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

Abstract Introduction Hypertension is one of the most common clinical features of patients with overt and subclinical hypercortisolism. Although previous studies have shown the coexistence of autonomous cortisol and aldosterone secretion, it is unclear whether aldosterone plays a role in hypertension among patients with hypercortisolism. Therefore, we examined the associations of plasma aldosterone concentrations (PACs) with hypertension among patients with overt and subclinical hypercortisolism. Methods This single-center retrospective cohort study included patients with adrenal tumor and serum cortisol levels after 1-mg dexamethasone suppression test >1.8 µg/dL (50 nmol/L). Using multivariable regression models adjusting for baseline characteristics, we investigated the association of PACs with systolic blood pressure and postoperative improvement of hypertension after the adrenalectomy. Results Among 89 patients enrolled in this study (median age, 51 years), 21 showed clinical signs of Cushing syndrome (overt hypercortisolism) and 68 did not show clinical presentations (subclinical hypercortisolism). We found that higher PACs were significantly associated with elevated systolic blood pressure among patients with subclinical hypercortisolism (adjusted difference [95% CI] = +0.59 [0.19-0.99], P = 0.008) but not among those with overt hypercortisolism. Among 33 patients with subclinical hypercortisolism and hypertension who underwent adrenalectomy, the postoperative improvement of hypertension was significantly associated with higher PACs at baseline (adjusted risk difference [95% CI] = +1.45% [0.35-2.55], P = 0.01). Conclusion These findings indicate that aldosterone may contribute to hypertension among patients with subclinical hypercortisolism. Further multi-institutional and population-based studies are required to validate our findings and examine the clinical effectiveness of the intervention targeting aldosterone for such patients. subclinical hypercortisolism, aldosterone, hypertension, adrenalectomy Issue Section: Clinical Research Article Cortisol production in the adrenal gland is regulated by the hypothalamus-pituitary-adrenal (HPA) axis. Subclinical hypercortisolism is a status characterized by the alteration of HPA axis secretion without typical signs or symptoms of overt hypercortisolism (eg, moon face, truncal obesity, easy bruising, thin extremities, proximal myopathy, cutaneous purple striae) [1, 2]. Although overt hypercortisolism can be detected by its clinical presentations or severe complications, it is sometimes challenging for clinicians to appropriately diagnose subclinical hypercortisolism because of the absence of such clinical presentations [2]. The 1-mg overnight dexamethasone suppression test (1-mg DST) measures the response of the adrenal glands to ACTH through the HPA axis and therefore has been widely used for screening and diagnosis of subclinical hypercortisolism [1, 3]. The European Society of Endocrinology Guideline has defined a partial suppression of the HPA axis (ie, serum cortisol levels after 1-mg DST [F-DST] > 1.8 µg/dL [50 nmol/L]) without clinical signs of overt cortisol hypersecretion as “possible autonomous cortisol secretion” and recommended screening these patients for metabolic disorders including hypertension and type 2 diabetes mellitus to offer appropriate treatment of these comorbidities [4]. Hypertension is one of the most common and distinguishing clinical features in patients with subclinical hypercortisolism [2] as well as overt hypercortisolism [5]. Although hypertension can be triggered by excess cortisol levels [5, 6], it is still unclear whether even slightly elevated cortisol levels among individuals with subclinical hypercortisolism contribute to the occurrence of hypertension. This raises another potential mechanism to cause hypertension such as the coexistence of hyperaldosteronism (ie, excess aldosterone that is an essential steroid hormone for sodium reabsorption, water retention, and blood pressure control) [7]. Previous studies have reported that 10% to 20% of primary aldosteronism is accompanied by cortisol-producing adenoma [8-10], and autonomous cortisol secretion was decreased after the resection of the aldosterone-producing adenoma (a subtype of primary aldosteronism) [11]. Furthermore, a previous mass spectrometry-based analysis revealed that cortisol secretion was frequently found in patients with primary aldosteronism [12]. Although these studies have examined cortisol biosynthesis in primary aldosteronism [13], evidence about whether aldosterone plays a role in the occurrence of hypertension among people with subclinical hypercortisolism is limited. To address this knowledge gap, we performed a cohort study examining the association between aldosterone and hypertension among patients with adrenal tumor and F-DST >1.8 µg/dL, stratified by whether patients had clinical signs of Cushing syndrome or not. We first analyzed the cross-sectional association between aldosterone and blood pressure at baseline. Then, we analyzed the longitudinal association between aldosterone at baseline and the improvement rate of hypertension after the adrenalectomy. Last, to further clarify the role of aldosterone in the regulation of blood pressure in subclinical hypercortisolism, we described the difference in aldosterone response to ACTH after the adrenalectomy according to the postoperative improvement of hypertension. Materials and Methods Data Sources and Study Participants A retrospective cohort study was designed to assess the clinical characteristics (focusing on aldosterone) among patients with hypercortisolism at the Yokohama Rosai Hospital from 2008 to 2017. We enrolled 89 patients with adrenal tumor and F-DST > 1.8 µg/dL (50 nmol/L) [3, 4, 14]. We then categorized them into 2 groups: (1) overt hypercortisolism (F-DST > 5.0 µg/dL [138 nmol/L]) and having clinical signs of Cushing syndrome (moon face, central obesity, dorsocervical fat pad [buffalo hump], purple striae, thin skin, easy bruising, and proximal myopathy] [15]) and (2) subclinical hypercortisolism (not having such clinical signs). All patients with overt hypercortisolism in this study showed F-DST > 5.0 µg/dL (138 nmol/L). The study was approved by the research ethics committee of the Yokohama Rosai Hospital, and all participants provided written informed consent. Measurements Demographic characteristics were self-reported, and body mass index (BMI) was calculated using measured weight and height. Systolic blood pressure was measured in the sitting position using a standard upper arm blood pressure monitor after a 5-minute rest in a quiet place [16]. The mean of 2 measurements was recorded. If the measurement was done only once on a given occasion, the level obtained was recorded. When the patients were already taking antihypertensives at enrollment, they were asked to report their blood pressure levels at the diagnosis of hypertension (ie, systolic blood pressure before starting antihypertensives). Blood samples were collected at 8:00 AM after the patient had rested in the supine position for 30 minutes. We measured F (µg/dL, × 27.6 for nmol/L) and ACTH (pg/mL, × 0.220 for pmol/L) using chemiluminescent enzyme immunoassay and electrochemiluminescent immunoassay, respectively. Plasma aldosterone concentrations (PACs; ng/dL, × 27.7 for pmol/L) and plasma renin activities (PRAs; ng/mL/h) were measured using radioimmunoassay. Any antihypertensive drugs were replaced with calcium channel antagonists (including dihydropyridine calcium channel antagonists) and/or α blocker several weeks before the measurement of PACs and PRAs according to the clinical guideline of the Japan Endocrine Society [17]. We also measured urine aldosterone (µg/day × 2.77 for nmol/d) and urine cortisol (µg/day, × 2.76 for nmol/d) using radioimmunoassay. The tumor size was estimated using contrast-enhanced thin-section computed tomography scans of the adrenal glands. To evaluate whether the patients had autonomous cortisol secretion, we performed 1-mg DST, in which dexamethasone (1 mg) was administered at 11:00 PM, and blood samples were drawn at 8:00 AM the following morning. F and ACTH were measured in 1-mg DST. The total or partial adrenalectomy was performed in all cases with overt hypercortisolism. For patients with subclinical hypercortisolism, the adrenalectomy was recommended to those who showed F-DST > 5.0 µg/dL (138 nmol/L) accompanying metabolic disorders [3]. It was also recommended to those who were expected to improve their clinical symptoms and/or metabolic disorders by the tumor resection, which included patients with hypertension possibly resulting from autonomous aldosterone secretion as well as autonomous cortisol secretion from the adrenal gland. The adrenalectomy was conducted when patients agreed with the treatment plan through informed consent. To evaluate whether patients had autonomous aldosterone secretion, we used the screening criterion of primary aldosteronism (ie, PAC/PRA ratio; aldosterone-to-renin ratio [ARR] > 20), followed by the confirmatory tests of primary aldosteronism that included the saline infusion test, captopril challenge, and/or furosemide stimulation test [17]. For patients who were considered to receive a benefit by the adrenalectomy and who agreed with the examination, we performed the segment-selective adrenal venous sampling to assess the laterality of hyperaldosteronism [18-20]. First, blood samples were collected from the bilateral central adrenal veins before ACTH stimulation. Then, we collected samples from the superior, lateral, and inferior tributaries of the right central adrenal vein and the superior and lateral tributaries of the left central adrenal vein after ACTH stimulation. Aldosterone excess (ie, hyperaldosteronism) was considered when the effluent aldosterone concentrations were > 250 ng/dL before ACTH stimulation and 1400 ng/dL after ACTH stimulation, respectively [18-20]. We used the absolute value instead of the lateralization index because individuals included in our study had elevated cortisol concentrations given the inclusion criteria (ie, F-DST >1.8 µg/dL [50 nmol/L]). For 9 patients with subclinical hypercortisolism who showed bilateral adrenal nodules, the side of adrenalectomy was determined by the nodule size and the results of adrenal venous sampling (ie, laterality of hyperaldosteronism). The adrenalectomy was conducted when patients agreed with the treatment plan through informed consent. Immunohistochemical evaluation of aldosterone synthase cytochrome P450 (CYP11B2) was conducted for some resected nodules. To evaluate the postoperative cortisol responsiveness to ACTH, we performed an ACTH stimulation test a year after the adrenalectomy, in which blood samples were collected and PAC and F were measured 30 and 60 minutes after ACTH administration. Postoperative improvement of hypertension was defined as blood pressure <140/90 mmHg without antihypertensives or the reduction of the number of antihypertensives to maintain blood pressure <140/90 mmHg after the adrenalectomy. Statistical Analyses We describe the demographic characteristics and endocrine parameters at baseline comparing patients with overt hypercortisolism and those with subclinical hypercortisolism using the Fisher exact test for categorical variables and Mann-Whitney U test for continuous variables. Second, for each group, we investigated the association between the baseline characteristics and systolic blood pressure using ordinary least-squares regression models. The model included age, sex, BMI, serum potassium levels, estimated glomerular filtration rate, tumor size, and F and PAC at 8:00 AM. Third, we estimated the risk difference and 95% CI of the improvement rate of hypertension after the adrenalectomy according to these baseline characteristics (including systolic blood pressure) using a modified least-squares regression model with a Huber-White robust standard error [21]. Last, to evaluate whether the improvement of hypertension is related to postoperative cortisol and aldosterone secretion, we compared PAC and F responsiveness to ACTH from peripheral blood samples between patients who improved hypertension and those who did not using the Mann-Whitney U test. The longitudinal and postoperative analyses were performed among patients with subclinical hypercortisolism because only 2 cases with overt hypercortisolism failed to show the improvement of hypertension after the adrenalectomy. To assess the robustness of our findings, we conducted the following 2 sensitivity analyses. First, we replaced F at 8:00 AM with F after DST in our regression models. Second, we estimated the risk difference of the improvement rate of hypertension after the adrenalectomy according to the postoperative F and PAC levels after ACTH stimulation, adjusting for the baseline characteristics included in our main model. We also conducted several additional analyses. First, to investigate the relationship of change in PAC after adrenalectomy with the improvement rate of hypertension, we included decrease in PAC between before and after adrenalectomy instead of PAC at baseline in the model. Second, to assess the relationship between aldosterone and hypertension among patients with subclinical hypercortisolism without primary aldosteronism, we reran the analyses excluding patients who met the diagnostic criteria of primary aldosteronism. Third, to understand the overall association, we reran the analyses using all samples as a single group to assess the relationship among people with overall (ie, overt and subclinical) hypercortisolism. Last, we compared PAC and F responsiveness with ACTH during adrenal venous sampling between patients with and without postoperative improvement of hypertension. All statistical analyses were performed using Stata, version 15. Results Among the 89 enrolled patients, 21 showed clinical signs of overt Cushing syndrome and 68 did not. The flow of the study population is shown in Fig. 1. Among 21 patients with overt hypercortisolism, 19 patients had hypertension. All patients underwent adrenalectomy, and 16 patients showed improved hypertension levels after the surgery (1 patient was referred to another hospital; therefore, no information is available). Among 68 patients with subclinical hypercortisolism, 63 had hypertension. After the evaluation of autonomous aldosterone secretion as well as autonomous cortisol secretion, of 33 patients who underwent adrenalectomy, 23 (70%) showed improved hypertension levels after the adrenalectomy (10 patients in the surgery group decided not to undergo