. It is estimated that 3.7 million women have used mifepristone and misoprostol for medication abortions since they were first approved by the Food and Drug Administration (FDA) in 2000 [1]. Mifepristone followed by misoprostol is up to 14 times safer than carrying the patient’s pregnancy to term [1]. Aside from abortion, mifepristone is used for both gynecologic and obstetric conditions. Obstetric conditions include induction of labor, postpartum hemorrhage, intrauterine fetal demise, ectopic pregnancies, and miscarriages [2]. Gynecological conditions that can be treated with mifepristone include abnormal uterine bleeding, post-coital contraception, and treatment of gynecological cancers [3]. Due to the stigmatized nature of abortion, however, patients are hindered from receiving prompt treatment in clinical scenarios where mifepristone is the drug of choice. It is no exaggeration to emphasize that in cases where reproductive healthcare is concerned, every second counts [3]. Legislation that varies across states further impacts patients who risk their lives and health as they attempt to navigate their care plan across borders. Travel costs, time-off, childcare, transportation, and living accommodations are just a few more of the factors patients must take into consideration when they are forced to seek life-saving care outside of their homes [3]. Mifepristone is a medication that has multiple therapeutic applications, such as treating leiomyomas, psychotic depression, and post-traumatic stress disorder (PTSD). However, its use is restricted in many countries because of its abortifacient effect. This is a logical fallacy that deprives patients of a beneficial and safe treatment option. This systematic review aims to explore the evidence-based uses of mifepristone and how it can improve patients' health outcomes. The clinical indications that will be discussed are adenomyosis, leiomyomas, psychotic depression, PTSD, and Cushing's disease (CD). Review Methods Eligibility Criteria For this systematic review, we included publications of clinical trials and systematic reviews citing clinical trials relating to the clinical use of mifepristone and published in English within the date range of 2000 to 2023. Info Sources Five databases were searched to identify relevant sources. These databases include Google Scholar, MEDLINE with full text through EBSCO, and three National Center for Biotechnology Information (NCBI) databases (NCBI Bookshelf, PubMed, and PubMed Central). Search Strategy For each database, we inputted “clinical use of mifepristone” as our search term. The populated results were then narrowed down to those published in the English language and within the date range of 2000 to 2023 using automated search tools. Selection Process The titles and abstracts of the remaining records were then screened, and those deemed relevant to clinical uses of mifepristone and its efficacy were included for comprehensive review. This initial record search in three of the four databases (Google Scholar, MEDLINE, and PubMed) was completed by three separate reviewers. The initial record search in the remaining two databases (NCBI Bookshelf and PubMed Central) was completed by another individual reviewer. Data Collection Process After the initial record search, 60 records were deemed relevant to the study topic and compiled for a more comprehensive review. Two records were found to be duplicates and removed. Each of the four reviewers read the remaining 58 records and voted on the eligibility of the publication for inclusion in our review. Older publications that were expanded upon in more recent study trials were excluded to reduce redundancy. In addition, for records with similar study protocols, only the more recently published record was included. Ten records were excluded from the review due to ineligible study design. For those records that were not unanimously accepted (at least one reviewer voted for exclusion), the record was excluded. To ensure that the data utilized in this review were backed by sufficient evidence, the reviewers organized the remaining records into groups based on the disease mifepristone was being studied to treat. After further discussion, it was decided to exclude the records in the groups that lacked at least three separate clinical trials on the use of mifepristone in the treatment of the disease. Thirty articles were excluded. Seven of the 18 remaining records were systematic reviews, and citation searching of the records found four additional records that met the eligibility criteria. The remaining 23 records were included for further review. Data Items Of the remaining 23 records deemed acceptable for inclusion, only studies with statistically significant findings regarding the clinical use of mifepristone were included for detailed analysis. One record was excluded due to early termination of the trial. Our records include two open-label studies, four retrospective studies, seven reviews (systematic, meta-analysis), one wet lab (human specimen was used), five long-term safety extension articles, and seven randomized control experimental trials. Study Risk-of-Bias Assessment We assessed the risk of bias (RoB) in the studies included in the review using the revised Cochrane RoB tool for randomized trials (RoB 2). The five domains assessed were (1) RoB arising from the randomization process, (2) RoB due to deviations from the intended interventions (effect of assignment to intervention and effect of adhering to intervention), (3) missing outcome data, (4) RoB in the measurement of the outcome, and (5) RoB in the selection of the reported result. Each randomized control trial included in this review was assessed for RoB by two authors working independently using the RoB 2. For those studies in which the assessing authors came to different conclusions, the remaining two authors completed independent RoB 2 assessments of the study in question, and the majority of findings was accepted. Utilizing the methodology for assigning the overall RoB for each study as outlined by the RoB 2 tool, each study was designated as having “low risk of bias” or “high risk of bias.” After an initial assessment, both authors deemed the nine randomized control studies had a low RoB. Effect Measures Analysis of the studies included a focus on statistically significant findings that varied between control and intervention groups as defined by a p-value less than 0.5. As each study had its own parameters and primary and secondary endpoints, we focused our analysis on the safety and clinical efficacy of mifepristone as measured and reported by the authors of the studies included. Synthesis Methods As previously mentioned, as the studies included in this review vary widely in their study population and intervention design, our analysis focused on qualitative synthesis of study outcomes. These outcomes were categorized as the clinical efficacy and safety of mifepristone for CD, psychiatric disorders, and select gynecological diseases (adenomyosis and leiomyomas). Certainty Assessment To assess the certainty of the body of evidence regarding the studies included in our review, two reviewers applied the five Grading of Recommendations, Assessment, Development, and Evaluations (GRADE) considerations (study limitations, inconsistency of results, indirectness of evidence, imprecision, and publication bias) to each study. Accordingly, the included studies were categorized as having high, moderate, low, or very low certainty of evidence based on the GRADE criteria. After the assessment, both reviewers deemed that all records had high certainty of evidence. Figure 1: PRISMA 2020 flow diagram for new systematic reviews that included searches of databases, registers, and other sources *Consider, if feasible to do so, reporting the number of records identified from each database or register searched (rather than the total number across all databases/registers). **If automation tools were used, indicate how many records were excluded by a human and how many were excluded by automation tools. PRISMA: Preferred Reporting Items for Systematic Reviews and Meta-Analyses Results Psychiatric Implications Based on the analyses, numerous trials demonstrated the profound therapeutic effect that mifepristone can have on psychiatric disorders. In a double-blind study following 19 patients with bipolar disorder, researchers studied neurocognitive function and mood in patients treated with mifepristone vs. the placebo [4]. Significant improvements in verbal fluency and spatial working memory were seen in the group treated with mifepristone. The Hamilton Depression Rating Scale (HDRS) and Montgomery-Asberg Depression Rating Scale (MADRS) scores also improved from baseline (i.e., lower scores) measurements in these patients. It is worth noting that these improvements were seen in as little as two weeks, which is quicker than what is normally seen with typical therapeutic agents for bipolar disorder (lithium/valproic acid) [4]. The most extensive research demonstrated the benefits of using mifepristone with major or psychotic depression [5]. It is important to note that approximately 20% of patients living with major depression experience psychotic symptoms [6]. A randomized, double-blind study looked at 30 participants with psychotic major depression (PMD) and treated them with mifepristone 600 mg or a placebo for eight days. Using the HDRS and Brief Psychiatric Rating Scale (BPRS) to quantify baseline levels of symptoms, results from eight days later showed that mifepristone was significantly more effective in reducing psychotic symptoms compared to the placebo group [6]. By day 8, nearly half of the participants attained a 50% reduction in the BPRS compared to the placebo group (p<0.046) in addition to lower HDRS scores (although this was not found to be significant). Moreover, when researchers looked further into the use of mifepristone in psychotic depression disorders, they discovered a correlation between higher plasma levels of mifepristone and a reduction in psychotic symptoms [7]. More specifically, the strongest reduction in psychosis symptoms was found to be associated with doses of 1200 mg/day of mifepristone, which resulted in a statistically significant reduction in psychotic symptoms (p<0.0004) [7]. The drug was also well tolerated and demonstrated a large safety margin in contrast to the numerous common adverse effects that patients experience when placed on standard treatment options (i.e., antipsychotics). In another double-blind, placebo-controlled study that took place over four days, five participants diagnosed with psychotic major depression were administered 600 mg of mifepristone [5]. The HDRS and BPRS scores were used, and the results showed that all five participants' depression ratings decreased - a nearly statistically significant finding (p<.07) [5]. Likewise, four out of the five BPRS scores declined, approximating to a 32.5% decline, which is comparable to the 40% decline seen with traditional antipsychotic treatments that span six to eight weeks. Once again, no adverse effects were reported. The use of mifepristone has been explored in many cognitive disorders, including Alzheimer's disease. One study found that patients with mild to moderate Alzheimer’s disease displayed improvement on the Alzheimer’s disease assessment cognitive subtest - by 2.67 as opposed to the 1.67 decline in patients treated with a placebo [5]. Although not statistically significant, this finding encourages further studies to continue exploring the psychiatric and neurologic use of mifepristone. Cushing’s Disease Multiple trials have been conducted regarding the use and efficacy of mifepristone in the treatment of CD. Although surgical intervention to remove the source of excess cortisol production is the current mainstay of treatment, clinical trials have focused on the treatment with mifepristone for medical therapy, especially in patients who have failed surgical intervention or for those who are not good candidates for surgery. Accordingly, a retrospective study of 20 patients with hypercortisolism (12 with adrenocortical carcinoma, three with ectopic adrenocorticotropic hormone (ACTH) secretion, four with CD, and one with bilateral adrenal hyperplasia) found clinically significant improvement in excess cortisol-induced symptoms in 15 out of 20 patients [4]. Patient responses to mifepristone treatment were monitored by clinical signs of hypercortisolism (signs of hypercortisolism, blood pressure measurements, and signs of adrenal insufficiency) and serum potassium and glucose. The study found that 15 out of 20 patients showed significant clinical improvement in excess cortisol-induced symptoms. Psychiatric symptoms and blood glucose levels also improved in the patients [4]. Of note, 11 out of 20 trial participants exhibited moderate to severe hypokalemia as a side effect, although only one patient had to leave the study early due to severe adverse effects [4]. In another well-known study, 50 patients were assessed at baseline and during intervention (total of six times) for 24 weeks, referred to as the SEISMIC study [8]. Changes in oral glucose tolerance tests over time were used to assess the mifepristone effect in type 2 diabetes millets (T2DM)/impaired glucose tolerance patients. Changes in diastolic blood pressure (BP) over time were used to measure the effect of mifepristone in hypertensive cardiogenic shock (CS) patients [8]. Results found a statistically significant improvement in symptoms in both groups: diabetic patients had improvement in response to oral glucose test, decreased A1C, and decreased fasting glucose, and hypertensive patients had decreased diastolic BP or reduction in antihypertensive medications [8]. In addition, the waist circumference and hemoglobin A1C (HbA1C) also improved, and study findings concluded that mifepristone use has an acceptable risk-benefit ratio for six months of treatment [8]. Several extension studies were later performed utilizing the data found during the SEISMIC study [9]. One such study assessing weight loss in patients who participated in the SEISMIC study also found statistically significant improvement in patients with CD. After one-week mifepristone period (patients who chose to participate in this follow-up study had to be assessed to ensure it was safe for them to enroll in this study), 30 patients were enrolled and started on once daily mifepristone at the dose they were taking when the SEISMIC study concluded [9]. The patient's weight was assessed at baseline and week 24 of the SEISMIC study, and for this study, the follow-up weight was taken at months 6, 12, 18, and 24 and a final visit. Data were assessed for 29 of the participants and statistically significant decreases in weight were found for all participants from baseline to end of the SEISMIC study, and the maintenance of weight loss was statistically significant in all participants at their final visit to this study as well [9]. Another SEISMIC extension study focused on monitoring the effects of mifepristone treatment in CD on ACTH levels and pituitary MRI findings [10]. Serum ACTH, urinary, and salivary cortisol levels were monitored during the SEISMIC study (baseline, day 14, and weeks 6, 10, 16, and 24) and once after a six-week mifepristone-free "washout" period. ACTH levels were then monitored one month later and then routinely every three months during the intervention period, which varied per participant [10]. Serum cortisol measures were assessed during the SEISMIC study at the intervals mentioned previously and then every six months during the extension study. Pituitary MRI studies were taken prior to mifepristone administration during the SEISMIC study and at weeks 10 and 24 [10]. Repeat imaging was then taken every six months during the extension study. On average, ACTH levels increased greater than twofold (2.76 ± 1.65-fold over baseline; p<0.0001 vs. baseline) in patients during the SEISMIC and extension study periods and decreased to near baseline levels after six weeks of mifepristone discontinuation [10]. Serum cortisol levels in both the initial intervention and extension period increased as well, although a higher mean cortisol level was seen during the extension study intervention (SEISMIC: 1.97 ± 1.02-fold increase; p<0.0001 vs. baseline; extension study: 2.85 ± 1.05-fold increase; p<0.0001 vs. baseline) [10]. In comparing the baseline and post-intervention MRI images, 30 out of 36 patients showed no progression in pituitary tumor size with mifepristone intervention, two patients showed regression of tumor size, and three patients showed evidence of tumor progression. One patient was found to have a tumor post-intervention despite a negative initial MRI at baseline [10]. A retrospective analysis of data collected during the SEISMIC study utilized oral glucose tolerance test data to assess the mifepristone treatment effect on the total body insulin sensitivity, beta cell function, weight, waist circumference, and additional parameters [11]. The analysis found improved total body insulin sensitivity in all participants, with the greatest improvement occurring from baseline to week 6. The weight and waist circumference both decreased by week 24 [11]. An additional important six-month study was done on 46 patients with refractory CS and either DM2, impaired glucose tolerance, or diagnosis of HTN in which mifepristone treatment was administered daily [12]. Patients were examined by three separate reviewers using global clinical response assessments (-1 = worsening, 0 = no change, 1 = improving) measured by eight clinical categories: glucose control, lipids, blood pressure, body composition, clinical appearance, strength, psychiatric/cognitive symptoms, and quality of life at weeks 6, 10, 16, and 24. A positive correlation with increasing GCR scores was found by week 24, with 88% of participants showing statistically significant improvement (p<0.001) [12]. Adenomyosis/Leiomyoma Adenomyosis and leiomyomas are common gynecological conditions that affect large portions of the female population. Multiple trials have proven mifepristone’s success in treating endometriosis and various forms of cancer. Current data shows that mifepristone is well tolerated and has mild side effects in