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Rie Hagiwara Department of Endocrinology and Metabolism, Hirosaki University Graduate School of Medicine, Hirosaki 036-8562, Japan Kazunori Kageyama Department of Endocrinology and Metabolism, Hirosaki University Graduate School of Medicine, Hirosaki 036-8562, Japan Yasumasa Iwasaki Suzuka University of Medical Science, Suzuka 510-0293, Japan Kanako Niioka Department of Endocrinology and Metabolism, Hirosaki University Graduate School of Medicine, Hirosaki 036-8562, Japan Makoto Daimon Department of Endocrinology and Metabolism, Hirosaki University Graduate School of Medicine, Hirosaki 036-8562, Japan Keywords: Cushing’s disease, Adrenocorticotropic hormone, Proopiomelanocortin, Corticotroph tumor, Histone deacetylase https://doi.org/10.1507/endocrj.EJ21-0778 Abstract Cushing’s disease is an endocrine disorder characterized by hypercortisolism, mainly caused by autonomous production of ACTH from pituitary adenomas. Autonomous ACTH secretion results in excess cortisol production from the adrenal glands, and corticotroph adenoma cells disrupt the normal cortisol feedback mechanism. Pan-histone deacetylase (HDAC) inhibitors inhibit cell proliferation and ACTH production in AtT-20 corticotroph tumor cells. A selective HDAC6 inhibitor has been known to exert antitumor effects and reduce adverse effects related to the inhibition of other HDACs. The current study demonstrated that the potent and selective HDAC6 inhibitor tubastatin A has inhibitory effects on proopiomelanocortin (Pomc) and pituitary tumor-transforming gene 1 (Pttg1) mRNA expression, involved in cell proliferation. The phosphorylated Akt/Akt protein levels were increased after treatment with tubastatin A. Therefore, the proliferation of corticotroph cells may be regulated through the Akt-Pttg1 pathway. Dexamethasone treatment also decreased the Pomc mRNA level. Combined tubastatin A and dexamethasone treatment showed additive effects on the Pomc mRNA level. Thus, tubastatin A may have applications in the treatment of Cushing’s disease. Access the PDF at https://www.jstage.jst.go.jp/article/endocrj/advpub/0/advpub_EJ21-0778/_pdf/-char/en

https://doi.org/10.1016/j.ajoc.2022.101455 Abstract Purpose To report the clinical course of a patient with central serous chorioretinopathy (CSCR) secondary to subclinical hypercortisolism before and after adrenalectomy. Observations A 50-year-old female patient with multifocal, chronic CSCR was found to have an adrenal incidentaloma and was diagnosed with subclinical hypercortisolism. Patient elected to undergo minimally-invasive adrenalectomy and presented at 3 months after surgery without subretinal fluid. Conclusions and Importance Subclinical Cushing's Syndrome (SCS) may present an underrecognized risk factor for developing chronic CSCR. Further investigation is needed to determine the threshold of visual comorbidity that may influence surgical management. Keywords Central serous chorioretinopathy Subclinical Cushing's syndrome Hypercortisolism Adrenalectomy Retina Surgical intervention 1. Introduction Central serous chorioretinopathy (CSCR) is characterized by accumulation of fluid in the subretinal or sub-RPE space, often with consequential visual impairment. Chronic CSCR has been reported as a manifestation of hypercortisolism due to Cushing's syndrome and subclinical hypercortisolism.1,2 However, the latter is often underrecognized owing to its inherently subtle nature and the optimal threshold for intervention based on associated comorbidities remains unclear. Herein we report the clinical course of a patient with CSCR secondary to subclinical hypercortisolism before and after adrenalectomy. 2. Case report A 50-year-old female presented with blurred, discolored spots in the right eye for several months. Her past medical history included mild hypertension treated with amlodipine. Past ocular and family history were noncontributory. On exam, Snellen visual acuity was 20/50 OD, 20/25 OS. Her pupils, intraocular pressure, and anterior segment exam were within normal limits. Dilated fundus exam revealed bilateral, multifocal areas of subretinal fluid and mottled pigmentary changes (Fig. 1A). Optical coherence tomography confirmed areas of subretinal fluid and other areas of outer retinal atrophy (Fig. 1B). Fundus autofluorescence revealed areas of hyperautofluorescence that highlighted the fundoscopic findings (Fig. 1C). Fluorescein angiography showed multifocal areas of expansile dot leakage (Fig. 1D). Altogether these findings were consistent with multifocal, chronic CSCR. Download : Download high-res image (1MB) Download : Download full-size image Fig. 1. Multimodal imaging of bilateral multifocal central serous chorioretinopathy. Fundus photographs reveal multifocal subretinal fluid and pigmentary changes (Fig. 1A). Optical coherence tomography demonstrates subretinal fluid and outer retinal atrophy (Fig. 1B). Areas of hyperautofluorescence highlight the fundoscopic findings of subretinal fluid (Fig. 1C). Fluorescein angiography showing multiple areas of expansile dot leakage (Fig. 1D). On further clinical follow-up, an adrenal incidentaloma (AI) was detected when the patient underwent imaging for back pain. Subsequently she saw an endocrinologist; she had a normal serum cortisol, but low ACTH and an abnormal dexamethasone suppression test. This led to a diagnosis of subclinical hypercortisolism and provided a reason for her hypertension and chronic CSCR. Since the blur and relative scotomata interfered with her daily activities, she elected to try eplerenone, which yielded a moderate but suboptimal therapeutic response at 50 mg daily. While contemplating photodynamic therapy, in discussion with her endocrinologist, the patient opted to undergo minimally-invasive adrenalectomy. At last follow-up 3 months after surgery and 6 years after her initial presentation, she has been off eplerenone and without subretinal fluid (Fig. 2). Download : Download high-res image (1MB) Download : Download full-size image Fig. 2. Optical coherence tomography imaging at presentation and at last follow-up 3 months after adrenalectomy. There is a significant improvement in subretinal fluid in both eyes, though outer retinal irregularity remains. 3. Discussion CSCR has previously been associated with many risk factors including exposure to excess steroid. A recent study analyzing a nationally representative dataset of 35,000 patients found that patients with CSCR had a higher odds of Cushing's syndrome (OR 2.19, 95% CI 1.33 to 3.59, p = 0.002) than exposure to exogenous steroids (OR 1.14, 95% CI 1.09 to 1.19, p < 0.001)1 Our case highlights the importance of thorough medication reconciliation and careful consideration of comorbid conditions in patients with chronic CSCR. In recent years, subtle endogenous hypercortisolism, termed subclinical Cushing's syndrome (SCS), has been of particular interest in the endocrinology literature because it can be a challenging diagnosis and the most appropriate management remains controversial.3 In general, SCS is comprised of: 1) the presence of an adrenal incidentaloma or mass, 2) biochemical confirmation of cortisol excess, and 3) no classic phenotypic manifestations of Cushing's syndrome.4 Since adrenal incidentaloma has an estimated prevalence of 1–8% of the population,5 it is quite possible that SCS is an underrecognized risk factor for CSCR. SCS may potentiate metabolic syndrome and osteoporosis; studies have found that surgical resection of adrenal incidentalomas improve weight, blood pressure, and glucose control. Consequently, some authors recommend those with SCS-associated comorbidities be considered for resection.6 An important consideration in these patients is how visual comorbidity factors into intervention. In our patient's case, the recurrent CSCR, hypertension, and increased risk of metabolic syndrome were sufficient reasons to elect to have surgery. 4. Conclusion In summary, SCS is a condition of subtle cortisol dysregulation that may represent an underrecognized risk factor for chronic CSCR. Further investigation is needed to determine the threshold of visual comorbidity that may influence surgical management. Patient consent Consent to publish the case report was not obtained. This report does not contain any personal information that could lead to the identification of the patient. Acknowledgments and Disclosures Grant support was from the J. Arch McNamara Retina Research Fund. The following authors have no financial disclosures: RRS, AS, AC All authors attest that they meet the current ICMJE criteria for Authorship. No other contributions to acknowledge. References 1 M. Zhou, S.J. Bakri, S. Pershing Risk factors for incident central serous retinopathy: case-control analysis of a US national managed care population Br J Ophthalmol, 103 (12) (2019), pp. 1784-1788, 10.1136/bjophthalmol-2018-313050 View PDF View Record in ScopusGoogle Scholar 2 S.N. Appa Subclinical hypercortisolism in central serous chorioretinopathy Retin Cases Brief Rep, 8 (4) (2014), pp. 310-313, 10.1097/ICB.0000000000000036 View PDF View Record in ScopusGoogle Scholar 3 I. Chiodini, A. Albani, A.G. Ambrogio, et al. Six controversial issues on subclinical Cushing's syndrome Endocrine, 56 (2) (2017), pp. 262-266, 10.1007/s12020-016-1017-3 View PDF View Record in ScopusGoogle Scholar 4 M.A. Zeiger, G.B. Thompson, Q.-Y. Duh, et al. American association of clinical endocrinologists and American association of endocrine surgeons medical guidelines for the management of adrenal incidentalomas: executive summary of recommendations Endocr Pract Off J Am Coll Endocrinol Am Assoc Clin Endocrinol, 15 (5) (2009), pp. 450-453, 10.4158/EP.15.5.450 ArticleDownload PDFGoogle Scholar 5 M. Terzolo, A. Stigliano, I. Chiodini, et al. AME position statement on adrenal incidentaloma Eur J Endocrinol, 164 (6) (2011), pp. 851-870, 10.1530/EJE-10-1147 View PDF View Record in ScopusGoogle Scholar 6 L.B. Hsieh, E. Mackinney, T.S. Wang When to intervene for subclinical cushing's syndrome Surg Clin North Am, 99 (4) (2019), pp. 747-758, 10.1016/j.suc.2019.04.011 ArticleDownload PDFView Record in ScopusGoogle Scholar © 2022 The Authors. Published by Elsevier Inc. From https://www.sciencedirect.com/science/article/pii/S2451993622002018?via%3Dihub#!

Sorry guys - I just report the information as I get it I'm in the same "remission boat". This study was done in Sweden so maybe if we stay away from there it will all be good? BTW - welcome, Jazzy - sorry this was your first post.

An analysis of nationwide data from Sweden provides an overview of the increased risk of death associated with Cushing's disease was present even after biochemical remission. New data from an analysis of patient data over nearly 30 years suggests the increased risk of mortality associated with Cushing’s disease persists even after treatment. A 4:1 matched analysis comparing data from 371 patients with Cushing’s disease with 1484 matched controls, indicated risk of mortality was 5-fold greater among those not in remission compared to matched controls, but even those in remission at the last follow-up were at a 50% greater risk of mortality compared to controls. “To our knowledge, this is the first study that investigated mortality in an unselected cohort of patients treated for Cushing’s disease and followed up in comparison to mortality in matched controls. The mortality rate was more than doubled in patients with Cushing’s disease, and not being in remission was a strong predictor of premature death,” wrote investigators. With a lack of consensus surrounding the impact of biochemical remission on life expectancy in patients with Cushing’s disease, a team of investigators from multiple institutions in Sweden designed their study with the intent of assessing this association with mortality in a time-to-event analysis of an unselected nationwide Cushing’s disease cohort. Using the Swedish Pituitary Registry, investigators identified 371 patients with Cushing’s disease for inclusion in their analysis. The Swedish Pituitary Register is a nationwide registry that collected data on the majority of Swedish patients with Cushing’s disease. For the current study, investigators included all patients with Cushing’s disease from the register diagnosed between May 1991-September 2018 and followed these patients until the date of death, date of emigration, or December 26, 2018. From the register, investigators obtained data related to date of diagnosis, age, sex, treatment, and biochemical remission status evaluations. The median age at diagnosis was 44 (IQR, 32-56) years and the median follow-up was 10.6 (IQR, 5.7-18) years. The remissions rates for the study cohort were 80%, 92%, 96%, 91%, and 97% at the 1-, 5-, 10-, 15- and 20-year follow-ups, respectively. These patients were matched in a 4:1 based on age, sex, and residential area at the diagnosis data, yielding a cohort of 1484 matched controls. Upon analysis, the overall risk of mortality was greater among those with Cushing’s disease compared to the matched controls (HR, 2.1 [95% CI, 1.5-2.8]). Investigators pointed out increased risk was observed among patients in remission at the last follow-up (n=303; HR, 1.5 [95% CI, 1.02-2.2]), those in remission after a single pituitary surgery (n=177; HR, 1.7 [95% CI, 1.03-2.8]), and those not in remission (n=31; HR, 5.6 [95% CI, 2.7-11.6]). Additionally, results indicated cardiovascular disease and infections were the most overrepresented cases of death, accounting for 32 and 12 of the 66 total instances of mortality. “The findings of the present study confirm and complement previous findings of increased overall mortality in Cushing’s disease patients, having a more than doubled HR for death compared to matched controls. Most importantly, an increased HR persisted among patients who had been successfully treated and reached a Cushing’s disease biochemical cure,” investigators added. This study, “Increased mortality persists after treatment of Cushing’s disease: A matched nationwide cohort study,” was published in the Journal of the Endocrine Society. From https://www.endocrinologynetwork.com/view/medicaid-expansion-under-aca-may-have-reduced-rate-of-major-diabetes-related-amputations

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

Although Dr. Friedman is at the forefront of Cushing’s Disease, he was not invited to be part of the Pituitary Society Consensus Guidelines on Cushing’s Disease published in Lancet Diabetes and Endocrinology in 2021, many of his ideas on Cushing’s Disease that he has been advocating for years were included in the recent guidelines. In this informative webinar, Dr. Friedman will discuss The use of imaging for the diagnosis of Cushing’s Disease The need for multiple testing to diagnose episodic Cushing’s Disease The importance of UFC and salivary cortisol testing The use of medication trial prior to surgery The use of ketoconazole for the medication trial and longer-term treatment Dr. Friedman will also discuss new Cushing’s medications. Sunday • April 3 • 6 PM PST Via Zoom Click here to join the meeting orhttps://us02web.zoom.us/j/4209687343?pwd=amw4UzJLRDhBRXk1cS9ITU02V1pEQT09OR+16699006833,,4209687343#,,,,*111116#Slides will be available on the day of the talk here. You can also click to read the consensus guidelines There will be plenty of time for questions using the chat button. For more information, email us at mail@goodhormonehealth.com