adrenalectomy). Patients with subclinical hypercortisolism who underwent adrenalectomy showed lower PRA and higher ARR than those without adrenalectomy (Supplementary Table S1) [22]. Figure 1. Open in new tabDownload slide Enrollment and follow-up of the study population after the adrenalectomy. aThe prevalence of patients with overt hypercortisolism and hypertension among this study population may be higher than in the general population and therefore needs to be carefully interpreted given that the study institute is one of the largest centers for adrenal diseases in Japan. bAll patients in this category showed autonomous cortisol secretion (ie, serum cortisol levels >5.0 µg/dL [138 nmol/L] after a 1-mg dexamethasone suppression test). cOne case underwent adrenalectomy at another hospital and therefore no information was available after the adrenalectomy. dThe adrenalectomy was performed for 33 patients who were expected to improve their clinical symptoms and/or metabolic disorders, including hypertension. This assessment was mainly based on autonomous cortisol secretion evaluated by a 1-mg dexamethasone suppression test, complicated metabolic disorders, and autonomous aldosterone secretion evaluated by adrenal venous sampling for patients who were positive for the screening and confirmatory tests of primary aldosteronism. Details in the assessment can be found in the Methods section or elsewhere [18-20]. Demographic Characteristics and Endocrine Parameters Among Patients With Overt and Subclinical Hypercortisolism The median age (interquartile range) was 51 years (46, 62 years), and 72% were female. Patients with overt hypercortisolism were relatively younger and showed a higher estimated glomerular filtration rate and larger tumor size compared with patients with subclinical hypercortisolism (Table 1). Other demographic characteristics were similar between these groups. Patients with overt hypercortisolism showed higher F with undetected low ACTH, higher F after DST, and higher urine cortisol levels compared with those with subclinical hypercortisolism who instead showed higher PAC and ARR. Among patients with subclinical hypercortisolism, 9/68 (13.2%) showed undetectable ACTH levels and 25/68 (36%) were positive for PA screening criterion (ie, ARR > 20) followed by at least 1 positive confirmatory test. Based on the results of adrenal venous sampling of these cases, 9 showed aldosterone excess in the right nodules, 6 showed aldosterone excess in the left nodules, and 7 showed aldosterone excess on both sides, respectively (3 cases did not show aldosterone excess on both sides). Immunohistochemical evaluation of CYP11B2 was examined for 6 resected adrenal glands, and all of them showed positive expression. Patients’ characteristicsa Patients with overt hypercortisolism (N = 21) Patients with subclinical hypercortisolism (N = 68) P Age, y 46 [38-52] 54 [47-63] 0.002 Female, n (%) 18 (85.7) 46 (67.7) 0.11 Body mass index, kg/m2 23.4 [20.6-26.2] 23.1 [21.7-25.1] 0.94 Systolic blood pressure, mm Hg 156 [140-182] 162 [151-191] 0.29 Diastolic blood pressure, mm Hg 98 [92-110] 100 [90-110] 0.73 Serum potassium, mEq/Lb 3.9 [3.5-4.0] 3.8 [3.6-4.0] 0.98 eGFR, mL/min/1.73 m2 86.7 [77.3-123.0] 82.1 [69.8-87.7] 0.02 Tumor size by CT scan, mm 28 [25-30] 22 [17-26] 0.001 ACTH, 8:00 AM − c 6.6 [2.4-11.8] — F, 8:00 AM 16.6 [12.5-18.8] 9.5 [7.7-12.0] <0.001 PRA, 8:00 AM 0.7 [0.4-1.3] 0.5 [0.2-1.0] 0.10 PAC, 8:00 AM 8.3 [7.2-9.8] 9.2 [7.2-16.2] 0.09 ARR, 8:00 AM 10.0 [6.4-16.7] 21.0 [9.8-46.5] 0.02 F after DST 16.5 [14.4-18.7] 5.1 [3.2-7.5] <0.001 Urine cortisol 220.0 [105.0-368.0] 49.5 [37.4-78.5] <0.001 Urine aldosterone 5.7 [3.9-10.1] 7.2 [4.8-13.1] 0.16 Conversion to SI units: ACTH, pg/mL × 0.220 for pmol; F, µg/dL × 27.6 for nmol/L; PAC, ng/dL × 27.7 for pmol/L; urine aldosterone, μg/day × 2.77 for nmol/d; Urine cortisol, μg/day × 2.76 for nmol/d. Abbreviations: ARR, aldosterone-to-renin ratio; CRH, corticotropin-releasing hormone; CT, thin-section computed tomography; DST, 1-mg dexamethasone suppression test; eGFR, estimated glomerular filtration rate; F, serum cortisol; PRA, plasma renin activity; PAC, plasma aldosterone concentration. a Data are presented as median (interquartile range) or count (proportions) unless otherwise indicated. b Serum potassium levels were controlled using potassium supplement/tablets at enrollment. c Undetected in all cases. Open in new tab Association of Demographic Characteristics and Endocrine Parameters With Systolic Blood Pressure Among patients with overt hypercortisolism, we did not find a significant association of demographic characteristics and endocrine parameters with systolic blood pressure (Table 2). However, among patients with subclinical hypercortisolism, we found that higher PACs at 8:00 AM were significantly associated with systolic blood pressure (adjusted coefficient [95% CI] = +0.59 [0.19-0.99], P = 0.008). The results did not change when we used F after DST instead of F at 8:00 AM (Supplementary Table S2) [22]. Table 2. Cross-sectional association of demographic characteristics and endocrine parameters with systolic blood pressure among patients with overt and subclinical hypercortisolism Outcome Systolic blood pressure at baseline Groups Patients with overt hypercortisolism Patients with subclinical hypercortisolism Parameters Adjusted coefficient (95% CI) P Adjusted coefficient (95% CI) P Age, y +1.73 (0.17-3.30) 0.03 +0.49 (−0.13 to 1.10) 0.12 Female −7.48 (−76.75 to 61.79) 0.81 +15.38 (−0.83 to 31.59) 0.06 Body mass index +5.47 (−2.4 to 13.33) 0.15 +1.07 (−0.49 to 2.63) 0.17 Serum potassium +11.29 (−23.42 to 45.99) 0.48 −9.61 (−26.38 to 7.15) 0.26 eGFR −0.12 (−1.00 to 0.77) 0.77 −0.44 (−0.89 to 0.01) 0.06 Tumor size −2.39 (−6.92 to 2.14) 0.26 +0.40 (−0.46 to 1.26) 0.35 F, 8:00 AMa,b +1.96 (−1.27 to 5.18) 0.20 +1.26 (−1.00 to 3.52) 0.27 PAC, 8:00 AMa −2.86 (−7.38 to 1.66) 0.18 +0.59 (0.19-0.99) 0.008 Abbreviations: DST, 1-mg dexamethasone suppression test; eGFR, estimated glomerular filtration rate; F, serum cortisol; PRA, plasma renin activity; PAC, plasma aldosterone concentration. a ACTH and PRA were not included in the main model because they have strong correlation with F and PAC, respectively (ie, multicollinearity). The results did not change when additionally adjusting for ACTH and PRA. b The results did not change when we replaced F at 8:00 AM with F after DST (Supplementary Table S2). Open in new tab Association of Demographic Characteristics and Endocrine Parameters With Hypertension Improvement After the Adrenalectomy Among Patients With Subclinical Hypercortisolism Among 33 patients with subclinical hypercortisolism and hypertension who underwent the adrenalectomy, we found that age and higher PAC were significantly associated with a higher improvement rate of hypertension after the adrenalectomy (age, adjusted risk difference [95% CI] = +2.36% [1.08-3.64], P = 0.001; PAC, adjusted risk difference [95% CI] = +1.45% [0.35-2.55], P = 0.01; Table 3). The results did not change when we used F after DST instead of F at 8:00 AM (Supplementary Table S3) [22]. Patients with improved hypertension after the surgery showed significantly lower PACs 60 minutes after a postoperative ACTH stimulation test than those without the improvement of hypertension (P = 0.05), although F and PAC/F ratio were not significantly different between these 2 groups (Table 4). The association between lower PACs after postoperative ACTH stimulation and higher improvement rate of hypertension was also found in the multivariable regression analysis adjusting for baseline characteristics (adjusted risk difference [95% CI] = −1.08% [−1.92 to −0.25], P = 0.01; Supplementary Table S4) [22]. Table 3. Longitudinal association of demographic characteristics and endocrine parameters with hypertension improvement after the adrenalectomy among patients with subclinical hypercortisolisma Outcome Hypertension improvement after the adrenalectomy Parameters Adjusted risk difference (95% CI) P Age +2.36% (1.08-3.64) 0.001 Sex (female) −11.32% (−61.37 to 38.73) 0.64 Body mass index −5.08% (−10.29 to 0.13) 0.06 Systolic blood pressure −0.67% (−1.77 to 0.43) 0.22 Serum potassium −0.06% (−31.84 to 31.71) 1.00 eGFR +0.53% (−0.36 to 1.42) 0.23 Tumor size +0.79% (−1.35 to 2.93) 0.45 F, 8:00 AMb,c −2.81% (−7.43 to 1.81) 0.22 PAC, 8:00 AMb +1.45% (0.35-2.55) 0.01 Abbreviations: eGFR, estimated glomerular filtration rate; F, serum cortisol; PRA, plasma renin activity; PAC, plasma aldosterone concentration. a Analysis was not performed for patients with overt hypercortisolism because only 2/18 cases failed to show improved hypertension after the adrenalectomy. b ACTH and PRA were not included in the main model because they have strong correlation with F and PAC, respectively (ie, multicollinearity). The results did not change when additionally adjusting for ACTH and PRA. c The results did not change when we replaced F at 8:00 AM with F after DST (Supplementary Table S3). Open in new tab Table 4. Aldosterone and cortisol response to ACTH a year after the adrenalectomy according to hypertension improvement status among patients with subclinical hypercortisolisma Outcome: hypertension improvement status after the adrenalectomy Improvement (+) (N = 23) Improvement (−) (N = 10) Parameters Median [IQR] Median [IQR] P PAC 60 min after ACTH stimulation 13.6 [10.0-16.7] 15.5 [13.7-43.1] 0.05b F 60 min after ACTH stimulation 16.9 [13.7-20.6] 18.5 [13.5-24.7] 0.61 PAC/F ratio 60 min after ACTH stimulation 0.70 [0.52-1.39] 1.27 [0.50-5.44] 0.26 Conversion to SI units: F, µg/dL × 27.6 for nmol/L; PAC, ng/dL × 27.7 for pmol/L. Abbreviations: F, serum cortisol; PAC, plasma aldosterone concentration. a Analysis was not performed for patients with overt hypercortisolism because only 2/18 cases failed to show improved hypertension after the adrenalectomy. b The association was also observed after adjusting for baseline characteristics (eg, age, sex, body mass index, systolic blood pressure, serum potassium, estimated glomerular filtration rate, tumor size) and F 60 min after ACTH stimulation a year after the adrenalectomy (Supplementary Table S4). Open in new tab Additional Analyses Decreased PAC between before and after adrenalectomy was significantly associated with hypertension improvement (Supplementary Table S5) [22]. When we restricted samples to those without primary aldosteronism, PACs at baseline tended to be associated with systolic blood pressure but the 95% CI included the null (Supplementary Table S6) [22]. Decreased PAC after adrenalectomy was associated with hypertension improvement after the adrenalectomy, whereas PAC at baseline was not associated with that outcome (Supplementary Table S7) [22]. When we analyzed the entire sample (ie, both overt and subclinical hypercortisolism), PAC at baseline was associated with systolic blood pressure at baseline (Supplementary Table S8) [22] and hypertension improvement after the adrenalectomy (Supplementary Table S9) [22]. We also found the higher median value of PAC response to ACTH during adrenal venous sampling at the remained (ie, not resected by the adrenalectomy) side of adrenal gland among patients whose hypertension did not improve compared with those whose hypertension improved after the surgery, but the difference was not statistically significant (Supplementary Table S10) [22]. Discussion In this retrospective cohort study, we found that higher aldosterone levels were associated with higher systolic blood pressure among patients with possible autonomous cortisol secretion and without clinical signs of overt Cushing syndrome (ie, subclinical hypercortisolism). In this group, higher aldosterone before the adrenalectomy was associated with the postoperative improvement of hypertension. Moreover, we found that patients with postoperative improvement of hypertension showed lower aldosterone response to ACTH after the adrenalectomy compared with those without the improvement of hypertension. Decrease in PACs after the adrenalectomy was associated with improved hypertension even among patients with subclinical hypercortisolism who did not have primary aldosteronism at baseline, whereas baseline PAC was not associated with that outcome. We found no evidence that aldosterone is associated with systolic blood pressure among patients with overt hypercortisolism. These findings indicate that elevated aldosterone may contribute to the presence of hypertension and its improvement rate after the adrenalectomy for patients with subclinical hypercortisolism. To the best of our knowledge, this is one of the first studies to assess the potential role of aldosterone in hypertension among patients with overt and subclinical hypercortisolism, during both pre- and postoperative phases. Since aldosterone- and cortisol-producing adenoma was reported in 1979 [23, 24], several studies have assessed the cortisol production in aldosterone-producing adenoma clinically and histologically [8-10, 25] and showed the correlation between the degree of glucocorticoid excess levels and metabolic markers including BMI, waist circumference, blood pressure, insulin resistance, and high-density lipoprotein [12]. Prior research suggested that aldosterone-producing adenoma might produce cortisol as well as aldosterone even when serum cortisol levels after DST is less than 1.8 µg/dL (50 nmol/L) [11]. Although these studies have focused on cortisol synthesis among patients with aldosterone-producing adenoma, little is known about aldosterone synthesis among patients with cortisol-producing adenoma. Given that patients with hypercortisolism tend to have therapy-resistant hypertension and electrolyte disorders [8], our findings may generate the hypothesis that aldosterone contributes to the incidence and severity of hypertension in patients with possible autonomous cortisol secretion; this warrants further investigation. There are several mechanisms by which cortisol excess leads to hypertension, such as regulating endothelial nitric oxide synthase expression modulated by 11β-hydroxysteroid dehydrogenases [26], activating the mineralocorticoid receptor [27] and upregulating vascular endothelin-1 [28]. Moreover, hypercortisolism impairs the production of endothelial vasodilators, including prostacyclin, prostaglandins, and kallikreins [29]. Despite these potential mechanisms, the direct effect of cortisol may not be sufficient to explain hypertension in patients with hypercortisolism, particularly subclinical hypercortisolism, and the presence of cortisol and aldosterone coproducing adenoma indicates another potential pathway to induce hypertension through aldosterone excess. Aldosterone is a steroid