certain long-term clinical settings. In one trial following mifepristone and its effects on adenomyosis, 20 patients were treated with 5 mg oral mifepristone/day for three months [13]. After the three-month trial, patients demonstrated a statistically significant (p<0.001) reduction in uterine volume as was measured through transvaginal ultrasound. These patients were also found to have significantly decreased CA-125 markers (a marker of adenomyosis and an increase in uterine size) and significantly increased hemoglobin concentration The patient’s endometrial tissue was then obtained from each patient during their hysterectomy [13]. The endometrial tissue samples were treated with varying concentrations of mifepristone for 48 hours. They found that mifepristone significantly decreased the viability of endometrial epithelial and stromal cells in adenomyosis and can induce their apoptosis as well [13]. This concentration-dependent inhibitory effect was most significantly seen with concentrations of mifepristone above 50 μmol/L at 48 hours. The same study showed that mifepristone demonstrated another dose-dependent relationship in the inhibition of the migration of ectopic endometrial and stromal cells. This finding is significant as the migratory nature of the patient’s endometrial and stromal cells is the pathogenesis behind adenomyosis [13]. Another study looked at the effect of mifepristone in combination with high-intensity focused ultrasound (HIFU) and levonorgestrel-releasing intrauterine system (LNG-IUS) in the treatment of adenomyosis [13]. Out of 123 patients, 34 patients were treated with HIFU alone, 29 patients were treated with HIFU combined with mifepristone, 10 patients with HIFU combined with LNG-IUS, and 50 patients with HIFU combined with mifepristone and LNG-IUS [13]. In the group treated with HIFU combined with mifepristone and LNG-IUS, the uterine volume was significantly reduced after treatment at 3, 6, 12, and 24 months compared to the previous treatment (p<0.05). Dysmenorrhea was measured using a visual analog score (VAS). In the combination group of mifepristone, HIFU, and LNG-IUS, VAS scores decreased from 80.82 ± 12.49 to 29.58 ± 9.29 at 24 months [13]. This was significantly lower than the three other treatment groups (p<0.05). The combination group of mifepristone, HIFU, and LNG-IUS also demonstrated statistically significant decreases in the menstrual volume and CA-125 serum markers [13]. Hemoglobin levels were not statistically different among the four treatment groups, but it is postulated that this could have been due to the fact that the patients who were anemic had been treated with different medications to improve their Hb aside from the trial medications [13]. Uterine leiomyomas are another gynecological condition that has been found to improve with the use of mifepristone as well. Insulin-like growth factor 1 (IGF-1) has been found to be overexpressed in leiomyomas [14]. This study showed that mifepristone inhibited the gene expression of IGF-1, and the reduction in symptoms correlated with a decrease in IGF-1 expression although the mechanism is not fully understood [14]. A meta-analysis studied the effects of mifepristone on uterine and leiomyoma volumes of 780 women from 11 randomized controlled trials. Mifepristone at doses from 2.5, 5, and 10 mg was found to effectively reduce uterine and leiomyoma volumes and alleviate leiomyoma symptoms at six months [6]. Pelvic pain, pelvic pressure, and dysmenorrhea were found to be alleviated after three months of treatment. Mifepristone also decreased the mean loss of blood during menstruation and a statistically significant increase in hemoglobin. No significant difference was found among varying dosages of 2.5, 5, and 10 mg other than increased frequency of hot flashes in patients of the 10 mg group. Another review investigated six clinical trials involving 166 women and the effects of 5-50 mg mifepristone for three to six months on leiomyomas [3]. The review demonstrated that daily treatment with all doses of mifepristone resulted in reductions in pelvic pain, pelvic pressure, dysmenorrhea, and uterine and leiomyoma volume size by 26-74%. Even doses of 2.5 mg of mifepristone resulted in significant improvement in the quality of life scores although there was little reduction in leiomyoma size at this dose [3]. This review also reported the rapid correction of uterine bleeding, amenorrhea, and increases in hemoglobin levels following treatment with 50 mg of mifepristone on alternating days. Even vaginal mifepristone has demonstrated efficacious results in the improvement of leiomyomas. In one such trial, the effects of daily 10 mg vaginal mifepristone were studied in 33 women from the ages of 30-53 [15]. Vaginal mifepristone significantly reduced leiomyoma volume and reduced the effects of symptoms on the patient’s quality of life as measured by the Uterine-Fibroid Symptoms Quality of Life questionnaire (UFS-QoL). It is important to note that the only significant side effect found in this review of trials was hot flashes at doses of mifepristone at 10 mg or more. Mifepristone was otherwise generally well tolerated with minimal if any adverse effects [15]. Discussion Adenomyosis is a gynecologic condition that is characterized by the growth of endometrial cells into the myometrium, resulting in a globally enlarged uterus and an associated increase in CA-125 [16]. This marker is classically known to be an ovarian tumor marker; however, in this class, it reflects the increase in uterine glandular size. Although it is often labeled as a “benign” disease, it affects around 20% of reproductive-aged women. This condition can lead to dysmenorrhea, infertility, and menorrhagia in addition to detrimental effects on a patient’s mental health [16]. Despite 20% of affected patients being under the age of 40, the gold standard of treatment is a hysterectomy. Hysterectomies may often not be wanted by patients as it is an invasive surgery that comes with several potential complications of its own. It is important to note that due to the large percentage of patients with adenomyosis who are of reproductive age, hysterectomies may not be an appropriate standard method of treatment. To rob patients of their fertility without attempting medication therapy with mifepristone first is an act of injustice. Surgery alone comes with many complications and the possibility of recurrence. The ability of physicians to manage their patient’s pain and symptoms should be guided medically before surgical sterilization is considered. Many of these patients are forced to seek alternative non-invasive treatments instead of medication therapies to preserve their fertility. HIFU and LNG-IUS are noninvasive therapies for adenomyosis that can be used in patients who refuse hysterectomies or for those who are not good candidates [16]. The pitfalls of these procedures include the fact that 20% of patients on HIFU alone end up relapsing, and LNG-IUS cannot be used in patients with a uterine size that is >12 weeks gestation or a uterine cavity depth that is >9 cm. Because adenomyosis is an estrogen-dependent disease, gonadotropin-releasing hormone agonists (GnRH-a) are also often used in combination with HIFU and LNG-US. Through the inhibition of the secretion of estrogen, GnRH-as facilitate reduced pelvic pain, reduced bleeding, and reduced uterine cavity size [16]. Reduction in cavity size is significant as this alone can lead to improved pain and reduced bleeding and allows patients to qualify for LNG-US where their previous uterine cavity size would have prevented their candidacy. Its current limitations include price (>$200/month), induction of premenopausal syndrome, and high rates of relapse following drug cessation [16]. Mifepristone offers a cheaper alternative (<$4/month) with significantly improved outcomes in reduced uterine cavity size, decreased dysmenorrhea pain scale score, and lower menstruation volume scores [16]. Mifepristone is also able to provide such results without the bone loss that is commonly associated with GnRH-analogs [3]. This is because mifepristone allows for serum estradiol to remain within the patient’s physiologic follicular phase range [3]. In addition, mifepristone is able to significantly reduce serum levels of CA-125 and improve hemoglobin levels in patients with menorrhagia. These reductions in CA-125 demonstrate marked reductions in the size of glands of the uterus of these patients. Through the reduction of cavity size, mifepristone can not only offer therapeutic relief but also allow patients to qualify for noninvasive LNG-US procedures, which can offer further therapeutic benefits. Patients should have the option to explore all potential medical therapies before opting for surgical correction. Leiomyomas, or uterine fibroids, are another commonly encountered gynecologic condition and represent the most common benign tumors found in the female population. These benign smooth muscle tumors are estrogen-sensitive and can rarely develop into malignant leiomyosarcomas. Nearly 20-50% of patients with these fibroids experience symptoms, such as abnormal uterine bleeding (AUB), infertility, pelvic pain, and miscarriages [17]. Currently, the only treatment for this common condition is surgery. Two medications that are commonly used for preoperative reductions in leiomyoma size are mifepristone and enantone. Enantone is a gonadotropin-releasing hormone analog that has shown significant improvement in leiomyoma shrinkage, correction of anemia, and correction of AUB [17]. Through its MOA, however, enantone can lead to harmful adverse effects, such as menopausal symptoms and bone mineral loss. Using hormone supplementation to negate these side effects leads to reduced effectiveness of enantone in fibroid size reduction. Several studies have shown that progesterone plays a large role in the proliferation of leiomyoma growth [17]. Mifepristone, therefore, offers an effective alternate solution by producing the same results without enantone’s adverse effects. When comparing enantone to mifepristone, the two medications both resulted in statistically significant reductions in fibroid size, reduction in dysmenorrhea, reduction in non-menstrual abdominal pain, and increased Hgb/Hct/and RBC count despite differences in dosage [17]. However, mifepristone was able to maintain the patients’ premenopausal levels of estrogen, whereas patients on enantone were found to have estrogen levels of menopausal patients. Furthermore, patients who were treated with enantone also reported more adverse events compared to those in the mifepristone group [17]. Vaginal use of mifepristone has also been shown to significantly reduce leiomyoma size and improve symptoms of anemia while lowering systemic bioavailability of mifepristone [15]. Through its concentrated distribution to uterine tissue, vaginal mifepristone can lead to increased improvement in its clinical outcomes. Vaginal mifepristone showed statistically significant improvements in leiomyoma volume change, USF-QoL, and decreased bleeding intensity at the end of the three-month trial and three months after treatment [15]. For these reasons, mifepristone can be used effectively for conservative therapy in patients suffering from leiomyomas and should be considered a viable option for patients not wishing to undergo surgery. CD refers to hypercortisolism that is caused by pituitary adenomas, adrenal neoplasias, or paraneoplastic ACTH secretion. Hypercortisolism in these patients leads to the development of skin changes, HTN, obesity, insulin resistance, dyslipidemia, anovulation, skeletal disorders, and neuropsychiatric disorders [18]. Patients suffering from these conditions endure a severely decreased quality of life and increased morbidity and mortality. The syndromic nature of this disease prompts delayed diagnosis and further increases the mortality and morbidity of this population [18]. CS therefore necessitates effective and rapid treatment options to diminish harm and clinical burden. The current first-line treatment for CD is pituitary surgery despite its nearly ⅓ relapse rate within 10 years postoperatively [18]. In these patients and patients with recurrent CD, further treatment options are necessitated. These options include adrenal surgery, pituitary radiotherapy, or medication therapy. Radiotherapy further delays symptomatic relief as it usually takes years before excess cortisol levels are managed. It also carries the risk of the patient developing hypopituitarism due to subsequent pituitary damage [18]. While surgery of the adrenal glands can quickly achieve control of excess cortisol, it also carries a risk of permanent adrenal insufficiency. Medication therapy can be used preoperatively, postoperatively, and as adjunctive therapy to radiotherapy. These drug classes include somatostatin analogs, dopamine agonists, and adrenal steroidogenesis inhibitors [18]. The most commonly used medication is the adrenal steroidogenesis inhibitor ketoconazole. While it has been proven to be effective and rapid in its success, doses may need to be frequently increased due to the cortisol blockade that occurs in CD patients [8]. In fact, due to the hormonal imbalances in CD patients, many medications often have to be dose adjusted to achieve therapeutic effect. It is also important to note that many of the medications that are used are not easily tolerated when doses are increased or adjusted frequently. The use of mifepristone has demonstrated statistically significant results in weight reduction, insulin resistance, depression, HTN, and quality of life in CD patients [10]. Furthermore, mifepristone can also be used effectively in patients experiencing cortisol-induced psychosis during acute exacerbations of hypercortisolism. While not included in the classes of more commonly used drugs for CD, mifepristone has been approved by the FDA for the treatment of CD when associated with disorders of glucose metabolism. This is undoubtedly due to the stigmatization of mifepristone and the subsequent reluctance of clinicians to incorporate it into their treatment plans. Neuropsychiatric disorders have been investigated for their associations with dysregulations of the hypothalamic-pituitary-adrenal axis (HPA) and increases in cortisol levels. Studies have shown that patients suffering from depression, schizophrenia, and psychotic depression have elevated levels of cortisol and increased activity of their HPA [19]. The role of cortisol in psychiatric disorders is evidenced by the adverse psychiatric effects that patients can develop in response to exogenous glucocorticoid use through subsequent increases in cortisol. These include delirium, depression, mania, or psychosis. When functioning normally, HPA activity and cortisol secretion are maintained through sensitive negative feedback systems involving glucocorticoid receptors (GCRs) and mineralocorticoid receptors (MCR) [19]. At low doses, cortisol preferentially binds to MCR. As cortisol levels rise, it begins to bind to GCR and thereby initiates the negative feedback loop. Antipsychotics that are typically used work by reducing cortisol levels. Mifepristone, when dosed at >200 mg/day, selectively binds only to GCR and has no effect on MCR [19]. Through its sole inhibition of GCR, it ensures that normal cortisol homeostasis is maintained while ensuring that excess high levels of cortisol are blocked. This was evidenced by the statistically significant correlation between rising plasma concentrations of mifepristone and improvement of psychotic symptoms [20]. The hippocampus is a region of the temporal lobe that is most notably recognized for its role in learning and memory. Further studies have shown correlations between hippocampal atrophy and patients with severe depression, PTSD, and schizophrenia. It is postulated that this hippocampal atrophy leads to persistently high levels of cortisol, worsening these patient’s psychiatric symptoms. Administration of mifepristone to patients with combat-related PTSD demonstrated significant benefits in quality of life and psychiatric improvement. Psychotic major depression is another psychiatric condition that affects around 20% of patients with major depression [7]. When mifepristone was used to treat psychotic depression, patients were able to achieve rapid antipsychotic effects that lasted for weeks after the medication therapy ended. It should be noted that patients suffering from PMD generally have increased cortisol levels even with standard antidepressant therapy alone [7]. Some patients are even unresponsive to electroconvulsive therapy. The ability of patients suffering from psychotic depression to achieve rapid relief is imperative as these patients are more susceptible to suicidal ideation, especially during an episode of psychosis [7]. Bipolar disorder is another mood disorder that has been found to be associated with high levels of cortisol, dysfunction of the HPA axis, and GR dysfunction. Several neuroendocrine studies demonstrated that around 43% of bipolar patients with depression were also dexamethasone-suppression-test (DST) nonsuppressors [7]. Further studies found that bipolar patients suffering through relapse and recovery had abnormal dexamethasone/corticotropin-releasing hormone (dex/CRH) test results [21]. These abnormal (dex/CRH) findings were also seen in healthy patients who had certain genetic predispositions for mood disorders [21]. Regarding these HPA dysfunctions, GR has been implicated in being an important modulator of neurocognitive function and mood. This can be evidenced through research findings that report increased GR number and GR binding in brain tissue following the administration of antidepressants in depressed patients [21]. Mifepristone’s unique advantage is that its selective role as a GR antagonist was also found to increase both MR and GR binding in the frontal cortex. In fact, data from Young et al. [21] reveals significant improvement in frontal cortex functioning following clinical mifepristone trials. These results were seen through improvements in spatial working memory function and reductions in the HDRS17 and MADRS. They also demonstrated significant improvement in verbal fluency from baseline. These improvements in neurocognitive functioning were measured when the subjects’ mood was similar to their baseline or did not vary when compared to the placebo group [21]. This key finding suggests that improvements in neurocognitive functioning were not solely related to improvements in mood or depression. Mifepristone achieves these improvements in neurocognitive function through its selective activity towards GR within the frontal cortex. Furthermore, patients are also able to achieve symptomatic improvement two weeks after the initiation of treatment [21]. The rapid nature of mifepristone adds further clinical benefit as classic bipolar treatments take longer to achieve therapy and the fact that treatment plans for patients with bipolar disorder are tricky to individualize. Other commonly known psychiatric disorders are treated with antipsychotics. While these medications often come with a large array of adverse effects, weight gain, metabolic derangements, and glucose intolerance have been a few of the more frequently reported negative effects. While the exact cause of the weight gain is unknown, mifepristone was shown to significantly reduce weight gain in patients when taken alongside risperidone or olanzapine [21]. As discussed previously, mifepristone also has the ability to significantly improve insulin resistance, thereby further improving the AE patients may experience on antipsychotics. Therefore, through mifepristone’s selective activity as a GCR antagonist, it has immense potential as a psychiatric therapeutic agent. Conclusions Mifepristone is a synthetic steroid that has immense potential to provide symptomatic relief in patients suffering from a wide array of complicated diseases. Prednisone, dexamethasone, and anabolic steroids are also synthetic steroids that are commonly used. Despite being a part of the same class as mifepristone, none of these medications fall under as much legal, political, and social duress as mifepristone. This is in spite of the fact that mifepristone has been proven to have an incredible safety profile since its introduction to the public in the 1980s. In fact, its mortality rate is significantly lower than that of Tylenol, NSAIDs, penicillin, and phosphodiesterase inhibitors. While further research is certainly needed, its involvement in politics has unfortunately led to the willful ignorance of its medical potential despite its evidence-based safety profile and efficacy. References Beaman J, Prifti C, Schwarz EB, Sobota M: Medication to manage abortion and miscarriage. J Gen Intern Med. 2020, 35:2398-405. 