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

Abstract Corticotroph pituitary adenomas commonly cause Cushing’s disease (CD), but some of them are clinically silent. The reason why they do not cause endocrinological symptoms remains unclear. We used data from small RNA sequencing in adenomas causing CD (n = 28) and silent ones (n = 20) to explore the role of miRNA in hormone secretion and clinical status of the tumors. By comparing miRNA profiles, we identified 19 miRNAs differentially expressed in clinically functioning and silent corticotroph adenomas. The analysis of their putative target genes indicates a role of miRNAs in regulation of the corticosteroid receptors expression. Adenomas causing CD have higher expression of hsa-miR-124-3p and hsa-miR-135-5p and lower expression of their target genes NR3C1 and NR3C2. The role of hsa-miR-124-3p in the regulation of NR3C1 was further validated in vitro using AtT-20/D16v-F2 cells. The cells transfected with miR-124-3p mimics showed lower levels of glucocorticoid receptor expression than control cells while the interaction between miR-124-3p and NR3C1 3′ UTR was confirmed using luciferase reporter assay. The results indicate a relatively small difference in miRNA expression between clinically functioning and silent corticotroph pituitary adenomas. High expression of hsa-miR-124-3p in adenomas causing CD plays a role in the regulation of glucocorticoid receptor level and probably in reducing the effect of negative feedback mediated by corticosteroids. Keywords: neuroendocrine pituitary tumors; Cushing’s disease; silent corticotroph adenoma; miRNA; hsa-miR-124-3p; NR3C1; glucocorticoid receptor 1. Introduction Pituitary adenomas (also referred to as pituitary neuroendocrine tumors, PitNETs) represent about 10–20% of intracranial neoplasms in adults. They may originate from different kinds of secretory pituitary cells including corticotroph ACTH-secreting cells. Corticotroph adenomas commonly cause ACTH-dependent Cushing’s disease, but a significant proportion of these tumors are endocrinologically non-functioning and classified as subclinical/silent corticotroph adenomas (SCAs) [1]. CD-causing ACTH tumors are commonly small microadenomas with approximately 50% being smaller than 5 mm, which is challenging for MRI diagnostics [2]. In contrary, SCAs are commonly diagnosed due to neurological symptoms related to tumor mass at the stage of large macroadenomas. Frequently they show invasive growth and increased proliferation index [1]. According to current recommendations, SCAs are now referred to as “high-risk” pituitary adenomas which refers to their fast and invasive growth, high risk of recurrence and resistance to medical therapy [3,4]. They are recognized to be more aggressive than other clinically nonfunctioning pituitary tumors such as those of gonadotroph origin or null-cell adenomas [5]. The mechanism underlying the difference in secretory activity of CD-causing and subclinical tumors is unclear and only a few studies focused on this issue were published. The results indicated a role of the expression levels of particular genes/proteins involved in the regulation of POMC expression and pro-hormone conversion into ACTH as well as genes involved in pituitary differentiation [6,7,8,9,10,11,12,13]. However, it also appears that both active and silent corticotroph adenomas share a similar overall gene expression profile [14,15]. The aim of this study was to compare the profiles of microRNA (miRNA) expression in clinically functioning and silent corticotroph adenomas and to identify miRNAs that play a role in different ACTH secretory activity. 2. Results 2.1. Patients Characteristics The study included 28 patients with CD and 20 patients suffering from SCA. All patients with CD had clear clinical signs and symptoms of hypercortisolism verified according to biochemical criteria including elevated midnight cortisol levels and 24 h urinary free cortisol (UFC). Patients with SCA had no clinical or biochemical signs of hypercortisolism and showed normal levels of midnight cortisol and 24 h UFC. Patients with CD had significantly higher morning serum cortisol levels than patients with SCAs (p = 0.0002) while no significant difference was observed in the morning serum ACTH levels. No difference in cortisol/ACTH ratio was observed between CD and SCA patients. All the adenoma samples were ACTH-positive upon immunohistochemical staining against pituitary hormones (ACTH, GH, TSH, FSH, LH, α-subunit) and had characteristic ultrastructural features of corticotroph adenoma. Forty-one adenomas were positive only for ACTH, while seven ACTH-positive adenomas showed additional moderate/weak immunoreactivity for α-subunit. Increased proliferation assessed by Ki67 index ≥ 3% was observed in a similar proportion of CD and SCA patients, seven tumors causing CD and five SCAs. A higher proportion of sparsely vs. densely granulated adenomas was observed in SCAs than in CD-related adenomas, but the difference did not cross a significance threshold (p = 0.0787). No difference in the proportion of invasive/noninvasive adenomas was observed in clinically functioning and silent corticotroph adenomas. All SCAs were macroadenomas, while tumors causing CD included 17 macroadenomas and 11 microadenomas. No significant differences in preoperative clinical parameters, including 24 h UFC, morning serum ACTH level, morning and midnight serum cortisol level, cortisol/ACTH ratio, were observed between CD patients with micro- and macroadenomas. Irrespectively, a correlation between tumors size and ACTH level (Spearman R= 0.4678; p = 0.0121) and a negative correlation between cortisol/ACTH ratio (Spearman R= −0.4015; p = 0.0342) was observed in CD patients. No correlation was found between the remaining biochemical parameters and tumor size. Overall, the patients’ characteristics are presented in Table 1, while details including both the clinical and histopathological data are shown in Supplementary Table S1. Table 1. Summary of clinical features of patients with Cushing’s disease and silent corticotroph adenomas. 2.2. Identification of miRNAs Differentially Expressed in Corticotroph Adenomas Causing CD and Subclinical Cortiotroph Adenomas NGS data on miRNA expression of 48 corticotroph adenomas from previous investigation were used to compare miRNA expression levels between adenomas causing CD (n = 24) and subclinical corticotroph adenomas (n = 20). Sequencing of small RNA libraries produced approximately 2,497,367 reads per sample, which were mapped to the human genome (hg19) and used for quantification of expression levels of known miRNAs, according to miRBase 22 release. Sequencing reads were annotated to 1917 miRNAs. Measurements of 1902 mature miRNAs expression were included in the analysis, after filtering out the miRNAs with low expression. When miRNA profiles of adenomas causing CD and SCAs were compared, a total of 19 differentially expressed miRNAs were found that met the criteria of adjusted p-value < 0.05. This set included 16 miRNAs with higher expression in tumors causing CD: hsa-miR-129-2-3p, hsa-miR-129-5p, hsa-miR-124-3p, hsa-miR-132-5p, hsa-miR-129-1-3p, hsa-miR-135b-5p, hsa-miR-27a-3p, hsa-miR-10b-5p, hsa-miR-9-3p, hsa-miR-6506-3p, hsa-miR-6864-5p, hsa-let-7b-5p, hsa-miR-670-3p, hsa-miR-22-5p, hsa-miR-346 and hsa-miR-9-5p, Three miRNAs with lower expression in CD patients were found: hsa-miR-1909-3p, hsa-miR-4319 and hsa-miR-181b-3p. Details are presented in Table 2 and Figure 1A,B. Figure 1. MiRNA expression profiling in corticotroph adenomas. (A). Difference in miRNA expression between functioning and silent corticotroph adenomas. Volcano plot showing differentially expressed miRNAs. Significance and fold change thresholds are marked with dashed lines. (B). Heat map representing the expression of differentially expressed miRNAs and clustering the samples of adenomas causing Cushing’s disease (CD) and silent corticotroph adenomas (SCA). (C). The correlation between the expression levels of differentially expressed miRNAs and POMC expression or hormonal laboratory measurements in patients: morning plasma ACTH level, morning and midnight plasma cortisol levels and 24 h urinary free cortisol; * indicate p-value < 0.05; ** indicate p-value < 0.01; *** indicate p-value < 0.001 Table 2. The list of miRNAs differentially expressed in corticotroph pituitary adenomas causing CD and silent corticotroph adenomas. 2.3. The Correlation of miRNA Expression and Patients’ Clinical Data Since the clustering of the tumors based on the expression of differentially expressed miRNAs did not clearly separate functioning and silent adenomas, we determined whether the expression of the identified differentially expressed miRNAs is directly related to the results of patients’ laboratory tests as well as POMC expression, measured in tumor samples with qRT-PCR. For this purpose, Spearman’s correlation was applied to calculate a correlation matrix. We observed a significant positive correlation between 13 miRNAs out of 19 differentially expressed miRNAs and at least one of clinical laboratory parameters: serum ACTH, morning cortisol level, midnight cortisol level or 24 h UFC. For 11 miRNAs, with higher expression in patients with CD a positive correlation was observed, while a negative correlation was observed for 3 miRNAs that have lower expression in patients with CD. Four of the differentially expressed miRNAs, hsa-miR-9-3p, hsa-miR-9-5p, hsa-miR-27a-3p and hsa-miR-6506-3p, are correlated with POMC expression level in tumor tissue. The absolute value of correlation coefficient ranged between 0.31 and 0.55 which indicates a weak/moderate relationship. Details are presented in Figure 1C. 2.4. Funtional Enrichment Analysis of Differentially Expressed miRNAs To investigate the possible functional role of the identified miRNAs with different expression levels in CD tumors and SCAs, we used the information on experimentally validated miRNA targets gathered in the miRtarbase release 8.0 database. High confidence known miRNA targets that were validated with luciferase reporter assay, reported in miRtarbase, were included in the analysis. The enrichment of the genes reported as miRNA targets of our 19 miRNAs of interest was determined with gene set over-representation analysis (GSOA) based on Gene Ontology (GO) Molecular Function and GO Biological Processes. The list of all the genes reported in miRTarbase as validated with reporter gene assay was used as reference. As a result, we found 30 GO Molecular Function terms and 293 GO Biological Processes terms as significantly enriched with genes that are targets of the 19 differentially expressed miRNAs. Top 10 enriched terms were related mainly to steroid hormone activity, regulation of transcription and regulation of stem cell differentiation, as shown in Figure 2. Details are presented in Supplementary Table S2. We paid special attention to the terms that refer to steroid hormone action, i.e., steroid hormone receptor activity (GO:0003707), nuclear receptor activity (GO:0004879), ligand-activated transcription factor activity (GO:0098531), as well as steroid hormone-mediated signaling pathway (GO:0043401) and hormone-mediated signaling pathway (GO:0009755). Importantly, the miRNA target genes that were overrepresented in these terms included NR3C1 and NR3C2 that encode for adrenal hormones glucocorticoid receptor (GR) and mineralocorticoid receptor (MR), respectively. According to the miRtarbase 9.0 database, hsa-miR-124-3p is a negative regulator of NR3C1 gene [16] while both hsa-miR-124-3p and hsa-miR-135b-5p downregulate MR [17]. Figure 2. Gene set over-representation analysis of putative target genes of miRNAs differentially expressed in clinically functioning and silent corticotroph adenomas. Using the PubMed search, we found additional evidence strongly supporting the role of hsa-miR-124-3p in the regulation of NR3C1 [18,19,20,21] as well as the role of hsa-miR-135b-5p in downregulating NR3C2 [22,23]. 2.5. Comparison of the Expression of NR3C1 and NR3C2 in Corticotroph Adenomas Causing CD and Silent Adenomas We determined the expression levels of NR3C1 and NR3C2 in corticotroph adenomas with qRT-PCR. We observed a significantly lower expression of both genes in samples from CD patients (n = 24) as compared to SCAs (n = 24); fold change (FC) 0.49 p = 0.0166 and FC 0.37 p = 0.0132, for NR3C1 and NR3C2, respectively. However, the observed difference is rather slight and a notable dispersion of the results was observed (Figure 3). The differences in NR3C1 and NR3C2 expression correspond to the differences in hsa-miR-124-3p and hsa-miR-135b-5p levels. Patients with CD have higher levels of both miRNAs and lower levels of NR3C1 and NR3C2 mRNA (Figure 3). Unfortunately, we did not find a direct correlation between the expression levels of hsa-miR-124-3p and NR3C1 or hsa-miR-135b-5p and NR3C2. Figure 3. The expression levels of NR3C1 and NR3C2 measured with qRT-PCR as well as hsa-miR-124-3p and hsa-miR-135b-5p measured with small RNA sequencing in tumor samples from CD patients and silent corticotroph adenomas; * indicate p-value < 0.05 2.6. Investigtion of miRNA-Related Regulation of NR3C1 In Vitro Transfecting the cultured cells with miRNA mimics is the commonly used approach of in vitro validation of specific miRNA–mRNA interaction. We used mice corticotroph tumor AtT-20/D16v-F2 cells for in vitro experiment and initially verified whether these cells do express Nr3c1 and Nr3c2 genes using deposited RNAseq data from a previous experiment on AtT-20 cells (GSE132324; Gene Expression Omnibus) and qRT-PCR. This showed that the AtT-20/D16v-F2 have relatively high expression of Nr3c1 but do not express Nr3c2. Thus, we focused on the regulatory role of miR-124-3p on Nr3c1 expression. We used miRBase [24] and Targetscan [25] to determine whether miR-124-3p is evolutionarily conserved in humans and mice and whether it targets NR3C1 in both species. It confirmed that miR-124-3p is broadly conserved and it shares the same sequence of mature miRNA in humans and mice. Importantly, GR is among highly rated miR-124-3p predicted targets in both humans and mice and two highly conserved miR-124-3p binding motifs in 3′UTR of this gene were identified in these two species (Figure 4A). Figure 4. Role of mir-124-3p in regulation of glucocorticoid receptor gene. (A). Putative hsa-mir-124-3p target sites in 3′UTR of NR3C1. (B). Reduced expression of Nr3c1 gene expression and glucocorticoid receptor (GR) protein level in AtT-20/D16v-F2 cells treated with hsa-miR-124-3p mimics. (C). Results of luciferase reporter gene assay, showing the interaction between Nr3c1 3′UTR site 2 and mir-124-3p; * indicate p-value < 0.05; ns—not significant. When we transfected AtT-20/D16v-F2 cells with miR-124-3p miRNA mimic and unspecific negative control miRNA mimic, we observed a significant decrease in Nr3c1 expression in cells treated with miR-124-3p miRNA mimic (Figure 4B). It was significantly lower than in cells treated with unspecific miRNA mimic. This difference was also clearly visible at the protein level. The GR level was reduced in cells treated with miR-124-3p miRNA mimic as compared to control (Figure 4B). Two fragments of Nr3c1 3′UTR including each of putative miR-124-3p binding motifs were cloned in plasmid vector into 3′ region of the firefly luciferase gene. AtT-20/D16v-F2 cells were transfected with empty vector, vector with miR-124-3p binding site 1 and vector miR-124-3p binding site 2. Each of the three variants of the cells were cotransfected with miR-124-3p miRNA mimic or unspecific miRNA mimic that served as a negative control. Luminescence was developed 48 h after transfection and detected with microplate reader. As a result, we observed a significant decrease in luminescence in the cells with introduced plasmid with miR-124-3p binding site 2 treated with miR-124-3p mimic as compared to the cells transfected with the same plasmid construct but with control miRNA mimic. This observation confirms the interaction between miR-124-3p and 3′ UTR of Nr3c1 at putative binding site 2 (Figure 4C). The experiment did not confirm an interaction between miR-124-3p and 3′ UTR of Nr3c1 at binding site 1 since no significant difference of luminescence was found in cells transfected with plasmid vector harboring this binding motif treated with miR-124-3p mimic and the same cells treated with negative miRNA mimic (Figure 4C). 3. Discussion Based on the clinical manifestation and biochemical tests results, pituitary corticotroph adenomas can be divided into functioning adenomas causing Cushing’s disease and SCAs. These two subtypes of tumors also differ in terms of some characteristics in MRI [2,26] and pathological features [27]. In contrast to CD-causing adenomas which are commonly small microadenomas, SCAs are diagnosed as macroadenomas due to neurological symptoms related to tumor mass. They are characterized by invasive growth, high risk of recurrence and resistance to medical therapy and are therefore referred to as “high-risk” pituitary adenomas according to current classification [3,4]. In our study, the SCAs were larger than functioning counterparts, as expected. A clear prevalence of women is observed among CD patients according to literature data [28], while it is not observed in patients suffering from SCAs. Our SCA group contained near equal representation of women and men as in previous reports [29,30]; however, some studies indicated female prevalence in SCAs [31]. Comparing functioning and silent corticotroph adenomas, we did not observe difference in patients’ age as well as differences in invasive growth status, ratio of adenomas with increased proliferation index and proportions of sparsely and densely granulated adenomas that may suggest the lack of difference in the tumors’ “aggressiveness”. Importantly, limitations for generalization of our results should be noted. The number of patients included in the analysis is relatively low and the group is not representative of the general population, especially in the case of patients suffering from Cushing’s disease. Since the main goal of our study was a molecular profiling of tumor tissue, we intentionally preselected large adenomas, which allowed us to have enough tissue for DNA/RNA isolation and successful molecular procedures. In our investigation, we observed a negative correlation between cortisol/ACTH ratio and tumor volume in functioning corticotroph adenomas as described previously [32]. However, we did not observe any difference between micro- and macroadenomas causing CD as compared to SCAs (data not shown) as was found in previous studies [12]. The reason why some of corticotroph adenomas exhibit excessive hormone secretion and the others remain clinically silent is unclear and only few attempts have been made to determine the possible molecular mechanism underlying this difference in secretory activity. They were mainly focused on investigating the expression of the selected genes or proteins by comparing subclinical and functioning corticotroph adenomas. These studies indicated different expression levels of prohormone convertase 1/3 POMC, genes encoding somatostatin receptors, corticotropin releasing hormone receptor 1, vasopressin receptor (V1BR), corticosteroid 11-beta-dehydrogenase as well as NEUROD1 and TPIT [6,7,8,9,10,11,12,13]. However, whole transcriptome studies indicated that adenomas causing CD and subclinical corticotroph adenomas share a very common gene expression profile and a very low number of differentially expressed genes can be found by comparing transcriptome of silent and CD-causing ACTH tumors [14,15]. In our study, we determined the miRNA expression profile of 28 clinically functioning adenomas and 20 SCAs with next-generation sequencing of small RNA fraction. This allowed for the quantification of over 1900 miRNA annotated to current version of miRbase database and comparing their expression in two groups of tumor samples. We found a significant difference only in the expression levels of 19 miRNAs, that represent less than 1% of the miRNAs included in the analysis. This result resembles the observation from previous comparison of whole transcriptome profiles in functioning adenomas and SCAs where only 34 differentially expressed genes were found. Generally, both observations indicate a very common molecular profile of corticotroph adenomas, regardless of the functional status. In our study, the expression levels of 13 out of 19 identified differentially expressed miRNAs were also correlated with peripheral ACTH/cortisol levels, further supporting the role of these miRNAs in secretory activity of corticotroph adenomas. The possible role of miRNA in subclinical nature of SCAs was addressed in only one previous study by García-Martínez A et al. [33]. The authors compared the expression of 5 miRNAs in 24 functioning and 23 silent adenomas and observed a difference in hsa-miR-200a and hsa-miR-103 levels [33]. Their results were not confirmed by our investigation since these two miRNAs were not found among differentially expressed miRNAs. In our data, very a similar expression level of hsa-miR-200a was observed in clinically functioning and silent adenomas. In turn, a slightly higher expression of hsa-miR-103a-3p was observed in SCAs as previously reported, but the difference did not cross the significance threshold level. We should note that different methods were used for these two studies and technical and analytical differences could result in this discrepancy. Since miRNAs play a role in gene regulation, their effect should be investigated in the context of the function of targeted genes. The interaction between miRNA and its target mRNA 3′UTR can be predicted with in silico tools. Unfortunately, prediction results can be very difficult to interpret since a huge number of predicted interactions can be found for some miRNAs. For example, when using the Targetescan (http://www.targetscan.org; accessed on 28 February 2022) prediction tool [25], over 4000 target genes