hormone not only promoting sodium reabsorption and volume expansion but also activating the mineralocorticoid receptor in the kidney and nonepithelial tissues (eg, adipose tissue, heart, endothelial cells, and vascular smooth muscle cells) [30]. It also induces oxidative stress, inflammation, fibrosis, vascular tone, and endothelial dysfunction [31]; therefore, aldosterone excess could induce hypertension even when it is slightly elevated [32]. A recent multiethnic study showed that aldosterone levels within the reference range were associated with subclinical atherosclerosis partially mediated through elevated blood pressure [33]. These mechanisms support our results indicating the potential contribution of aldosterone to hypertension among patients with subclinical hypercortisolism. This study had several limitations. First, we did not have information on the duration of cortisol excess and therefore the estimated effect of cortisol on hypertension in our study might have been underestimated. The duration of exposure to mild hypercortisolism may be one of the important drivers of cardiovascular and metabolic disorders including irreversible vasculature remodeling in patients with subclinical hypercortisolism [2]. Second, we did not have the genetic information of adrenal tumors including aldosterone-producing adenoma. Given the heterogeneity of aldosterone responsiveness to ACTH [34] and postoperative hypertension resolution rate across genetic mutations (eg, KCNJ5, ATP1A1, ATP2B3, CACNA1D, CTNNB1) [35], such information might affect our findings. Third, because of the nature of an observational study, we cannot rule out the unmeasured confounding. Fourth, because aldosterone and cortisol levels were measured at a single point, we may have a risk of mismeasurement. Moreover, when evaluating aldosterone levels, we used dihydropyridine calcium channel blockers to control hypertension based on the clinical guideline of primary aldosteronism in Japan; this might lower serum aldosterone levels. Fifth, because the present study was conducted at a single center, selection bias is inevitable [13]. Given that primary aldosteronism—one of the major causes of secondary hypertension—has still been underdiagnosed, partially because of insufficient recognition of clinical guidelines [36], our findings may indicate the importance of considering aldosterone when evaluating patients with subclinical hypercortisolism accompanied by hypertension. However, we need to carefully interpret the observed “prevalence” in this study because individuals potentially having subclinical hypercortisolism were likely to come to our hospital, which specializes the adrenal disorders, and thus the numbers do not reflect the prevalence in general population. The small number of resected adrenal glands with the evaluation of CYP11B2 expression in this study cohort also limits the prevalence estimation of primary aldosteronism. Finally, as we only followed up 1 year after the adrenalectomy, we could not evaluate the long-term resolution rate of hypertension. To overcome these limitations and generalize our findings, future molecular studies and multicenter longitudinal studies with sufficient individual datasets and longer follow-up are required. In conclusion, plasma aldosterone concentrations were associated with systolic blood pressure and improvement rate of hypertension after the adrenalectomy among patients with subclinical hypercortisolism—possible autonomous cortisol secretion without clinical signs of overt Cushing syndrome. Our findings underscore the importance of considering aldosterone when patients have an adrenal tumor with possible autonomous cortisol secretion complicated with hypertension. Future molecular and epidemiological studies are warranted to identify the potential role of aldosterone in hypertension among patients with subclinical hypercortisolism, clarify how often these patients also have primary aldosteronism, and examine the clinical effectiveness of the intervention targeting aldosterone for such patients. Funding K.I. was supported by the Japan Society for the Promotion of Science (JSPS; 21K20900 and 22K17392) and The Japan Endocrine Society. Study sponsors were not involved in study design, data interpretation, writing, or the decision to submit the article for publication. The funders had no role in the design and conduct of the study; collection, management, analysis, and interpretation of the data; preparation, review, or approval of the manuscript; and decision to submit the manuscript for publication. Conflicts of Interest All of authors confirm that there is no conflict of interest in relation to this work. Data Availability Restrictions apply to the availability of some data generated or analyzed during this study to preserve patient confidentiality or because they were used under license. The corresponding author will on request detail the restrictions and any conditions under which access to some data may be provided. Abbreviations ARR aldosterone-to-renin ratio BMI body mass index DST dexamethasone suppression test F serum cortisol level HPA hypothalamus-pituitary-adrenal PAC plasma aldosterone concentration PRA plasma renin activity © The Author(s) 2022. Published by Oxford University Press on behalf of the Endocrine Society. This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs licence (https://creativecommons.org/licenses/by-nc-nd/4.0/), which permits non-commercial reproduction and distribution of the work, in any medium, provided the original work is not altered or transformed in any way, and that the work is properly cited. For commercial re-use, please contact journals.permissions@oup.com © The Author(s) 2022. Published by Oxford University Press on behalf of the Endocrine Society. From https://academic.oup.com/jes/article/7/1/bvac167/6782230?login=false

Cortisol isn’t bad; you need it to help regulate your responses to life. Regulation involves a very complex interplay of feedback loops between the hypothalamus, pituitary gland, and adrenal glands, says Dr. Singh. “In general, cortisol levels tend to peak in the late morning and gradually decline throughout the day,” he explains. “When a stressful event occurs, the increased cortisol will work alongside our ‘fight or flight’ mechanisms to either upregulate or downregulate bodily functions. [Affected systems include] the central nervous system, cardiovascular system, gastrointestinal system, or immune system.” In addition to normal processes that trigger or suppress cortisol release, levels can also be affected by different medical conditions, Dr. Singh says. For example, if someone has abnormally high levels of cortisol, this is called Cushing’s syndrome, which is typically caused by a tumor affecting any of the glands that take part in the process of cortisol production. When people suffer from abnormally low levels of cortisol, it’s called Addison’s disease. It generally occurs due to adrenal gland dysfunction, but could also be the result of abnormal functioning of any of the other glands in the cortisol production process. Finally, if you use corticosteroid medications such as prednisone or dexamethasone, prolonged use will result in excessive cortisol production, Dr. Singh says. “If the medication is not adequately tapered down when discontinued, the body’s ability to create cortisol can become permanently impaired,” he says. From https://www.yahoo.com/lifestyle/manage-pesky-stress-hormone-cortisol-184900397.html

Abstract MiRNAs are important epigenetic players with tissue- and disease-specific effects. In this study, our aim was to investigate the putative differential expression of miRNAs in adrenal tissues from different forms of Cushing’s syndrome (CS). For this, miRNA-based next-generation sequencing was performed in adrenal tissues taken from patients with ACTH-independent cortisol-producing adrenocortical adenomas (CPA), from patients with ACTH-dependent pituitary Cushing’s disease (CD) after bilateral adrenalectomy, and from control subjects. A confirmatory QPCR was also performed in adrenals from patients with other CS subtypes, such as primary bilateral macronodular hyperplasia and ectopic CS. Sequencing revealed significant differences in the miRNA profiles of CD and CPA. QPCR revealed the upregulated expression of miR-1247-5p in CPA and PBMAH (log2 fold change > 2.5, p < 0.05). MiR-379-5p was found to be upregulated in PBMAH and CD (log2 fold change > 1.8, p < 0.05). Analyses of miR-1247-5p and miR-379-5p expression in the adrenals of mice which had been exposed to short-term ACTH stimulation showed no influence on the adrenal miRNA expression profiles. For miRNA-specific target prediction, RNA-seq data from the adrenals of CPA, PBMAH, and control samples were analyzed with different bioinformatic platforms. The analyses revealed that both miR-1247-5p and miR-379-5p target specific genes in the WNT signaling pathway. In conclusion, this study identified distinct adrenal miRNAs as being associated with CS subtypes. Keywords: cortisol; ACTH; miRNA; Cushing’s; hypercortisolism; pituitary 1. Introduction Cushing’s syndrome (CS) results from the excessive secretion of cortisol, leading to visceral obesity, resistance to insulin, osteoporosis, and altered lipid and glucose metabolism [1,2]. Excessive production of cortisol by the adrenal glands can be either ACTH-dependent or -independent. In the majority of patients, hypercortisolism is due to ACTH secretion by corticotroph adenomas of the pituitary gland (Cushing’s disease, CD) or by ectopic tumors [3]. Approximately 20% of cases are ACTH-independent, where cortisol is secreted autonomously by the adrenal cortex. The pathology of ACTH-independent cases is diverse; they are most often caused by unilateral cortisol-producing adrenocortical adenomas (CPA). Rare causes are cortisol-secreting adrenocortical carcinomas (ACC), primary bilateral macronodular adrenocortical hyperplasia (PBMAH), bilateral CPAs, and primary pigmented nodular adrenal disease (PPNAD) [4,5]. Irrespective of the subtype, prolonged exposure to cortisol in CS is associated with increased mortality and cardiovascular morbidity in its patients [6]. Treatment is based on the underlying cause of hypercortisolism, with pituitary surgery or adrenalectomy being the preferred choice. Medical therapy options in CS are few and consist of pituitary-directed drugs, steroid synthesis inhibitors, and glucocorticoid receptor antagonists [7]. For the timely diagnosis and targeted management of CS and its subtypes, a comprehensive understanding of cortisol secretion, in terms of canonical signaling pathways as well as upstream epigenetic factors, is needed. MiRNA molecules have emerged as key epigenetic players in the transcriptional regulation of cortisol production. Briefly, the deletion of Dicer in adrenals, a key miRNA processing enzyme, revealed diverse expression changes in miRNAs along with related changes in steroidogenic enzymes such as Cyp11b1 [8]. Furthermore, key enzymes in the cortisol biosynthesis pathway, namely Cyp11a1, Cyp21a1, Cyp17a1, Cyp11b1, and Cyp11b2, were also found to be regulated by various miRNAs (miRNA-24, miRNA-125a-5p, miRNA-125b-5p, and miRNA-320a-3p) in in vitro studies [9]. Consequently, various studies have also characterized miRNA expression profiles in CS subtypes. Importantly, miRNA expression in the corticotropinomas of CD patients was found to vary according to USP8 mutation status [10]. Other studies have also identified specific miRNA candidates and associated target genes in the adrenals of patients with PPNAD [11], PBMAH [12,13], and massive macronodular adrenocortical disease [14]. Interestingly, no common miRNA candidates were found among these studies, indicating the specificity of miRNAs to the different underlying pathologies in CS. There are limited studies directly comparing miRNA expression profiles of ACTH-dependent and ACTH-independent CS patients. Consequently, in our previous study, we found differences in expression profiles when comparing circulating miRNAs in CD and CPA patients [15]. We hypothesized that the presence of ACTH possibly influences the miRNA profile in serum due to the upstream differential expression in the origin tissues. In this study, we aim to further explore this hypothesis by comparing the miRNA expression profile of adrenal tissues in ACTH-dependent and ACTH-independent CS. In brief, miRNA specific sequencing was performed in two prevalent subtypes of CS: in CD, the most prevalent ACTH-dependent form; and in CPA, the most prevalent ACTH-independent form. Specific miRNA candidates related to each subtype were further validated in other forms of CS. To further investigate our hypothesis, the response of miRNA candidates following ACTH stimulation was assessed in mice, and the expression of miRNAs in murine adrenals was subsequently investigated. Finally, an extensive targeted gene analysis was performed based on in silico predictions, RNA-seq data, and luciferase assays. 2. Results 2.1. Differentially Expressed miRNAs NGS revealed differentially expressed miRNAs between the different groups analyzed (Figure 1). CD and CPA taken together as CS showed a differentially expressed profile (42 significant miRNAs) in comparison to controls. Moreover, individually, CPA and CD were found to show a significantly different expression profile in comparison to controls (n = 38 and n = 17 miRNAs, respectively). Interestingly, there were no significantly upregulated genes in the adrenals of patients with CD in comparison to the control adrenals. A comparative analysis of the top significant miRNAs (log2 fold change (log2 FC) > 1.25 & p < 0.005) between the two groups was performed and the representative Venn diagrams are given in Figure 2. Briefly, miR-1247-5p, miR-139-3p, and miR-503-5p were significantly upregulated in CPA, in comparison to both CD and controls. Furthermore, miR-150-5p was specifically upregulated in CPA as compared to CD. Several miRNAs (miR-486-5p, miR-551b-3p, miR-144-5p, miR-144-3p, and miR-363-3p) were found to be significantly downregulated in the groups of CPA and CD in comparison to controls. MiR-19a-3p and miR-873-5p were found to be commonly downregulated in CPA in comparison to both CD and controls. Principal component analyses based on miRNA sequencing did not identify any major clusters among the samples. Furthermore, the miRNA profile was not different among the CPA samples based on the mutation status of PRKACA (Supplementary Materials Figure S1). Figure 1. Differentially expressed miRNAs from sequencing. Volcano plot showing the relationship between fold change (log2 fold change) and statistical significance (−log10 p value). The red points in the plot represent significantly upregulated miRNAs, while blue points represent significantly downregulated miRNAs. CPA, cortisol producing adenoma; CD, Cushing’s disease; Cushing’s syndrome represents CPA and CD, taken together. Figure 2. Venn analyses of the common significant miRNAs from each group. The significantly expressed miRNAs from each sequencing analysis were shortlisted and compared between the groups. CPA, cortisol producing adenoma; CD, Cushing’s disease. 