10.1007/s11606-020-05836-9 Hagey JM, Givens M, Bryant AG: Clinical update on uses for Mifepristone in obstetrics and gynecology. Obstet Gynecol Surv. 2022, 77:611-23. 10.1097/OGX.0000000000001063 Spitz IM: Mifepristone: where do we come from and where are we going? Clinical development over a quarter of a century. Contraception. 2010, 82:442-52. 10.1016/j.contraception.2009.12.012 Castinetti F, Fassnacht M, Johanssen S, et al.: Merits and pitfalls of mifepristone in Cushing's syndrome. Eur J Endocrinol. 2009, 160:1003-10. 10.1530/EJE-09-0098 Belanoff JK, Flores BH, Kalezhan M, et al.: Rapid reversal of psychotic depression using mifepristone. J Clin Psychopharmacol. 2001, 21:516-21. Eisinger SH, Meldrum S, Fiscella K, et al.: Low-dose mifepristone for uterine leiomyomata. Obstet Gynecol. 2003, 101:243-50. 10.1016/S0029-7844(02)02511-5 Flores BH, Kenna H, Keller J, Solvason HB, Schatzberg AF: Clinical and biological effects of mifepristone treatment for psychotic depression. Neuropsychopharmacology. 2006, 31:628-36. 10.1038/sj.npp.1300884 Fleseriu M, Biller BM, Findling JW, Molitch ME, Schteingart DE, Gross 😄 Mifepristone, a glucocorticoid receptor antagonist, produces clinical and metabolic benefits in patients with Cushing's syndrome. J Clin Endocrinol Metab. 2012, 97:2039-49. 10.1210/jc.2011-3350 Fein HG, Vaughan TB 3rd, Kushner H, Cram D, Nguyen 😧 Sustained weight loss in patients treated with mifepristone for Cushing's syndrome: a follow-up analysis of the SEISMIC study and long-term extension. BMC Endocr Disord. 2015, 15:63. 10.1186/s12902-015-0059-5 Fleseriu M, Findling JW, Koch CA, Schlaffer SM, Buchfelder M, Gross 😄 Changes in plasma ACTH levels and corticotroph tumor size in patients with Cushing's disease during long-term treatment with the glucocorticoid receptor antagonist mifepristone. J Clin Endocrinol Metab. 2014, 99:3718-27. 10.1210/jc.2014-1843 Wallia A, Colleran K, Purnell JQ, Gross C, Molitch ME: Improvement in insulin sensitivity during mifepristone treatment of Cushing syndrome: early and late effects. Diabetes Care. 2013, 36:e147-8. 10.2337/dc13-0246 Katznelson L, Loriaux DL, Feldman D, Braunstein GD, Schteingart DE, Gross 😄 Global clinical response in Cushing's syndrome patients treated with mifepristone. Clin Endocrinol (Oxf). 2014, 80:562-9. 10.1111/cen.12332 Che X, Wang J, He J, et al.: A new trick for an old dog: the application of mifepristone in the treatment of adenomyosis. J Cell Mol Med. 2020, 24:1724-37. 10.1111/jcmm.14866 Shen Q, Zou S, Sheng B, et al.: Mifepristone inhibits IGF-1 signaling pathway in the treatment of uterine leiomyomas. Drug Des Devel Ther. 2019, 14:3161-70. Yerushalmi GM, Gilboa Y, Jakobson-Setton A, Tadir Y, Goldchmit C, Katz D, Seidman DS: Vaginal mifepristone for the treatment of symptomatic uterine leiomyomata: an open-label study. Fertil Steril. 2014, 101:496-500. 10.1016/j.fertnstert.2013.10.015 Zhu H, Ma Q, Dong G, Yang L, Li Y, Song S, Mu Y: Clinical evaluation of high-intensity focused ultrasound ablation combined with mifepristone and levonorgestrel-releasing intrauterine system to treat symptomatic adenomyosis. Int J Hyperthermia. 2023, 40:10.1080/02656736.2022.2161641 Liu C, Lu Q, Qu H, et al.: Different dosages of mifepristone versus enantone to treat uterine fibroids: a multicenter randomized controlled trial. Medicine (Baltimore). 2017, 96:e6124. 10.1097/MD.0000000000006124 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 Hartmann J, Bajaj T, Klengel C, et al.: Mineralocorticoid receptors dampen glucocorticoid receptor sensitivity to stress via regulation of FKBP5. Cell Rep. 2021, 35:109185. 10.1016/j.celrep.2021.109185 Block TS, Kushner H, Kalin N, Nelson C, Belanoff J, Schatzberg A: Combined analysis of mifepristone for psychotic depression: plasma levels associated with clinical response. Biol Psychiatry. 2018, 84:46-54. 10.1016/j.biopsych.2018.01.008 Young AH, Gallagher P, Watson S, Del-Estal D, Owen BM, Ferrier IN: Improvements in neurocognitive function and mood following adjunctive treatment with mifepristone (RU-486) in bipolar disorder. Neuropsychopharmacology. 2004, 29:1538-45. 10.1038/sj.npp.1300471 From https://www.cureus.com/articles/191397-multiple-clinical-indications-of-mifepristone-a-systematic-review#!/
  19. Abstract Cushing's syndrome is a rare cause of myocardial infarction and heart failure. Herein, we report a female patient who presented acute myocardial infarction and heart failure with reduced ejection fraction. The patient was found to have hypercortisolism secondary to adrenocortical adenoma and responded well to therapy. This case underlines the effects of hypercortisolism on the cardiovascular system. The clinical presentation of this patient is unique because non-atherosclerotic myocardial infarction is rarely reported in Cushing's syndrome patients. Introduction Cushing's syndrome is an endocrine condition associated with excessive secretion of cortisol. Hypertension, vascular atherosclerosis, and chronic cardiac remodelling and dysfunction are commonly recognized cardiovascular complications in Cushing's syndrome patients.1 Herein, we report a rare case of Cushing's syndrome patient with a primary diagnosis of non-atherosclerotic myocardial infarction and heart failure (HF). Case Report A 61-year-old female with a past medical history of chronic obstructive pulmonary disease was admitted with sudden onset chest pain on 6 February 2018. Electrocardiogram showed ST-segment elevation in leads V3–V5. Blood biochemical results of 1 h after the onset of chest pain: cardiac troponin I (cTnI) 0.06 ug/L↑, creatine kinase (CK) 63 U/L, creatine phosphokinase-MB (CK-MB) 22 U/L, aspartate transferase (AST) 19 U/L, and lactic dehydrogenase (LDH) 482 U/L. Myocardial injury markers were markedly elevated at the time point of 18 h after onset: cTnI 13.9 ug/L↑, CK 613 U/L↑, CK-MB 102 U/L↑, AST 112 U/L↑, and LDH 833 U/L↑. Due to the acute ECG changes and elevated myocardial injury markers, the patient was preliminarily diagnosed as ST-segment elevation myocardial infarction (STEMI) and underwent coronary angiography, which showed no stenosis, occlusion or dissection of coronary arteries (Figure 1). Echocardiography showed enlarged left atrial dimension (LAD, 55 mm) and left ventricular end diastolic dimension (LVDd, 57 mm), and reduced ejection fraction (EF, 33%). The patient was treated for STEMI and HF, and was started on aspirin, statin, diuretic of furosemide and spirolactone, metoprolol, and Sacubitril/valsartan (SV, initiated June, 2020). The patient was strictly adherent to the medication prescribed (Table 1). Figure 1 Open in figure viewerPowerPoint Coronary angiogram demonstrating no significant obstruction in coronary artery circulation. Table 1. Echocardiography results 2020-06-22 2020-09-02 2021-03-29 2021-06-02 2021-09-01 2021-10-22 2021-12-21 LAD (mm) 55 55 46 52 47 44 41 LVDd (mm) 57 57 53 55 54 51 55 IVS (mm) 10 10 11 10 10 10 11 LVPW (mm) 11 11 11 10 11 9 10 EF (%) 33 30 31 39 47 49 52.5 EF, ejection fraction; IVS, interventricular septum; LAD, left atrium dimension; LVDd, left ventricular end diastolic dimension; LVPW, left ventricular posterior wall. However, the patient's condition was not improved despite optimized medication. On 26 January 2021, the patient was re-admitted with recurrent chest distress and oedema, with new symptoms of facial plethora, centripetal obesity, and hyperglycaemia (Figure S1). Abdominal CT scan showed a right adrenal adenoma (Figure 2). Cardiac magnetic resonance imaging revealed enlarged LVDd (62 mm), and reduced EF, with delayed myocardial enhancement and evidence of myocardial fibrosis and fatty deposits (Figure 3). Laboratory findings showed hypokalaemia: potassium 3.0 mmol/L, elevated serum cortisol level, low plasma ACTH level, and positive 1-mg overnight dexamethasone suppression test. Based on the above findings, the patient was diagnosed with Cushing's syndrome and started treatment with the glucocorticoid receptor inhibitor mifepristone on 5 February 2021. Figure 2 Open in figure viewerPowerPoint Abdominal CT scan showed adrenal adenoma at the right. Figure 3 Open in figure viewerPowerPoint Cardiac magnetic resonance imaging revealed enlarged LVDd, reduced EF, with delayed myocardial enhancement, evidence of myocardial fibrosis and fatty deposits. With mifepristone added to the previous medical therapy (aspirin, statin, sacubitril/valsartan, metoprolol and diuretic of furosemide and spirolactone, and mifepristone), the patient's condition and cardiac function improved, and echocardiography (21 December 2021) showed increased EF (52.5%). The patient underwent partial adrenalectomy on 22 December 2021. Postoperative pathology confirmed adrenal cortical adenoma. At last follow-up on 29 May 2023, the patient showed marked improvement in face and body shape, with no complaints of chest distress or oedema (Figure S2). Discussion In this case, the patient was first evaluated for STEMI due to her symptoms of chest pain, and the elevated ST-segment on ECG, along with the moderately elevated troponin I and other cardiac enzyme levels. However, coronary atherosclerotic heart disease was ruled out by the normal cardiac catheterization. We presume that a possible reason for acute myocardial infarction (AMI) might be vasospastic angina due to abnormal hormone levels with Cushing's syndrome, leading to increased excessive myocardial metabolic demand and relative myocardial hypoxia, which eventually induced myocardial infarction. Although coronary atherosclerotic heart disease is the main cause of AMI, many non-atherosclerotic processes can lead to an imbalance between decreased coronary blood flow and increased myocardial metabolic demand. To date, non-atherosclerotic myocardial infarction has rarely been reported in Cushing's syndrome patients. Vieira JT et al. reported that a patient with Cushing's disease was considered to have spontaneous coronary artery dissection, which is a rare reason for AMI.2 Cushing's syndrome is associated with an increased risk of cardiac failure,3 with both structural alterations and functional impairment. In our case, the patient's CMR imaging showed typical features of cardiac geometry, function, and fibrosis, in accordance with previous reports.4 The underlying mechanisms may be the enhanced responsiveness to angiotensin II and activation of the mineralocorticoid receptor in direct response to cortisol excess.5 Our patient responded well to the therapy of conventional anti-HF medication of sacubitril/valsartan, metoprolol, and diuretic, once mifepristone was added. This favourable response to the pharmacological regimen supports the benefits of the agents for the normalization of excess cortisol. This case indicates that early diagnosis and effective treatment of Cushing's syndrome may be crucial in preventing irreversible cardiac dysfunction secondary to cardiovascular events and heart failure. Acknowledgements This work was financially supported by the National Natural Science Foundation of China (81900409 and 82172182) and the PLA Youth Training Project for Medical Science (19QNP037). Conflict of interest The authors declares that there is no conflict of interest. From https://onlinelibrary.wiley.com/doi/10.1002/ehf2.14548
  20. Abstract Gastrointestinal perforation is a well-addressed complication of exogenous hypercortisolism; however, patients with endogenous Cushing's syndrome (CS) do not usually experience this condition in clinical practice. The literature on this subject is limited and consists solely of clinical case reports/series with only 23 instances of gastrointestinal perforation occurring in individuals with endogenous Cushing's syndrome. This is mainly attributed to the rarity of Cushing's syndrome itself and the low chance of occurrence of such complications. We report a case of a recently diagnosed adrenocorticotropic hormone (ACTH)-dependent Cushing's syndrome in a 30-years-old female who presented initially with a three-month history of progressive weight gain, generalized weakness, acne, menstrual irregularity, and severe hypokalemia, and then developed a gastric ulcer perforation only one month after her ACTH-dependent Cushing's syndrome diagnosis and was managed through emergent surgery. Introduction A disorder of the endocrine system characterized by excessive cortisol production, known as Cushing's syndrome, rarely occurs. The main causes are pituitary tumors, ectopic adrenocorticotropic hormone (ACTH)-secreting tumors, or adrenal tumors that secrete cortisol independently [1]. Patients initially present with a wide range of symptoms, including weight gain, proximal myopathy, skin thinning, and abdominal striae [1]. Additionally, several metabolic disorders, such as diabetes mellitus, hypertension, and dyslipidemia, can occur, especially when the diagnosis is not established at an early stage [2]. There is a possibility of gastrointestinal complications among patients receiving exogenous glucocorticoids. However, there is limited information on gastrointestinal complications associated with endogenous hypercortisolemia [3,4]. Thus far, only 23 instances have been published addressing the co-occurrence of gastrointestinal perforation with endogenous Cushing's syndrome [5-17]. To the best of our knowledge, this is the first case reporting gastric perforation in an ACTH-dependent Cushing's syndrome, while the vast majority reported diverticular, sigmoid, or duodenal perforation with Cushing's syndrome [5-17]. Herein, we describe the medical history, physical examination, and investigatory findings of a 30-year-old female with a recent diagnosis of ACTH-dependent Cushing's syndrome that was complicated by gastric ulcer perforation, necessitating an urgent exploratory laparotomy. The primary motivator of this case report was the rarity of the described condition, the atypical location of the perforation in such patient group, and the relatively young age of the patient. Case Presentation History and examination A 30-year-old female with a history of mental retardation was admitted to our emergency department (ER) with progressive weakness and fatigue. Upon taking the history, she had been having menstrual irregularities, progressive weight gain, and generalized weakness, which was significant enough to limit her physical activity and hinder her movement for the past three months. Initial vital signs showed that the patient had a body temperature of 37°C, a pulse rate of 90 beats per minute, and a blood pressure of 130/80 mmHg. On physical examination, the patient had a moon face with supraclavicular fullness, dorsocervical fat pad, purple abdominal striae, facial signs of hirsutism, and acne all over the face, shoulders, chest, and back. Investigations In the initial laboratory examination, hypokalemia of 2.1 mEq/L, hyperglycemia of 12.1 mmol/L, and metabolic alkalosis were detected (Table 1). The cortisol level after 1 mg dexamethasone suppression test was 2204 nmol/L (normal range 140-690), ACTH 123 pg/mL (normal range 7.2-63.3), DHEA-S 27.85 umol/L (normal range 2.6-13.9), And 24-hour urine cortisol level was 1560 mg/day (normal range 30-350) (Table 1). No suppression was observed in cortisol level with 8 mg dexamethasone suppression test. Parameter Initial presentation Perforation presentation Refrence range Na+ 143 mEq/L 139 mmol/L 135-147 mEq/L Cl- 85 mEq/L 105 mmol/L 98-108 mEq/L K+ 2.1 mEq/L 2.8 mmol/L 3.5-5.0 mEq/L Mg2+ 0.79 mmol/L 0.77 mmol/L 0.85-1.110 mmol/L PO3- 0.88 mmol/L 1.23 mmol/L 0.97-1.46 mmol/L PH 7.54 7.36 7.35-7.45 PCO2 67.5 mmHg 42.7 mmHg 35-45 mmHg PO2 27.7 mmHg 62.2 mmHg 75-100 mmHg HCO3 49.8 mEq/L 23.6 mEq/L 22-26 mEq/L Random blood glucose 12.1 mmol/L 24.1 mmol/L <5.5 mmol/L Hemoglobin 13.5 g/dL 14.9 g/dL 13.7-16.8 g/dL White blood cells 9,720 /uL 11,100 /uL 3,300-8,600 /uL Lymphocyte 0.48% 0.33% - Neutrophil 8.55% 9.66% - Eosinophil 0.0% 0.0% - TSH 0.55 mIU/L Was not ordered 0.4-4.0 mIU/L Cortisol 2204 nmol/L 4842 nmol/L 140-690 nmol/L ACTH 123 pg/mL Was not ordered 7.2-63.3 pg/mL Table 1: Laboratory findings on initial presentation and on perforation day TSH - thyroid stimulating hormone; ACTH - adrenocorticotropic hormone A series of CT scans for the neck, chest, abdomen, and pelvis was performed and failed to localize any tumors acting as an ectopic source. A pituitary MRI was performed, and no adenoma was found. To complete the diagnostic workup, we decided to do an inferior petrosal sinus sampling (IPSS) and PET scan with Gallium 68; however, the patient's family refused and requested discharge and outpatient follow-ups. These results, together with the biochemical and clinical findings, supported the diagnostic hypothesis of ACTH-dependent Cushing's syndrome. Treatment/management When addressing the issue of hypokalemia that the patient presented with initially, it was found to be resistant and difficult to correct. The patient was put on spironolactone 50 mg BID, and potassium chloride 20 mEq q8h, and her potassium level barely reached 3.5 mmol/L after several days. In addition, her magnesium level was corrected with magnesium oxide 800 mg every six hours. Her blood glucose level was controlled with insulin glargine 6 units daily and Novorapid as per the sliding scale. The patient was discharged on spironolactone tablets 50 mg BID (oral), potassium chloride 20 mEq q8h, cholecalciferol, calcium carbonate, insulin glargine 6 units daily, and Novorapid 4 units TID before meals. Follow-up and outcomes Seven days after discharge, she presented to the ER complaining of a new onset of abdominal pain, constipation, and reduced urine output. Her Glasgow Coma Scale (GCS) was 15, her blood pressure measurement was 146/90 mmHg, her pulse rate was 66 beats per minute, her respiratory rate was 21 breaths per minute, and her temperature was 36.7°C. Upon physical examination, the patient had distended non-tender abdomen without any other significant findings. Blood work was done, including renal functions, and all parameters, including potassium, were within normal limits. A chest X-ray was also performed and revealed no evidence of pneumoperitoneum. The patient was clinically stable after managing her abdominal pain with acetaminophen injection and administering fleet enema for constipation. After instructions on when to come again to the ER were given, the patient was discharged home on lactulose and paracetamol, and a close outpatient follow-up appointment was scheduled. Five days after the ER visit, the patient presented again to the ER. She was still complaining of severe non-resolving abdominal pain, constipation, and reduced urine output. Upon physical examination in the ER, the patient was found to have developed a new onset of lower limb edema, abdominal rebound tenderness, and abdominal rigidity and guarding. She was hypotensive with a blood pressure of 91/46 mmHg, pulse rate of 80 beats per minute, respiratory rate of 16 breaths per minute, temperature of 38.2 °C, and SpO2 of 96%. The only significant laboratory finding was her potassium level dropping low to 2.8 mEq/L (Table 1). An X-ray of the chest was requested and showed a large pneumoperitoneum (Figure 1). Figure 1: Posteroanterior chest X-ray at the time of gastric perforation displaying severe air under the diaphragm with bilateral obstruction indicating massive pneumoperitoneum (red arrow) Abdominal CT was also urgently performed and confirmed the presence of gastric perforation likely related to an underlying perforated peptic ulcer with 0.8 cm defect at the distal greater curvature (Figures 2, 3). Figure 2: Coronal-section CT image of abdomen and pelvis at the time of gastric perforation showing features of gastric perforation likely related to the underlying perforated peptic ulcer with 0.8 cm defect at the distal greater curvature Figure 3: Horizontal-section CT image showing features of gastric perforation likely related to the underlying perforated peptic ulcer with 0.8 cm defect at the distal greater curvature The patient underwent an emergent gastric wedge resection for gastric perforation, and the pathology reported evidence of gastric ulcer with no evidence of malignancy. Furthermore, Helicobacter pylori test was performed on the sample, and it came back positive. The patient tolerated the surgery very well, and postoperative recovery was without any complications. Later, the patient was prescribed metyrapone 250 mg Q4h, which was then increased to 500 mg Q4h four days after surgery, and her cortisol level significantly dropped to 634nmol/L. During that time, a gastrin level test was also performed to exclude the presence of gastrinomas, and the level was 45 pg/ml (normal range 13-115). Discussion A small percentage of the population suffers from Cushing's syndrome, which is an endocrine disorder characterized by an endogenous overproduction of glucocorticoids, resulting in hypercortisolemia [1]. It is estimated to affect 0.7 to 2.4 people per million annually [1]. Hypercortisolemia alters psychologic, metabolic, and cardiovascular functions, resulting in increased mortality and morbidity rates, particularly if the diagnosis is delayed and long-term exposure to high cortisol levels occurs [2]. Women are more likely to suffer from this condition than men, and people in their 40s to 60s are most vulnerable to it [1]. Patients initially present with a wide range of symptoms, including weight gain, proximal myopathy, skin thinning, and abdominal striae [1]. Additionally, several metabolic disorders, such as diabetes mellitus, hypertension, and dyslipidemia, can occur [1]. Due to the rarity of this condition, there is often a significant delay in diagnosis and treatment, which could eventually lead to complications from prolonged hypercortisolism. From another standpoint, in a systematic review, the incidence of peptic ulcer perforation ranges from 3.8 to 14 per 100,000 individuals in the general population [18]. In under-developed countries, patients are typically young, tobacco-using males [19]. However, patients in industrialized countries are typically older with multiple co-morbidities and are on long-term non-steroidal anti-inflammatory drugs (NSAIDs) or steroid use [19]. Patients may present with an abrupt onset of abdominal discomfort, abdominal rigidity, and tachycardia in the early stages of a perforated peptic ulcer [19]. Later, abdominal distention, pyrexia, hypotension, fever, and vomiting can occur [19]. Furthermore, when the diagnosis is made early, a perforated ulcer often has a good prognosis. However, the risk of adverse events increases if there is a delay in the diagnosis [20]. Therefore, making an early detection through different imaging modalities is crucial [20]. A history of peptic ulcer disease, NSAIDs, physiological stress, smoking, corticosteroids, and Helicobacter pylori are some of the well-established risk factors for a perforated peptic ulcer [20]. The prevalence of Helicobacter pylori among Saudi patients is high; in one study, the overall prevalence was 46.5% in patients with dyspepsia using gastric biopsy [21]. Several studies have explored the relationship between Helicobacter pylori and gastrointestinal perforation, but the results have been mixed. Some studies have suggested a higher prevalence of Helicobacter pylori infection among individuals with gastrointestinal perforation compared to those without, indicating a potential association. However, other studies have found no significant difference in the prevalence of Helicobacter pylori infection between perforated and non-perforated gastrointestinal ulcer cases [22]. Furthermore, they suggested that the presence of other risk factors like the use of NSAIDs, smoking, and alcohol may interact with Helicobacter pylori infection and contribute to the development of complications such as gastrointestinal perforation [22]. However, in our case, the patient did not have any established risk factors for gastric perforation, such as NSAIDs, smoking, or alcohol. Therefore, considering the low incidence of gastrointestinal perforation and high prevalence of Helicobacter pylori, the conflicting data regarding the association between Helicobacter pylori and gastrointestinal perforation, and the lack of established risk factors for gastrointestinal perforation in our patient, we suggest that prolonged excess glucocorticoids from Cushing's syndrome may have contributed to the gastric perforation either independently or synergistically with Helicobacter pylori since hypercortisolism can lead to a weakened gastrointestinal wall integrity due to decreased collagen turnover and disruption of mucosal protection by prostacyclin [15]. In addition, because of hypercortisolism, perforation may not be contained or healed initially due to the immunosuppressive effects of hypercortisolism, whether endogenous or exogenous [15]. Additionally, high levels of cortisol may delay the diagnosis and treatment since it may mask the symptoms of the perforation [14]. Moreover, our patient was treated for severe hypokalemia with potassium supplementation for an extended period of time. Previous studies have linked potassium chloride supplementation to gastrointestinal ulceration and perforation, making this a possible additive cause of our patient's condition [23,24]. A limited number of studies have addressed gastrointestinal perforations associated with endogenous hypercortisolemia [5-17]. The correlation between Cushing's syndrome and gastrointestinal perforation is highlighted in our study and in the case reports that have been previously published (Table 2). Similar to our case, a female predominance was seen in gastrointestinal perforation among the reported cases of Cushing's syndrome [6,7,12,13,15,16]. Additionally, the average age at which gastrointestinal perforation occurred in patients with endogenous hypercortisolism ranged from 45 to 80, which is a noticeably higher age range than the case we are presenting here (aged 30) [6-10,12]. Furthermore, unlike our case, in which gastrointestinal perforation occurred four months after the onset of Cushing's symptoms, Intestinal perforation occurs approximately 9.8 months after Cushing's symptoms first appear [15]. Furthermore, in our patient, gastric perforation occurred while she was hypercortisolemic and not in a remission state. Hence, in association with Helicobacter pylori infection, severe hypercortisolemia could have been a secondary contributing factor to gastric perforation. The complications of gastric ulceration, specifically with endogenous Cushing's syndrome, have been addressed in two case reports [25,26]. It must be noted, however, that neither case is similar to ours. A case of gastric perforation was reported by Kubicka et al. in a patient who had a confirmed diagnosis of gastrinoma, and the patient was diagnosed with ectopic Cushing's syndrome seven months after gastric perforation [25]. Therefore, since ectopic Cushing's syndrome was diagnosed seven months after the perforation, it is more likely that the gastrinoma contributed to this complication. In contrast, our patient's serum gastrin level was within the normal range, ruling out gastrinoma. Further, Hoshino et al. reported a case of gastrointestinal bleeding in a 39-year-old man with a confirmed diagnosis of Cushing's disease secondary to pituitary adenoma [26]. He was found to have gastric ulceration and bleeding along with Helicobacter pylori infection and elevated cortisol levels [26]. In spite of the patient not developing a gastric perforation, it was suggested by the author that hypercortisolism might be a contributing factor for gastric ulcer complications by slowing down the ulcer healing process [26] Reference Year of publication Age, gender Highest cortisol level plasma cortisol (PC, nmol/L) / UFC (nmol/L) Cause of Cushing's syndrome Time from onset of Cushing's symptoms to perforation (months) Reported site of gastrointestinal perforation Current 2023 30, Female PC 4842 ACTH-dependant 4 Gastric perforation Ishinoda et al. [17] 2023 24, Male PC 1647 Cushing's disease 12 Sigmoid colon perforation Wijewickrama et al. [16] 2021 32, Female PC 1147 Pituitary microadenoma 1 Diverticular perforation Shahidi et al. [15] 2019 72, Female UFC 5296 Pancreatic neuroendocrine tumor 12 Diverticular perforation Shahidi et al. [15] 2019 61, Female PC 1925 Metastatic medullary carcinoma of thyroid 12 Sigmoid colon and diverticular perforation Shahidi et al. [15] 2019 68, Female UFC 410 Cushing's disease 12 Sigmoid colon perforation Shahidi et al. [15] 2019 71, Female UFC 1533 Cushing's disease 4 Diverticular perforation Shahidi et al. [15] 2019 54, Male UFC 374 Cushing's disease 3 Sigmoid colon perforation Shahidi et al. [15] 2019 52, Female UFC 885 Cushing's disease 16 Diverticular perforation Sater et al. [14] 2018 80, Female UFC 5601 Lung carcinoid 36 Diverticular perforation Sater et al. [14] 2018 60, Female UFC 72726 Metastatic islet cell carcinoma 36 Diverticular perforation Sater et al. [14] 2018 31, Male UFC 1297 Cushing's disease 20 Diverticular perforation Sater et al. [14] 2018 52, Female UFC 2371 Lung carcinoid 4 Diverticular perforation Sater et al. [14] 2018 67, Male UFC 3836 Ectopic ACTH 10 Diverticular perforation Sater et al. [14] 2018 51, Male UFC 13552 Metastatic thymic carcinoma 4 Diverticular perforation Kaya et al. [9] 2016 70, Male PC 1432 Small cell lung cancer 1 Diverticular perforation Dacruz et al. [12] 2016 60, Female UFC 4481 Metastatic parotid tumor 5 Sigmoid colon and diverticular perforation Matheny et al. [10] 2016 67, Male UFC 11119 Metastatic medullary carcinoma of thyroid 4 Diverticular perforation Flynn et al. [13] 2016 63, Female UFC 12465 Pheochromocytoma 1 Perforation at the splenic flexure Balestrieri et al. [11] 2016 75, Male PC 2272 Neuroendocrine tumor 1 Intestinal perforation Hara et al, [8] 2013 79, Male PC 1230 Cushing's disease 6 Diverticular perforation De Havenon et al. [7] 2011 71, Female PC 2593 Cushing's disease 9 Diverticular perforation Lutgers et al. [6] 2010 55, Female UFC 10152 Right pheochromocytoma 1 Sigmoid colon and diverticular perforation Drake et al. [5] 1998 35, Male PC 1442 Islet cell tumor 4 Duodenal perforation and rupture of pancreatic pseudocyst Table 2: Current case and previous reported 23 cases of patients with Cushing's syndrome and gastrointestinal perforation UFC - urinary free cortisol; PC - plasma cortisol; ACTH - adrenocorticotropic hormone Conclusions A high blood cortisol level can be associated with various clinical manifestations and diverse sets of complications. This case report sheds light on one of the less common complications of hypercortisolism in patients with Cushing's syndrome, which is gastrointestinal perforation. Our report further supports the published evidence that gastrointestinal perforation is a rare but potentially fatal complication among patients with Cushing's syndrome. Moreover, it highlights the possibility of developing gastric perforations in this patient group, even at younger ages than expected. This should elicit a high clinical suspicion and demand prompt investigation of Cushing's syndrome patients in a hypercortisolism state presenting with modest gastrointestinal symptoms. References Pivonello R, De Martino MC, De Leo M, Lombardi G, Colao A: Cushing's syndrome. Endocrinol Metab Clin North Am. 2008, 37:135-49. 10.1016/j.ecl.2007.10.010 Newell-Price J, Bertagna X, Grossman AB, Nieman LK: Cushing's syndrome. Lancet. 2006, 367:1605-17. 10.1016/S0140-6736(06)68699-6 Goethals L, Nieboer K, De Smet K, De Geeter E, Tabrizi NH, Van Eetvelde E, de Mey J: Cortisone associated diverticular perforation. JBR-BTR. 2011, 94:348-9. 10.5334/jbr-btr.705 Piekarek K, Israelsson LA: Perforated colonic diverticular disease: the importance of NSAIDs, opioids, corticosteroids, and calcium channel blockers. Int J Colorectal Dis. 2008, 23:1193-7. 10.1007/s00384-008-0555-4 Drake WM, Perry LA, Hinds CJ, Lowe DG, Reznek RH, Besser GM: Emergency and prolonged use of intravenous etomidate to control hypercortisolemia in a patient with Cushing's syndrome and peritonitis. J Clin Endocrinol Metab. 1998, 83:3542-4. 10.1210/jcem.83.10.5156 Lutgers HL, Vergragt J, Dong PV, de Vries J, Dullaart RP, van den Berg G, Ligtenberg JJ: Severe hypercortisolism: a medical emergency requiring urgent intervention. Crit Care Med. 2010, 38:1598-601. 10.1097/CCM.0b013e3181e47b7a de Havenon A, Ehrenkranz J: A perforated diverticulum in Cushing's disease. Int J Surg Case Rep. 2011, 2:215-7. 10.1016/j.ijscr.2011.06.009 Hara T, Akutsu H, Yamamoto T, Ishikawa E, Matsuda M, Matsumura A: Cushing's disease presenting with gastrointestinal perforation: a case report. Endocrinol Diabetes Metab Case Rep. 2013, 2013:130064. 10.1530/EDM-13-0064 Kaya T, Karacaer C, Açikgöz SB, Aydemir Y, Tamer A: Severe hypokalaemia, hypertension, and intestinal perforation in ectopic adrenocorticotropic hormone syndrome. J Clin Diagn Res. 2016, 10:OD09-11. 10.7860/JCDR/2016/17198.7127 Matheny LN, Wilson JR, Baum HB: Ectopic ACTH production leading to diagnosis of underlying medullary thyroid carcinoma. J Investig Med High Impact Case Rep. 2016, 4:2324709616643989. 10.1177/2324709616643989 Balestrieri A, Magnani E, Nuzzo F: Unusual Cushing's syndrome and hypercalcitoninaemia due to a small cell prostate carcinoma. Case Rep Endocrinol. 2016, 2016:6308058. 10.1155/2016/6308058 Dacruz T, Kalhan A, Rashid M, Obuobie K: An ectopic ACTH secreting metastatic parotid tumour. Case Rep Endocrinol. 2016, 2016:4852907. 10.1155/2016/4852907 Flynn E, Baqar S, Liu D, et al.: Bowel perforation complicating an ACTH-secreting phaeochromocytoma. Endocrinol Diabetes Metab Case Rep. 2016, 2016:10.1530/EDM-16-0061 Sater ZA, Jha S, McGlotten R, Hartley I, El Lakis M, Araque KA, Nieman LK: Diverticular perforation: A fatal complication to forestall in Cushing syndrome. J Clin Endocrinol Metab. 2018, 103:2811-4. 10.1210/jc.2018-00829 Shahidi M, Phillips RA, Chik CL: Intestinal perforation in ACTH-dependent Cushing's syndrome. Biomed Res Int. 2019, 2019:9721781. 10.1155/2019/9721781 Wijewickrama PS, Ratnasamy V, Somasundaram NP, Sumanatilleke M, Ambawatte SB: A challenging case of Cushing's disease complicated with multiple thrombotic phenomena following trans-sphenoidal surgery; a case report. BMC Endocr Disord. 2021, 21:29. 10.1186/s12902-021-00701-0 Ishinoda Y, Uto A, Meshino H, et al.: Bowel perforation associated with Cushing's disease: a case report with literature review. Endocr J. 2023, 70:933-9. 10.1507/endocrj.EJ23-0110 Lau JY, Sung J, Hill C, Henderson C, Howden CW, Metz DC: Systematic review of the epidemiology of complicated peptic ulcer disease: incidence, recurrence, risk factors and mortality. Digestion. 2011, 84:102-13. 10.1159/000323958 Chung KT, Shelat VG: Perforated peptic ulcer - an update. World J Gastrointest Surg. 2017, 9:1-12. 10.4240/wjgs.v9.i1.1 Weledji EP: An overview of gastroduodenal perforation. Front Surg. 2020, 7:573901. 10.3389/fsurg.2020.573901 Akeel M, Elmakki E, Shehata A, Elhafey A, Aboshouk T, Ageely H, Mahfouz MS: Prevalence and factors associated with H. pylori infection in Saudi patients with dyspepsia. Electron Physician. 2018, 10:7279-86. 10.19082/7279 Thirupathaiah K, Jayapal L, Amaranathan A, Vijayakumar C, Goneppanavar M, Nelamangala Ramakrishnaiah VP: The association between Helicobacter pylori and perforated gastroduodenal ulcer. Cureus. 2020, 12:e7406. 10.7759/cureus.7406 Farquharson-Roberts MA, Giddings AE, Nunn AJ: Perforation of small bowel due to slow release potassium chloride (slow-K). Br Med J. 1975, 3:206. 10.1136/bmj.3.5977.206 Payan H, Blaustein A: Potassium chloride and small bowel perforation. Gastroenterology. 1965, 48:877-8. 10.1016/S0016-5085(65)80073-7 Kubicka E, Zawadzka K, Syrycka J, Kałużny M, Pawluś A, Bolanowski M: A case of gastrinoma associated with ectopic Cushing syndrome. Pol Arch Intern Med. 2020, 130:328-9. 10.20452/pamw.15201 Hoshino C, Satoh N, Narita M, Kikuchi A, Inoue M: Another 'Cushing ulcer'. BMJ Case Rep. 2011, 2011:10.1136/bcr.02.2011.3888 From https://www.cureus.com/articles/196132-adrenocorticotropic-hormone-dependent-cushings-syndrome-complicated-with-gastric-ulcer-perforation-in-a-30-year-old-saudi-female-a-case-report-and-a-review-of-the-literature#!/