were predicted for each hsa-miR-9-3p, hsa-miR-1909-3p, hsa-miR-22-5p and hsa-miR-181b-3p that we found as differentially expressed in CD and SCA. Therefore, to investigate a possible functional relevance of differentially expressed miRNAs we used a database of experimentally validated miRNA targets [34]. Gene set over-representation analysis of miRNA target genes indicated their enrichment in the pathways of steroid hormone nuclear receptors functioning. This result indicates that miRNAs that have different expression levels in CD and SCAs play a role in the regulation of expression of genes involved in steroid hormone signaling at hormone receptor level. It is especially interesting since this group of compounds includes adrenal hormones that play a role in the regulation of the hypothalamic–pituitary–adrenal (HPA) axis. The particular enriched miRNA target genes included NR3C1 and NR3C2 that encode for corticosteroid hormone receptors (GR and MR, respectively). Both receptors are located in the cytoplasm where they bind glucocorticoids. Upon ligand binding, they are translocated to nucleus where they form dimers on DNA at glucocorticoid response elements (GREs). Glucocorticoid and mineralocorticoid receptors directly regulate the expression of target genes and/or influence the expression indirectly through the interaction with other transcription factors [35]. Glucocorticoids play a role in the basic mechanism of negative feedback of HPA axis. They act on hypothalamus, where high cortisol levels reduce secretion of corticotropin-releasing hormone (CRH), thus they directly reduce stimulation of ACTH secretion by anterior pituitary lobe. Glucocorticoids also inhibit the activity of pituitary cells indirectly. Corticotroph cells express GRs and their activation results in the reduction of POMC expression and secretion of ACTH [36,37]. In pituitary corticotroph adenomas, NR3C1 point mutations and loss of heterozygosity in NR3C1 locus were identified [38]. These mutations seem to affect the secretory activity and result in tumor resistance to corticosteroids [39]. Reduced expression of corticosteroid receptors in corticotroph adenomas has been reported in patients with resistance to high doses of dexamethasone [40]. These data indicate a role of GR in secretory activity of clinically functioning corticotroph adenomas. The expression of corticosteroid genes was previously investigated in CD-causing tumors and SCAs and no significant differences were found. However, it is worth noting that a low number of SCA patients was included in these studies: n = 9 [13], n = 8 [11] and n = 2 [41]. According to previously published results, hsa-miR-124-3p is a negative regulator of NR3C1 [16,18,19,20,21]. This was observed in acute lymphoblastic leukemia [19], adipocytes [20] and human embryonic kidney cells [21], where the reduced expression of NR3C1 upon an increase in hsa-miR-124-3p as well as a direct interaction between this miRNA and 3′UTR of GR gene were observed. Some additional clinical observations also suggest the role of hsa-miR-124-3p in the regulation of the response to cortiosteroids in patients with acute-on-chronic liver failure [18] and lymphoblastic leukemia [19]. Hsa-miRNA-124 also mediates corticosteroid resistance in T-cells of sepsis patients through the downregulation of GR [42]. Our analysis of the expression level of NR3C1 in corticotroph adenomas showed that tumors causing CD have lower gene expression and accordingly they exhibit higher levels of hsa-miR-124-3p. Subsequently, the role of hsa-miR-124-3p in NR3C1 downregulation was confirmed in mice AtT-20/D16v-F2 corticotroph cells using miRNA mimics and reporter gene assay. Transfection of AtT-20/D16v-F2 cells with hsa-miR-124-3p mimics resulted in reduced NR3C1 mRNA expression and GR protein level. We also confirmed the interaction between hsa-miR-124-3p and one of two predicted binding motifs in 3′UTR of NR3C1 with luciferase reporter gene assay. Since sequences of hsa-miR-124-3p and target sequence in 3′UTR of NR3C1 mRNA are the same in mice and in humans, we believe that results showing the regulation of the GR-encoding gene in mice AtT-20/D16v-F2 cells are also relevant to humans. Together, the available data indicate that in pituitary corticotrophs, hsa-miR-124-3p downregulates the expression of the GR gene. Since this receptor mediates the response of pituitary cells to cortisol, the expression of hsa-miR-124-3p appears to be an important element in the regulation of secretory activity of corticotroph cells. Based on these results, we can hypothesize that in CD, a high level of hsa-miR-124-3p contributes to lowering of GR expression and in consequence it plays a role in lowering the effect of glucocorticoid feedback on the activity of corticotroph adenoma. Hsa-miR-124-3p and hsa-miR-135b-5p can downregulate the expression level of MR, as proven in model HeLa cells [17]. Expression of both miRNAs is higher in corticotroph adenomas causing CD which corresponds to the lower expression of the NR3C2 gene in these tumors as compared to SCAs. Since the role of the MR receptor expression in pituitary cells is poorly understood, the functional implication of this observation is much less clear than in the case of GR downregulation. MR and GR have similar amino acid sequences, especially in DNA-binding domain, but they differ in affinity to corticosteroids. MR is specific for both mineralocorticoids and glucocorticoids while GR is specific predominantly for glucocorticoids. MRs have much higher affinity for glucocorticoids than GRs and are activated at basal glucocorticoid conditions, while GR occupancy is increased when glucocorticoid levels rise during the circadian peak or stress. Due to these differences, these two receptors play slightly different roles, despite the fact that they share a number of target genes [43]. MR expression is considered more tissue-specific than GR and was reported to be the most prevalent in kidney and adipose tissue but also in the hippocampus and hypothalamus [44]. However, the available databases of human expression pattern such as the Genotype-Tissue Expression project (https://gtexportal.org; accessed on 10 December 2021) or Protein atlas (https://www.proteinatlas.org; accessed on 10 December 2021) indicate that MR is widely expressed in multiple human tissues and organs including the pituitary gland. Unfortunately, a role of MR receptor in pathogenesis of pituitary tumors remains unknown. AtT-20 cells, which are the only available cell line model of corticotroph adenoma, do not express MR receptor, thus the procedure of experimental validation of the role of miRNA in NR3C2 silencing is not applicable. With a lack of experimental data on the exact role of MR, we can only hypothesize that miRNA-mediated silencing of NR3C2 may have the similar effect on HPA axis feedback as silencing of NR3C1. It may enhance ACTH secretion by reducing the direct inhibitory effect of glucocorticoids on neoplastic pituitary corticotrophs. The difference in expression of hsa-miR-124-3p and hsa-miR-135b-5p between subclinical and CD-causing adenomas is not big, thus we suppose that high expression of these miRNAs is not the only cause of difference in ACTH secretion. Presumably this is one of the mechanisms in the regulation of corticotrophs’ secretory activity. The model of miRNA-based corticosteroid receptor regulation does not undermine the role of previously described differences in the expression of convertase 1/3, POMC, somatostatin receptors or corticotropin releasing hormone receptor 1 or genes involved in differentiation of pituitary cells [6,7,8,9,10,11,12,13]. When considering the complex nature of the regulation of ACTH secretion, it can be assumed that multiple mechanisms may be involved in the silent character of subclinical adenomas. The low number of identified differentially expressed miRNAs or genes in silent and clinically functioning adenomas probably results from the intertumoral molecular heterogeneity of SCAs. This is also in line with clinical evidence indicating that some silent corticotroph adenomas can transform into clinically functioning ones while the others remain silent [1]. The misregulation of GR expression or NR3C1 mutation may have important therapeutical implications in CD patients. Non-selective GR antagonist Mifepristone was officially approved for treatment in patients with Cushing’s syndrome [45] while another new GR inhibitor, Relacorilant (CORT125134), is under clinical investigation for its use in this group of patients [46]. The further studies will be required to assess the role of GR abnormalities in response to GR-targeting treatment in CD. In our study, we focused mainly on the role of hsa-miR-124-3p and hsa-miR-135b-5p in the regulation of corticosteroid receptors, but the role of other differentially expressed miRNAs can also be elucidated, based on the function of putative target genes. In the pathways enrichment analysis of the putative targets, molecular functions related to transcriptional regulation were found among the top processes. Interestingly, five miRNAs, i.e., hsa-miR-132-5p, hsa-miR-135b-5p, hsa-miR-27a-3p, hsa-miR-9-3p and hsa-miR-9-5p, were previously reported to downregulate the expression of FOXO1 transcription factor [47,48,49,50,51]. FOXO1 plays an important role in the differentiation of pituitary cells [52] and secretion of gonadotropic hormones [53,54] and prolactin [55]. The role of FOXO1 in pituitary corticotroph cells was not investigated but it was shown to regulate POMC expression in POMC hypothalamic neurons [56]. In POMC, neurons of arcuate nucleus FOXO1 directly suppresses POMC expression. A similar mechanism was also observed in prolactin pituitary adenomas where FOXO1 suppresses the promoter of PRL gene [55]. It is possible that high expression of hsa-miR-132-5p, hsa-miR-135b-5p, hsa-miR-27a-3p, hsa-miR-9-3p and hsa-miR-9-5p in pituitary corticotroph adenomas reduces the level of FOXO1 and eventually contributes to the upregulation of POMC expression. In our data from corticotroph adenomas, we observed the correlation between levels of hsa-miR-9-3p/hsa-miR-9-5 and POMC expression, which also supports this concept, but the exact role of miRNAs in possible FOXO1-related regulation of secretory activity of corticotroph cells requires further functional investigation. 4. Materials and Methods 4.1. Patients and Tissue Samples Pituitary tumor samples from 48 patients were collected during transsphenoidal surgery. Formalin-fixed and paraffin-embedded (FFPE) tissue samples, including 28 samples from patients with Cushing’s disease and 20 samples of SCA were used for the study. Diagnosis of hypercortisolism was based on standard hormonal criteria: increased UFC in three 24 h urine collections, disturbances of cortisol circadian rhythm, increased serum cortisol levels accompanied by increased or not suppressed plasma ACTH levels at 8.00 and a lack of suppression of serum cortisol levels to <1.8 µg/dL during an overnight dexamethasone suppression test (1 mg at midnight). The pituitary etiology of Cushing’s disease was confirmed based on the serum cortisol levels or UFC suppression < 50% with a high-dose dexamethasone suppression test (2 mg q.i.d. for 48 h) or a positive result of a corticotrophin-releasing hormone stimulation test (100 mg i.v.) and positive pituitary magnetic resonance imaging. ACTH levels were assessed using IRMA (ELSA-ACTH, CIS Bio International, Gif-sur-Yvette Cedex, France). The analytical sensitivity was 2 pg/mL (reference range: 10–60 pg/mL). Serum cortisol concentrations were determined by the Elecsys 2010 electrochemiluminescence immunoassay (Roche Diagnostics, Mannheim, Germany). Sensitivity of the assay was 0.02 μg/dL (reference range: 6.2–19.4 μg/dL). UFC was determined after extraction (liquid/liquid with dichloromethane) by electrochemiluminescence immunoassay (Elecsys 2010, Roche Diagnostics)—reference range: 4.3–176 μg/24 h. All the tumors underwent detailed histopathological diagnosis including immunohistochemical staining with antibodies against particular pituitary hormones (ACTH, GH, TSH, FSH, LH, α-subunit) and Ki67 as well as ultrastructural analysis with electron microscopy. The SCAs were characterized by the following clinicopathological criteria: positive immunohistochemical staining for ACTH, lack of signs and symptoms of hypercortisolism (Cushing’s syndrome), negative hormonal evaluation and non-compliance with diagnostic criteria of the CD. Macroadenoma was defined as an adenoma with at least one diameter exceeding 10 mm, and the tumor volume was assessed with the diChiro Nelson formula (height × length × width × π/6). Invasive growth of the tumors was evaluated using Knosp grading [57]. Adenomas with Knosp grades 0, 1 and 2 were considered non-invasive, while those with Knosp 3 and 4 were considered invasive. Forty-three patients had a clear history of not using any drugs that control the overproduction of the cortisol or ACTH (ketoconazole, mitotane, metyrapone, osilodrostat, mifepristone, pasireotide) before surgical treatment. The information on preoperative pharmacological treatment was not available for 5 patients. Tumor tissue content of each FFPE sample ranged between 80 and 100% (median 99%), as assessed with histopathological examination. Patients’ characteristics are presented in Table 1 and details on each patient’s data are available in Supplementary Table S1. The study was approved by the local Ethics Committee of Maria Sklodowska-Curie National Research Institute of Oncology in Warsaw, Poland. Each patient provided informed consent for the use of tissue samples for scientific purposes. Total RNA from FFPE samples was purified with RecoverAll™ Total Nucleic Acid Isolation Kit for FFPE tissue (Thermo Fisher Scientific, Waltham, MA, USA) and measured using NanoDrop 2000 (Thermo Fisher Scientific). RNA was stored at −70 °C. 4.2. Micro RNA Expression Profiling For comparing the miRNA expression profiles in CD-causing and clinically silent adenomas, NGS data from our previous investigation of miRNA expression in corticotroph adenomas were used. The dataset is available at Gene Expression Omnibus, accession no GSE166279. Sequencing of small RNA fraction was performed in 48 tumor samples (28 CD patients and 20 SCA patients) with ion semiconductor sequencing technology, as described previously [58]. Briefly, Ion Total RNA-Seq Kit v2 (Thermo Fisher Scientific) was used for sequencing library construction, Ion Xpress™ RNA-Seq Barcode Kit was used for hybridization and ligation of RNA adapters. RNA reverse transcription and subsequent cDNA purification and library size selection were performed using Nucleic Acid Binding Beads and verified using Bioanalyzer 2100 with High Sensitivity DNA Kit (Agilent, Santa Clara, CA, USA). Ion Chef instrument, with Ion PI™ Hi-Q™ Chef Kit (Thermo Fisher Scientific) and Ion Proton sequencer (Thermo Fisher Scientific) were used for library preparation and sequencing, respectively. BamToFastq package was applied for converting unmapped bam files into fastq files. miRDeep2 was applied for read mapping to known human miRNAs (according to miRBase release 22) and reads quantification. Data normalization and differential expression analysis were performed using DESeq2. Filtration for low-expression miRNAs was applied as described previously. FC of expression calculated as the ratio of the normalized read-count value in CD-causing and silent adenomas was used as a measure of expression difference. Adjusted p-value < 0.05 was used as significance threshold. MiRtarbase release 9.0 database [34] was used to identify known miRNA target genes. PANTHER (http://pantherdb.org; accessed on 10 December 2021) [59] was used for gene set over-representation analysis. 4.3. qRT-PCR gene Expression Analysis One microgram of RNA was subjected to reverse transcription with Transcriptor First Strand cDNA Synthesis Kit (Roche Diagnostics). qRT-PCR reaction was carried out in 384-well format using 7900HT Fast Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) and Power SYBR Green PCR Master Mix (Thermo Fisher Scientific) in a volume of 5 μL, containing 2.25 pmol of each primer. The samples were amplified in triplicates. GAPDH was used as reference gene. Delta Ct method was used to calculate the relative expression level. PCR primers’ sequences are presented in Supplementary Table S3. 4.4. Cell Line Culture and miRNA Mimic Transfection AtT-20/D16v-F2 cells were purchased from ATCC collection and cultured in DMEM medium supplemented with 10% FBS, as recommended. MiRCURY LNA miRNA Mimics including hsa-miR-124-3p mimic (YM00471256, Qiagen, Hilden, Germany) and negative control mimic (YM00479902-ADB, Qiagen) were used. AtT-20/D16v-F2 cells were seeded at 5 × 104 per well of a 24-well plate in culture medium and transfected with 50 nM miRNA with 1% (v/v) HiPerFect Transfection Reagent (Qiagen), according to the manufacturer’s instructions. The next day, the culture medium was changed. In total, 48 h after transfection the cells were harvested and subjected to isolation of total RNA with RNeasy Mini Kit (Qiagen). The expression of the putative hsa-miR-124-3p target gene was determined with qRT-PCR. 4.5. Luciferase Reporter Gene Assay Hsa-miR-124-3p target sites in 3′UTR of NR3C1 were determined with Targetscan [25]. Each of two predicted hsa-miR-124-3p target sites were cloned into pmirGLO Dual-Luciferase miRNA Target Expression Vector (Promega, Madison, WI, USA). AtT-20/D16v-F2 cells (2 × 104/well) were seeded onto a 96-well plate in 100 µL culture medium. The next day, the cells were transfected with 100 ng of each plasmid vector, independently using 0.25% (v/v) lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA) in 10 µL of DMEM. The cells were subsequently transfected with either hsa-miR-124-3p mimic (YM00471256, Qiagen) or negative control mimic (YM00479902-ADB, Qiagen) in a final concentration of 50 nM using HiPerfectReagent (Qiagen). Culture medium was changed on the next day. Luciferase activity was measured with One-Glo Luciferase Assay System (Promega) 48 h after transfection. 4.6. Western Blotting Cells were lysed in ice cold RIPA buffer, incubated for 30 min in 4 °C and centrifuged at 12,500× g rpm for 20 min at 4 °C. Samples were resolved using SDS-PAGE and electrotransferred to polyvinylidene fluoride membranes (PVDF) (Thermo Fisher). GR protein was detected with monoclonal anti-Glucocorticoid Receptor antibody (ab183127, Abcam, Cambridge, UK), and secondary anti-rabbit antibody conjugated to HRP (#7074, Cell Signaling, Beverly, MA, USA). Glyceraldehyde-3-Phosphate Dehydrogenase (#MAB374, Millipore, Bedford, MA, USA) detected with mouse HRP-conjugated antibody (#7076 Cell Signaling) served as control. Visualization was performed with SuperSignal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific) and CCD digital imaging system Alliance Mini HD4 (UVItec Limited, Cambridge, UK). 4.7. Statistical Analysis A two-sided Mann–Whitney U-test was used for analysis of continuous variables. The Spearman correlation method was used for correlation analysis. Significance threshold of α = 0.05 was adopted. Data were analyzed using GraphPad Prism 6.07 (GraphPad Software, La Jolla, CA, USA). Hierarchical clustering analysis was carried out with Cluster 3.0, and the results were visualized using TreeView 1.6 software (Stanford University School of Medicine, Stanford, CA, USA). Supplementary Materials The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/ijms23052867/s1. Author Contributions Conceptualization, M.M. and M.B.; Methodology, M.B. and B.J.M.; Software, J.B.; Formal analysis, P.K., B.J.M. and M.B.; Investigation, B.J.M., P.K., N.R., M.B. and M.P.; Resources, J.K., G.Z., A.S. and T.M.; Data curation, J.B., B.J.M. and M.B.; Writing—original draft preparation, M.B., P.K. and B.J.M.; Writing—review and editing, all the authors; Visualization, M.B. and B.J.M.; Supervision, M.M.; Project administration M.B.; Funding acquisition, M.M. All authors have read and agreed to the published version of the manuscript. Funding This research was funded by National Science Centre, Poland, grant number 2021/05/X/NZ5/01874. Institutional Review Board Statement The study was conducted in accordance with the Declaration of Helsinki, and approved by the local Ethics Committee of Maria Sklodowska-Curie Institute—Oncology Center in Warsaw, Poland; approval no. number 44/2018, date of approval 26 July 2018. Informed Consent Statement Informed consent was obtained from all subjects involved in the study. Data Availability Statement Data from next-generation sequencing of small RNA fraction of 48 corticotroph adenoma samples are available at Gene Expression Omnibus, accession no GSE166279. Conflicts of Interest The authors declare no conflict of interest. References Ben-Shlomo, A.; Cooper, O. Silent Corticotroph Adenomas. Pituitary 2018, 21, 183–193. 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@happygirl - this looks perfect for you with all your videos! If you or anyone else from these boards does this, please let me know Help Rare Patient Voice spread the word to other patients and caregivers about RPV by submitting a short video on your experience with us. Check out the growing group of patients and caregivers who have recorded stories: https://rarepatientvoice.com#sharevoice. As a thank you for recording a video, we will send you a Rarity zebra plushie AND enter you in a raffle to win a $100 Amazon gift card. Follow these steps to record and submit your own video! Step 1: Scan with code below with the camera app from your Apple/Android mobile device or click the link below! https://admin.storyvine.com/app_users/sign_up/Sharing_My_Voice Step 2: Download the Storyvine app from the App Store or Google Play Step 3: Film and upload your video! To thank you for recording a video, we will send you a Rarity zebra plushie AND enter you in a raffle to win a $100 Amazon gift card. Congratulations to Natalie of California, our January 3 raffle winner! Our next raffle will be held in early February.