2.2. Validation and Selection of Candidate miRNAs For validation by QPCR, the most significant differentially expressed miRNAs (log2 FC > 1.25 & p < 0.005) among the groups were chosen (Table S1). According to the current knowledge, upregulated miRNAs are known to contribute more to pathology than downregulated miRNAs [16]. Since the total number of significantly upregulated miRNAs was six, all these miRNAs were chosen for validation. Contrarily, 25 miRNAs were significantly downregulated among the groups. In particular, miR-486-5p, miR-551b-3p, miR-144-5p, miR-144-3p, and miR-363-3p were found to be commonly downregulated in the CS group in comparison to controls; therefore, these miRNAs were chosen for validation. Among the upregulated miRNA candidates, miR-1247-5p QPCR expression confirmed the NGS data (Figure 3A, Table S1). Moreover, miR-150-5p and miR-139-3p were upregulated in CPA specifically in comparison to CD, and miR-379-5p was upregulated in CD in comparison to controls by QPCR. In the case of downregulated genes, none of the selected miRNAs could be confirmed by QPCR (Figure 3B). Thus, analysis of the six upregulated and five downregulated miRNAs from NGS yielded two significantly upregulated miRNA candidates, miR-1247-5p in CPA and miR-379-5p in CD, when compared to controls. These miRNA candidates were taken up for further QPCR validation in an independent cohort of other subtypes of CS (Figure 4), namely ACTH-dependent ectopic CS (n = 3) and ACTH-independent PBMAH (n = 10). The QPCR analysis in the other subtypes revealed miR-1247-5p to be consistently upregulated in ACTH-independent CS (PBMAH and CPA) in comparison to ACTH-dependent CS (CD and ectopic CS) and controls. On the other hand, miR-379-5p was upregulated in CD and PBMAH in comparison to controls. Figure 3. QPCR analyses of significant miRNAs from sequencing analyses. Data are represented as mean ± standard deviation (SD) of −dCT values: (A) Expression analysis of significantly upregulated miRNAs; (B) Expression analysis of common significantly downregulated miRNAs. Housekeeping gene: miR-16-5p. Statistics: ANOVA test with Bonferroni correction to detect significant differences between patient groups with at least a significance of p-value < 0.05 (*). Figure 4. QPCR analyses of significantly upregulated miRNAs from validation QPCR. Data are represented as mean ± standard deviation (SD) of −dCT values. Housekeeping gene: miR-16-5p. Statistics: ANOVA test with Bonferroni correction to detect significant differences between patient groups with at least a significance of p-value < 0.05 (*). 2.3. In Vivo Assessment of ACTH-Independent miR-1247-5p To analyze the influence of ACTH on miRNA expression, the expression of miR-1247-5p and miR-379-5p were assessed in the adrenal tissues of ACTH stimulated mice at different time points. For this analysis, miR-96-5p was taken as a positive control, as it has previously been reported to be differentially expressed in ACTH stimulated mice [17]. The analyses revealed that the expression of miR-1247-5p and miR-379-5p did not change at different timepoints of the ACTH stimulation (Figure 5). Meanwhile, the positive control of mir-96-5p showed a dynamic expression pattern with upregulation after 10 min, followed by downregulation at the subsequent 30 and 60 min time points, in concordance with previously reported findings [18]. Figure 5. Analysis of miRNA expression in ACTH stimulated mice tissue. QPCR analyses of positive controls, miR-96-5p, and candidates miR-379-5p and miR-1247-5p. Mice were injected with ACTH, and adrenals were collected at different timepoints to assess the impact of ACTH on miRNA expression. Data are represented as mean ± standard deviation (SD) of −dCT values. Housekeeping gene: miR-26a-5p. Statistics: ANOVA test with Bonferroni correction to detect significant differences between patient groups with at least a significance of p-value < 0.05 (*). 2.4. In Silico Analyses of miRNA Targets Two diverse approaches were employed for a comprehensive in silico analysis of the miRNA targets. First, the predicted targets of miR-1247-5p and miR-379-5p were taken from the TargetScan database, which identified miRNA–mRNA target pairs based on sequence analyses [19]. The expression status of these targets was then checked in the RNA sequencing data from CPA vs. controls (miR-1247-5p) and PBMAH vs. controls (miR-379-5p). Targets that showed significant expression changes in the sequencing data were shortlisted (Figure 6A). Among the 1061 predicted miR-1247-5p targets, 28 genes were found to show significant expression changes in CPA (20 upregulated, 8 downregulated). On the other hand, for 124 predicted miR-379-5p targets, 23 genes were found to show significant expression changes in PBMAH (20 upregulated, 3 downregulated). Interestingly, the selected targets were found to be unique for each miRNA, except for FICD (FIC domain protein adenylyltransferase) (Figure 6B). Figure 6. (A) Differentially expressed target genes of miRNAs from sequencing. Data are represented as log2 fold change in comparison to the controls. Statistics: ANOVA test with Bonferroni correction to detect significant differences between patient groups with at least a significance of p-value < 0.05. (B) Venn analyses of common significant miRNA target genes and related pathways. The significantly expressed targets from each sequencing analysis were shortlisted and compared between the groups. Predicted pathways of the targets from the Panther database were shortlisted and compared between the groups. 2.5. In Vitro Analyses of miR-1247-5p Targets For in vitro analyses, we focused on downregulated targets, as we expect our upregulated miRNA candidates to cause a downregulation of the target mRNAs. For our downregulated mRNAs, only targets of miR-1247-5p were found to have published links to CS, namely Cyb5a, Gabbr2, and Gnaq (Table 1). Therefore, these three targets were then verified by QPCR in the groups of CPA, CD, PBMAH, ectopic CS, and controls (Figure 6). Only Cyb5A was found to be significantly downregulated in ACTH-dependent forms (ectopic CS and CD) as well as in ACTH-independent CS (PBMAH and CPA) in comparison to controls. Consequently, to assess whether Cyb5a is indeed regulated by miR-1247-5p, a dual luciferase assay was performed. To prove our hypothesis, treatment of Cyb5a-WT cells with miR-1247-5p mimic was expected to lead to a reduced luminescence, whereas no effects were expected in cells treated with the miR-1247-5p inhibitor or the Cyb5a-mutant (with a mutation in the miR-1247-5p binding site). As shown in Figure 7, transfection of miR-1247-5p significantly reduced luminescence from Cyb5a-WT in comparison to cells transfected with Cyb5a-WT and miR-1247-5p inhibitors. However, these predicted binding results were not found to be specific, as there were no significant differences when compared to wells transfected with Cyb5a-WT alone (Figure 8). Consecutively, when the mutated Cyb5a-Mut were transfected along with the mimics and inhibitors, no significant differences in luminescence were observed in the transfected population. Thus, direct interaction between miR-1247-5p and its predicted target gene Cyb5A could not be conclusively proven using this luciferase assay. Figure 7. QPCR analyses of the top predicted targets of miR-1247-5p. Data are represented as mean ± standard deviation (SD) of −dCT values. Housekeeping gene: PPIA. Statistics: ANOVA test with Bonferroni correction to detect significant differences between patient groups with at least a significance of p-value < 0.05 (*). Figure 8. Results of dual luminescence assay on cells transfected with miR-1247-5p mimics and related controls. Cells were transfected with plasmids containing either the WT or Mut miRNA binding sequence in Cyb5a. Either miR-1247-5p mimics or miR-1247-5p inhibitors were transfected together with the respective plasmids. Data are represented as mean ± standard error of mean (SEM) of relative luminescence unit values. Statistics: ANOVA test with Bonferroni correction to detect significant differences between patient groups with at least a significance of p value < 0.05 (*). Table 1. Analysis of the predicted targets of miR-1247-5p and their expression levels in comparison to controls (log2 fold change). Published literature on target genes in reference to CS is highlighted in bold. 2.6. Pathway Analyses of miRNA Targets For the pathway analysis (Reactome) we used the 28 predicted miRNA-1247-5p targets and the 23 predicted miRNA-379-5p targets from TargetScan, which were significantly differently expressed in the RNA-seq (Figure 6). Concurrently, the pathways commonly enriched by both miRNAs included the WNT signaling pathway and N-acetyl-glucosamine synthesis (Figure 9A). As a complementary approach, in silico analyses were also performed based on the targets from miRTarBase. In this database, targets are shortlisted based on published experimental results. In this analysis, miR-1247-5p (n = 21) and miR-379-5p targets (n = 85) were unique. While the validated targets of miR-379-5p were found to show significant changes in expression in the RNA-seq data from PBMAH (n = 12), none of the validated miR-1247-5p targets were found to show significant expression changes in the RNA-seq data from CPA. Therefore, all the validated targets of the miRNAs were subjected to pathway analyses (Panther). Interestingly, the WNT signaling pathway was also found to be commonly regulated by both miRNAs using this approach (Figure 9B). Finally, the expression status of target genes related to WNT signaling pathways were checked in our RNA-seq data (Figure S2). Given the upregulated status of the miRNAs, a downregulated expression of its target genes was expected. However, a significantly upregulated expression was observed for DVL1 in CPA in comparison to controls and for ROR1 in PBMAH in comparison to controls. Figure 9. Pathway analyses of miRNA target genes. (A) The predicted targets were matched with the RNA-seq expression data. For miR-1247-5p, CPA vs. controls expression data; and for miR-379-5p, PBMAH vs. controls expression data. The significantly expressed target genes were then subjected to pathway analyses by Reactome. The significantly enriched pathway networks (p < 0.05) and their related genes are given. (B) The experimentally validated target genes from miRTarBase were analyzed for their role in pathways by the Panther database. The target genes and their related pathways are given. The commonly represented pathways are marked in bold. 3. Discussion MiRNAs are fine regulators of both physiology and pathology and have diverse roles as diagnostic biomarkers as well as therapeutic targets. While circulating miRNAs have been investigated as potential biomarkers for hypercortisolism in CS subtypes (36), comprehensive analyses of their pathological role in CS subtypes have not yet been performed. This study hoped to uncover the pathological role of miRNAs in different CS subtypes as well as identify unique epigenetic targets contributing to hypercortisolism in these subtypes. As such, miRNA sequencing was performed in the ACTH-independent CPA and ACTH-dependent CD, with additional QPCR validation in PBMAH and ectopic CS. As expected, miRNA expression profiles in CD and CPA were very different. 3.1. ACTH-Independent Upregulated miRNAs in CS Among the analyzed miRNAs, only miR-1247-5p and miR-379-5p showed the most prominent changes in expression levels. Briefly, miR-1247-5p was significantly upregulated in ACTH-independent forms of CS, CPA, and PBMAH (Figure 1 and Figure 3) while miR-379-5p was found to be upregulated in CD and PBMAH, in comparison to controls. Even though CD and PBMAH represent CS subtypes with different ACTH dependence, albeit both with hyperplastic tissue, it is interesting to find a shared miRNA expression status. Concurrently, miRNAs have been identified as dynamic players in regulating the acute effect of ACTH on adrenal steroidogenesis in in vivo murine studies [20,21]. Further diverse miRNAs have been characterized to regulate steroidogenesis in ACTH and dexamethasone treated rats [22] (suppressed ACTH) as well as in in vitro studies [20]. It is possible that miR-379-5p contributes to the adrenal hyperplasia present in both PBMAH and CD by targeting specific genes related to hyperplasia, and miR-1247-5p by contributing to cortisol production independent of ACTH regulation in CPA and PBMAH. Interestingly, the miRNA candidates (mir-1247-5p and miR-379-5p) in our study have not been previously characterized in any of these studies. Furthermore, the expression of mir-1247-5p and miR-379-5p were found to be independent of ACTH stimulation, underlying their role in CS independently of the HPA axis control and postulating specific regulatory processes. 3.2. Target Genes of miRNAs in CS Initially, we focused on the selection of known CS specific target genes that could be directly repressed by miRNAs, thereby contributing to pathology. The predicted target genes of miR-1247-5p and miR-379-5p were assessed for their downregulated expression status in the RNA-seq data. Among the selected target genes, only Cyb5a was found to be significantly downregulated in all forms of CS (Figure 6). Cytochrome b5 (CYB5A) is a marker of the zona reticularis and is an important regulator of androstenedione production [23,24]. Based on its role in adrenal steroidogenesis, it is possible that Cyb5a is downregulated by miR1247-5p. To prove our hypothesis, a dual luciferase assay was performed in HELA cell line to identify a direct interaction between Cyb5a and miR-1247-5p (Figure 7). Unfortunately, a direct interaction could not be proven, indicating that miR-1247-5p perhaps regulates its target genes in different ways. 3.3. Pathway Analyses of miRNA Targets To identify miRNA specific regulatory processes, comprehensive target and pathway analyses were performed. Independent pathway analyses of the respective targets with two different databases of Reactome and Panther showed the WNT signaling pathway as a common targeted pathway of both mir-1247-5p and miR-379-5p (Figure 8). The WNT signaling pathway represents a crucial regulator in diverse developmental as well as pathological processes with tissue-specific effects [25,26]. Consequently, the WNT pathway has been largely characterized as one of the dysregulated pathophysiological mechanisms in CPA. Mutations in PRKACA, one of the WNT signaling proteins, are present in approximately 40% of CPA [27]. In the case of CD, dysregulated WNT signaling has been characterized as promoting proliferation in ACTH-secreting pituitary adenomas [28]. Moreover, activating mutations in beta catenin, one of the WNT signaling pathways, has been characterized as driving adrenal hyperplasia through both proliferation-dependent and -independent mechanisms [29]. Thus, it could be hypothesized that by targeting specific genes in the pathway, miRNAs drive specific pathophysiological processes in diverse CS subtypes. 