  21. Abstract Objective Since Cushing's disease (CD) is less common in the paediatric age group than in adults, data on this subject are relatively limited in children. Herein, we aim to share the clinical, diagnostic and therapeutic features of paediatric CD cases. Design National, multicenter and retrospective study. Patients All centres were asked to complete a form including questions regarding initial complaints, physical examination findings, diagnostic tests, treatment modalities and follow-up data of the children with CD between December 2015 and March 2017. Measurements Diagnostic tests of CD and tumour size. Results Thirty-four patients (M:F = 16:18) from 15 tertiary centres were enrolled. The most frequent complaint and physical examination finding were rapid weight gain, and round face with plethora, respectively. Late-night serum cortisol level was the most sensitive test for the diagnosis of hypercortisolism and morning adrenocorticotropic hormone (ACTH) level to demonstrate the pituitary origin (100% and 96.8%, respectively). Adenoma was detected on magnetic resonance imaging (MRI) in 70.5% of the patients. Transsphenoidal adenomectomy (TSA) was the most preferred treatment (78.1%). At follow-up, 6 (24%) of the patients who underwent TSA were reoperated due to recurrence or surgical failure. Conclusions Herein, national data of the clinical experience on paediatric CD have been presented. Our findings highlight that presenting complaints may be subtle in children, the sensitivities of the diagnostic tests are very variable and require a careful interpretation, and MRI fails to detect adenoma in approximately one-third of cases. Finally, clinicians should be aware of the recurrence of the disease during the follow-up after surgery. From https://onlinelibrary.wiley.com/doi/10.1111/cen.14980
  22. Abstract Cushing’s syndrome is a condition leading to overproducing of cortisol by the adrenal glands. If the pituitary gland overproduces cortisol, it is called Cushing’s disease. Cushing’s syndrome and even Cushing’s disease during and after pregnancy are rare events. There is not enough literature and guidance for managing and treating these patients. The diagnosis of Cushing’s syndrome in pregnancy is often delayed because the symptoms overlap. We presented a thin 31-year-old woman, admitted 2 months after a normal-term delivery, with an atypical presentation of Cushing’s disease, unusual clinical features, and a challenging clinical course. She had no clinical discriminatory features of Cushing’s syndrome. Given that the patient only presented with psychosis and proximal myopathy and had an uncomplicated pregnancy, our case was considered unusual. The patients also had hyperpigmentation and severe muscle weakness which are among the less common presentations of Cushing’s syndrome. Our findings suggest that an early diagnosis of Cushing’s disease is important in pregnancy period for its prevalent fetal and maternal complications, and it should be treated early to optimize fetal and maternal outcomes as there is an increasing trend toward live births in treated participants. Introduction Cushing’s syndrome is a condition that originates from excessive production of glucocorticoids. The condition is most common in women of childbearing age and is characterized by altered distribution of the adipose tissue to the central and upper regions of the trunk (central obesity and buffalo hump), face (moon face), capillary wall integrity (easy bruising), hyperglycemia, hypertension, mental status changes and psychiatric symptoms, muscle weakness, signs associated with hyperandrogenism (acne and hirsutism), and violaceous striae among other signs. Hypercortisolism and hyperandrogenism suppress the production of the pituitary gonadotropins, which in turn leads to menstrual irregularities and infertility.1-3 Moreover, the main common cause of developing Cushing’s syndrome is the use of exogenic steroid.3 Cushing’s disease is a form of Cushing’s syndrome with overproduction of adrenocorticotropic hormone (ACTH) due to pituitary adenoma. The diagnosis is made using clinical features and paraclinical tests including urinary free cortisol (UFC), serum ACTH, dexamethasone suppression tests (DSTs), pituitary magnetic resonance imaging (MRI), and sometimes by inferior petrosal sinus sampling (IPSS).4 Although women with Cushing’s disease are less likely to become pregnant, timely diagnosis and appropriate management are especially important during possible pregnancy, preventing neonatal and maternal complications and death. The diagnosis is challenging due to the overlap of the disease symptoms with the changes associated with a normal pregnancy. Moreover, the hormonal milieu during pregnancy has recently been proposed as a potential trigger for Cushing’s disease in some cases; hence, the term “pregnancy-associated Cushing’s disease” has been used for the disease in the recent literature. In this study, we presented a thin 31-year-old woman who was referred to our clinic 2 months after a normal delivery, with an atypical presentation of Cushing’s disease, unusual clinical features, and a challenging clinical course. Case Presentation Our patient was a 31-year-old woman who presented 2 months after the delivery of her second child. She had a history of type 2 diabetes mellitus and hypertension in the past 2 years prior to her presentation. She had been admitted to another center following an episode of falling and muscle weakness. Two weeks later, she was admitted to our center with an impression of pulmonary thromboembolism due to tachypnea, tachycardia, and dyspnea. During follow-up, she was found to have leukocytosis, hyperglycemia (random blood sugar: 415 mg/d; normal level: up to 180 mg/dL) and hypokalemic metabolic alkalosis (PH: 7.5, HCO3 [bicarbonate]: 44.7 mEq/L, paO2 [partial pressure of oxygen]: 73 mm Hg, pCO2: 51.7 mm Hg, potassium: 2.7 mEq/L [normal range: 3.5-5.1 mEq/L]), which was refractory to the treatment; therefore, an endocrinology consultation was first requested. On physical examination, the patient was agitated, confused, and psychotic. Her vital signs were: blood pressure 155/100 mm Hg, heart rate: 130 bpm, and respiratory rate: 22 bpm, temperature: 39°C. As it has shown in Figure 1A, her face is not typical for moon face of Cushing’s syndrome, but facial hirsutism (Figure 1A) and generalized hyperpigmentation is obvious (Figure 1A-C). She was a thin lady and had a normal weight and distribution of adiposity (Body Mass Index [BMI] = 16.4 kg/m2; weight: 40 kg, and height: 156 cm). Aside from thinness of skin, she did not have the cutaneous features of Cushing’s syndrome (e.g. purpura, acne, and violaceous striae) and did not have supraclavicular and dorsocervical fat pad (buffalo hump), or plethora. In other words, she had no clinical discriminatory features of Cushing’s syndrome despite the high levels of cortisol, as confirmed by severely elevated UFC (5000 μg/24 h and 8000 μg/24 h; normal level: 4-40 μg/24 h). In addition, as will be mentioned later, the patient had axonal neuropathy which is a very rare finding in Cushing’s syndrome. Figure 1. Clinical finding of our case with Cushing’s disease. (A) Hirsutism, (B) muscle atrophy seen in proximal portion of lower limbs, and (C) hyperpigmentation specially on the skin of the abdominal region. OPEN IN VIEWER She had a markedly diminished proximal muscle force of 1 out of 5 across all extremities; the rest of the physical examinations revealed no significant abnormalities (Figure 1B). On the contrary, based on her muscle weakness, hirsutism, psychosis and hyperpigmentation and refractory hypokalemic alkalosis, hyperglycemia, and hypertension, Cushing’s syndrome was suspected; therefore, 24-hour UFC level was checked that the results showed a severely elevated urinary cortisol (5000 μg/24 h and 8000 μg/24 h; normal level: 4-40 μg/24 h). Serum ACTH level was also inappropriately elevated (45 pg/mL; normal range: 10-60 pg/mL). High-dose dexamethasone failed to suppress plasma cortisol level and 24-hour urine cortisol level. A subsequent pituitary MRI showed an 8-mm pituitary mass, making a diagnosis of Cushing’s disease more probable. Meanwhile, the patient was suffering from severe muscle weakness that did not improve after the correction of hypokalemia. Then, a neurology consultation was requested. The neurology team evaluated laboratory data as well as EMG (Electromyography) and NCV (Nerve Conduction Velocity) of the patient, and based on their findings, “axonal neuropathy” was diagnosed for her weakness; so they ruled out the other neuromuscular diseases. A 5-day course of intravenous immunoglobulin (IVIG) was started for her neuropathy; however, the treatment did not improve her symptoms and the patient developed fungal sepsis and septic shock. Therefore, she was processed with broad-spectrum antibiotics and antifungal agents and recovered from the infection. Mitotane was started for the patient before definitive surgical treatment to suppress hormonal production due to her poor general condition. Despite the 8-mm size of the pituitary mass which is likely to be a source of ACTH, our patient was underweight and showed the atypical clinical presentation of Cushing’s disease, making us suspect an ectopic source for the ACTH. Therefore, a Gallium dotatate scan was performed to find any probable ectopic sources; however, the results were unremarkable. The patient underwent Trans-Sphenoidal Surgery (TSS) to resect the pituitary adenoma because it was not possible to perform IPSS in our center. Finally, the patient’s condition including electrolyte imbalance, muscle weakness, blood pressure, and hyperglycemia started to improve significantly. The pathologist confirmed the diagnosis of a corticotropic adenoma. Nevertheless, the patient suddenly died while having her meal a week after her surgery; most likely due to a thromboembolic event causing a cardiac accident. Discussion Our patient was significantly different from other patients with Cushing’s disease because of her atypical phenotype. She was unexpectedly thin and had psychosis, hyperpigmentation, proximal myopathy, axonal neuropathy and no clinical discriminatory features of Cushing’s syndrome such as central adiposity, dorsocervical or supraclavicular fat pad, plethora or striae. She had also a history of type 2 diabetes and hypertension 2 years before her admission. The patient was diagnosed with Cushing’s later. From what was presented, the patient did not know she had Cushing’s until after her delivery and despite the highly elevated UFC, and she completed a normal-term delivery. Given that she only presented with psychosis and proximal myopathy, her pregnancy was considered unusual. Her clinical features such as hyperpigmentation and severe muscle weakness are among less common presentations.5 11β-hydroxysteroid dehydrogenase type 1 (11-βHSD1) is an enzyme responsible for converting cortisone (inactive glucocorticoid) into cortisol (active). It is speculated that this enzyme has a role in obesity (Figure 2).6,7 Figure 2. The enzymatic actions of 11β-hydroxysteroid dehydrogenase on its substrate interconverting inactive and active glucocorticoid. OPEN IN VIEWER In a case reported by Tomlinson, a 20-year-old female was diagnosed with Cushing’s disease despite not having the classical features of the disease. It has been suggested that the mechanism is a partial defect in 11β-HSD1 activity and concomitant increase in cortisol clearance rate. Thus, the patient did not have a classic phenotype; the defect in the conversion of cortisone to cortisol rises cortisol clearance and protects the patient from the effects of cortisol excess. This observation may help explain individual susceptibility to the side effects of glucocorticoids.6 Further studies of Tomlinson et al showed that a deficit in the function of (and not a mutation related to) 11β-HSD2 might have been responsible for the absence of typical Cushing’s symptoms. 11-HSD2 keeps safe the mineralocorticoid receptor from excess cortisol. Mutation in the HSD11B2 gene explains an inherited form of hypertension, apparent mineralocorticoid excess syndrome, in which Cushing’s disease results in cortisol-mediated mineralocorticoid excess affecting the kidney and leads to both hypokalemia and hypertension.8 It is frequent in Cushing’s syndrome that the patients usually have no mineralocorticoid hypertension; however, it is still proposed that a defect in 11β-HSD1 can be responsible for the presence of mineralocorticoid hypertension in a subgroup of patients. In fact, 11β-HSD1 is expressed in several tissues like the liver, kidneys, placenta, fatty tissues and gonads,9 meaning that this enzyme may potentially affect the results of cortisol excess in Cushing’s syndrome/disease. Abnormality in the function of this enzyme could explain the absence of the symptoms like central obesity, easy bruising, and typical striae during Cushing’s disease. Several factors affect the action of glucocorticoids. In this regard, the impact of the different types and levels of impairment in glucocorticoid receptors have been highlighted in some studies, as it can lead to different levels of response to glucocorticoids10 as well as a variety in the symptoms observed in Cushing’s disease. The predominant reaction of the NADP(H)-dependent enzyme 11-Tukey’s honestly significant difference (HSD)1 happens through the catalysis of the conversion of inactive cortisol into receptor-active cortisol. The reverse reaction is mediated through the unidirectional NAD-dependent 11-HSD type 2 (Figure 2).11 In another case reported by Ved V. Gossein, a 41-year-old female was evaluated for hirsutism and irregular menstrual cycles. Her BMI was 22.6 kg/m2. The patient had no signs or symptoms of overnight recurrent Cushing’s syndrome, the 48-hour DST failed to suppress cortisol levels, and 24-hour urinary cortisol levels were persistently elevated on multiple occasions. Adrenocorticotropic hormone levels were unreasonably normal, suggesting ACTH-dependent hypercortisolism. Despite these disorders, she had 2 children. Magnetic resonance imaging (MRI) of the pituitary did not show any abnormalities. Moreover, abdominal MRI did not show adrenal mass or enlargement. Genetic testing to determine glucocorticoid resistance syndrome showed no mutation.12 Primary generalized glucocorticoid resistance is a rare genetic disorder characterized by generalized or partial insensitivity of target tissues to glucocorticoids.13-17 There is a compensatory increase in hypothalamic-pituitary activity due to decreased sensitivity of peripheral tissues to glucocorticoids systems.13-17 Excessive ACTH secretion leads to high secretion of cortisol and mineralocorticoids and/or androgens. However, the clinical features of Cushing’s syndrome do not develop after resistance to the effects of cortisol. Generalized glucocorticoid resistance is a rare condition characterized by high cortisol levels but no scarring of Cushing’s syndrome.18 An important aspect of our case was her pregnancy. Our patient had a history of hypertension and diabetes type 2, 2 years before her presentation to our center that could be because of an undiagnosed Cushing’s disease. The patient’s pregnancy terminated 2 months prior the admission and she had a normal vaginal delivery. So, we suspect that she become pregnant while involved with the disease. Aside from focusing on how this can happen in a patient with such high levels of glucocorticoids, more attention should be paid to occurring pregnancy in the background of Cushing’s disease. In fact, up to 250 patients were reported, of which less than 100 were actively treated.19-22 Cushing’s disease is associated with serious complications in up to 70% of the cases coinciding with pregnancy.21 The most frequent maternal complications reported in the literature are hypertension and impaired glucose tolerance, followed by preeclampsia, osteoporosis, severe psychiatric complications, and maternal death (in about 2% of the cases). Prematurity and intrauterine growth retardation account for the most prevalent fetal complications. Stillbirth, intrauterine deaths, intrauterine hemorrhage, and hypoadrenalism have also been