Abstract Summary The pandemic caused by severe acute respiratory syndrome coronavirus 2 is of an unprecedented magnitude and has made it challenging to properly treat patients with urgent or rare endocrine disorders. Little is known about the risk of coronavirus disease 2019 (COVID-19) in patients with rare endocrine malignancies, such as pituitary carcinoma. We describe the case of a 43-year-old patient with adrenocorticotrophic hormone-secreting pituitary carcinoma who developed a severe COVID-19 infection. He had stabilized Cushing’s disease after multiple lines of treatment and was currently receiving maintenance immunotherapy with nivolumab (240 mg every 2 weeks) and steroidogenesis inhibition with ketoconazole (800 mg daily). On admission, he was urgently intubated for respiratory exhaustion. Supplementation of corticosteroid requirements consisted of high-dose dexamethasone, in analogy with the RECOVERY trial, followed by the reintroduction of ketoconazole under the coverage of a hydrocortisone stress regimen, which was continued at a dose depending on the current level of stress. He had a prolonged and complicated stay at the intensive care unit but was eventually discharged and able to continue his rehabilitation. The case points out that multiple risk factors for severe COVID-19 are present in patients with Cushing’s syndrome. ‘Block-replacement’ therapy with suppression of endogenous steroidogenesis and supplementation of corticosteroid requirements might be preferred in this patient population. Learning points Comorbidities for severe coronavirus disease 2019 (COVID-19) are frequently present in patients with Cushing’s syndrome. ‘Block-replacement’ with suppression of endogenous steroidogenesis and supplementation of corticosteroid requirements might be preferred to reduce the need for biochemical monitoring and avoid adrenal insufficiency. The optimal corticosteroid dose/choice for COVID-19 is unclear, especially in patients with endogenous glucocorticoid excess. First-line surgery vs initial disease control with steroidogenesis inhibitors for Cushing’s disease should be discussed depending on the current healthcare situation. Keywords: Adult; Male; Other; Belgium; Pituitary; Adrenal; Neuroendocrinology; Oncology; Insight into disease pathogenesis or mechanism of therapy; February; 2022 Background The pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has had a significant impact on the health care systems to date. The clinical presentation of coronavirus disease 2019 (COVID-19) is diverse, ranging from asymptomatic illness to respiratory failure requiring admission to the intensive care unit (ICU). Risk factors for severe course include old age, male gender, comorbidities such as arterial hypertension, diabetes mellitus, chronic lung-, heart-, liver- and kidney disease, malignancy, immunodeficiency and pregnancy (1). Little is known about the risk of COVID-19 in patients with rare endocrine malignancies, such as pituitary carcinoma. Case presentation This case concerns a 43-year-old man with adrenocorticotrophic hormone (ACTH)-secreting pituitary carcinoma (with cerebellar and cervical drop metastases) with a severe COVID-19 infection. He had previously received multiple treatment modalities including surgery, radiotherapy, ketoconazole, pasireotide, cabergoline, bilateral (subtotal) adrenalectomy and temozolomide chemotherapy as described elsewhere (2). His most recent therapy was a combination of immune checkpoint inhibitors consisting of ipilimumab (3 mg/kg) and nivolumab (1 mg/kg) (anti-CTLA-4 and anti-PD-1, respectively) every 3 weeks for four cycles, after which maintenance therapy with nivolumab (240 mg) every 2 weeks was continued. Residual endogenous cortisol production was inhibited with ketoconazole 800 mg daily. He had stabilized disease with a decrease in plasma ACTH, urinary free cortisol and stable radiological findings (2). Surgical resection of the left adrenal remnant was planned but was not carried out due to the development of a COVID-19 infection. In March 2021, he consulted our emergency department for severe respiratory complaints. He had been suffering from upper respiratory tract symptoms for one week, with progressive dyspnoea in the last three days. He tested positive for SARS-CoV-2 the day before admission. On examination, his O2 saturation was 72%, with tachypnoea (40/min) and bilateral pulmonary crepitations. His temperature was 37.2°C, blood pressure 124/86 mmHg and pulse rate 112 bpm. High-flow oxygen therapy was initiated but yielded insufficient improvement (O2 saturation of 89% and tachypnoea 35/min). He was urgently intubated for respiratory exhaustion. Investigation Initial investigations showed type 1 respiratory insufficiency with PaO2 of 52.5 mmHg (normal 75–90), PaCO2 of 33.0 mmHg (normal 36–44), pH of 7.47 (normal 7.35–7.45) and a P/F ratio of 65.7 (normal >300). His inflammatory parameters were elevated with C-reactive protein level of 275.7 mg/L (normal <5·0) and white blood cell count of 7.1 × 10⁹ per L with 72.3% neutrophils. His most recent morning plasma ACTH-cortisol level (measured using the Elecsys electrochemiluminescence immunoassays on a Cobas 8000 immunoanalyzer [Roche Diagnostics]) before his admission was 213 ng/L (normal 7.2–63) and 195 µg/L (normal 62–180) respectively, while a repeat measurement 3 weeks after his admission demonstrated increased cortisol levels of 547 µg/L (possibly iatrogenic due to treatment with high-dose hydrocortisone) and a decreased ACTH of 130 ng/L. Treatment On admission, he was started on high-dose dexamethasone therapy for 10 days together with broad-spectrum antibiotics for positive sputum cultures containing Serratia, methicillin-susceptible Staphylococcus aureus and Haemophilus influenzae. Thromboprophylaxis with an intermediate dose of low molecular weight heparin (tinzaparin 14 000 units daily for a body weight of 119 kg) was initiated. A ‘block-replacement’ regimen was adopted with the continuation of ketoconazole (restarted on day 11) in view of his endocrine treatment and the supplementation of hydrocortisone at a dose depending on the current level of stress. The consecutive daily dose of hydrocortisone and ketoconazole is shown in Fig. 1. View Full Size Figure 1 ‘Block-replacement’ therapy with ketoconazole and hydrocortisone/dexamethasone. Dexamethasone 10 mg daily was initially started as COVID-19 treatment, followed by hydrocortisone at a dose consistent with current levels of stress. Ketoconazole was restarted on day 11 and titrated to a dose of 800 mg daily to suppress endogenous glucocorticoid production. Citation: Endocrinology, Diabetes & Metabolism Case Reports 2022, 1; 10.1530/EDM-21-0182 Download Figure Download figure as PowerPoint slide Outcome and follow-up He developed multiple organ involvement, including metabolic acidosis, acute renal failure requiring continuous venovenous hemofiltration, acute coronary syndrome type 2, septic thrombophlebitis of the right jugular vein, and critical illness polyneuropathy. He was readmitted twice to the ICU, for ventilator-associated pneumonia and central line-associated bloodstream infection respectively. He eventually recovered and was discharged from the hospital to continue his rehabilitation. Discussion We describe the case of a patient with severe COVID-19 infection with active Cushing’s disease due to pituitary carcinoma, who was treated with high-dose dexamethasone followed by ‘block-replacement’ therapy with hydrocortisone in combination with off-label use of ketoconazole as a steroidogenesis inhibitor. His hospitalization was prolonged by multiple readmissions to the ICU for infectious causes. Our case illustrates the presence of multiple comorbidities for a severe and complicated course of COVID-19 in a patient with active Cushing’s disease. Dexamethasone was initially chosen as the preferred corticosteroid therapy, in analogy with the RECOVERY trial, in which dexamethasone at a dose of 6mg once daily (oral or i.v.) resulted in lower 28-day mortality in hospitalized patients with COVID-19 requiring oxygen therapy or invasive mechanical ventilation (3). However, the optimal dose/choice of corticosteroid therapy is unclear, especially in a patient population with pre-existing hypercortisolaemia. A similar survival benefit for hydrocortisone compared to dexamethasone has yet to be convincingly demonstrated. This may be explained by differences in anti-inflammatory activity but could also be due to the fact that recent studies with hydrocortisone were stopped early and were underpowered (4, 5). Multiple risk factors for a complicated course of COVID-19 are present in patients with Cushing’s syndrome and might increase morbidity and mortality (6, 7). These include a history of obesity, arterial hypertension and impaired glucose metabolism. Prevention and treatment of these pre-existing comorbidities are essential. Patients with Cushing’s syndrome also have an increased thromboembolic risk, which is further accentuated by the development of severe COVID-19 infection (6, 7). Thromboprophylaxis with low molecular weight heparin is associated with lower mortality in COVID-19 patients with high sepsis‐induced coagulopathy score or high D-dimer levels (8) and is presently widely used in the treatment of severe COVID-19 disease (9). Subsequently, this treatment is indicated in hospitalized COVID-19 patients with Cushing’s syndrome. It is unclear whether therapeutic anticoagulation dosing could provide additional benefits (6, 7). An algorithm based on the International Society on Thrombosis and Hemostasis-Disseminated Intravascular Coagulation score was proposed to evaluate the ideal anticoagulation therapy in severe/critical COVID-19 patients, with an indication for therapeutic low molecular weight heparin dose at a score ≥5 (9). Furthermore, the chronic cortisol excess induces suppression of the innate and adaptive immune response. Patients with Cushing’s syndrome, especially when severe and active, should be considered immunocompromised and have increased susceptibility for viral and other (hospital-acquired) infections. Prophylaxis for Pneumocystis jirovecii with trimethoprim/sulfamethoxazole should therefore be considered (6, 7). Additionally, there is a particular link between the pathophysiology of COVID-19 and Cushing’s syndrome. The SARS-CoV-2 virus (as well as other coronaviruses) enter human cells by binding the ACE2 receptor. The transmembrane serine protease 2 (TMPRSS2), expressed by endothelial cells, is additionally required for the priming of the spike-protein of SARS-CoV-2, leading to viral entry. TMPRSS2 was studied in prostate cancer and found to be regulated by androgen signalling. Consequently, the androgen excess frequently associated with Cushing’s syndrome might be an additional risk factor for contracting COVID-19 via higher TMPRSS2 expression (10), especially in women, in whom the effect of excess androgen would be more noticeable compared to male patients with Cushing’s syndrome. Treating Cushing’s syndrome with a ‘block-replacement’ approach, with suppression of endogenous steroidogenesis and supplementation of corticosteroid requirements, is an approach that should be considered, especially in severe or cyclic disease. The use of this method might decrease the need for monitoring and reduce the occurrence of adrenal insufficiency (7). Our patient was on treatment with ketoconazole, which was interrupted at initial presentation and then restarted under the coverage of a hydrocortisone stress regimen. Ketoconazole was chosen because of its availability. Advantages of ketoconazole over metyrapone include its antifungal activity with the potential for prevention of invasive pulmonary fungal infections, as well as its antiandrogen action (especially in female patients) and subsequent inhibition of TMPRSS2 expression (10). Regular monitoring of the liver function (every month for the first 3 months, at therapy initiation or dose increase) is necessary. Caution is needed due to its inhibition of multiple cytochrome P450 enzymes (including CYP3A4) and subsequently greater risk of drug-drug interactions vs metyrapone (7, 10). Another disadvantage of ketoconazole is the need for oral administration. In our patient, ketoconazole was delivered through a nasogastric tube. i.v. etomidate is an alternative in case of an unavailable enteral route. Finally, as a general point, the first-line treatment of a patient with a novel diagnosis of Cushing’s disease is transsphenoidal surgery. Recent endocrine recommendations pointed out the possibility of initial disease control with steroidogenesis inhibitors in patients without an indication for urgent intervention during a high prevalence of COVID-19 (7). This would allow the optimalization of metabolic parameters; emphasizing that the short-to mid-term prognosis is related to the cortisol excess and not its cause. Surgery could then be postponed until the health situation allows for safe elective surgery (7). This decision depends of course on the evolution of COVID-19 and the healthcare system in each country and should be closely monitored by policymakers and physicians. Declaration of interest The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported. Funding This work did not receive any specific grant from any funding agency in the public, commercial, or not-for-profit sector. Patient consent Written informed consent for publication of their clinical details and/or clinical images was obtained from the patient. Author contribution statement J M K de Filette is an endocrinologist-in-training and was the main author. All authors were involved in the clinical care of the patient. All authors contributed to the reviewing and editing process and approved the final version of the manuscript. 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by Valentina Guarnotta, Francesca Di Gaudio and Carla Giordano 1 Department of Health Promotion, Maternal-Infantile Care, Excellence Internal and Specialist Medicine “G. D’Alessandro” [PROMISE], Section of Endocrine Disease and Nutrition, University of Palermo, 90127 Palermo, Italy 2 Biochemistry Head CQRC Division (Quality Control and Biochemical Risk), Department of Health Promotion, Maternal-Infantile Care, Excellence Internal and Specialist Medicine “G. D’Alessandro” [PROMISE], University of Palermo, 90127 Palermo, Italy Author to whom correspondence should be addressed. Academic Editor: Edgard Delvin Nutrients 2022, 14(5), 973; https://doi.org/10.3390/nu14050973 Abstract Background: The primary objective of the study was to assess serum 25-hydroxyvitamin D [25(OH)D] values in patients with Cushing’s disease (CD), compared to controls. The secondary objective was to assess the response to a load of 150,000 U of cholecalciferol. Methods: In 50 patients with active CD and 48 controls, we evaluated the anthropometric and biochemical parameters, including insulin sensitivity estimation by the homeostatic model of insulin resistance, Matsuda Index and oral disposition index at baseline and in patients with CD also after 6 weeks of cholecalciferol supplementation. Results: At baseline, patients with CD showed a higher frequency of hypovitaminosis deficiency (p = 0.001) and lower serum 25(OH)D (p < 0.001) than the controls. Six weeks after cholecalciferol treatment, patients with CD had increased serum calcium (p = 0.017), 25(OH)D (p < 0.001), ISI-Matsuda (p = 0.035), oral disposition index (p = 0.045) and decreased serum PTH (p = 0.004) and total cholesterol (p = 0.017) values than at baseline. Multivariate analysis showed that mean urinary free cortisol (mUFC) was independently negatively correlated with serum 25(OH)D in CD. Conclusions: Serum 25(OH)D levels are lower in patients with CD compared to the controls. Vitamin D deficiency is correlated with mUFC and values of mUFC > 240 nmol/24 h are associated with hypovitaminosis D. Cholecalciferol supplementation had a positive impact on insulin sensitivity and lipids. Keywords: glucocorticoid; hypercortisolism; 25-hydroxyvitamin D; cholecalciferol 1. Introduction Vitamin D is the precursor of a hormone with pleiotropic effects. Its deficiency has been largely investigated and shown to be associated with many complications including diabetes mellitus, adrenal insufficiency, cardiovascular disease, neurological disorders and other endocrinopathies [1,2,3]. Vitamin D, also known as cholecalciferol, is first formed in the skin by the photolysis of 7-dehydrocholesterol and after hydroxylated in the liver to 25-hydroxyvitamin D [25(OH)D]. It is further transformed in the kidney into 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) (calcitriol) that is the active form [4]. Cushing’s disease (CD) is characterized by a cortisol excess due to autonomous pituitary ACTH secretion. Patients with CD show many comorbidities such as cardiovascular disease, metabolic disease, diabetes mellitus, metabolic syndrome, dyslipidemia, obesity, osteoporosis/osteopenia and infections that contribute to increasing the mortality risk for these patients [5,6,7,8,9,10,11]. Indeed, GCs are key regulators of intermediary metabolism promoting hepatic gluconeogenesis and glycogenosis and on lipid metabolism favouring the deposition of fat to the upper trunk and the face [12]. They stimulate water diuresis, glomerular filtration rate and renal plasma flow and these effects result in arterial hypertension and atherosclerosis. GCs reduce bone remodelling, augment urinary calcium excretion and decrease the intestinal calcium absorption. In addition, they act on immune and hematological systems inhibiting the secretion of interleukins and increasing the red blood cell count, respectively [12]. An interesting relationship exists between glucocorticoids (GCs) and vitamin D values [13,14,15,16]. Indeed, exogenous steroid therapy has been reported to be associated with vitamin deficiency [13]. The mechanism by which GCs reduce 25(OH)D levels is not direct, but indirect, regulating vitamin D receptor expression in many tissues and cells [17,18]. Some authors have shown that treatment with dexamethasone in mice was associated with a decrease in 1α-hydroxylase which is involved in the conversion from 25(OH)D3 to the active metabolite 1,25(OH)2D3 and an increase in 24-hydroxylase, able to break down the active form of calcitriol, in inactive, reducing circulating 25(OH)D levels [19]. In a clinical setting, controversial data have been reported on GCs effects on serum 1,25(OH)2D concentrations [20,21,22,23]. A likely reason for these discrepancies might be the marked heterogeneity of the studied groups. Some of these studies were performed in humans [23,24,25,26], and others in animal models [27,28], but only a few studies were conducted in subjects with endogenous hypercortisolism. Low serum 25(OH)D levels have significant skeletal and extra-skeletal consequences such as myopathy, high risk of fractures and also affect the immune system and metabolism. All of these systems are impaired in patients with hypercortisolism and a vitamin D deficiency may provide a further aggravation of CD comorbidities. Indeed, it may cause a reduced intestinal calcium absorption resulting in secondary hypocalcemia and hyperparathyroidism leading to a bone demineralization. Its deficiency can contribute to obesity and metabolic syndrome due to the lack of antiadipogenic effect of vitamin D and to cardiovascular disease by a deregulation of the renin–angiotensin–aldosterone system, cardiac contractility and increase in cytokine release [29]. In the end, vitamin D deficiency causes impaired insulin sensitivity and immune system [30]. The discrepancies that emerge in the above-mentioned studies suggest a need to investigate the role of 25(OH)D in patients with CD. Therefore, the primary objective of the study was to evaluate serum 25(OH)D levels in patients with CD, compared to a control group matched for age, BMI and gender, and search for a possible correlation with the degree of hypercortisolism. The secondary objective was to evaluate the response to a course of 150,000 U of cholecalciferol on metabolic and hormonal parameters 6 weeks after the administration in patients with CD. 2. Materials and Methods 2.1. Subjects and Study Design Fifty patients with active CD, 43 of them women (86%) and 7 of them men (20%) (mean age 50.9 ± 17.4 years; mean duration of disease 32.5 ± 22.4 years), followed from January 2016 to December 2020, by the Endocrinology of the University of Palermo, were included in the current study. Clinical practice guidelines and a recent consensus statement were used to diagnose CD [31,32]. We recruited a control group matched for age, BMI and gender in the same temporal period. It was composed of 48 patients, 33 women (82.5%) and 7 men (17.5%) (mean age 48.5 ± 13.4 years) were evaluated by our team for a suspicion not biochemically confirmed of Cushing’s syndrome (CS). In all patients, we evaluated phenotypic characteristics including moon face, facial rubor, dorsal fat pad or buffalo hump, defined as a fatty tissue deposit between the shoulders, purple striae, defined as wide, reddish-purple streaks, and myopathy defined as muscle weakness at the proximal level. We also assessed cardiovascular, metabolic and bone comorbidities. The diagnosis of metabolic syndrome was based on National Cholesterol Education Program Adult Treatment Panel (NCEP ATP III) criteria, while the diagnosis of diabetes mellitus and prediabetes were based on the American Diabetes Association (ADA, Arlington, VA, USA) criteria [33,34]. Among patients with diabetes mellitus (18 out of 50), 16 were treated with metformin alone, while 2 were treated with a combination of metformin and GLP-1 agonist receptors. Metformin and GLP-1 agonist receptors were discontinued 24 h and 2 weeks before metabolic evaluations, respectively, to avoid any interference with metabolic parameters. Diabetic patients were on good metabolic control (HbA1c ≤ 7%). Both CD patients and the controls were naïve to cholecalciferol. In CD and the controls, BMI and waist circumference (WC), fasting serum lipids (total cholesterol (TC), HDL cholesterol, LDL cholesterol and triglycerides (TG), HbA1c, glycaemia, insulinaemia, albumin corrected calcium, phosphorus and parathyroid hormone (PTH) were assessed. To avoid seasonal influences, serum 25(OH)D levels were only assayed between winter and spring seasons (November–April). We evaluated urinary free cortisol (UFC) as the mean of three 24 h urine collections (mUFC), cortisol after a low dose of dexamethasone suppression test and plasma ACTH. We defined patients with mild hypercortisolism when mUFC levels not exceeding twice the upper limit of normal (ULN), moderate hypercortisolism by a level of mUFC more than 2 to 5 times the ULN and severe hypercortisolism by a mUFC level more than 5 times the ULN, as previously reported [35]. As defined by the Endocrine Society guidelines, we considered 25(OH)D deficiency for values < 20 ng/mL (50 nmol/L), insufficiency as levels of 20–30 ng/mL (50–75 nmol/L) and sufficiency for values ≥ 30 ng/mL (≥75 nmol/L) [36]. In addition, severe 25(OH)D deficiency was defined by levels < 10 ng/mL (<25 nmol/L) [37]. As markers of insulin sensitivity, we calculated the homeostatic model of insulin resistance (HOMA2-IR) [38], and in 32 patients with CD and in 40 controls who had no previous diagnosis of diabetes, we also evaluated the Matsuda index of insulin sensitivity (ISI-Matsuda) [39], the oral disposition index (DIo) [40] and the area under the curve for insulin (AUC2h insulinemia) and glucose (AUC2h glycaemia). At the baseline visit, we assessed patients’ lifestyle habits: physical activity level, balanced diet (consumption of dairy products, meat, coffee, soft drinks), exposure to ultraviolet (UV) radiation, smoking status and alcohol use. We excluded patients with adrenal-dependent hypercortisolism, pregnancy, taking oral contraceptives, liver or renal disease, cholecalciferol supplementation within 3 months before the study, malabsorption syndrome and exposure to ultraviolet (UV) radiation (solarium and sunscreen usage). Patients with CD received an oral load dose of cholecalciferol of 150,000 UI [41,42] and biochemical parameters (metabolic and hormonal) were assayed 6 weeks after administration. The study protocol was approved by the Ethics Committee of the Policlinico Paolo Giaccone hospital. All patients signed a written informed consent. 