3.4. MiRNA Target Genes in WNT Signaling DVL1 (TargetScan) and DVL3 (miRTar) are the shortlisted target genes of miR-1247-5p in the WNT signaling pathway. These genes are members of canonical WNT pathways and, importantly, activation of the cytoplasmic effector Dishevelled (Dvl) is a critical step in WNT/β-catenin signaling initiation [30,31]. Interestingly, no difference in DVL1 and DVL3 gene expression was found in the analyses of ACTH-secreting pituitary adenomas [32]. Therefore, it could be possible that DVL1 and DVL3 are only targeted by miR-1247-5p specifically in the adrenal of CPA and PBMAH patients, leading to its characterized tumor progression. EDN1, TGFBR1 (TargetScan), and ROR1 (miRTar) were the target genes of miR-379-5p related to the WNT pathway. In epithelial ovarian cancer, Endothelin-1 (EDN-1) was found to regulate the epithelial–mesenchymal transition (EMT) and a chemoresistant phenotype [33]. In the case of receptor tyrosine kinase-like orphan receptor 1 (ROR1), higher expression of the gene was associated with a poor prognosis in ovarian cancer [34]. Concurrently, suppression of TGFBR1-mediated signaling by conditional knockout in mice was found to drive the pathogenesis of endometrial hyperplasia, independent of the influence of ovarian hormones [35]. Therefore, it could be hypothesized that the dysregulated expression of these factors in adrenals could trigger similar hyperplastic effects mediated by these factors, as in ovarian tissues. 3.5. Bottlenecks and Future Outlook Interestingly, among these genes, only DVL1 and ROR1 were found to be significantly upregulated in the RNA-seq data (Figure S2). The major regulatory role of miRNAs in gene expression come from their ability to repress gene expression at the level of transcription and translation. There are also reports of miRNA-mediated gene upregulation; however, the physiological evidence of the role is still unresolved [36]. Therefore, it is interesting to see the selected targets of miR-1247-5p and miR-379-5p upregulated. Moreover, it should be noted that most of the experimentally validated miRNA targets were identified by CLIP methods [37]. Crosslinking immunoprecipitation (CLIP) are binding assays that provide genome-wide maps of potential miRNA-target gene interactions based on sequencing. Moreover, these assays do not make functional predictions on the outcome of miRNA binding, and neither do upregulation or downregulation [38,39]. Therefore, in our current experimental setting, we could only identify potential miRNA target genes and speculate on their pathological role based on the published literature and in silico analyses. Furthermore, extensive mechanistic analyses based on these potential targets could help in elaborating the specific epigenetic pathways that fine-tune CS pathology in different subtypes. 4. Materials and Methods 4.1. Sample Collection and Ethics Approval All patients were registered in the German Cushing’s Registry, the ENS@T or/and NeoExNET databases (project number protocol code 379-10 and 152-10). The study was approved by the Ethics Committee of the University of Munich. All experiments were performed according to relevant guidelines and protocols, and written informed consent was obtained from all patients involved. The adrenal samples used in the sequencing (miRNA and RNA) belong to the same patient. For miRNA-specific next-generation sequencing (NGS), a total of 19 adrenocortical tissue samples were used. The cohort consisted of the following patient groups: ACTH-independent CPA, n = 7; ACTH-dependent hypertrophic adrenals of CD patients after bilateral adrenalectomy, n = 8; normal adjacent adrenal tissue from patients with pheochromocytoma as controls, n = 8. For QPCR validation, the patient groups included adrenal tissue from ACTH-independent PBMAH, n = 10, and ACTH-dependent ectopic CS, n = 3. In the case of mRNA sequencing, a total of 23 adrenocortical tissue samples were used. This includes the following patient groups: CPA, n = 7; PBMAH, n = 8; normal adjacent adrenal tissue from patients with pheochromocytoma as controls, n = 8. The clinical characteristics of the patients are given in Table 2. Furthermore, of the eight CPA samples in the study, three of them carried known somatic driver mutations in the PRKACA gene and in the ten PBMAH samples, two carried germline mutations in the ARMC5 gene. Table 2. Clinical characteristics of the patient groups. Data are given as median with 25th and 75th percentiles in brackets. CPA, cortisol producing adenoma; CD, Cushing’s disease. The adrenal tissues were stored at −80 °C. Total RNA isolation was carried out from all adrenal cortex samples by an RNeasy Tissue Kit (Qiagen, Hilden, Germany). The isolated RNA was kept frozen at −80 °C until further use. 4.2. MiRNA and RNA Sequencing RNA integrity and the absence of contaminating DNA were confirmed by Bioanalyzer RNA Nano (Agilent Technologies, Santa Clara, CA, USA) and by Qubit DNA High sensitivity kits, respectively. Sequencing libraries were prepared using the Illumina TruSeq Small RNA Library Preparation Kit. NGS was performed on 2 lanes of an Illumina HiSeq2500 (Illumina, CA, USA) multiplexing all samples (paired end read, 50 bp). The quality of sequencing reads was verified using FastQC0.11.5 (http://www.bioinformatics.babraham.ac.uk/projects/fastqc, date last accessed: 13 March 2020) before and after trimming. Adapters were trimmed using cutadapt [40]. Reads with <15 bp and >40 bp insert sequences were discarded. An alignment of reads was performed using miRBase V21 [41,42] with sRNAbench [43]. EdgeR and DeSeq in R were used for further analyses [44,45]. MiRNAs with at least 5 raw counts per library were included. RNA-seq was performed by Qiagen, Hilden, Germany. For mRNA, sequencing was performed on Illumina NextSeq (single end read, 75 bp). Adapter and quality trimming were performed by the “Trim Reads” tool from CLC Genomics Workbench. Furthermore, reads were trimmed based on quality scores. The QC reports were generated by the “QC for Sequencing Reads” tool from CLC Genomics Workbench. Read mapping and gene quantification were performed by the “RNA-seq Analysis” tool from CLC Genomics Workbench [46]. The miRNA-seq data generated in this study have been submitted to the NCBI (PRJNA847385). 4.3. Validation of Individual miRNAs MiRNAs and genes significantly differentially expressed by NGS were validated by QPCR. Reverse transcription of miRNA-specific cDNA was performed by using the TaqMan MicroRNA Reverse Transcription Kit (Thermo Fisher Scientific, Munich, Germany), and the reverse transcription of RNA genes was done by using the Superscript VILO cDNA synthesis Kit (Thermo Fisher Scientific, Munich, Germany). 50 ng of RNA was used for each of the reverse transcription reactions. Quantitative real-time PCR was performed using the TaqMan Fast Universal PCR Master Mix (2×) (Thermo Fisher Scientific, Munich, Germany) on a Quantstudio 7 Flex Real-Time PCR System (Thermo Fisher Scientific, Munich, Germany) in accordance with the manufacturer’s protocol. All QPCR reactions were performed in a final reaction volume of 20 μL and with 1 μL of 1:5 diluted cDNA. Negative control reactions contained no cDNA templates. Gene expression was quantified using the relative quantification method by normalization with reference gene [47]. Statistical analysis using the bestkeeper tool was used to compare and select the best reference gene with stable expression across the human adrenal samples [48]. Reference genes with significantly different Ct values (p-value < 0.01) between the samples were excluded. Furthermore, primer efficiency and the associated correlation coefficient R2 of the selected reference gene were determined by the standard curve method in serially diluted cDNA samples [49]. In the case of miRNA reference genes, miR-16-5p [48,50,51] and RNU6B [52] previously used in similar studies were compared. MiR-16-5p was found to show the most stable expression levels across the samples with a p-value of 0.452 in comparison to RNU6B which had a p-value of 0.001. In the case of RNA reference genes, PPIA [53] and GAPDH [54] were compared. Here, PPIA was found to show the most stable expression levels across the samples with a p-value of 0.019 in comparison to GAPDH which had a p-value of 0.003. Therefore, these genes were used for the normalization of miRNA and RNA expression in human adrenal samples. 4.4. Target Screening In silico prediction of the possible miRNA targets was performed using the miRNA target database, TargetScan, and miRTarBase [19,37]. The top predicted targets were further screened based on their expression status in the RNA-seq data from the adrenocortical tissues of CPA, PBMAH, and controls (unpublished data). Pathway analyses of the targets were performed using Reactome [55] and Panther [56] online platforms. The selected downregulated targets were analyzed by QPCR in the adrenocortical samples to confirm their expression status. The successfully validated candidates were then analyzed for regulation by the miRNA using a dual luciferase assay [57]. 4.5. Dual Luciferase Assay The interaction between the predicted 3′-UTR region of Cyb5a and miR-1247-5p was detected using a luciferase activity assay. The 3′UTR sequences of Cyb5a (129 bp) containing the predicted miR-1247-5p binding sites (psiCHECK-2 Cyb5a 3′UTR WT) were cloned into the psiCHECK-2 vector (Promega, Fitchburg, WI, USA). A QuikChange Site-Directed Mutagenesis kit (Agilent Technologies, CA, USA) was used to mutate the miR-1247-5p binding site (psiCHECK-2 Cyb5a 3′UTR mutant) according to the manufacturer’s protocol. All the sequences were verified by Sanger sequencing. Then, 200 ng of the plasmid was used for each transfection. Synthetic miR-1247-5p mimics and specific oligonucleotides that inhibit endogenous miR-1247-5p (miR-1247-5p inhibitors) were purchased from Promega and 100 nmol of the molecules were used for each transfection according to the manufacturer’s protocol. For the assay, HeLa cells were seeded in 96-well plates and incubated for 24 h. The following day, cells were transfected using the following different conditions: (1) psiCHECK-2 Cyb5a 3′UTR WT + miR-1247-5p mimic; (2) psiCHECK-2 Cyb5a 3′UTR WT + miR-1247-5p inhibitor; (3) psiCHECK-2 Cyb5a 3′UTR WT + water; (4) psiCHECK-2 Cyb5a 3′UTR mutant + miR-1247-5p mimic; (5) psiCHECK-2 Cyb5a 3′UTR mutant + miR-1247-5p inhibitor; (6) psiCHECK-2 Cyb5a 3′UTR mutant + water. Forty-eight hours later, luciferase activity in the cells was measured using the dual luciferase assay system (Promega, Fitchburg, WI, USA) in accordance with the manufacturer’s instructions. Renilla luciferase activity was normalized to firefly luciferase activity. Each treatment was performed in triplicate. Any interaction between the cloned gene, Cyb5a (WT and mutant), and miR-1247-5p mimic is accompanied by a decrease in luminescence. This decrease in luminescence would not be observed when the plasmids are transfected with the miR-1247-5p inhibitor, indicating that observed luminescence differences are caused by specific interactions between the plasmid and the miR-1247-5p mimic. Transfection of the plasmid with water corrects any background interactions between the cloned gene and endogenous miRNAs in the culture. 4.6. In Vivo ACTH Stimulation Experiments were performed on 13-week-old C57BL/6 J female mice (Janvier, Le Genest-Saint-Isle, France). Mice were intraperitoneally injected with 1 mg/kg of ACTH (Sigma Aldrich, Munich, Germany) and adrenals were collected after 10, 30, and 60 min of injections. In addition, control adrenals were collected from mice at baseline conditions (0 min). Mice were killed by cervical dislocation and adrenals were isolated, snap-frozen in liquid nitrogen, and stored at −80 °C for later RNA extraction. MiR-26a was taken as a housekeeping gene in the QPCR [58]. All mice were maintained in accordance with facility guidelines on animal welfare and approved by Landesdirektion Sachsen, Chemnitz, Germany. 4.7. Statistical Analysis and Software R version 3.6.1 was used for the statistical analyses. To identify RNAs differentially expressed, a generalized linear model (GLM, a flexible generalization of ordinary linear regression that allows for variables that have distribution patterns other than a normal distribution) in the software package edgeR (Empirical Analysis of DGE in R) was employed to calculate p-values [45,59]. p-values were adjusted using the Benjamin–Hochberg false discovery rate (FDR) procedure [60]. Disease groups were compared using the unpaired Mann–Whitney test, and to decrease the false discovery rate a corrected p-value was calculated using the Benjamin–Hochberg method. p adjusted < 0.05 and log2 fold change >1.25 was applied as the cut-off for significance for NGS data. GraphPad Prism Version 8 was used for the statistical analysis of QPCR. To calculate differential gene expression, the dCt method (delta Ct (cycle threshold) value equals target miRNA’s Ct minus housekeeping miRNA’s Ct) was used (Microsoft Excel 2016, Microsoft, Redmond, WA, USA). For QPCR, an ANOVA test with Bonferroni correction was used [61] to assess significance; p-values < 0.05 were considered significant. 