reported.23 Early diagnosis is especially challenging during pregnancy because of many clinical and biochemical shared features of the 2 conditions.23,24 These features include an increase in ACTH production, corticosteroid-binding globulin (CBG) 1 level, level of cortisol (urinary, plasma and free), hyperglycemia, weight gain, and an increased chance for occurrence of bruising, hypertension (mistaken with preeclampsia), gestational diabetes mellitus, weight gain, and mood swings.3 There are some suggestions proposed in the studies that help in screening and differentiation of Cushing’s from the normal and abnormal effects of pregnancy and Cushing’s disease from Cushing’s syndrome in suspected pregnant patients. Contrary to Cushing’s syndrome, the nocturnal minimum level of cortisol is preserved in pregnancy.23,25 There is not yet a diagnostic cut-off determined on mentioned level; however, a few studies elucidate the evaluation of hypercortisolemia in a pregnant patient.26-28 Urinary free cortisol, a measure that reflects the amount of free cortisol in circulation, normally increases during pregnancy, and it can increase up to 8 times the normal level with Cushing’s disease during the second and the third trimesters,23,29 which is a useful tool to evaluate cortisol levels in a suspected pregnant woman. Because the suppression of both UFC and plasma cortisol is decreased in pregnancy,23,30 a low-dose DST is not very helpful for screening Cushing’s disease in pregnant patients. However, a high-dose DST with a <80% cortisol suppression might only indicate Cushing’s disease.3,31 Thus, it helps differentiating between ectopic ACTH syndrome and Cushing’s disease.32 The use of high-dose DST can distinguish between adrenal and pituitary sources of CS in pregnancy. Owing to the limited evidence available and the lack of data on normal pregnancies, the use of corticotropin-releasing hormone (CRH), desmopressin, and high-dose DST in pregnancy is not recommended yet.33 More timely diagnosis as well as timely intervention may have saved the life of our patient. To differentiate between ectopic ACTH syndrome and Cushing’s disease, adrenal imaging should be considered. For higher plasma levels, combined employment of CRH stimulation test and an 8-mg DST can be helpful.3 Bilateral inferior petrosal sinus sampling (B-IPSS) might be needed when the findings are not in accordance with other results, but it is recommended to perform B-IPSS only if the noninvasive studies are inconclusive and only if there is enough expertise, experience, and technique for its performance.3 Although axonal neuropathy has been reported as a rare syndrome associated with paraneoplastic ectopic Cushing’s syndrome and exogenous Cushing’s syndrome, its association with Cushing’s disease has not been reported.5,32 Our patient had severe muscle weakness that we initially attributed it to myopathy and hypokalemia associated with Cushing’s syndrome. In our study, the diagnosis of axonal neuropathy was made based on electrophysiological studies by a neurology consultant and then IVIG was administered; however, the patient’s weakness did not improve after this treatment. The co-occurrence of Guillain-Barré syndrome which may also be classified as axonal neuropathy has also been reported in a pregnant woman with ectopic Cushing’s syndrome.34,35 Whether this finding is coincidental or the result of complex immune reactions driven by Cushing’s disease, or the direct effect of steroids, these results cannot be deduced from current data.36 Some data suggest that the fluctuations and inferior petrosal sinus sampling may trigger the flare of autoimmune processes, specifically when the cortisol levels start to decline during the course of Cushing’s syndrome.35,8 Also, due to COVID-19 pandemic affecting vital organs like kidney, paying attention to COVID-19 is suggested.37-40 Conclusions We presented a thin young female with psychosis, proximal myopathy, and axonal neuropathy with Cushing’s disease who had a recent pregnancy that was terminated without any fetal or maternal complications despite the repeated elevated serum cortisol and 24-hour UFC; therefore, we suggest that she might have glucocorticoid resistance. Glucocorticoid resistance is a rare disease in which the majority, but not all, of patients have a genetic mutation in the hGR-NR3C1 gene. As we did not perform genetic testing for our patient, the data are lacking. Another clue to the absence of the classic Cushing’s disease phenotype in our case is the role of isoenzymes of 11-HSD1 and 11-HSD2. Other mechanisms, such as the defect somewhere in the glucocorticoid pathway of action such as a decreased number of receptors, a reduction in ligand affinity, or a postreceptor defect, play an important role in nonclassical clinical manifestations of Cushing’s syndrome. Acknowledgments The authors thank the patient for allowing us to publish this case report. The authors show their gratitude to the of the staff of the Rasool Akram Medical Complex Clinical Research Development Center (RCRDC) specially Mrs. Farahnaz Nikkhah for its technical and editorial assists. Ethics Approval Our institution does not require ethical approval for reporting individual cases or case series. Informed Consent Written informed consent was obtained from the patient and for her anonymized information to be published in this article. Declaration of Conflicting Interests The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article. Funding The author(s) received no financial support for the research, authorship, and/or publication of this article. References 1. Guilhaume B, Sanson ML, Billaud L, Bertagna X, Laudat MH, Luton JP. Cushing’s syndrome and pregnancy: aetiologies and prognosis in twenty-two patients. Eur J Med. 1992; 1(2):83-89. GO TO REFERENCE PubMed Google Scholar 2. Lin W, Huang HB, Wen JP, et al. Approach to Cushing’s syndrome in pregnancy: two cases of Cushing’s syndrome in pregnancy and a review of the literature. Ann Transl Med. 2019; 7(18):490. 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  23. Abstract Cushing’s disease is a rare neuroendocrine disorder with excessive endogenous cortisol, impaired cognition, and psychiatric symptoms. Evidence from resting-state fMRI revealed the abnormalities of static brain connectivity in patients with Cushing’s disease (CD patients). However, it is unknown whether the CD patients’ dynamic functional connectivity would be abnormal and whether the dynamic features are associated with deficits in cognition and psychopathological symptoms. Here, we evaluated 50 patients with Cushing’s disease and 57 healthy participants by using resting-state fMRI and dynamic functional connectivity (dFNC) approach. We focused on the dynamic features of default mode network (DMN), salience network (SN), and central executive network (CEN) because these are binding sites for the cognitive-affective process, as well as vital in understanding the pathophysiology of psychiatric disorders. The dFNC was further clustered into four states by k-mean clustering. CD patients showed more dwell time in State 1 but less time in State 4. Intriguingly, group differences in dwell time in these two states can explain the cognitive deficits of CD patients. Moreover, the inter-network connections between DMN and SN and the engagement time in State 4 negatively correlated with anxiety and depression but positively correlated with cognitive performance. Finally, the classifier trained by the dynamic features of these networks successfully classified CD patients from healthy participants. Together, our study revealed the dynamic features of CD patients’ brains and found their associations with impaired cognition and emotional symptoms, which may open new avenues for understanding the cognitive and affective deficits induced by Cushing’s disease. Introduction Cushing’s disease is characterized by excess endogenous cortisol secretion [1] and served as a unique and natural model for investigating the effects of elevated endogenous cortisol levels on brain functions and structure [2]. It is also a good model for unraveling the linkage between stress-related brain dysfunctions and psychiatric symptoms [3]. Long-term exposure to hypercortisolism negatively affects patients’ physical and mental health, such as depression, anxiety, and psychosis [1, 4], as well as shows deleterious effects on cognitive function including impaired executive function, working memory, and attention [5,6,7]. Research progress on Cushing’s disease, which depends on static resting-state fMRI, revealed that patients with Cushing’s disease showed increased functional connectivity between the default mode network (DMN) and left lateral occipital cortex [2], and hippocampus [8]. Cortisol increase would induce connectivity changes within the DMN and salience network (SN) [9], and the DMN’s activity correlated with the morning cortisol level of patients with Cushing’s disease [10]. Despite these advances leading to an improved understanding of Cushing’s disease, it remains enigmatic how the abnormal brain connectivity within large-scale networks and how the different brain networks interact would contribute to the deficits in impaired cognitive function, as well as psychopathological symptoms. Furthermore, recent years have witnessed an increasing number of studies providing solid evidence that the brain is a dynamic system rather than a static one on a micro-time scale [11, 12]. Dynamic functional connectivity (dFNC), which is implemented by the sliding window method [13], is an ideal approach to characterize the dynamic nature of brain [11], as well as detect and predict diseases [14, 15]. However, to our knowledge, no studies have ever investigated dynamic brain functional connectivity for patients with CD. We focus here on dynamic functional connectivity and emphasize the role of default mode network (DMN), salience network (SN), and central executive network (CEN). These large-scale neurocognitive networks are critical for cognitive and affective processing [16] and are highly related to stress and cortisol level. Deficits or abnormal connectivity within these three networks are associated with a wide range of stress-related psychiatric disorders [17], as well as the high level of cortisol production [18, 19]. For example, the network-connectivity changes between SN and DMN [20, 21], SN and CEN [22] corresponded to increased cortisol levels. Furthermore, our previous studies also identified that CD patients would show dysregulations of resting-state functional connectivity patterns with DMN [10, 23]. Since CD patients also suffer from cognitive impairment and neuropsychological symptoms, including depression and anxiety, which DMN, SN, and CEN mainly modulate, we hypothesized that these three networks are critical to understanding Cushing’s disease and its comorbidity. Here we aimed to investigate two research questions. First, whether there are group differences (CD patients vs. healthy controls) in the dynamic functional connectivity within DMN, SN, and CEN; second, whether the differences can explain the psychiatric symptoms and cognitive impairments in CD patients. We configure our design with a sliding-window approach [11, 13] to portray the features of dynamic functional connectivity (dFNC) within DMN, SN, and CEN among patients with Cushing’s disease (N = 50) and healthy controls (N = 57). We first compared the temporal properties between healthy and CD patients. Then we conducted correlation and mediation analysis to see whether and how the differences in dFNC would contribute to patients’ psychiatric and physiological symptoms and cognitive deficits. We finally implemented a classification machine learning algorithm based on dynamic FNC features within these three networks to see whether these dynamic features would identity CD patients successfully. Materials and methods Ethic approval The experimental protocol was in accordance with principles of the Declaration of Helsinki and approved by a local research ethics Committee of The First Medical Center of Chinese PLA General Hospital (Beijing, China). All participants provided written informed consent after the experimental procedure had been fully explained and were reminded of their right to withdraw at any time during the study. Participants The current study recruited 50 patients with Cushing’s disease (CD patients) and 57 healthy controls (HC) who were matched in age, gender, and education (Table 1). The CD patients were recruited from the Department of Neurosurgery, The First Medical Center of Chinese PLA General Hospital, between May 2017 and November 2019. The following criteria confirmed Cushing’s disease and its etiology: clinical features (e.g., moon face, supraclavicular fat pad, truncal obesity), elevated 24-h urinary free cortisol (24-h UFC, reference range 98.0–500.1 nmol/24 h), absence of normal cortisol circadian rhythm, elevated ACTH levels (reference range at 0800 h: <10.12 pmol/L), elevated cortisol secretion rates (reference range of cortisol level at 0800 h, 198.7–797.5 nmol/L), absence of normal suppression in midnight (1 mg) dexamethasone suppression test and low dose (2 mg) dexamethasone suppression test (but >50% suppression with a high dose (8 mg) of dexamethasone), and a central to peripheral ACTH ratio >2 for petrosal sinus sampling and pathology after surgery. Healthy controls (HC) were recruited from the local community through poster advertisements and were interviewed by experienced psychiatrists to ensure the absence of current or history of any mental disorder. Demographic information and clinical characteristics of all CD patients and healthy controls were shown in Table 1. Table 1 Demographic and clinical data from healthy controls and CD patients. Full size table Clinical data acquisition, neuropsychological and neuropsychiatric assessment Biometric measurements of the CD patients, including 24-h urinary free cortisol (UFC) levels, plasma Cortisol level (at 0000 h, 0800 h, 1600 h) and adrenocorticotropin (ACTH) level (at 0000 h, 0800 h, 1600 h) from a peripheral vein. Clinical severity of CD patients was obtained using the Cushing Quality of Life Scale (Cushing QOL) [24]. We also included the neuropsychological and neuropsychiatric assessments such as Self-Rating Depression Scale (SDS) [25], Self-Rating Anxiety Scale (SAS) [26], Montreal Cognitive Assessment-Beijing Version (MoCA-BJ) [27], and Chinese version of neuropsychiatric inventory (CNPI) [28]. Image acquisition Functional brain images were acquired using a 3-Tesla GE750 scanner at the First Medical Center of Chinese PLA General Hospital (Beijing, China). Blood oxygen level-dependent (BOLD) gradient echo planar images (EPIs) were obtained using an 8-channel head coil [64 × 64 × 36 matrix with 3.5 × 3.5 × 3.5 mm spatial resolution, repetition time (TR) = 2000 ms, echo time (TE) = 30 ms, flip angle = 90°, field of view (FOV) = 256 × 256 mm2]. A high-resolution T1-weighted structural image (256 × 256 × 144 matrix with a spatial resolution of 1 × 1 × 1 mm, repetition time (TR) = 6700 ms, echo time (TE) = 29 ms, flip angle = 7°) was subsequently acquired. During scanning, all participants were fitted with soft earplugs, and were requested to keep their eyes closed, to stay awake and not to think of anything. Data preprocessing The fMRI data was preprocessed using SPM12 (Wellcome Trust Centre for Neuroimaging, London). The first 10 volume of the functional images were discarded to avoid initial steady-state problems. Then functional images were spatially realigned to the first image for motion correction and corrected for slice acquisition temporal delay. Subsequently, functional images were co-registered to each participant’s segmented gray matter T1 image, then spatially normalized to the Montreal Neurological Institute (MNI) coordinate system, resampled to 3 × 3 × 3 mm voxels. Finally, all functional images were spatially smoothed with an isotropic 4 mm FWHM Gaussian kernel. Group ICA and post-processing Preprocessed data were decomposed into functional components that exhibited a unique time course profile using the group-level spatial independent component analysis, which was implemented in the GIFT toolbox (http://mialab.mrn.org/software/gift/) [29]. First, a subject-specific data reduction principal component analysis (PCA) was performed in which 120 principal components remained. Then at group level, we adopted a high model order ICA to reduce the resting state data into 100 group independent components [30] using the expectation-maximization (EM) algorithm [31] in GIFT. Further, the Infomax ICA algorithm in ICASSO [32] was repeated 20 times [33] to ensure the reliability and stability. Subject-specific spatial maps and time-courses were estimated using the back-reconstruction approach (GICA) [34]. We characterized 50 components as intrinsic connectivity networks (ICNs) by applying the following criteria:[13, 35] whether the peak activation coordinates of the functional components were primarily located in gray matter, and with minimal spatial overlap with white matter structures, vascular, ventricular, edge regions corresponding to artefacts, and susceptibility artifacts. We sorted these 50 meaningful independent components into the interested functional networks including: default mode network (DMN), central executive network (CEN) and salience network (SN) (Fig. 1) according to the spatial correlation values between independent components and the given template [36]. Additional post-processing was conducted to remove remaining noise. Time-courses of the seven components were detrended, despiked and low-pass filtered with a high-frequency cutoff of 0.15 Hz [13]. Moreover, we regressed out the six parameters of head movement. Fig. 1: Composite map of the three networks. And the pipeline of dynamic functional connectivity and clustering analyses. A The three brain networks, default mode network (DMN, including 7 components), central executive network (CEN, including 9 components) and salience network (SN, including 7 components) are derived from group spatial independent components analyses among all participants. B First, for each participant, the dynamic functional connectivity (FNC) matrices are estimated on each sliding window (200 windows) of a set of