2.2. Assays Biochemical parameters were measured by standard methods (Modular P800, Roche, Milan, Italy), as previously reported [9]. Hormonal parameters were measured by electrochemiluminescence immunoassay (ECLIA, Elecsys, Roche, Milan, Italy) following the manufacturer’s instructions, as previously reported [9]. Mean UFC was measured by mass spectrometry, as previously reported [35]. Normal values for hormonal markers were defined as follows: ACTH 2.2–14 pmol/L and UFC 59–378 nmol/24 h. 2.3. Statistical Analysis We used statistical Packages for Social Science SPSS version 19 (SPSS, Inc., Chicago, IL, USA) for data analysis. The normality of quantitative variables was tested with the Shapiro–Wilk test. We calculated mean ± SD for continuous variables and rates and proportions for categorical variables. The differences between paired continuous variables (CD vs. controls) were analysed using one-way ANOVA. We used univariate Pearson correlation to evaluate the relations with the outcome parameters. For those variables which were significant at univariate correlation, we performed multiple linear regression analysis to identify independent predictors of the dependent variable 25(OH)D. A p-value of 0.05 was considered statistically significant. A receiver operating characteristic (ROC) analysis was performed to investigate the diagnostic ability of significantly associated risk factors to predict 25(OH)D deficiency. The ROC curve is plotted as sensitivity versus 1-specificity. The area under the ROC curve (AUC) was estimated to measure the overall performance of the predictive factors for serum 25(OH)D deficiency. 3. Results At baseline, patients with CD had a higher frequency of arterial hypertension (p = 0.009), osteoporosis/osteopenia (p = 0.002), hypercholesterolemia (p = 0.002), diabetes mellitus (p = 0.026), myopathy (p < 0.001), facial rubor (p = 0.005), buffalo hump (p = 0.002) and hypovitaminosis deficiency (p = 0.001) than the controls (Table 1). Table 1. Comorbidities of patients with CD and controls at baseline. By contrast, the controls had a higher frequency of vitamin D sufficiency (p = 0.004). Patients with CD also had higher WC (p = 0.031), PTH (p = 0.003), glycaemia (p = 0.010), HbA1c (p = 0.004), total cholesterol (p < 0.001), LDL cholesterol (p = 0.002), ACTH (p < 0.001), mUFC (p = 0.001), cortisol after a low dose of dexamethasone suppression test (p = 0.001) and lower 25(OH)D (p < 0.001), ISI-Matsuda (p = 0.007) and DIo (p = 0.003) than the controls (Table 2). Table 2. Anthropometric and biochemical parameters of patients with CD and controls at baseline. Six weeks after cholecalciferol treatment, patients with CD showed increased serum calcium (p = 0.017), 25(OH)D (p < 0.001), ISI-Matsuda (p = 0.035), DIo (p = 0.045) and a decrease in PTH (p = 0.004) and total cholesterol (p = 0.017) levels than at baseline (Table 3). Table 3. Anthropometric and biochemical parameters at baseline and 6 weeks after cholecalciferol supplementation in patients with CD. Considering the degree of hypercortisolism, in patients with severe hypercortisolism we observed 25(OH)D deficiency in 73.1% of cases (53.8% of them had a severe deficiency), insufficiency in 12.5% of cases and sufficiency in 6.3% of cases. In patients with moderate hypercortisolism, we observed 25(OH)D deficiency in 64.7% of cases (29% of them had a severe deficiency), insufficiency in 23.5% of cases and sufficiency in 11.8% of cases. In patients with mild hypercortisolism, we observed deficiency in 52.9% of cases (20% of them had a severe deficiency), insufficiency in 41.1% of cases and sufficiency in 6% of cases. At univariate correlation, in patients with CD at baseline, serum 25(OH)D was inversely correlated with glycaemia (r = −0.385, p = 0.019), HbA1c (r = −0.391, p = 0.017), WC (r = −0.373, p = 0.023), mUFC (r = −0.466, p = 0.033) and cortisol after a low dose of dexamethasone suppression test (r = −0.299, p = 0.049) (Table 4). In the controls, at baseline, 25(OH)D was inversely correlated with WC (r = −0.130, p = 0.042) (Table 4). Table 4. Correlation of serum 25-hydroxyvitamin D [25(OH)D] levels at baseline in patients with Cushing’s disease and controls. Multivariate analysis showed that mUFC was independently inversely associated with 25(OH)D (p = 0.010) in patients with CD (Figure 1). In the controls, no significant associations were found. Figure 1. Independent variables associated with serum 25(OH)D in patients with active CD at multivariate analysis. mUFC: mean urinary free cortisol. The ROC analysis showed that a cut-off of mUFC > 240 nmol/24 h was associated with 25(OH)D deficiency with a specificity of 100% and a sensitivity of 56.9%, AUC 0.803 (Figure 2). Figure 2. 25(OH)D status and mUFC. ROC curve showed that a cut-off of mUFC > 240 nmol/24 h could be associated with 25(OH)D deficiency. Statistical analysis was performed using the chi-square test and receiver operator characteristic (ROC) curve analysis. 4. Discussion The present study shows that patients with active CD have lower serum 25(OH)D values than the controls and that serum 25(OH)D levels are inversely correlated with mUFC in CD. In addition, a cholecalciferol load is associated after 6 weeks from the administration with an improvement of serum 25(OH)D and glycometabolic and lipid parameters in patients with CD. Furthermore, we found that higher values of mUFC than 240 nmol/24 h are predictive of 25(OH)D deficiency. The degree of hypercortisolism evaluated by UFC levels is a useful parameter to quantify the “amount” of cortisol secretion, even though it is not sufficiently exhaustive to assess the aggressiveness of the disease [35]. Indeed, a combination of several factors, including the degree of hypercortisolism, but also the duration of the disease, age and other individual predisposing factors, contribute to the aggressiveness of the disease. Long-standing studies were conducted on vitamin D levels in patients with CD. Patients with CD, with and without osteopenia, were compared before and after oral calcium load showing that serum 1,25 (OH)2D3 plasma levels were higher in subjects with osteopenia than in those without it, likely due to a secondary increase in PTH levels as an effect of hypercortisolism [19]. Another study investigated the effect of hypercortisolism and eucortisolism, showing a reduction in serum 25(OH)D levels, but not in 1,25 (OH)2D3 in patients with hypercortisolism. By contrast, two other studies found normal serum 25(OH)D values in patients with CD [23,24]. However, all the above-mentioned studies were conducted on a small sample of patients. Recently, a meta-analysis conducted on the studies that evaluated serum 25(OH)D levels in patients treated with GCs reported lower serum 25(OH)D levels in these patients compared to healthy subjects [16]. A hypothetical reason was that patients with CD had low 24-hydroxylase levels than the controls, causing an alteration of vitamin D catabolism. An interesting in vitro study in NCI-H295R cells found that treatment with 1,25(OH)2D3 decreased corticosterone secretion without affecting cortisol levels [43]. As expected, in the current study, we showed that treatment with cholecalciferol is associated with an improvement in insulin sensitivity and total cholesterol values in patients with CD. Indeed, cholecalciferol supplementation has been reported to be associated with improved peripheral insulin sensitivity and secretion in patients at high risk of diabetes or with type 2 diabetes [44]. A recent meta-analysis on 41 randomized controlled studies showed a significant improvement in total cholesterol levels after cholecalciferol supplementation. In addition, this improvement was more pronounced in patients with vitamin D deficiency [45,46]. A recent study compared the metabolism of vitamin D in patients with CD and controls after cholecalciferol treatment, showing that patients with CD had a higher 25(OH)D/24,25(OH)2D ratio than healthy controls, likely due to a decrease in 24-hydroxylase activity. The authors concluded that this alteration of vitamin D catabolism might have an influence on the effectiveness of cholecalciferol therapy in CD [47]. There are some limitations in the current study. First, the study is not randomized. Second, the dose of cholecalciferol administered is the same independently of the baseline serum 25(OH)D values. Third, we did not register the intake of milk and dairy products of the patients included in the study. In conclusion, serum 25(OH)D levels are lower in subjects with active CD compared to controls matched for age, BMI and gender. Vitamin D deficiency is correlated with mUFC and values of mUFC > 240 nmol/24 h are predictive of 25(OH)D deficiency. In addition, cholecalciferol supplementation has a positive impact on insulin sensitivity and lipids and therefore should be considered part of the treatment of patients with CD at diagnosis, in order to improve the comorbidities. However, further studies are needed to evaluate a possible effect of cholecalciferol supplementation on the aggressiveness of CD. Author Contributions Conceptualization, V.G. and F.D.G.; methodology, V.G.; software, V.G.; validation, V.G., F.D.G. and C.G.; formal analysis, V.G.; investigation, V.G.; resources, F.D.G.; data curation, V.G.; writing—original draft preparation, V.G.; writing—review and editing, V.G.; visualization, V.G.; supervision, C.G.; project administration, C.G.; funding acquisition, C.G. 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Each month, The Clinical Advisor makes one new clinical feature available ahead of print. Don’t forget to take the poll. The results will be published in the next month’s issue. A 35-year-old woman is seen in the outpatient clinic for evaluation of an incidental pituitary macroadenoma. Her medical history is significant for hypertension, diabetes, hyperlipidemia, polycystic ovary syndrome, and obesity. She initially presented to the emergency department (ED) a week ago after an episode of right visual field changes that she described as waviness in her right eye and right hemibody sensory changes without motor deficits. While in the ED, she underwent a full workup for possible stroke, which was negative. Magnetic resonance imaging (MRI) of her brain without contrast revealed a 12-mm pituitary lesion; a repeat MRI with contrast was then ordered (Figure). No serum hormonal panel was available for review from ED records. Figure. Magnetic resonance imaging of the case patient. Left image: sagittal view. Right image: coronal view with contrast. Credit: Melissa Wasilenko, MSN, RN Upon further questioning of her medical history during the clinic visit, the patient notes that a few years ago she was attempting to become pregnant and was evaluated by her gynecologist for amenorrhea. At that time, she reportedly completed an endocrine laboratory workup that showed a slightly elevated prolactin level between 30 and 40 ng/mL (normal level in nonpregnant women, <30 ng/mL). Per the patient, the minimal elevation was not enough to concern the gynecologist and no MRI was ordered at that time. Her gynecologist recommended that she lose weight. Her menses returned to normal with weight loss. With a history of disrupted menstrual cycles, infertility, and patient reported elevated prolactin level, there is high suspicion for endocrine disruption. A complete pituitary panel is ordered again to examine the current hormone function considering the recent MRI findings. This revealed a prolactin of 33.7 ng/ml, and all other hormonal levels were within normal limits. Because the patient reports multiple episodes of visual disturbances and the size of the pituitary adenoma on MRI, a neuro-ophthalmology referral is initiated for visual field testing and to determine if the pituitary macroadenoma is causing mass effect and compressing the optic nerve. The neuro-ophthalmologist found she had no visual field defect from her adenoma on visual field testing and believed that her visual disturbances were probably migraine in nature. Discussion Pituitary gland tumors are usually found incidentally on imaging studies obtained for other reasons or in workup of patients with abnormal endocrine hormone levels (both decreased and increased levels) or with symptoms of mass effect from the lesions.1 These tumors are typically benign in nature; cases with malignancy are extremely rare.1 The exact pathophysiology of pituitary adenomas remains unknown but is thought to be linked to heredity, hormonal influences, and genetic mutations.1 Pituitary tumors are commonly found in adults between the ages of 35 and 60 years of age.2,3 The estimated prevalence of pituitary adenomas varies widely by study and findings are typically based on autopsy and radiology data. Surveillance, Epidemiology, and End Results (SEER) Program data from 2004 to 2018 show an incidence rate of pituitary adenomas and pituitary incidentalomas of 4.28 ± 0.04 and 1.53 ± 0.02 per 100,000 population.4 Pituitary tumors have been found in 14.4% of unselected autopsy cases and 22.5% of radiology tests.1 The SEER data suggest that incidence rates are similar among women and men but are higher among women in early life and higher among males in later life.5 Rates of prolactinomas (prolactin-secreting tumors) and corticotropinomas (adrenocorticotropic hormone-secreting tumors; Cushing disease) are higher in women than men.6 Earlier SEER data showed a significantly higher incidence of pituitary adenomas in Black individuals compared with other racial/ethnic groups; several factors may account for this discrepancy such as the higher stroke rate in this population, which leads to a greater likelihood for brain imaging that detects incident pituitary tumors.5 Incidental findings of pituitary adenoma may be found during workup related to hormonal dysfunction (amenorrhea, galactorrhea, fertility disorders, sexual dysfunction), noticeable vision change, new-onset headaches, or imaging performed for other diagnostic purposes.7 Pituitary Types Pituitary tumor types are differentiated by location, size, and functional status. Pituitary tumors commonly arise from the anterior portion of the gland (adenohypophysis) and rarely from the posterior portion (neurohypophysis).2 Both adenohypophyseal and neurohypophyseal tumors are commonly benign and slow-growing.1 Malignant pituitary tumors account for less than 1% of pituitary lesions and are usually metastases from breast and lung cancers.3 Adenohypophyseal carcinoma is rare, with less than 140 reported cases.2 Pituitary tumors are categorized by the size1,2: Microadenomas (<10 mm) Macroadenomas (>10 mm to 40 mm) Giant adenomas (>40 mm) Pituitary adenomas are further classified as functioning (hormone-secreting) or nonfunctioning (nonsecreting).1,6 If the adenoma is functioning, hormone levels will be found in excess. If the levels are within normal limits, a nonfunctioning pituitary adenoma is suspected. Functioning Tumors Approximately 65% of all pituitary adenomas are functioning tumors.2 Functioning pituitary adenomas present in various ways depending on which hormone is involved and the level of hormone secretion. Prolactinomas are the most common type of functioning adenomas followed by growth hormone-secreting and adrenocorticotropic hormone-secreting pituitary tumors. Adenomas secreting thyrotropin and follicle-stimulating hormone are less commonly found.2 Clinical features of functional pituitary adenomas are outlined in Table 1.2.8 Table 1. Clinical Features and Laboratory Findings of Functioning Pituitary Adenomas Nonfunctioning Tumors Approximately 20% to 30% of pituitary adenomas are nonfunctional.3 These tumors may go undiagnosed for years until the mass of the tumor starts to effect surrounding structures and causing secondary symptoms such as compression of the optic chiasm causing vision impairments. Nonfunctioning pituitary adenomas and prolactinomas (functioning) are the 2 most common types of pituitary adenomas.2,3 The consulting clinician must understand the difference in pathology of these 2 types of lesions, what diagnostic test to order, how to interpret the test results, and which specialty to refer the patient to best on the initial workup findings. Initial Workup Proper baseline workup should be initiated before referring patients with incidental pituitary adenoma to a specialist. The initial workup includes imaging, blood work to determine if the pituitary adenoma is causing hormonal dysfunction, and neuro-ophthalmology referral for visual field testing to determine if the optic nerve/chiasm is impacted. Imaging The most accurate diagnostic modality of pituitary gland pathology is MRI with and without contrast. The MRI should focus on the hypothalamic-pituitary area and include contrasted imaging to evaluate the soft tissue within the intracranial structure.9 The coronal and sagittal views are the best to display the pituitary gland width and height and identify abnormalities.9 The MRI provides a detailed evaluation of the pituitary gland related to adjacent structures within the skull, which helps to detect microalterations of the pituitary gland.10 If a pituitary adenoma is an incidental finding on another imaging modality (such as a computed tomography scan or MRI without contrast), an MRI with and without contrast that focuses on the pituitary gland should be obtained. Pituitary Laboratory Panel A complete pituitary panel workup should be obtained including prolactin, thyrotropin, free thyroxine, cortisol (fasting), adrenocorticotropic hormone, insulinlike growth factor 1, growth hormone, follicle-stimulating hormone, luteinizing hormone, estradiol in women, and total testosterone in males.1 Tests should be completed in the morning while fasting for the most accurate results. For instance, normally cortisol levels drop during fasting unless there is abnormality. Table 2 below shows normal laboratory ranges for a complete pituitary panel. Serum prolactin levels can slightly increase in response to changes in sleep, meals, and exercise; emotional distress; psychiatric medications; and oral estrogens. If the initial prolactin level is borderline high (21-40 ng/mL), the test should be repeated. Normal levels are higher in women than in men. Microadenomas may cause slight elevations in prolactin level (ie, <200 ng/mL), while macroadenomas are likely to cause greater elevations (ie, >200 ng/mL).1 Patients with giant prolactinomas typically present with prolactin levels ranging from 1000 ng/mL to 100,000 ng/mL.11 Perimetry Pituitary adenomas may cause ophthalmologic manifestations ranging from impaired visual field to diplopia because of upward displacement of the optic chiasm. The optic chiasm is located above the pituitary gland and a pituitary tumor that grows superiorly can cause compression in this area.12 Optic chiasm compression from a pituitary adenoma commonly causes bitemporal hemianopsia.2 If the tumor volume is promptly reduced by surgical resection or medication (in the case of prolactinomas), initial vision changes due to compression may be reversible.12 Baseline and routine follow-up perimetry are important in patients with pituitary adenoma, as symptoms of optic chiasm compression may go unnoticed by patients as visual field deficits often develop gradually. Also, post-treatment perimetry assessments can be used to compare the initial testing to evaluate reversible visual field deficits. It is recommended that patients with pituitary adenomas (both function and nonfunctiong) receive neuro-ophthalmologic evaluations twice a year to ensure no visual changes have occurred.12 Referral to a Specialist Management of pituitary adenomas requires a multidisciplinary team of specialists including endocrinologists, neurosurgeons, and neuro-ophthalmologists. The type of adenoma governs which specialist patients with incidental adenoma should see first. Patients with functioning pituitary adenomas should be referred to an endocrinologist before a neurosurgeon. The most prevalent functioning adenomas, prolactinoma, are initially treated with dopamine agonist medications.1,6 A patient with prolactinoma would only need to see a neurosurgeon if they have a macroadenoma that is not responsive or only partially responsive to dopamine agonists therapy or is causing vision deficits related to compression of the optic chiasm.2 Patients with nonfunctioning pituitary adenomas should first be referred to a neurosurgeon to discuss surgical options versus observation. The recommended treatment for patients with nonfunctioning adenomas and clinical features of mass effect (ie, visual deficits) is surgery.1,6 If the patient is asymptomatic with no signs of visual field deficits, the neurosurgery team may recommend continued surveillance with serial imaging and serial perimetry screenings.12 The patient in the case was found to have a nonfunctioning pituitary adenoma (prolactin was 33.7 ng/mL). Neuro-ophthalmology did not find any visual field defect upon initial assessment; the patient decided to continue observation with serial imaging (MRI) and serial neuro-ophthalmology assessments. Serial imaging with MRI brain revealed slow but real progression of the pituitary macroadenoma (12 mm initially; 13 mm 6 months later; and 14 mm 1 year from initial MRI findings). Although the patient still did not have any visual field defects per the neuro-ophthalmology reassessments, the documented growth on MRI over a short period of time was enough to make the patient more amendable to surgical resection. The patient underwent trans-sphenoidal resection of the pituitary lesion approximately 16 months after discovery of the tumor. Conclusion A thorough workup including laboratory testing, imaging, and vision field testing is the foundation of an effective referral process for pituitary adenomas and guides which specialist is consulted first. If patients are referred before initial workup is completed, delays in care, unnecessary specialty visits, and increased overall health care costs may occur. Melissa Wasilenko, MSN, RN, is a registered nurse at Lyerly Neurosurgery in Jacksonville, Florida. She is currently pursuing a doctorate in nursing practice with a focus in family medicine at the University of North Florida in Jacksonville. References 1. Russ S, Anastasopoulou C, Shafiq I. Pituitary adenoma. 2021 Jul 18. In: StatPearls. StatPearls Publishing; 2022 Jan–. Updated July 18, 2021. 2. Greenberg MS. Tumors of non-neural origin. In: Handbook of Neurosurgery, 9th ed. Thieme Medical Publishers: 2019; 1655-1755 3. Yeung M, Tahir F. The pathology of the pituitary, parathyroids, thyroid and adrenal glands. Surgery. 2020;38(12):747-757. 4. Watanabe G, Choi SY, Adamson DC. Pituitary incidentalomas in the United States: a national database estimate. World Neurosurg. 2021:S1878-8750(21)01780-0. doi:10.1016/j.wneu.2021.11.079 5. McDowell BD, Wallace RB, Carnahan RM, Chrischilles EA, Lynch CF, Schlechte JA. Demographic differences in incidence for pituitary adenoma. Pituitary. 2011;14(1):23-30. doi:10.1007/s11102-010-0253-4 6. Molitch ME. Diagnosis and treatment of pituitary adenomas: a review. JAMA. 2017;317(5):516-524. doi:10.1001/jama.2016.19699 7. Yao S, Lin P, Vera M, et al. Hormone levels are related to functional compensation in prolactinomas: a resting-state fMRI study. J Neurol Sci. 2020;411:116720. doi:10.1016/j.jns.2020.116720 8. Beck-Peccoz P, Persani L, Lania A. Thyrotropin-secreting pituitary adenoma. In: Feingold KR, Anawalt B, Boyce A, et al, ed. Endotext. MDText.com, Inc.; 2019. 9. Yadav P, Singhal S, Chauhan S, Harit S. MRI evaluation of size and shape of normal pituitary gland: age and sex related changes. J Clin Diagnostic Research. 2017;11(12):1-4. doi:10.7860/JCDR/2017/31034.10933 10. Varrassi M, Cobianchi Bellisari F, Bruno F, et al. High-resolution magnetic resonance imaging at 3T of pituitary gland: advantages and pitfalls. Gland Surg. 2019;8(Suppl 3):S208-S215. doi:10.21037/gs.2019.06.08 11. Shimon I. Giant prolactinomas. Neuroendocrinology. 2019;109(1):51-56. doi:10.1159/000495184 12. Vié AL, Raverot G. Modern neuro-ophthalmological evaluation of patients with pituitary disorders. Best Pract Res Clin Endocrinol Metab. 2019;33(2):101279. doi:10.1016/j.beem.2019.05.003 From the March/April 2022 Issue of Clinical Advisor