5. Conclusions In conclusion, while comprehensive information regarding the role of miRNAs in acute and chronic phases of steroidogenesis is available, there is little known about the pathological independent role of miRNAs in the pathology of CS. In our study, we have described ACTH-independent miR-1247-5p and miR-379-5p expression in CS for the first time. Thus, by regulating different genes in the WNT signaling pathway, the miRNAs may individually contribute to the Cushing’s pathology in specific subtypes. Supplementary Materials The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/ijms23147676/s1. Author Contributions Conceptualization, S.V., A.C. and A.R.; methodology, S.V., R.Z. and M.E.; software, S.V. and M.E.; validation, R.Z., A.O., D.W. and B.W.; formal analysis, S.V.; investigation, S.V., R.Z., M.E., A.O. and D.W.; resources, A.C., B.W., M.R. and A.R.; data curation, S.V. and R.Z.; writing—original draft preparation, S.V., R.Z. and A.R.; writing—review and editing, S.S., M.R. and A.R.; visualization, S.V.; supervision, A.R.; project administration, A.R.; funding acquisition, A.C., S.S., M.R. and A.R. All authors have read and agreed to the published version of the manuscript. Funding This work was supported by a grant from the Deutsche Forschungsgemeinschaft (DFG) (within the CRC/Transregio 205/1 “The Adrenal: Central Relay in Health and Disease”) to A.C., B.W., S.S., M.R. and A.R., and individual grant SB 52/1-1 to S.S. This work is part of the German Cushing’s Registry CUSTODES and has been supported by a grant from the Else Kröner-Fresenius Stiftung to MR (2012_A103 and 2015_A228). A.R. was supported by the FöFoLe Program of the Ludwig Maximilian University, Munich. We thank I. Shapiro, A. Parl, C. Kühne, and S. Zopp for their technical support. Institutional Review Board Statement The study was conducted according to the guidelines of the Declaration of Helsinki and approved by the Ethics Committee of the Ludwig Maximilian University, Munich (protocol code 379-10, 152-10 and 20 July2021). Informed Consent Statement Informed consent was obtained from all subjects involved in the study. Data Availability Statement The miRNA-seq data generated in this study have been submitted to the NCBI (PRJNA847385). Conflicts of Interest The authors declare no conflict of interest. References Kotłowska, A.; Puzyn, T.; Sworczak, K.; Stepnowski, P.; Szefer, P. 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The Carling Adrenal Center, a worldwide destination for the surgical treatment of adrenal tumors, becomes the first center to offer the use of amniotic membrane during adrenal surgery which saves functional adrenal tissue in patients undergoing adrenal surgery. This novel technique enables more patients to have a partial adrenalectomy thereby preserving some normal adrenal physiology, potentially eliminating life-long adrenal hormone replacement. Preliminary clinical data from the Carling Adrenal Center suggest that the use of a human amniotic membrane allograph on the adrenal gland remnant following partial adrenal surgery leads to faster recovery of normal adrenal gland function. Rather than removing the entire adrenal gland—which has been standard of care for decades—a portion of the adrenal gland is able to be salvaged with amniotic membrane placed upon the remnant as a biologic covering. The preliminary data from an ongoing clinical trial shows this technique translates into fewer patients needing steroid hormone replacement following adrenal surgery, and if they do, it is for a significantly shorter period of time. "Sometimes it is possible, and preferable, to remove the adrenal tumor without removing the entire adrenal gland. This is called partial adrenal surgery and our study shows this technique is more successful when amniotic membrane is used," said Dr. Carling. He further stresses that "removing only part of the adrenal gland is a more advanced operation and is typically only performed by expert adrenal surgeons. The goal is to leave some normal adrenal tissue so that the patient can avoid adrenal insufficiency which requires a daily dose of several adrenal hormones and steroids. Partial adrenal surgery is especially beneficial for patients with pheochromocytoma, as well as Conn's and Cushing's syndrome. Avoiding daily steroids is life-changing for these patients so this is a major breakthrough." So how does it work? The increased viability of the adrenal gland remnant is presumed to be related to the release of growth factors known to be present in amniotic tissue which is in direct contact with the adrenal gland remnant as a covering. The results are improved rates of viable adrenal cortical tissues with faster regeneration and recovery to normal endocrine physiology by the adrenal cortical cells. These findings come during Adrenal Disease Awareness Month. Adrenal gland diseases cause many debilitating symptoms like chronic headaches, anxiety, depression, fatigue, brain fog, memory loss, dangerously high blood pressure, heart arrythmia, weight gain, tremors, and more, yet they are often misdiagnosed or improperly treated. Since many doctors are inexperienced in the workup of adrenal hormone problems and only see a handful of adrenal tumors during their careers, it is important for patients to know about the symptoms of adrenal tumor disease and request their doctor measure adrenal hormones. Adrenal.com is the leading resource for adrenal gland function, tumors and cancers, and an award-winning resource for adrenal gland surgery. The diagnosis and surgical treatment of all types of adrenal tumor types are discussed. Adrenal.com is edited by Dr. Tobias Carling who has performed more adrenal surgery than any other surgeon and has published some of the most important scientific studies of adrenal disease and adrenal surgery including the understanding of the pathogenesis of pheochromocytoma and adrenal tumors causing Conn's and Cushing's syndrome. Established by Dr. Tobias Carling in 2020, the Carling Adrenal Center located at the Hospital for Endocrine Surgery in Tampa FL, is the highest volume adrenal surgical center in the world. The Center now averages nearly 20 adrenal tumor patients every week. Dr Carling was the Director of Endocrine Surgery at Yale University prior to opening the Center in Tampa. At the new Hospital for Endocrine Surgery, Dr Carling joins the Norman Parathyroid Center, the Clayman Thyroid Center and the Scarless Thyroid Surgery Center as the highest volume endocrine surgery center in the world. About the Carling Adrenal Center: Founded by Dr. Tobias Carling, one of the world's leading experts in adrenal gland surgery, the Carling Adrenal Center is a worldwide destination for the surgical treatment of adrenal tumors. Dr. Carling spent nearly 20 years at Yale University, including 7 as the Chief of Endocrine Surgery before leaving in 2020 to open to Carling Adrenal Center, which performs more adrenal operations than any other hospital in the world. (813) 972-0000. More about partial adrenalectomy for adrenal tumors can be found at the Center's website www.adrenal.com. From https://www.streetinsider.com/PRNewswire/Novel+application+of+amniotic+membrane+saves+adrenal+tissue+in+patients+undergoing+adrenal+surgery/19915274.html

Highlights • Cushing syndrome (CS) is a rare disorder with a variety of underlying etiologies. • CS is expected to affect 0.2 to 5 people per million per year. • Adrenal-dependent CS is an uncommon variant of CS. • This study reports a rare occurrence of pituitary and adrenal adenoma with CS. Abstract Introduction Cushing syndrome is a rare disorder with a variety of underlying etiologies, that can be exogenous or endogenous (adrenocorticotropic hormone (ACTH)-dependent or ACTH-independent). The current study aims to report a case of ACTH-independent Cushing syndrome with adrenal adenoma and nonfunctioning pituitary adenoma. Case report A 37–year–old female presented with amenorrhea for the last year, associated with weight gain. She had a moon face, buffalo hump, and central obesity. A 24-hour urine collection for cortisol was performed, revealing elevated cortisol. Cortisol level was non-suppressed after administering dexamethasone. MRI of the pituitary revealed a pituitary microadenoma, and the CT scan of the abdomen with adrenal protocol revealed a left adrenal adenoma. Discussion Early diagnosis may be postponed due to the variety of clinical presentations and the referral of patients to different subspecialists based on their dominant symptoms (gynecological, dermatological, cardiovascular, psychiatric); it is, therefore, critical to consider the entire clinical presentation for correct diagnosis. Conclusion Due to the diversity in the presentation of CS, an accurate clinical, physical and endocrine examination is always recommended. Keywords Cushing syndrome Cushing's disease Adrenal adenoma Pituitary adenoma Urine free cortisol 1. Introduction Cushing syndrome (CS) is a collection of clinical manifestations caused by an excess of glucocorticoids [1]. CS is a rare disorder with a variety of underlying etiologies that can be exogenous due to continuous corticosteroid therapy for any underlying inflammatory illness or endogenous due to either adrenocorticotropic hormone (ACTH)-dependent or ACTH-independent [2], [3]. Cushing syndrome is expected to affect 0.2 to 5 people per million per year. Around 10% of such cases involve children [4], [5]. ACTH-dependent glucocorticoid excess owing to pituitary adenoma accounts for the majority (60–70%) of endogenous CS, with primary adrenal causes accounting for only 20–30% and ectopic ACTH-secreting tumors accounting for the remaining 5–10% [6]. Adrenal-dependent CS is an uncommon variant of CS caused mostly by benign (90%) or malignant (8%) adrenal tumors or, less frequently, bilateral micronodular (1%) or macronodular (1%) adrenal hyperplasia [7]. The current study aims to report a case of ACTH-independent Cushing syndrome with adrenal adenoma and nonfunctioning pituitary adenoma. The report has been arranged in line with SCARE guidelines and includes a brief literature review [8]. 2. Case report 2.1. Patient's information A 37–year–old female presented with amenorrhea for the last year, associated with weight gain. She denied having polyuria, polydipsia, headaches, visual changes, dizziness, dryness of the skin, cold intolerance, or constipation. She had no history of chronic disease and denied using steroids. She visited an internist, a general surgeon, and a gynecologist and was treated for hypothyroidism. She was put on Thyroxin 100 μg daily, and oral contraceptive pills were given for her menstrual problems. Last time, the patient was referred to an endocrinology clinic, and they reviewed the clinical and physical examinations. 2.2. Clinical examination She had a moon face, buffalo hump, central obesity, pink striae over her abdomen, and proximal weakness of the upper limbs. After reviewing the history and clinical examination, CS was suspected. 2.3. Diagnostic assessment Because the thyroid function test revealed low thyroid-stimulating hormone (TSH), free T3, and freeT4, the patient was sent for a magnetic resonance imaging (MRI) of the pituitary, which revealed a pituitary microadenoma (7 ∗ 6 ∗ 5) mm (Fig. 1). Since the patient was taking thyroxin and oral contraceptive pills, the investigations were postponed for another six weeks due to the contraceptive pills' influence on the results of the hormonal assessment for CS. After six weeks of no medication, a 24-hour urinary free cortisol (UFC) was performed three times, revealing elevated cortisol levels (1238, 1100, and 1248) nmol (normal range, 100–400) nmol. A dexamethasone suppression test was done (after administering dexamethasone tab 1 mg at 11 p.m., serum cortisol was measured at 9 a.m.). The morning serum cortisol level was 620 nmol (non-suppressed), which normally should be less than 50 nmol. The ACTH level was below 1 pg/mL. Download : Download high-res image (103KB) Download : Download full-size image Fig. 1. Contrast enhanced T1W weighted MRI (coronal section) showing small 7 mm hypo-enhanced microadenoma (yellow arrow) in right side of pituitary gland with mild superior bulge. Based on these findings, ACTH independent CS was suspected. The computerized tomography (CT) scan of the abdomen with adrenal protocol revealed a left adrenal adenoma (33 mm × 25 mm) without features of malignancy (Fig. 2). Download : Download high-res image (168KB) Download : Download full-size image Fig. 2. Computed tomography scan of the abdomen with IV contrast, coronal section, showing 33 mm × 25 mm lobulated enhanced left adrenal tumor (yellow arrow), showing absolute washout on dynamic adrenal CT protocol, consistent with adrenal adenoma. 2.4. Therapeutic intervention The patient was referred to the urologist clinic for left adrenalectomy after preparation for surgery and perioperative hormonal management. She underwent laparoscopic adrenalectomy and remained in the hospital for two days. The histopathology results supported the diagnosis of adrenal adenoma. 2.5. Follow-up She was released home after two days on oral hydrocortisone 20 mg in the morning and 10 mg in the afternoon. After one month of follow-up, serum cortisol was 36 nmol, with the resolution of some features such as weight reduction (3 kg) and skin color (pink striae became white). 