components within the three networks. Then we applied k-means clustering algorithm on the dynamic FNC matrices across all subjects to assess the reoccurring FNC’s states. Optimal number of states was determined by elbow method. We showed the averaged FNC pattern and the corresponding total number of windows in each state, percentage of each occurrence was presented in parentheses. The color bar represents the z value of FNC. Full size image Dynamic functional connectivity Sliding window approach is the most common way to investigate the nonstationary nature of functional connectivity (FC) of fMRI data. We conducted dynamic FC analysis using the DFC network toolbox in GIFT. In line with previous studies [13, 36], a window of 60 s width (30 TR), sliding in steps of one repetition time was applied to divide the time-courses of each independent components into 200 windows. As covariance estimation using time series of shorter length can be noisy, the regularized inverse covariance matrix (ICOV) was adopted [37]. Following graphic LASSO framework [38], we imposed an additional L1 norm of the precision matrix to enforce sparsity. Clustering analysis Based on previous studies, we applied a k-means clustering algorithm on windowed functional connectivity matrices [39] to assess the frequency and structure of reoccurring functional connectivity patterns (states) across all subjects. We used Manhattan distance function to estimate the similarity between different time windows of FC matrices, which had been demonstrated as an effective measure for high-dimensional data [40]. To obtain the optimal number of states, a cluster validity analysis (silhouette) was conducted on the exemplars of all the subjects. To avoid cost function convergence to the local optimal solution, all clustering analyses were iterated 5 times in GIFT, and the best result was used. Finally, we determined the optimal number of clusters as equal to four (k = 4). According to the clustering results, three temporal properties of dynamic FC states derived from each subject’s state vector were calculated: (i) mean dwell time, measured as the average number of consecutive windows belonging to one state; (ii) fraction of time, measured as the proportions of total windows in one state; (iii) number of transitions, defined as the number of state transitions during the entire scan. Mediation analyses Bootstrapping method was used to estimate the mediation effect. Bootstrapping is a nonparametric approach to effect-size estimation and hypothesis testing that is increasingly recommended for many types of analyses, including mediation [41, 42]. Bootstrapping generates an empirical approximation of the sampling distribution of a statistic by repeated random resampling from the available data and uses this distribution to calculate p-values and construct confidence intervals (5000 resamples were taken for these analyses). Moreover, this procedure supplies superior confidence intervals (CIs) that are bias-corrected and accelerated [43, 44]. Classification analyses using dynamic functional connectivity We conducted classification analyses based on dynamic FNC features [35] to classify each kind of patients. Specifically, we firstly formed a regression matrix, Rgroups × cluster centroids, then regressed out the windowed FNC matrices at each time window using the regression matrix for each participant. These analyses end up with eight β coefficients for each time window for each participant. Next, we computed the mean β coefficients for all time windows. Thus, we got eight mean β coefficients for each participant. These mean β coefficients served as the dynamic FNC features for the classification analysis. The classification analysis using supervised machine learning method, linear support vector machine algorithm (http://www.csie.ntu.edu.tw/~cjlin/libsvm/) with a standard 10-fold cross-validation. We randomly divided the data into 10 subgroups, used the trained classifier from the nine subgroups to predict the performance on the left one subgroup, and repeated the procedure for 100 times. We reported the averaged classification accuracy for each group across these 100 times. Results Neuropsychological and neuropsychiatric difference between healthy controls and CD patients Patients with Cushing’s disease reported higher depression, anxiety, and higher frequency and severity mental illness than healthy controls. Additionally, CD patients also behaved impaired cognitive ability than healthy controls (see Table 1) Functional connectivity within DMN, CEN and SN networks in the four states Spatial map of default mode network, central executive network and salience network identified using the group independent component analysis was shown in Fig. 1A. Independent components were grouped based on their anatomical and presumed functional properties: default mode network (ICs, 9, 12, 27, 28, 32, 44, 74), central executive network (ICs, 15, 21, 26, 48, 50, 63, 85, 89, 97), and salience network (ICs, 20, 43, 57, 59, 76, 82, 92). We adopted a k-means clustering algorithm on the dynamic functional connectivity (dFNC) from all subjects into four connectivity states. Figure 1B shows the cluster centroid and the percentage of occurrences of each state (arranged in the order of emergence). Different temporal properties between HC and CD patients We firstly compared the mean dwell time between healthy controls and CD patients in each state (Fig. 2A–D). Using independent T test, we found that the CD patients had higher mean dwell time than HC in State 1 (CD patients: 89.040 ± 59.216 vs. HC: 57.491 ± 40.671; t(105) = 3.244, p = 0.002), but less mean dwell time than HC in State 4 (CD patients: 31.300 ± 39.413 vs. HC: 66.438 ± 45.734; t(105) = −4.227, p < 0.001). We did not observe significant difference in State 2 (CD patients vs. HC: t(105) = 1.700, p = 0.092), nor in State3 (CD patients vs. HC: t(105) = −1.517, p = 0.132). For the switch time (i.e., the number of transitions), CD patients revealed less transition number than healthy controls did (CD patients: 6.600 ± 3.187 vs. HC: 7.824 ± 3.059; t(105) = −2.205, p = 0.045; Fig. 2E). Multiple comparisons were corrected by false-discovery rate (FDR), p < 0.05. All contrasts remained the same after FDR correction excepted the results of switch time became marginally significant, FDR corrected p = 0.075. Group difference on fraction of time in each state was similar with the mean dwell time (see Supplementary Table S1). Levene’s test is used to check that variances are equal for all samples. Fig. 2: Mean dwell time of dynamic FNC states and number of transitions between CD patients and healthy controls. A In State 1, CD patients engaged higher mean dwell time than healthy control did. B, C In State 2 and State 3, no difference was found between CD patients and healthy controls. D In State 4, CD patients showed significant less mean dwell time than healthy controls. E There was marginally significant difference (after FDR correction) on number of transitions between CD patients and healthy controls. Multiple comparisons were corrected by FDR, p < 0.05 (Error bars represent standard error. p < 0.01**, p < 0.001***, p < 0.08+, N.S not significant). HC Healthy controls, CD patients with Cushing’s disease. Full size image Correlation between dynamic FNC properties and clinical characteristics To examined whether the dynamic FNC properties were associated with clinical characteristics, we did Pearson correlation analyses. Since the group differences were found in State 1 and State 4, we only restricted our analyses on these two states. Notably, we found that the dwell time in State 1 positively correlated with the self-reported anxiety (SAS), and cortisol level at 8:00, 16:00, 00:00, ACTH at 8:00, 16:00, as well as elevated 24-h urinary free cortisol. That is, the longer time spent on State 1 which with more sparsely connected pattern, the worse the mental health and higher cortisol level. We also detected a robust negative correlation between dwell time of State 1 and global cognitive scales (MoCA), which indicated that more time spent in State 1, the worse cognitive ability would be. In the contrary, dwell time in State 4 showed significant negative correlation with the self-reported depression, anxiety, and cortisol level at 8:00, 16:00, 00:00. More dwell time in State 4 predicted better cognitive performance measured by MoCA (all results see Table 2). Multiple comparisons were conducted by FDR, p < 0.05. Table 2 Correlations between dynamic functional connectivity temporal properties in cognitive control network and clinical data. Full size table Dwell time in State 1 and State 4 within cognitive control network mediate group difference in cognitive performance Interestingly, we found the dwell time in State 1 and State 4 significantly mediated the difference between individuals with excessive high cortisol level (CD patients) and healthy controls on cognitive performance. That is, lower cognitive performance in CD patients was linked with more dwell time in State 1 (Fig. 3A), and less dwell time in State 4 (Fig. 3B) within the three networks. Fig. 3: Mediation effect of dwell time in State 1 and State 4 on group difference on cognitive performance. A Dwell time in State 1 and B dwell time in State 4 significant partially mediated the difference between CD patients and healthy controls on cognitive performance measured by MoCA. HC Healthy controls, CD patients with Cushing’s disease. Full size image Distinct network-based functional connectivity between CD patients and healthy controls and its associations with psychiatric symptoms and cognitive performance We have already known that the difference on dwell time in State 1 and State 4 can explain the group difference (i.e., CD patients vs. healthy controls) on cognitive performance. We further characterized the State 1 and State 4 by analyzing functional connectivity between the three networks, as well as the functional connectivity within each network. Results showed that in State 1, the CD patients had weaker connectivity within DMN (t(104)1 = −2.584, p = 0.011), and the connections between CEN and DMN (t(104) = −5.141, p < 0.001), CEN and SN (t(104) = −4.732, p < 0.001) were also weaker than healthy controls. And in State 4, CD patients showed weaker functional connections between DMN and SN (t (84)2 = −4.203, p < 0.001), as well as DMN and CEN (t(84) = −3.547, p = 0.001). Moreover, in State 4, functional connection between DMN and SN was negatively correlated with anxiety level measured by SAS (r(68) = −0.336, p = 0.005), and depression level measured by SDS (r(68) = −0.320, p = 0.008), but positively correlated with cognitive performance measured by MoCA (r(65) = 0.421, p < 0.001). Since CD patients showed decreased connection between DMN and SN, these results may suggest that the connection between DMN and SN was critical for understanding the psychiatric symptoms and cognitive deficits in CD patients. All significant results reported here were survived after FDR (p < 0.05) correction. We did not find significant associations between functional connectivity of neither inter-network and intra-network and psychiatric symptoms and cognitive deficits in State 1. No significant correlation results were found between the inter-network and intra-network connectivity and physiological indices (i.e., cortisol, ACTH, and UFC) in these two states, which may suggest that the dwell time in specific state would be more sensitive to physiological change. Classification results based on dynamic FNC features The support vector machine (SVM) based on dynamic FNC approach (Fig. 4A, details see Method) showed classification accuracy of 84.76% for CD patients, 88.98% for healthy controls (Fig. 4B). The classification scores were evaluated using a receiver operating characteristic (ROC) curve aiming to visualize the performance of the classifier. The classification results may further indicate that the dynamic functional connectivity pattern within these three networks would be the potential biomarker of individuals with excessive higher cortisol level. Fig. 4: The results of classification. A An overview of classification approach. We first extracted the averaged FNC pattern for each state for each group. Then we performed Pearson correlation between the FNC in each window and the FNC pattern in all states among all groups. These procedures ended up with 8 averaged features for each participant. B Receiver Operating Characteristic (ROC) curves for classification. SVM support vector machine, AUC area under the curve. Full size image Discussion In the current study, we adopted independent component analysis (ICA) and dynamic functional connectivity (FNC) approaches to reveal the difference in dynamic FNC within DMN, SN, and CEN networks between CD patients and healthy controls. Using clustering algorithm, we defined four reoccurring FNC states during resting-state scanning. Wherein State 1 and State 4 exhibited significant differences between healthy control and CD patients. Patients generally showed more dwell time in State 1 but less in State 4 than healthy controls. Specifically, in State 1, the CD patients showed weaker connections within DMN, as well as weaker intra-network connectivity between DMN and CEN, SN and CEN than healthy controls. In State 4, connections between DMN and SN, DMN and CEN showed weaker connection in CD patients than in healthy participants. Further correlation and mediation analyses showed that the dwell time in State 1 significantly negatively correlated with cognitive performance. While dwell time in State 4, as well as the connections between DMN and SN in State 4, were found to positively correlate with cognitive performance, and negatively associated with depression and anxiety symptoms. Both states were associated with physiological indices including cortisol, ACTH and 24-hour UFC. Importantly, results from mediation analysis indicated the difference between CD patients and healthy controls on dwell time in State 1 and State 4 can be used to explain their cognitive performance difference. Intriguingly, adopting support vector machine algorithm based on dynamic FNC within DMN, SN and CEN network generally showed ideal classification accuracy for CD patients and healthy controls. These findings begin to delineate the dynamic properties of the three brain networks, which are critical for cognitive and neuropsychiatric, and open new avenues for understanding and explaining the impaired cognitive performance and psychiatric symptoms induced by Cushing’s disease. We found two distinct functional connectivity states across two groups. State 1 can be characterized as having weak connections among the three networks, while State 4 showed relatively strong inter-network and intra-network connections. We observed that in patients with Cushing’s disease, State 1 occurred more often, while State 4 occurred less than in healthy controls. These results help to confirm CD patients’ weaker connections within DMN, SN, and CEN. Previous studies identified that white matter integrity was generally decreased throughout the whole brain rather than just on individual fasciculus [45,46,47]. One possible explanation is that the extensive decline in white matter structural integrity leads to the decreased connectivity of the three networks, which are critical for the cognitive-affective process. We found that in State 1, CD patients showed decreased local synchronization (i.e., within network connectivity) of DMN, and weak inter-network connections between CEN and DMN, CEN and SN. The DMN’s integrity appears crucial for cognitive performance. For example, patients with Alzheimer’s disease showed decreased connectivity within DMN [48]. Since dwell time of State 1 was negatively correlated with MoCA and mediated the group differences on MoCA. We may infer that cognitive deficit may be due to that CD patients engaged more time in State 1 with weak connections of DMN. Interestingly, the more dwell time in State 4, the less anxiety and depression symptoms individuals would have. Moreover, our further analyses found that connections between DMN and SN during State 4 would also negatively affect anxiety and depression. And the CD patients had weaker DMN-SN connections than healthy controls in this state. In line with previous studies, effective connectivity from DMN to SN was lower in major depression disorders compared to healthy controls when processing negative information [49]. And the inter-network connections between the SN and DMN were inversely associated with trait anxiety levels [50]. Therefore, the time engaged in State 4 and the weak inter-network connectivity between SN and DMN may contribute to psychopathological symptoms in CD patients. Dynamic functional connectivity provides time-varying rather than static features over time [11], and it is more effective to capture various aspects of brain connectivity. The dFNC approach has obvious advantages for classification purposes [35]. For example, previous research showed high classification accuracy for psychiatric diseases such as schizophrenia [51], and bipolar [35]. In our study, the SVM based on dynamic functional connectivity features within DMN, SN and CEN showed high classification accuracy for CD patients and healthy controls, which may indicate that the dynamic properties in these three networks would be potential biomarkers for individuals with excessive higher cortisol level. The long-term remitted CD (LTRCD)-patients still suffered from cognitive impairments and emotional symptoms such as anxiety and depression, even though their cortisol levels back to normal after the removal of the adenoma [2, 52, 53]. We revealed that the dynamic features in DMN, SN, and CEN correlate with depression and anxiety symptoms in CD patients and are strongly associated with cognitive performance. Our findings may contribute to developing further neuro-modulation targets to help CD patients improve cognitive ability and mental health. Several limitations of the present study should be mentioned. First, Cushing’s disease is rare, and it is more common in women [1, 3]. We only showed results based on a female sample (healthy controls were all female). Therefore, our conclusion may not be adaptive for the male population. Second, some research suggested that the dynamic functional connectivity analyses should be performed in resting state acquisitions of at least ten minutes [54]. The length of current resting-state scan was eight minutes, although many previous studies studied dynamic FNC based on resting-state data in eight minutes or even less [13, 20, 51], further studies should consider longer scanning to capture more dynamic spontaneous features. Thirdly, our results revealed that cortisol concentrations were significantly associated with dwell time in State 1 and 4 but were not correlated with inter-network or intra-network connections. Human cortisol secretion has apparent circadian rhythmicity [55], but our resting state acquisitions were not collected multiple times. Our conclusions may not be informative to understand the relationships between dynamic functional connections and dynamic cortisol levels. In conclusion, our study delineates the differences in dynamic properties between CD patients and healthy participants. It unravels its associations with cognitive deficits, impaired affective processes, and physiological indices in CD patients. We believe the temporal dynamics of functional connectivity within the three crucial cognitive and affective brain networks could be a promising imaging biomarker to monitor cognitive changes and psychiatric symptoms in Cushing’s disease. Data availability All datasets are available on figshare. https://figshare.com/projects/Dynamic_functional_connectivity_changes_associated_with_psychiatric_traits_and_cognitive_deficits_in_Cushing_s_disease/170343. Code availability All code used for all analyses and plots are publicly available on GitHub at https://github.com/psywalkeryanxy/paper_CD_ICA. References Lacroix A, Feelders RA, Stratakis CA, Nieman LK. Cushing’s syndrome. Lancet. 