https://doi.org/10.1002/ccr3.5337 Abstract A 50-year-old woman with adrenal Cushing's syndrome and chronic hepatitis C developed an acute exacerbation of chronic hepatitis C before adrenectomy. After administration of glecaprevir/pibrentasvir was started, her transaminase levels normalized promptly and a rapid virological response also was achieved. Laparoscopic left adrenectomy was then performed safely. 1 INTRODUCTION Reports of reactivation of hepatitis C virus (HCV) and acute exacerbation of chronic hepatitis C associated with immunosuppressive therapy and cancer drug therapy are rarer than for hepatitis B virus (HBV) but have been made occasionally. In HBV infection, viral reactivation and acute hepatitis caused by an excess of endogenous cortisol due to Cushing's syndrome have been reported, but no acute exacerbation of chronic hepatitis C has been reported so far. Here, we report a case of acute exacerbation of chronic hepatitis C during the course of adrenal Cushing's syndrome. 2 CASE REPORT A woman in her 50s underwent a CT scan at a nearby hospital to investigate treatment-resistant hypertension and was found to have a left adrenal mass. Her blood tests showed low ACTH and HCV antibody positivity, and she was referred to our hospital because she was suspected of having Cushing's syndrome and chronic hepatitis C. There is nothing special to note about her medical or family history. She had never smoked and drank very little. Her physical findings on admission were 164.5 cm tall, 92.6 kg in weight, and a BMI of 34.2 kg/m2. Her blood pressure was 179 / 73 mmHg, pulse 64 /min (rhythmic), body temperature 36.8°C, and respiratory rate 12 /min. She had findings of central obesity, moon face, buffalo hump, and red skin stretch marks. Her blood test findings (Table 1) showed an increase in ALT, HCV antibody positivity, and an HCV RNA concentration of 4.1 log IU/mL. The virus was genotype 2. Cortisol was within the reference range, but ACTH was as low, less than 1.5 pg/mL. Her bedtime cortisol level was 7.07 μg/dL, which was above her reference of 5 μg/dL, suggesting the loss of diurnal variation in cortisol secretion. Testing showed the amount of cortisol by 24-hour urine collection was 62.1 μg/day, and this level of cortisol secretion was maintained. In an overnight low-dose dexamethasone suppression test, cortisol after loading was 6.61 μg/dL, which exceeded 5 μg/dL, suggesting that cortisol was autonomously secreted. Her contrast-enhanced CT scan (Figure 1) revealed a tumor with a major axis of about 30 mm in her left adrenal gland. MRI scans showed mild hyperintensity in the “in phase” (Figure 2A) and decreased signal in the “out of phase” (Figure 2B), suggesting her adrenal mass was an adenoma. Based on the above test results, she was diagnosed with chronic hepatitis C and adrenal Cushing's syndrome. She agreed to receive treatment with direct acting antiviral agents (DAAs) after resection of the left adrenal tumor. However, two months later, she had liver dysfunction with AST 116 U/L and ALT 213 U/L (Figure 3). HBV DNA was undetectable at the time of liver injury, but the HCV RNA concentration increased to 6.4 logIU/mL. Therefore, an acute exacerbation of chronic hepatitis C was suspected, and a percutaneous liver biopsy was performed. The biopsy revealed an inflammatory cell infiltration, mostly composed of lymphocytes and plasma cells and mainly in the portal vein area (Figure 4). Fibrosis and interface hepatitis were also observed, and spotty necrosis was evident in the hepatic lobule. No clear fat deposits were found in the hepatocytes, ruling out NASH or NAFLD. According to the New Inuyama classification, hepatitis equivalent to A2-3/F1-2 was considered. Because HBV DNA was not detected, no new drug was used, and no cause of liver damage, such as biliary atresia, was found; the patient was diagnosed with liver damage due to reactivation of HCV, with acute exacerbation of chronic hepatitis C. The treatment policy was changed, in order to treat hepatitis C before the left adrenal resection, and administration of glecaprevir/pibrentasvir was started. A blood test two weeks after the start of treatment confirmed normalization of AST and ALT, and a rapid virological response was achieved (Figure 3). Subsequently, HCV RNA remained negative, no liver damage was observed, and laparoscopic left adrenectomy was safely performed nine months after the initial diagnosis. The pathological findings were adrenal adenoma, and no atrophy was observed in the attached normal adrenal cortical gland. After the operation, hypertension improved and weight loss was obtained (92.6 kg (BMI: 34.2 kg/m2) before the operation, but 77.0 kg (BMI: 28.5 kg/m2) one year after the operation). ACTH increased, and the adrenal Cushing's syndrome was considered to have been cured. Regarding HCV infection, the sustained virological response has been maintained to date, more than 2 years after the completion of DAA therapy, and the follow-up continues. TABLE 1. Laboratory data on admission Hematology Chemistry WBC 6100 /μL TP 8.2 g/dL DHEA-S 48 /μL RBC 526 x 104 /μL Alb 3.4 g/dL PRA 0.7 ng/mL/h Hb 15.8 g/dL T-Bil 0.3 mg/dL ALD 189 pg/mL Ht 49.1 % AST 33 U/L PLT 25.5 x 104 /μL ALT 46 U/L Serological tests LDH 201 U/L CRP <0.10 mg/dL ALP 292 U/L HBsAg (-) γ-GTP 77 U/L anti-HBs (-) Coagulation BUN 13 mg/dL anti-HBc (+) PT 126.1 % Cr 0.63 mg/dL HBeAg (-) APTT 27.5 sec HbA1c 6.2 % anti-HBe (+) Cortisol 7.46 μg/dL anti-HCV (+) ACTH <1.5 pg/mL FBS 82 mg/dL Genetic tests Na 138 mmol/L HBV DNA Undetectable Cl 105 mmol/L HCV RNA 4.1 LogIU/Ml K 3.6 mmol/L HCV genotype 2 Ca 9.0 mg/dL Abbreviations: Hematology: WBC, white blood cells; RBC, red blood cells; Hb, hemoglobin; Ht, hematocrit; PLT, platelets. Coagulation: PT, prothrombin time; APTT, activated partial thromboplastin time. Chemistry: TP, total protein; Alb, albumin; T-Bil, total bilirubin; AST, aspartate transaminase; ALT, alanine aminotransferase; LDH, lactate dehydrogenase; ALP, alkaline phosphatase; γGTP, γ-glutamyl transpeptidase; BUN, blood urea nitrogen; Cr, creatinine; HbA1c, Hemoglobin A1c; FBS, fasting blood sugar; Na, sodium; Cl, chlorine; K, potassium; Ca, calcium; DHEA-S, dehydroepiandrosterone sulfate; PRA, plasma renin activity; ALD, aldosterone. Serological tests: CRP, C-reactive protein; HBsAg, hepatitis B surface antigen; anti-HBs, hepatitis B surface antibody; anti-HBc, hepatitis B core antibody; HBeAg, hepatitis B e antigen; anti-HBe, hepatitis B e antibody; anti-HCV, hepatitis C virus antibody. Genetic tests: HBV DNA, hepatitis B virus deoxyribonucleic acid; HCV RNA, hepatitis C virus ribonucleic acid. FIGURE 1 Open in figure viewerPowerPoint Contrast-enhanced CT examination. Contrast-enhanced CT examination revealed a tumor (arrow) with a major axis of about 30 mm in the left adrenal gland FIGURE 2 Open in figure viewerPowerPoint MRI image of the adrenal lesion. MRI showed mild hyperintensity in the "in phase" (A) and decreased signal in the "out of phase" (B), suggesting adrenocortical adenoma (arrow) FIGURE 3 Open in figure viewerPowerPoint Changes in serum transaminase and HCV RNA levels. All showed rapid improvement by administration of direct acting antivirals. ALT: alanine aminotransferase, AST: aspartate transaminase, HCV RNA: hepatitis C virus ribonucleic acid FIGURE 4 Open in figure viewerPowerPoint Pathological findings of tissues obtained by percutaneous liver biopsy. Infiltration of inflammatory cells, which was mostly composed of lymphocytes and plasma cells and a small number of neutrophils, was observed mainly in the portal vein area. This was accompanied by fibrous enlargement and interface hepatitis. Although the arrangement of hepatocytes was maintained in the hepatic lobule, spotty necrosis was observed in some parts. No clear fat deposits were found in the hepatocytes, and NASH or NAFLD was a negative finding. According to the New Inuyama classification, hepatitis equivalent to A2-3/F1-2 was considered (a; ×100, b; ×200, scale bar = 500 µm) 3 DISCUSSION Reactivation of HBV can cause serious liver damage. Therefore, it is recommended to check the HBV infection status before starting anticancer chemotherapy or immunotherapy and to continue monitoring for the presence or absence of reactivation thereafter.1, 2 On the other hand, there are fewer reports of the reactivation of HCV, and many aspects of the pathophysiology of HCV reactivation remain unclear. In this case, it is possible that chronic hepatitis C was acutely exacerbated due to endogenous cortisol secretion in Cushing's syndrome. Although the definition of HCV reactivation has not been defined, several studies3-5 have defined an increase of HCVRNA of 1.0 log IU/ml or more as HCV reactivation. In addition, the definition of acute exacerbation of chronic hepatitis C is that ALT increases to more than three times the upper limit of the reference range.3, 4, 6 Mahale et al. reported a retrospective study in which acute exacerbation of chronic hepatitis C due to cancer medication was seen in 11% of 308 patients.3 Torres et al. also reported that, in a prospective study of 100 patients with cancer medication, HCV reactivation was found in 23%.4 Given these reports, HCV reactivation potentially could occur quite frequently. However, Torres et al. reported that only 10% of all patients had acute exacerbations, none of which led to liver failure.4 Such data suggest that HCV reactivation may often be overlooked in actual cases without aggravation. Thus, the frequency of aggravation due to hepatitis virus reactivation is thought to be lower for HCV than for HBV. However, there are some reports of deaths from acute exacerbation of chronic hepatitis C.7-10 In addition, if severe hepatitis develops following viral reactivation, mortality rates have been reported to be similar for HBV and HCV.8, 11 Thus, reactivation of HCV is considered to be a pathological condition that requires caution, similar to HBV. Torres et al. reported that administration of rituximab or corticosteroids is a significant independent risk factor.4 In addition, there are reports of acute exacerbation of chronic hepatitis C due to corticosteroids administered as antiemetics and as immunosuppressive therapy.12-14 Therefore, excess cortisol can reactivate not only HBV but also HCV. The mechanism by which HCV is reactivated with cortisol is assumed to be decreased cell-mediated immunity due to rapid apoptosis of circulating T cells caused by glucocorticoids,4 enhancement of HCV infectivity by upregulation of viral receptor expression on the hepatocyte surface,15 and enhanced viral replication.16 In addition, there is a report that genotype 2 is more common in cases with acute exacerbation of chronic hepatitis C,4, 13 which is consistent with this case. Regarding HBV reactivation due to Cushing's syndrome, three cases of acute exacerbation of chronic hepatitis B have been reported.17-19 It is believed that Cushing's syndrome caused a decrease in cell-mediated immunity and humoral immunity due to an endogenous excess of cortisol, resulting in an acute exacerbation of chronic hepatitis B.13 As described above, because an excess of cortisol can cause reactivation of HCV, it is considered that a decrease in immunocompetence due to Cushing's syndrome, which is an excess of endogenous cortisol, can also cause reactivation of HCV and acute exacerbation of chronic hepatitis. However, as far as we can determine, no cases of Cushing's syndrome causing HCV reactivation or acute exacerbation of chronic hepatitis C have been reported and similar cases may be latent. Among the reports of acute exacerbation of hepatitis B due to adrenal Cushing's syndrome, there is a case in which the liver damage and viral load were improved only by adrenalectomy.17 Therefore, it is also possible that hepatitis C was improved by adrenal resection in this case. However, general anesthesia associated with adrenalectomy and the use of various drugs used for postoperative physical management should be avoided, if possible, in situations where some severe liver damage is present. In addition, reactivation of immunity due to rapid depletion of glucocorticoid, following resection of an adrenal tumor, may lead to exacerbation of liver damage. In this case, the amount of HCV and hepatic transaminase levels were improved rapidly by glecaprevir/pibrentasvir treatment, and the operation could be performed safely. If Cushing's syndrome is complicated by an acute exacerbation of hepatitis C, clinicians should consider including treatment strategies such as in this case. Summarizing the above, when liver damage appears in HCV-infected patients with Cushing's syndrome, it will be necessary to distinguish the acute exacerbation and reactivation of chronic hepatitis C. Treatment with DAAs may then be considered to be effective for reactivation of HCV and acute exacerbation of chronic hepatitis. 4 CONCLUSION We report a case of chronic hepatitis C with acute exacerbation during the course of Cushing's syndrome. At the time of cancer drug therapy and in the state of endogenous and extrinsic corticosteroid excess, it is necessary to pay attention not only to acute exacerbation of chronic hepatitis B but also to hepatitis C. ACKNOWLEDGEMENTS All authors would like to thank the patient and his family for allowing this case study. CONFLICT OF INTEREST The authors have no conflict of interests. AUTHOR CONTRIBUTIONS TO and KM were collected and analyzed the data and wrote and edited the manuscript. KH, ST, HO, KT, KM, and JK were involved in the patient's care and provided advice on the preparation of this case report. ETHICAL APPROVAL This study complied with the standards of the Declaration of Helsinki and the current ethical guidelines. CONSENT Written informed consent was obtained from the patient to publish this report in accordance with the journal's patient consent policy. From https://onlinelibrary.wiley.com/doi/10.1002/ccr3.5337