3. Discussion Cushing's syndrome is a serious and well-known medical condition that results from persistent exposure of the body to excessive glucocorticoids, either from endogenous or, most frequently, exogenous sources [9]. The average age of diagnosis is 41.4 years, with a female-to-male ratio of 3:1 [10]. ACTH-dependent CS accounts for almost 80% of endogenous CS, while ACTH-independent CS accounts for nearly 20% [10]. This potentially fatal condition is accompanied by several comorbidities, including hypertension, diabetes, coagulopathy, cardiovascular disease, infections, and fractures [11]. Exogenous CS, also known as iatrogenic CS, is more prevalent than endogenous CS and is caused by the injection of supraphysiologic glucocorticoid dosages [12]. ACTH-independent CS is induced by uncontrolled cortisol release from an adrenal gland lesion, most often an adenoma, adrenocortical cancer, or, in rare cases, ACTH-independent macronodular adrenal hyperplasia or primary pigmented nodular adrenal disease [13]. The majority of data suggests that early diagnosis is critical for reducing morbidity and mortality. Detection is based on clinical suspicion initially, followed by biochemical confirmation [14]. The clinical manifestation of CS varies depending on the severity and duration of glucocorticoid excess [14]. Some individuals may manifest varying symptoms and signs because of a rhythmic change in cortisol secretion, resulting in cyclical CS [15]. The classical symptoms of CS include weight gain, hirsutism, striae, plethora, hypertension, ecchymosis, lethargy, monthly irregularities, diminished libido, and proximal myopathy [16]. Neurobehavioral presentations include anxiety, sadness, mood swings, and memory loss [17]. Less commonly presented features include headaches, acne, edema, abdominal pain, backache, recurrent infection, female baldness, dorsal fat pad, frank diabetes, electrocardiographic abnormalities suggestive of cardiac hypertrophy, osteoporotic fractures, and cardiovascular disease from accelerated atherosclerosis [10]. The current case presented with amenorrhea, weight gain, moon face, buffalo hump, and skin discoloration of the abdomen. Similar to the current case, early diagnosis may be postponed due to the variety of clinical presentations and the referral of patients to different subspecialists based on their dominant symptoms (gynecological, dermatological, cardiovascular, psychiatric); it is, therefore, critical to consider the entire clinical presentation for correct diagnosis [18]. Weight gain may be less apparent in children, but there is frequently an arrest in growth with a fall in height percentile and a delay in puberty [19]. The diagnosis and confirmation of the etiology can be difficult and time-consuming, requiring a variety of laboratory testing and imaging studies [20]. According to endocrine society guidelines, the initial assessment of CS must include one or more of the three following tests: 24-hour UFC measurement; evaluation of the diurnal variation of cortisol secretion by assessing the midnight serum or salivary cortisol level; and a low-dose dexamethasone suppression test, typically the 1 mg overnight test [21]. Although UFC has sufficient sensitivity and specificity, it does not function well in milder cases of Cushing's syndrome [22]. In CS patients, the typical circadian rhythm of cortisol secretion is disrupted, and a high late-night cortisol serum level is the earliest and most sensitive diagnostic indicator of the condition [23]. In the current case, the UFC was elevated, and cortisol was unsuppressed after administration of dexamethasone. All patients with CS should have a high-resolution pituitary MRI with a gadolinium-based contrast agent to prove the existence or absence of a pituitary lesion and to identify the source of ACTH between pituitary adenomas and ectopic lesions [24]. Adrenal CT scan is the imaging modality of choice for preoperatively localizing and subtyping adrenocortical lesions in ACTH-independent Cushing's syndrome [9]. MRI of the pituitary gland of the current case showed a microadenoma and a CT scan of the adrenals showed left adrenal adenoma. Surgical resection of the origin of the ACTH or glucocorticoid excess (pituitary adenoma, nonpituitary tumor-secreting ACTH, or adrenal tumor) is still the first-line treatment of all forms of CS because it leaves normal adjacent structures and results in prompt remission and inevitable recovery of regular adrenal function [12], [25]. Laparoscopic (retroperitoneal or transperitoneal) adrenalectomy has become the gold standard technique for adrenal adenomas since it is associated with fewer postoperative morbidity, hospitalization, and expense when compared to open adrenalectomy [17]. In refractory cases, or when a patient is not a good candidate for surgery, cortisol-lowering medication may be employed [26]. The current case underwent left adrenalectomy. Symptoms of CS, such as central obesity, muscular wasting or weakness, acne, hirsutism, and purple striae generally improve first and may subside gradually over a few months or even a year; nevertheless, these symptoms may remain in 10–30% of patients [27]. Glucocorticoid replacement is essential after adrenal-sparing curative surgery until the pituitary-adrenal function returns, which might take up to two years, especially if adrenal adenomas have been resected [25]. Chronic glucocorticoid excess causes lots of new co-morbidities, lowering the quality of life and increasing mortality. The most common causes of mortality in CS are cardiovascular disease and infections [28]. After one month of follow-up, serum cortisol was 36 nmol, and several features, such as weight loss (3 kg) and skin color, were resolved (pink striae became white). In conclusion, the coexistence of adrenal adenoma and pituitary adenoma with CS is a rare possibility. Due to the diversity in the presentation of CS, an accurate clinical, physical and endocrine examination is always recommended. Laparoscopic adrenalectomy is the gold standard for treating adrenal adenoma. Consent Written informed consent was obtained from the patient's family for publication of this case report and accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal on request. Provenance and peer review Not commissioned, externally peer-reviewed. Ethical approval Approval is not necessary for case report (till 3 cases in single report) in our locality. The family gave consent for the publication of the report. Funding None. Guarantor Fahmi Hussein Kakamad, Fahmi.hussein@univsul.edu.iq. Research registration number Not applicable. CRediT authorship contribution statement Abdulwahid M. Salh: major contribution of the idea, literature review, final approval of the manuscript. Rawa Bapir: Surgeon performing the operation, final approval of the manuscript. Fahmi H. Kakamad: Writing the manuscript, literature review, final approval of the manuscript. Soran H. Tahir, Fattah H. Fattah, Aras Gh. Mahmood, Rawezh Q. Salih, Shaho F. Ahmed: literature review, final approval of the manuscript. Declaration of competing interest None to be declared. References [1] S.M. Ahmed, S.F. Ahmed, S. Othman, B.A. Abdulla, S.H. Mohammed, A.M. Salih, et al. 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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/10.1002/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

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/

Justine Herndon, PA-C, and Irina Bancos, MD, on Post-Operative Cushing Syndrome Care – Curative procedures led to widespread resolution or improvement of hyperglycemia by Scott Harris , Contributing Writer, MedPage Today January 18, 2022 In a recent study, two-thirds of people with Cushing syndrome (CS) saw resolved or improved hyperglycemia after a curative procedure, with close post-operative monitoring an important component of the process. Among 174 patients with CS included in the longitudinal cohort study (pituitary in 106, ectopic in 25, adrenal in 43), median baseline HbA1c was 6.9%. Of these, 41 patients were not on any therapy for hyperglycemia, 93 (52%) took oral medications, and 64 (37%) were on insulin. At the end of the period following CS remission (median 10.5 months), 37 (21%) patients had resolution of hyperglycemia, 82 (47%) demonstrated improvement, and 55 (32%) had no change or worsened hyperglycemia. Also at the end of follow-up, HbA1c had fallen 0.84% (P<0.0001), with daily insulin dose decreasing by a mean of 30 units (P<0.0001). Justine Herndon, PA-C, and Irina Bancos, MD, both endocrinology researchers with Mayo Clinic in Minnesota, served as co-authors of the report, which was published in the Journal of the Endocrine Society. Here they discuss the study and its findings with MedPage Today. The exchange has been edited for length and clarity. What was the study's main objective? Herndon: As both a hospital diabetes provider and clinic pituitary/gonadal/adrenal provider, I often hear questions from colleagues about how to manage a patient's diabetes post-operatively after cure from CS. While clinical experience has been helpful in guiding these discussions, the literature offered a paucity of data on diabetes/hyperglycemia specifically after surgery. There was also a lack of data on specific subgroups of CS, whether by sub-type or severity. Therefore, we felt it was important to see what our past patient experiences showed in terms of changes in laboratory data, medications, and which patients were more likely to see improvement in their diabetes/hyperglycemia. The overall goal was to help clinicians provide appropriate patient education and care following a curative procedure. In addition to its primary findings, the study also identified several factors associated with resolution or improvement of hyperglycemia. What were these factors? Bancos: Both clinical and biochemical severity of CS, as well as Cushing subtype, were associated with improvement. We calculated severity based on symptoms and presence of comorbidities, and we calculated biochemical severity based on hormonal measurements. As clinical and biochemical scores were strongly correlated, we chose only one (biochemical) for multivariable analysis. In the multivariable analysis of biochemical severity of Cushing, subtype of Cushing, and subtype of hyperglycemia, we found that patients with a severe biochemical severity score were 2.4 fold more likely to see improved hyperglycemia than people with a moderate or mild severity score (OR 2.4 (95% CI 1.1-4.9). We also found that patients with the nonadrenal CS subtype were 2.9 fold more likely to see improved hyperglycemia when compared to people with adrenal CS (OR of 2.9 (95% CI 1.3-6.4). The type of hyperglycemia (diabetes versus prediabetes) was not found to be significant. Did anything surprise you about the study results? Herndon: I was surprised to see improvement in hyperglycemia in patients who were still on steroids, as you would expect the steroids to still have an impact. This shows how much a CS curative procedure truly leads to changes in the comorbidities that were a result of the underlying disease. Also, I was surprised that the type of hyperglycemia was not a predictor of improvement after cure, although it was quite close. We also had a few patients whose hyperglycemia worsened, and we could not find a specific factor that predicted which patients did not improve. What are the study's implications for clinicians who treat people with CS? Bancos: We think our study shows the clear need for closer follow-up -- more frequently than the typical three-to-six months for diabetes. This can be accomplished through review of more than just HbA1c, such as reviewing blood glucose logbooks, asking about hypoglycemia symptoms, and so forth. Patients with severe CS who are being treated with insulin or hypoglycemic medications are especially likely to decrease their medications to avoid hypoglycemia during postoperative period. Read the study here. Bancos reported advisory board participation and/or consulting with Strongbridge, Sparrow Pharmaceutics, Adrenas Therapeutics, and HRA Pharma outside the submitted work. Herndon did not disclose any relevant financial relationships with industry. Primary Source Journal of the Endocrine Society Source Reference: Herndon J, et al "The effect of curative treatment on hyperglycemia in patients with Cushing syndrome" J Endocrine Soc 2022; 6(1): bvab169. From https://www.medpagetoday.com/reading-room/endocrine-society/adrenal-disorders/96709

Extracted and adapted from this series: Post 1) I was officially diagnosed with Cushing's yesterday. I have a CT scan to check on my adrenal tumor and a meeting with my surgeon tomorrow. Hopefully they will schedule surgery for Monday or Tuesday. I have suffered over a year with this, been in congestive heart failure, and believe this cortisol caused my son to be stillborn in March. It's been the year from hell. Please pray that all goes well tomorrow and that I will be cured of this once and for all!! Post 2) Surgery set for the 23rd!!!!! He is planning a right adrenaltectomy. I am so darn excited... Post 3) I'm almost two weeks out of adrenal surgery. He removed the tumor & my gland. This has been the hardest and most painful two weeks of my life. I am already noticing little changes in my body. My skin is getting texture, my hair is not as brittle, my swelling goes down each day, and my nails are white instead of yellow and are stronger. I am getting hair back on my arms, legs, & feet too. I can't wait to continue to get well. I am ready to be able to get out and about. I am pretty much housebound now because of the pain of the withdrawal from the cortisol. I stay on my painkillers and rest in my recliner. Hubby bought it for me because I can't sleep in the bed comfortably. He's the best. He's been sleeping on our air mattress in the living room with me for almost 2 weeks now. He is always there to help me get out of the recliner when I need to. He is amazing. Just wanted to update you all. Getting better everyday. Post 4) I am on 40mg Hydrocortisone daily right now. I will have my first wean close to Christmas. I have an appt. on the 21st with my endo. She is fantastic and saved my life from this stuff. I am so blessed. Today is a rough day. I did have 2 good days in a row which was a huge blessing. Thanks for thinking of me! Post 5) Well, I just survived month 1 of recovery. It was HORRIBLE. I have never had so much pain in my life. I am still on 40 mg and my endo. wants me to wean 10 mg starting on the 27th. We'll see how it goes. I have so much pain, shaking, chills, no sleep NOW. I can't imagine how its going to be on a lower dose. My cortisol level was SO HIGH (2107) before surgery. I knew this withdrawal was going to be terrible. SHe had never seen a level as high as mine before. The lab actually tested my urine twice because they didn't believe it the first time. I am doing a lot of resting right now. I am very nervous about my mother leaving on New Year's Day. I don't know how I am going to handle my 3 year old on my own. I hurt so badly and my vision isn't the greatest yet. Thanks for thinking of me and writing me back. Post 6) We have another call into my endo about my suffering. I have done nothing but shake uncontrollably all day so far. I hurt so badly. I am up every hour at night writhing in pain. I refuse to suffer like this anymore. I want some relief. Thank you so much for all of the advice. It means the world to me. Great news is that I am off my BP meds as of today!! Cardiologist's office said I could quit them. I am thrilled. Now to get this pain under control. Post 7) Endo said we can do whatever I can tolerate. I am now doing 20/20/10 instead of 20/10/10. I am still in pain, but it's a little more tolerable. She said if I am just miserable and can't take the pain, then I can do a bedtime dose. I am going to try melatonin to help me sleep per her suggestion. She wants to see how I do on this new dose and start a slow wean in a few weeks. Post 8) Things have been getting better by the week. New years day was my best physical and mental day so far. I can actually feel my old self returning! !! Today I have lots of bone/muscle pain. Its better than a few weeks ago by far. Yesterday I was able to enjoy my son and play with him for the first time in a long time. I could even dance a little with him. He was so happy. I am down to 20/17.5/10& am handling it well. The pain is tolerable. My hump is almost gone, my stomach is mushy and shrinking, skin is peeling and improving, hair is growing in normally. I will be six weeks out this Wed.