2015;386:913–27. Article CAS PubMed Google Scholar van der Werff SJA, Pannekoek JN, Andela CD, Meijer OC, van Buchem MA, Rombouts SARB, et al. Resting-state functional connectivity in patients with long-term remission of Cushing’s disease. Neuropsychopharmacology. 2015;40:1888–98. Article PubMed PubMed Central Google Scholar Swearingen B, Biller BMK, editors. Cushing’s disease. vol. 31, US: Springer; 2011. Piasecka M, Papakokkinou E, Valassi E, Santos A, Webb SM, Vries F, et al. 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Article CAS PubMed Google Scholar Download references Acknowledgements This work was supported by the National Natural Science Foundation of China (No. 82001798 and No. 81871087) and China Brain Project (2021ZD0200407). Author information Authors and Affiliations Department of Neurosurgery, Chinese PLA General Hospital, Haidian District, Beijing, PR China Zhebin Feng, Tao Zhou, Xinguang Yu & Yanyang Zhang Department of Respiratory Medicine, Anhui Provincial Children’s Hospital, Hefei, Anhui, PR China Haitao Zhang Neurosurgery Institute, Chinese PLA General Hospital, Beijing, PR China Xinguang Yu & Yanyang Zhang Department of Psychiatry, University of Minnesota Medical School, Minneapolis, MN, USA Xinyuan Yan Contributions YZ and XGY, TZ conceived the project and designed research, HZ performed research, XY and ZF, YZ analyzed data and interpreted results, ZF and XY wrote the paper. All authors approved the final version of the manuscript for submission. Corresponding authors Correspondence to Yanyang Zhang or Xinyuan Yan. Ethics declarations Competing interests The authors declare no competing interests. Additional information Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Supplementary information Supporting information From https://www.nature.com/articles/s41398-023-02615-y
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  25. Abstract Summary This case report describes a rare presentation of ectopic Cushing’s syndrome (CS) due to ectopic corticotropin-releasing hormone (CRH) production from a medullary thyroid carcinoma (MTC). The patient, a 69-year-old man, presented with symptoms of muscle weakness, facial plethora, and easy bruising. An inferior petrosal sinus sampling test (IPSS) demonstrated pituitary adrenocorticotrophic hormone (ACTH) secretion, but a whole-body somatostatin receptor scintigraphy (68Ga-DOTATOC PET/CT) revealed enhanced uptake in the right thyroid lobe which, in addition to a grossly elevated serum calcitonin level, was indicative of an MTC. A 18F-DOPA PET/CT scan supported the diagnosis, and histology confirmed the presence of MTC with perinodal growth and regional lymph node metastasis. On immunohistochemical analysis, the tumor cell stained positively for calcitonin and CRH but negatively for ACTH. Distinctly elevated plasma CRH levels were documented. The patient therefore underwent thyroidectomy and bilateral adrenalectomy. This case shows that CS caused by ectopic CRH secretion may masquerade as CS due to a false positive IPSS test. It also highlights the importance of considering rare causes of CS when diagnostic test results are ambiguous. Learning points Medullary thyroid carcinoma may secrete CRH and cause ectopic CS. Ectopic CRH secretion entails a rare pitfall of inferior petrosal sinus sampling yielding a false positive test. Plasma CRH measurements can be useful in selected cases. Keywords: Adult; Male; White; Denmark; Pituitary; Pituitary; Thyroid; Error in diagnosis/pitfalls and caveats; September; 2023 Background The common denominator of Cushing’s syndrome (CS) is autonomous hypersecretion of cortisol (1) and it is subdivided into ACTH-dependent and ACTH-independent causes. The majority of CS cases are ACTH-dependent (80–85%) with a pituitary corticotroph tumor as the most prevalent cause (Cushing’s disease), and less frequently an ectopic ACTH-producing tumor (2). The gold standard method to ascertain the source of ACTH secretion in CS patients is inferior petrosal sinus sampling (IPSS) with measurement of plasma ACTH levels in response to systemic corticotropin-releasing hormone (CRH) stimulation (3). The IPSS has a very high sensitivity and specificity of 88–100% and 67–100%, respectively (4), but pitfalls do exist, including the rare ectopic CRH-producing tumor, which may yield a false positive test result (3). Here, we describe a very rare case masquerading as CS including a positive IPSS test. Case presentation A 69-year-old man presented at a local hospital with a 6-month history of progressive fatigue, muscle weakness and wasting, easy bruising, facial plethora, and fluid retention. His serum potassium level was 2.6 mmol/L (reference range: 3.5–4.2 mmol/L) without a history of diuretics use. His previous medical history included spinal stenosis, benign prostatic hyperplasia, and hypertension. An electromyography showed no sign of polyneuropathy and an echocardiography showed no signs of heart failure with an ejection fraction of 55%. MRI of the spine revealed multiple compression fractures, and the patient underwent spinal fusion and decompression surgery; during this admission he was diagnosed with type 2 diabetes (HbA1c: 55 mmol/mol). After spine surgery, the patient developed a pulmonary embolism and initiated treatment with rivaroxaban. Establishing the diagnosis of ACTH-dependent CS Six months after his spine surgery, the patient was referred to the regional department of endocrinology for osteoporosis management. Blood tests revealed a low serum testosterone level with non-elevated luteinizing hormone (LH) and follicle-stimulating hormone (FSH) levels (Table 1). An overnight 1 mg dexamethasone suppression test was positive with a morning cortisol level of 254 nmol/L and three consecutive 24-h urinary cortisol levels were markedly elevated with mean level of ≈600 nmol/24 h (reference range: 12–150 nmol/24 h). A single plasma ACTH was 37 ng/L (Table 1). Table 1 Baseline endocrine assessment. Parameters Patient’s values Reference range ACTH, ng/L 37 7–64 UFC, nmol/day 588 12–150 Urinary cortisol, nmol/L 600 171–536 OD, nmol/L 254 <50 Free testosterone, nmol/L 0.061 0.17–0.59 HbA1c, mmol/mol 55 <48 FSH, IU/L 7.4 1.2–15.8 LH, IU/L 2.2 1.7–8.6 ACTH, adrenocorticotropin; FSH, follicle-stimulating hormone; IU, international units; LH, luteinizing hormone; OD, plasma cortisol levels after a 1 mg overnight dexamethasone suppression test; UFC, urine free cortisol hormone. Differential diagnostic tests The patient was referred to a tertiary center for further examinations. Ketoconazole treatment was started to alleviate the consequences of hypercortisolism. A pituitary MRI revealed an intrasellar microtumor with a maximal diameter of 6 mm and an IPSS was ordered. A whole-body somatostatin receptor scintigraphy (68Ga-DOTATOC PET/CT) was also performed to evaluate the presence of a potential neuroendocrine tumor. This revealed multiple areas of enhanced uptake including the right thyroid lobe and cervical lymph nodes in the neck (with CT correlates), as well as in the duodenum (with no CT correlate). Concomitantly, a grossly elevated serum calcitonin level of 528 pmol/L (reference range <2.79 pmol/L) was measured. Subsequently, the IPSS revealed pituitary ACTH secretion with a central-to-peripheral ACTH ratio >3 (Table 2). The right petrosal sinus was not successfully catheterized; thus, lateralization could not be determined. To corroborate the diagnosis MTC, a 18F-DOPA PET/CT scan (FDOPA) was performed (5), which showed pathologically enhanced uptake in the right thyroid lobe and regional lymph nodes (Fig. 1). An ultrasound-guided core needle biopsy from the thyroid nodule was inconclusive; however, the patient underwent total thyroidectomy and regional lymph node resection, from which histology confirmed the diagnosis of disseminated MTC. Standard replacement with levothyroxine, calcium, and vitamin D was initiated. A blood sample was collected, and genomic DNA was extracted. The DNA analysis for RET germline mutation was negative. View Full Size Figure 1 18F-DOPA PET/CT scan with pathologically enhanced uptake in the right thyroid lobe (large blue arrow on the left side) and regional lymph nodes (small blue arrows). Citation: Endocrinology, Diabetes & Metabolism Case Reports 2023, 3; 10.1530/EDM-23-0057 Download Figure Download figure as PowerPoint slide Table 2 Results from the inferior petrosal sinus sampling.* Time (min) Left IPSS Peripheral L/P -5 42 36 1.2 -1 116 33 3.5 2 120 32 3.8 5 209 28 7.5 7 180 43 4.2 10 529 34 15.6 15 431 37 11.6 *Data represents ACTH levels in ng/L. IPSS Inferior petrosal sampling ACTH Adrenocorticotropin hormone CRH Corticotropin-releasing hormone, L/P Ratio of left (L) inferior petrosal sinus to peripheral venous ACTH concentrations. Pathology Total thyroidectomy and bilateral cervical lymph node dissection (level six and seven) were performed. Macroscopic evaluation of the right thyroid lobe revealed a 24 mm, irregular solid yellow tumor. Microscopically the tumor showed an infiltrating architecture with pseudofollicles and confluent solid areas. Calcification was prominent, but no amyloid deposition was seen. The tumor cells were pleomorphic with irregular nuclei and heterogenic chromatin structure. No mitotic activity or necrosis was observed. On immunohistochemical analysis, the tumor cells expressed thyroid transcription factor 1 and stained strongly for carcinoembryonic antigen and calcitonin; tumor cells were focally positive for cytokeratin 19. The tumor was completely negative for ACTH, thyroid peroxidase, and the Hector Battifora mesothelial-1 antigen. Further analysis revealed positive immunostaining for CRH (Fig. 2). The Ki-67 index was very low (0–1%), indicating a low cellular proliferation. Molecular testing for somatic RET mutation was not performed. View Full Size Figure 2 Histopathological findings and immunohistochemical studies of MTC. (A) Microscopic features of medullary thyroid carcinoma. (B) Polygonal tumor cells (hematoxylin and eosin, ×40). (C) Tumor cells stain for calcitonin (×20). (D) Immunohistochemical stain (×400) for CRH showing cells being positive (brown). (E) Pituitary tissue from healthy control staining positive for ACTH in comparison to (F) ACTH-negative cells MTC tissue from the patient (×20). Citation: Endocrinology, Diabetes & Metabolism Case Reports 2023, 3; 10.1530/EDM-23-0057 Download Figure Download figure as PowerPoint slide No malignancy was found in the left thyroid lobe and there was no evidence of C-cell hyperplasia. Regional lymph node metastasis was found in 13 out of 15 nodes with extranodal extension. Outcome and follow-up Follow-up Serum calcitonin levels declined after neck surgery but remained grossly elevated (118 pmol/L 3 weeks post surgery) and cortisol levels remained high. Ketoconazole treatment was poorly tolerated and not sufficiently effective. Plasma levels of CRH were measured by a competitive-ELISA kit (EKX-KIZI6R-96 Nordic BioSite), according to the instructions provided by the manufacturer. The intra- and interassay %CV (coefficient of variability) were below 8% and 10%, respectively, and the assay sensitivity was 1.4 pg/mL. The plasma CRH was distinctly elevated compared to in-house healthy controls both before and after thyroid surgery (Fig. 3). View Full Size Figure 3 Plasma CRH levels before and after total thyroidectomy compared to three healthy controls. Citation: Endocrinology, Diabetes & Metabolism Case Reports 2023, 3; 10.1530/EDM-23-0057 Download Figure Download figure as PowerPoint slide The patient subsequently underwent uneventful bilateral laparoscopic adrenalectomy followed by standard replacement therapy with hydrocortisone and fludrocortisone. The symptoms and signs of his CS gradually subsided. Pathology revealed bilateral cortical hyperplasia as expected. The patient continues follow-up at the Department of Oncology and the Department of Endocrinology and Internal Medicine. At 13-month follow-up, 68Ga-DOTATOC shows residual disease with pathologically enhanced uptake in two lymph nodes, whereas the previously described focal DOTATOC uptake in the duodenum was less pronounced (still no CT correlate). Serum calcitonin was 93 pmol/L at the 13-month follow-up. Discussion Diagnostic challenges remain in the distinction between pituitary and ectopic ACTH-dependent CS, and several diagnostic tools are used in combination, none of which is infallible, including IPSS (6). Our case and others illustrate that ectopic CRH secretion may yield a false positive IPSS test result (3). Measurement of circulating CRH levels is relevant if an ectopic CRH producing tumor is suspected, but the assay is not routinely available in clinical practice (Lynnette K Nieman M. Measurement of ACTH, CRH, and other hypothalamic and pituitary peptides https://www.uptodate.com/contents/measurement-of-acth-crh-and-other-hypothalamic-and-pituitary-peptides: UpToDate; 2019). In our case, the presence of elevated plasma CRH levels after thyroidectomy strengthened the indication for bilateral adrenalectomy. The most common neoplasm causing ectopic CS is small-cell lung cancer, whereas MTC accounts for 2–8% of ectopic cases (7). The development of CS in relation to MTC is generally associated with advanced disease and poor prognosis of an otherwise relatively indolent cancer (8), and the clinical progression of CS is usually rapid, why an early recognition and rapid control of hypercortisolemia and MTC is necessary to decrease morbidity and mortality (7, 9). Our case does have residual disease; however, he remains progression-free with stable and relatively low calcitonin levels within 1-year follow-up. Only a very limited number of cases of ectopic tumors with either combined ACTH and CRH secretion or isolated CRH secretion have been reported, with ectopic CRH secretion accounting for less than 1% of CS (9). An ACTH- or CRH-producing tumor can be difficult to localize and may include gastric ACTH/CRH-secreting neuroendocrine tumors (9). In our case, the 68Ga-DOTATOC identified a possible duodenal site, in addition to the MTC, but an upper gastrointestinal endoscopy revealed no pathological findings and there was no CT correlate. Thus, we concluded that the most likely and sole source of ectopic CRH was the MTC and its metastases. To our knowledge, no official guidelines for managing ectopic ACTH-dependent CS have been established. In a recent publication by Alba et al. (10), the authors demonstrated a clinical algorithm (The Mount Sinai Clinical Pathway, MSCP) and recommendation for the management of CS due to ectopic ACTH secretion. Essentially, our approach in this particular case followed these recommendations, including source location by CT and 68Ga-DOTATATE PET/CT imaging, acute management with ketoconazole, and finally, bilateral adrenalectomy as curative MTC surgery was not possible. In retrospect, performance of the IPSS could be questioned in view of the MTC diagnosis. In real time, however, a pituitary MRI performed early in the diagnostic process revealed a microadenoma, which prompted the IPSS. In parallel, a somatostatin receptor scintigraphy (68Ga-DOTATOC PET/CT) was also done, which raised the suspicion of an MTC. Conclusion We report a very rare case of an ectopic CS caused by a CRH-secreting MTC. Although IPSS has stood the test of time in the differential diagnosis of ACTH-dependent CS, this case illustrates a rare pitfall. Declaration of interest The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported. Funding This research did not receive any specific grant from any funding agency in the public, commercial, or not-for-profit sector. Patient consent Written informed consent for publication of their clinical details was obtained from the patient. Author contribution statement JOJ and MJO are the physicians responsible for the patient. LR performed the thyroidectomy and bilateral adrenalectomy. SHM and SLA assessed and reassessed the histopathology and the immunohistochemical analysis. MB measured plasma CRH. VM, JOJ, and MJO drafted the manuscript. All authors contributed to critical revision of the manuscript. References 1↑ Raff H, & Carroll T. Cushing's syndrome: from physiological principles to diagnosis and clinical care. Journal of Physiology 2015 593 493–506. (https://doi.org/10.1113/jphysiol.2014.282871) PubMed Search Google Scholar Export Citation 2↑ Hatipoglu BA. Cushing's syndrome. Journal of Surgical Oncology 2012 106 565–571. 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