This article was originally published here Front Surg. 2022 Feb 2;8:806855. doi: 10.3389/fsurg.2021.806855. eCollection 2021. ABSTRACT PURPOSE: Currently, endoscopic transsphenoidal surgery (ETS) and microscopic transsphenoidal surgery (MTS) are commonly applied treatments for patients with pituitary adenomas. This meta-analysis was conducted to evaluate the efficacy and safety of ETS and MTS for these patients. METHODS: A computer search of Pubmed, Embase, Cochrane library, Web of Science, and Google Scholar databases was conducted for studies investigating ETS and MTS for patients with pituitary adenomas. The deadline is March 01, 2021. RevMan5.1 software was used to complete this meta-analysis after literature screening, data extraction, and literature quality evaluation. RESULTS: A total of 37 studies including 5,591 patients were included. There was no significant difference in gross tumor removal (GTR) and hormone-excess secretion remission (HES remission) between two groups [RR = 1.10, 95% CI (0.99-1.22), P = 0.07; RR = 1.09, 95% CI (1.00-1.20), P = 0.05]. ETS was associated with lower incidence of diabetes insipidus (DI) [RR = 0.71, 95% CI (0.58-0.87), P = 0.0008], hypothyroidism [RR = 0.64, 95% CI (0.47-0.89), P = 0.007], and septal perforation [RR = 0.32, 95% CI (0.13-0.79), P = 0.01] than those with MTS. CONCLUSION: This meta-analysis indicated that ETS cannot significantly improve GTR and HES remission. However, ETS could reduce the incidence of DI, hypothyroidism, and septal perforation without increasing the rate of other complications. SYSTEMATIC REVIEW REGISTRATION: https://www.crd.york.ac.uk/prospero/#myprospero, identifier: CRD42021241217. PMID:35187049 | PMC:PMC8847202 | DOI:10.3389/fsurg.2021.806855

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

Cushing’s disease is a progressive pituitary disorder in which there is an excess of cortisol in the body. While the disease can be treated surgically, this option is not possible for all patients. This is one of the approved medications that assist in controlling cortisol levels in people with Cushing’s disease. Korlym (mifepristone), developed and marketed by Corcept Therapeutics, is an FDA-approved treatment for high blood sugar (hyperglycemia) in adults with Cushing’s syndrome who have type 2 diabetes or glucose intolerance, and for whom surgery is not an option, or failed to control their symptoms. Bios of Cushies who have taken Korlym. Korlym discussions on the Message Boards. Learn more here and here. How does Korlym work? Cushing’s syndrome is characterized by high levels of cortisol in the body. Cortisol is a hormone that helps control a wide range of bodily functions, including blood pressure, salt levels, and blood sugar (glucose) levels. Too much cortisol may cause blood sugar levels to rise — a hallmark of both type 2 diabetes and glucose intolerance. Cortisol exerts its effects by binding to glucocorticoid receptors on the surface of cells. Korlym works by blocking cortisol’s access to these receptors, thereby preventing the chain of events leading to elevated blood sugar levels and diabetes. The medication is specifically meant to treat patients with endogenous Cushing’s syndrome, in which the body’s own overproduction of cortisol — usually due to the presence of a tumor — is the reason why hormone levels rise above healthy limits. Korlym in clinical trials Corcept conducted a Phase 3 trial (NCT00569582) to evaluate the safety and efficacy of mifepristone in 50 adults with endogenous Cushing’s syndrome and type 2 diabetes or impaired glucose tolerance, or high blood pressure alone. In the group with diabetes, 60% of participants showed a clinically meaningful improvement in glucose control in a two-hour oral glucose test. In the high blood pressure group, an improvement in diastolic blood pressure — the pressure in the arteries while the heart rests between beats — was seen in 38% of participants. In addition, an overall clinical improvement was seen in 87% of participants, as assessed by an independent review board. Board members looked at a range of symptoms, including body weight and composition, Cushing-like appearance, and psychological symptoms. Common adverse events reported in the study included fatigue, nausea, headache, low potassium, joint pain, vomiting, and swelling, called edema. Thickening of the lining of the uterus was reported among female participants. A pilot Phase 4 trial (NCT01990560) also demonstrated that mifepristone may be helpful in managing mild autonomous cortisol secretion (ACS), a subclinical form of Cushing’s syndrome in which patients do not display typical signs and symptoms of Cushing’s, despite having high cortisol levels. That pilot trial enrolled eight patients who received 300 mg tablets once daily for six months. In two patients, this dose was upped to 600 mg after two months due to a lack of clinical response. Treatment led to significant reductions in fasting glucose levels and insulin resistance — when certain cells no longer respond well to insulin, a hormone that controls how cells store and use glucose. Another study also indicated that mifepristone can effectively treat patients with ectopic Cushing’s syndrome. This is a form of Cushing’s caused by tumors found outside the brain’s pituitary gland, in which case the condition is known as Cushing’s disease. Other details Korlym’s blood absorption is higher when the medication is given with food. Patients should, therefore, take the medication within one hour of having a meal, so as to increase its effectiveness. Importantly, eating grapefruit or drinking grapefruit juice should be avoided while taking the medication, since both may interfere with its absorption. Korlym also may interact with a variety of other prescription meds, including cholesterol-lowering medicines simvastatin and lovastatin, the immunosuppressant cyclosporine, headache treatments ergotamine and dihydroergotamine, and opioid fentanyl. The antifungal treatment ketoconazole (sold under the brand name Nizoral, among others), used off-label to treat Cushing’s in the U.S., also can change the way Korlym is absorbed in the body. Since both medications can be prescribed simultaneously to Cushing’s patients, doctors should carefully evaluate their benefits, taking into account the potential risks. Additionally, mifepristone — Korlym’s active ingredient — blocks the action of the hormone progesterone, which is important for maintaining pregnancy. Taking Korlym during pregnancy will result in pregnancy loss. Therefore, Korlym should never be taken by women who are pregnant or by those who may become pregnant. Treatment with Korlym also may cause blood potassium levels to drop, a condition known as hypokalemia. Potassium is a mineral that helps the body regulate fluid balance, nerve signals, and muscle contraction. As such, patients’ potassium levels should be monitored closely in the first weeks after starting or increasing Korlym’s dose, as well as periodically thereafter.

DOI: 10.7759/cureus.22044 Cite this article as: Pattipati M, Gudavalli G (February 09, 2022) Association Between Cushing’s Syndrome and Sleep Apnea: Results From the National Inpatient Sample. Cureus 14(2): e22044. doi:10.7759/cureus.22044 Abstract Background Cushing’s syndrome is a metabolic disorder related to excess cortisol production. Patients with Cushing’s syndrome are at risk for the development of other comorbid medical conditions such as hypertension, diabetes, obesity, and obstructive sleep apnea. Obstructive sleep apnea has been well associated with endocrine disorders such as acromegaly and hypothyroidism. However, its causal association with Cushing’s syndrome is still unclear. We utilized a national database to study the prevalence of sleep apnea in Cushing’s syndrome. Hypothesis We hypothesized that patients with Cushing’s syndrome might have an increased prevalence of sleep apnea. Methods Patients aged above 18 years from the NIS database between 2017 and 2018 with a diagnosis of Cushing’s syndrome and sleep apnea were extracted using the 10th revision of the International Classification of Diseases (ICD-10) codes, with code E24 representing Cushing’s syndrome and G47.3 representing sleep apnea. The prevalence of sleep apnea and other comorbid medical conditions were identified using the ICD-10 codes. Logistic regression analysis was performed to examine the association between Cushing’s syndrome and sleep apnea. Results Cushing’s syndrome was prevalent in 0.037% (2,248 of 6,023,852) of all inpatient hospitalizations. Patients with Cushing’s syndrome were slightly younger (mean age: 54 ± 16 versus 58 ± 20) and more likely to be females (76%, 1,715 out of 2,248) and had higher rates of sleep apnea (21.9% versus 8.7%, p < 0.000) and obstructive sleep apnea (OSA) (18.6% versus 7.2%, p < 0.000) when compared to the general population. Cushing’s syndrome is independently associated with sleep apnea, with an unadjusted odds ratio (OR) of 2.94 (p < 0.01) and an adjusted odds ratio (aOR) of 1.79 after adjusting for demographics and other risk factors for sleep apnea and comorbid medical conditions (p < 0.01). Conclusions Cushing’s syndrome is associated with increased prevalence of sleep apnea and independent predictor of sleep apnea. Further prospective studies are recommended to validate the causal association. The high prevalence and coexistence of both these disorders validate screening for sleep apnea as part of routine workup in patients with Cushing’s syndrome and vice versa. 20220209-420-10au3f.pdf

Diurnal’s pioneering phase 2 study evaluates modified-release hydrocortisone for adrenal insufficiency Diurnal has announced that the first patient has been dosed in its phase 2 European clinical trial of modified-release hydrocortisone. It is treating people with adrenal insufficiency (AI), also known as Addison’s disease, while the trial also represents a significant marketing opportunity for the company across Europe and throughout the UK. The CHAMPAIN phase 2 study aims to evaluate the efficacy, safety and tolerability of modified-release hydrocortisone versus Plenadren in AI. It is anticipated that it will take six months to reach completion. Modified-release hydrocortisone is a preparation of hydrocortisone that has been specifically designed for patients with diseases of cortisol deficiency–such as AI–and additionally for congenital adrenal hyperplasia (CAH). It is approved for the latter disease in Europe and the UK under the commercial name Efmody. AI is a long-term endocrine disorder, which affects approximately 298,000 patients in Europe and the UK. It is caused by inadequate production of steroid hormones in the cortex of the adrenal glands. AI can result in severe fatigue and–if left untreated–adrenal crisis may be life-threatening. Martin Whitaker, CEO of Diurnal, commented: “We are pleased to have dosed our first patient in the CHAMPAIN phase 2 study for adults with AI as we seek to explore the efficacy of modified-release hydrocortisone in diseases of cortisol deficiency. “There is a high unmet need for adult patients suffering from AI across Europe with current treatment options leading to poor quality of life. We believe modified-release hydrocortisone has the potential to replicate the physiological overnight rise of cortisol in these patients and we look forward to the data readout from the CHAMPAIN study in H2 2022,” he added. From https://www.pharmatimes.com/news/first_adrenal_insufficiency_patient_dosed_in_phase_ii_study_1387551

Cushing’s disease is a progressive pituitary disorder in which there is an excess of cortisol in the body. While the disease can be treated surgically, this option is not possible for all patients. This is one of the approved medications that assist in controlling cortisol levels in people with Cushing’s disease. sturisa was approved in 2020 to treat adults with Cushing’s disease for whom pituitary surgery is ineffective or not an option. The oral medication works by inhibiting an enzyme called 11-beta-hydroxylase, which is involved in cortisol production. Isturisa, also known as osilodrostat or LCI699, is an approved treatment originally developed by Novartis, but now acquired by Recordati to treat people with Cushing’s disease, a condition in which a pituitary tumor causes the body to produce excessive levels of the stress hormone cortisol. In 2020, the U.S. Food and Drug Administration (FDA) approved Isturisa to treat adults with Cushing’s disease for whom pituitary surgery was not an option, or ineffective. Earlier that same year, the European Commission (EC) approved Isturisa to treat people with endogenous Cushing’s syndrome. The medication also was approved for the same indication in Japan in 2021. How does Isturisa work? Isturisa is an oral medicine that inhibits an enzyme called 11-beta-hydroxylase, which is involved in cortisol production. Blocking the activity of this enzyme prevents excessive cortisol production, normalizing the levels of the hormone in the body and easing the symptoms of Cushing’s disease. Isturisa in clinical trials A Phase 2 clinical trial (NCT01331239) investigated the safety and efficacy of Isturisa as a Cushing’s disease treatment. The trial that concluded in October 2019 initially was named LINC-1, but, through a study protocol amendment, patients who completed the study could continue into a second phase called LINC-2. The company published findings that covered both patient groups in the journal Pituitary. Data showed that Isturisa reduced cortisol levels in the urine of all patients by week 22. Urine cortisol levels reached and remained within a normal range in 79% of the patients by then. Common adverse effects included nausea, diarrhea, lack of energy, and adrenal insufficiency — a condition in which the adrenal glands are unable to produce enough hormones. A Phase 3 clinical trial (NCT02180217) called LINC-3 also assessed the safety and efficacy of Isturisa in 137 patients with Cushing’s disease (77% female, median age 40 years). Participants were given Isturisa for 26 weeks, with efficacy-based dose adjustments during the first 12 weeks. Then, the 71 participants with a complete response (those whose urine cortisol levels were within normal limits) at week 26 and who did not require a dose increase after week 12, were assigned randomly to either continue treatment with Isturisa or switch to a placebo. After this 34-week period, 86% of Isturisa-treated patients had normal urinary cortisol levels, as compared to 29% of participants given placebo. All participants then were given Isturisa for an additional 12 weeks. At the end of the 48-week study, 66% of participants had normal urine cortisol levels. Results from LINC-3 formed the basis for regulatory approvals of Isturisa. Common adverse side effects in the trial included nausea, headache, fatigue, and adrenal insufficiency. A multi-center, randomized, double-blind, placebo-controlled Phase 3 trial (NCT02697734) called LINC-4 further confirmed the safety and efficacy of Isturisa as a Cushing’s disease therapy. During the trial, patients received Isturisa or a placebo through a 12-week period followed by treatment with Isturisa until week 48. Top-line results showed that 77% of patients on Isturisa experienced a complete response after the 12-week randomized period, as compared to 8% of those on placebo. No new safety data were noted. A roll-over, worldwide Phase 2 study (NCT03606408) is recruiting patients who have successfully completed any of the previous clinical trials. Patients can continue to take the dosage they received during the initial trial. The aim of this study is to assess the long-term effects of Isturisa for up to five years.

This article was originally published here Microvasc Res. 2022 Jan 21:104323. doi: 10.1016/j.mvr.2022.104323. Online ahead of print. ABSTRACT PURPOSE: Macrovascular alterations are prominent in Cushing’s syndrome (CS). Microvascular abnormalities are yet to be established. This cross-sectional observational study aimed to evaluate microvascular changes in nailfold capillaries and their association with disease status and carotid intima-media thickness (CIMT) as a marker of atherosclerosis. METHODS: A total of 70 patients with CS [46 (65.7%) ACTH-dependent pituitary adenoma and 24 (34.3%) adrenocortical adenomas] and 100 healthy controls were enrolled. The microvascular structure was evaluated using nailfold video-capillaroscopy (NVC). RESULTS: The median number of capillaries was less [10 mm (IQR: 2, min-max:7-14) vs. 11 mm (IQR: 2, min-max:9-19) (p < 0.001)], the median limb diameter and capillary width were wider in the CS group than in the controls (p = 0.016 and p = 0.002, respectively). Microhemorrhages within limited areas were more frequent in the CS group than in the controls (p = 0.046). Observed capillary changes were similar among the patients with CS with remission or active disease. CIMT levels were higher in the CS group than in the controls and similar in subjects with active disease and remission. Univariate logistic regression analyses revealed that the number of capillaries and capillary widths were associated with body mass index (BMI), the presence of type 2 diabetes mellitus, HbA1c, and CIMT. CONCLUSION: Morphologic alterations present similarly in nailfold capillaries in subjects with CS regardless of disease status, resembling changes in chronic atherosclerotic diseases. Microvascular changes in nailfold capillaries measured using NVC can be used as a marker in the assessment of cardiovascular risk in patients with CS. PMID:35074338 | DOI:10.1016/j.mvr.2022.104323 From https://www.docwirenews.com/abstracts/rheumatology-abstracts/capillary-microarchitectural-changes-in-cushings-syndrome/

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

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

1. In patients with benign adrenal tumors, women are more likely to be diagnosed with mild autonomous cortisol secretion (MACS). 2. Patients with MACS have a higher prevalence and severity of cardiometabolic disease, namely hypertension and type 2 diabetes. Evidence Rating Level: 2 (Good) Study Rundown: While benign adrenal tumors are routinely incidentally discovered by imaging, not all these tumors have pathological effects, existing as nonfunctional adrenal tumors (NFAT). However, others overproduce steroids resulting in mild autonomous cortisol secretion (MACS) or Cushing’s syndrome (CS) if severe. The clinical impact of these diseases on cardiometabolic disease is poorly described. This study, therefore, sought to characterize the cardiometabolic disease burden and steroid excretion in this population via a cross-sectional study. Patients with benign adrenal tumors were classified with NFAT, MACS-1 (possible), MACS-2 (definite), or CS based upon clinical assessment and 1-mg overnight dexamethasone suppression test. Results revealed that MACS-2 and CS were more prevalent among women. Compared to patients in the NFAT group, patients with MACS-2 and CS were more likely to have hypertension, require antihypertensives, type 2 diabetes, and require insulin therapy. Taken together, this study supports that women with benign adrenal tumors are more likely to be diagnosed with MACS and are consequently at greater risk for hypertension and type 2 diabetes, warranting regular cardiometabolic assessment for this population. This study was limited by its cross-sectional study design and predefined clinical outcomes biased for cardiometabolic outcomes. Click to read the study in Annals of Internal Medicine Relevant Reading: Natural History of Adrenal Incidentalomas With and Without Mild Autonomous Cortisol Excess: A Systematic Review and Meta-analysis In-Depth [cross-sectional study]: In this prospective, cross-sectional study, 1305 patients diagnosed with incidental benign adrenal adrenocortical adenoma were selected across 14 participating centers. Patients with other diagnoses of cortisol excess such as primary aldosteronism or on cortisol-altering medications were excluded. Following clinical assessment and 1-mg overnight dexamethasone-suppression, patients were categorized into having a nonfunctional adrenal tumor (NFAT) (morning serum cortisol <50 nmol/L), possible mild autonomous cortisol secretion (MACS-1) (morning serum cortisol: 50-138 nmol/L), definite MACS (MACS-2) (morning serum cortisol: >138 nmol/L), or Cushing’s syndrome (CS) (presence of overt clinical symptoms of CS). The results found that while women made up the majority of the study cohort (67.3%), the proportion of females was more pronounced in the MACS-2 (73.6%) and CS (86.2%) groups. With respect to cardiometabolic disease, patients in the MACS-2 group were more likely to have hypertension (adjusted prevalence ratio [aPR], 1.15; 95% confidence interval [CI], 1.04-1.27), require three or more hypertensives (aPR, 1.31; 95% CI, 1.02-1.68) and requirement for insulin therapy (aPR, 1.89; 95% CI, 1.01 – 3.52) when compared to patients in the NFAT group. The same trend was found with greater significance for those in the CS group. The prevalence of dyslipidemia was not found to be significantly different between all groups. Additionally, these findings were not found to be attributed to other factors such as 1-mg DSG results, the presence of bilateral tumor, or adrenal tumor size. Finally, urinary steroid profiling found that patients with MACS and CS were more likely to have lower excretion levels of androgen metabolites and increased excretion levels of glucocorticoids. Overall, this study supports increased cardiometabolic disease burden amongst women with MACS. RELATED REPORTS Autonomous cortisol secretion correlated with mortality for adrenal incidentalomas Mutations in PKA catalytic subunit associated with Cushing’s syndrome Image: PD ©2022 2 Minute Medicine, Inc. All rights reserved. No works may be reproduced without expressed written consent from 2 Minute Medicine, Inc. Inquire about licensing here. No article should be construed as medical advice and is not intended as such by the authors or by 2 Minute Medicine, Inc. Tags: adrenal incidentalomaautonomouscardiometabolic diseasecortisol secretioncushing's syndromedexamethasone suppression From https://www.2minutemedicine.com/women-with-mild-autonomous-cortisol-secretion-are-at-greater-risk-for-cardiometabolic-disease/