Single-cell transcriptome analysis identifies a unique tumor cell type producing multiple hormones in ectopic ACTH and CRH secreting pheochromocytoma Abstract Ectopic Cushing’s syndrome due to ectopic ACTH&CRH-secreting by pheochromocytoma is extremely rare and can be fatal if not properly diagnosed. It remains unclear whether a unique cell type is responsible for multiple hormones secreting. In this work, we performed single-cell RNA sequencing to three different anatomic tumor tissues and one peritumoral tissue based on a rare case with ectopic ACTH&CRH-secreting pheochromocytoma. And in addition to that, three adrenal tumor specimens from common pheochromocytoma and adrenocortical adenomas were also involved in the comparison of tumor cellular heterogeneity. A total of 16 cell types in the tumor microenvironment were identified by unbiased cell clustering of single-cell transcriptomic profiles from all specimens. Notably, we identified a novel multi-functionally chromaffin-like cell type with high expression of both POMC (the precursor of ACTH) and CRH, called ACTH+&CRH + pheochromocyte. We hypothesized that the molecular mechanism of the rare case harbor Cushing’s syndrome is due to the identified novel tumor cell type, that is, the secretion of ACTH had a direct effect on the adrenal gland to produce cortisol, while the secretion of CRH can indirectly stimulate the secretion of ACTH from the anterior pituitary. Besides, a new potential marker (GAL) co-expressed with ACTH and CRH might be involved in the regulation of ACTH secretion. The immunohistochemistry results confirmed its multi-functionally chromaffin-like properties with positive staining for CRH, POMC, ACTH, GAL, TH, and CgA. Our findings also proved to some extent the heterogeneity of endothelial and immune microenvironment in different adrenal tumor subtypes. Editor's evaluation The study described an extremely rare type of adrenal pheochromocytoma that secretes both ACTH and CRH, in addition to catecholamines. Single-cell RNA sequencing of the tumor and other tumors revealed a group of cells that are responsible for the hormone secretion. We believe that this work will provide an interesting example of functional endocrine tumors and how they are formed. https://doi.org/10.7554/eLife.68436.sa0 Introduction Cushing’s syndrome (CS) is a rare disorder caused by long-term exposure to excessive glucocorticoids, with an annual incidence of about 0.2–5.0 per million (Lacroix et al., 2015; Newell-Price et al., 2006; Lindholm et al., 2001; Steffensen et al., 2010; Bolland et al., 2011; Valassi et al., 2011). About 80% of CS cases are due to ACTH secretion by a pituitary adenoma, about 20% are due to ACTH secretion by nonpituitary tumors (ectopic ACTH syndrome [EAS]), and 1% are caused by corticotropin-releasing hormone (CRH)-secreting tumors (Alexandraki and Grossman, 2010; Ejaz et al., 2011; Ballav et al., 2012). Most EAS tumors (~60%) are more common intrathoracic tumors, only 2.5–5% of all EAS are caused by a pheochromocytoma (Alexandraki and Grossman, 2010; Isidori et al., 2006; Ilias et al., 2005; Aniszewski et al., 2001). Pheochromocytoma, a catecholamine-producing tumor, becomes even rarer when it is capable of both secreting ACTH and CRH (Lenders et al., 2005; Zelinka et al., 2007). By 2020, only two cases with pheochromocytoma secreted both ACTH and CRH were reported (Elliott et al., 2021; O’Brien et al., 1992; Jessop et al., 1987). As one of the largest adrenal tumor treatment centers in China, our hospital, Peking Union Medical College Hospital (PUMCH) receives more than 500 adrenal surgery performed per year, with almost 100 cases undergoing pheochromocytoma surgery. But so far, we have encountered only one case of pheochromocytoma secreting both ACTH and CRH, which was first reported in this study. Since the combination of dual ACTH/CRH secreting pheochromocytoma with CS is extremely rare, there is limited knowledge about the diagnosis and management of this disease. Ectopic secretion hormones ACTH and CRH may complicate the presentation of pheochromocytoma, and this tumor usually leads to CS, which can be fatal if not properly diagnosed and managed (Ballav et al., 2012; Ilias et al., 2005; Lenders et al., 2014; Lase et al., 2020). Surgical resection of the pheochromocytoma is the primary treatment option. Although previous studies have reported ectopic ACTH and CRH secreting pheochromocytomas, it was unclear whether a unique cell type that produces multiple hormones influences CS. The concept of ‘one cell, one hormone, and one neuron one transmitter,’ which is known as Dale’s Principle (Dale in 1934; for detailed discussion, see Burnstock, 1976), has dominated the understanding of neurotransmission for many years (Burnstock, 1976). Currently, single-cell RNA-sequencing (scRNA-seq) can examine the expression profiles of a single cell and is recognized as the gold standard for defining cell states and phenotypes (Tang et al., 2009; Tammela and Sage, 2020; Kolodziejczyk et al., 2015; Patel et al., 2014; Tirosh et al., 2016b; Tirosh et al., 2016a; Puram et al., 2017; Venteicher et al., 2017; Young et al., 2018; Bernard et al., 2019; Segerstolpe et al., 2016; Reichert and Rustgi, 2011). It can reveal the presence of rare and novel unique cell types, such as CFTR-expressing pulmonary ionocytes on lung airway epithelia (Montoro et al., 2018; Plasschaert et al., 2018). It also provides an unbiased method to better understand the diversity of immune cells in the complex tumor microenvironment (Papalexi and Satija, 2018; Stubbington et al., 2017). In this study, we reported a rare case of CRH/ACTH-secreting pheochromocytoma infiltrating the kidney and psoas muscle tissue. scRNA-seq identified a unique chromaffin-like cell type, called ACTH+&CRH + pheochromocyte, with both high expression of POMC (precursor for ACTH) and CRH pheochromocyte as well as TH (tyrosine hydroxylase, a key enzyme for catecholamine synthesization). Immunocytochemical and immunofluorescence staining showed all for these markers, which confirmed the tumor capable of multiple hormones secreting characteristics. We determined that the expression of POMC directly causes the secretion of ACTH, and the expression of CRH indirectly promotes the secretion of ACTH hormone, which ultimately leads to CS. After the tumor resection, clinical manifestations also showed complete remission of CS. For comparison, other adrenal tumor subtypes were also collected and studied, namely, a common pheochromocytoma (without ectopic ACTH or CRH secretion function) and two adrenocortical adenomas. We used a scRNA-seq approach to obtain transcriptomic profiles for all collected samples and identified a list of differentially expressed genes (DEGs) through cell clustering and markers finding. Notably, GAL, co-expressed with ACTH and CRH, could be a new candidate marker to detect the rare ectopic ACTH+&CRH + secreting pheochromocytes by comparing ACTH+&CRH + pheochromocyte with common pheochromocyte and cortical cell clusters. It suggested that GAL, which encodes small neuroendocrine peptides, may be locally involved in the regulation of the hypothalamic-pituitary-adrenal (HPA) axis. Results Single-cell profiling and unbiased clustering of collecting specimens We applied scRNA-seq methods to perform large-scale transcriptome profiling of seven prospectively collected samples from tumors and peritumoral tissue of three adrenal tumor patients (Figure 1A). Case 1 suffered from a rare pheochromocytoma with typical Cushingoid features. The laboratory results showed high levels of cortisol, ACTH, and catecholamines. The abdominal contrast-enhanced computer tomography scanning revealed bilateral adrenocortical hyperplasia and irregular tumor within the left adrenal. After the resection, we collected three dissected tumor specimens (esPHEO_T1, esPHEO_T2, and esPHEO_T3) from different anatomic sites of the tumor and an adrenal tissue adjacent to the tumor (esPHEO_Adj). For comparison, we also collected other adrenal tumors, namely, a common pheochromocytoma (PHEO_T) from Case 2 and two adrenocortical adenomas (ACA_T1 and ACA_T2) from Case 3. Case 2 showed elevated catecholamines and normal levels of cortisol and ACTH. Case 3 showed a high level of cortisol, a low level of ACTH, and an intermediate level of catecholamines. The detailed clinical information for the three cases was summarized in Appendix 1—table 1. To investigate the difference of the secretory function, we performed the immunohistochemistry (IHC) staining of selected markers, CgA (chromogranin A) and ACTH in esPHEO_T1, PHEO_T, and esPHEO_Adj samples (Figure 1B). We observed that CgA positive cells were present in both pheochromocytomas (esPHEO_T1 and PHEO_T), but ACTH positive cells were only observed in the rare pheochromocytoma (esPHEO_T1) with the ACTH-secreting cellular characteristics. As expected, there were no CgA and ACTH positive cells in the adjacent sample (esPHEO_Adj). Thus, at the clinical stage, our histopathology results confirmed that Case 1 was a rare ectopic ACTH secreting pheochromocytoma which stained positively for both ACTH and CgA. Figure 1 Download asset Open asset Clinical sample collection of adrenal tumor and adjacent specimen for scRNA-seq analysis. (A) scRNA-seq workflow for three tumor specimens (esPHEO_T1, esPHEO_T2, and esPHEO_T3) and one adjacent specimen (esPHEO_Adj) from the rare pheochromocytoma with ectopic ACTH and CRH secretion (Case … see more Then, we applied scRNA-seq approaches to selected seven specimen samples (six tumors and one sample adjacent to the tumor). The tissues after resection were rapidly digested into a single-cell suspension, and the 3′-scRNA-seq protocol (Chromium Single Cell 3′ v2 Libraries) was performed for each sample unbiasedly. After quality control filtering to remove cells with low gene detection, high mitochondrial gene coverage, and doublets filtration, we compiled a unified cells-by-genes expression matrix of a total of 44,511 individual cells (Supplementary file 1, Appendix 1—figure 2). Then the SCT-transformed normalization, principal component analysis (PCA), was employed to perform unsupervised dimensionality reduction. Then, the cells were clustered based on the graph-based clustering analysis, and visualized in the distinguished diagram using the Uniform Manifold Approximation and Projection (UMAP) method. The marker genes were calculated to identify each cell cluster by performing differential gene expression analysis (Supplementary file 2). As shown in Figure 2A, the distinct cell clusters were identified and the conventional cell lineage gene markers were employed to annotate the clusters, such as CHGA and CHGB for adrenal chromaffin cell, cytochrome P450 superfamily for adrenocortical cell, S100B for sustentacular cell, GNLY for NK cell, MS4A1 for B cell, CD8A for CD8+ T cell, and IL7R for CD4+ T cell. Based on the expression of gene markers, we recognized a total of 16 main cell groups: ACTH+&CRH + pheochromocyte, pheochromocyte, adrenocortical, sustentacular, erythroblast/granulosa, endothelial, fibroblast, neutrophil, monocyte, macrophage, plasma, B, NK, CD8+ T&NKT, CD8+ T, and CD4+ T, among which the endothelial cell group was composed of four endothelial cell subgroups. The heatmap showed the expression levels of specific cluster markers for each cell phenotype that we identified (Figure 2B). For this analysis, we specifically focused on the four types of adrenal cells and showed their markers in a heatmap (Appendix 1—figure 3). Additionally, we detected the transcription factors alongside their candidate target genes, which are jointly called regulons. The analysis scored the activity of regulon for each cell (Appendix 1—figure 4A) and yielded specific regulons for each cellular cluster (Appendix 1—figure 4B). We also specifically focused on the adrenal cells and found XBP1 as the top regulons for ACTH+&CRH + pheochromocyte and adrenocortical cell type (Appendix 1—figure 4C). Figure 2 Download asset Open asset Different cell types and their highly expressed genes through single-cell transcriptomic analysis. (A) The t-distributed stochastic neighbor embedding (t-SNE) plot shows 16 main cell types from all specimens. (B) Heatmap shows the scaled expression patterns of the top 10 marker genes in each cell … see more Identification of a previously unrecognized cell type The presence of heterogeneous cell populations in different adrenal tumor specimens and the peritumoral sample (Figure 3A) prompted us to investigate their cellular compositions and characteristics. As shown in Figure 3B, different sources of specimens represented distinct cell type compositions. Notably, although the size of the cell clusters of the adrenal gland was relatively small, four distinct subtypes of adrenal cells were observed, including ACTH+&CRH + pheochromocyte, pheochromocyte, adrenocortical cells, and sustentacular cells. The ACTH+&CRH + pheochromocytoma cell subtype was specific to three tumor samples, esPHEO_T1, esPHEO_T2, and esPHEO_T3 from Case 1, but was not observed in the peritumoral sample (esPHEO_Adj) and other adrenal tumor samples from Case 2 (PHEO_T) and Case 3 (ACA_T1 and ACA_T2). This result was consistent with the clinical symptoms in our earlier reports that ACTH was only over-secreted in pheochromocytoma of Case 1. The cell cluster of ACTH+&CRH + pheochromocyte was supported by the specific expression of the markers POMC (proopiomelanocortin) and CRH (corticotropin-releasing hormone) (Figure 3C). POMC is a precursor of ACTH, and CRH is the most important regulator of ACTH secretion. We also detected another specific expression signal, GAL, for the cell cluster of ACTH+&CRH + pheochromocyte (Figure 3C). GAL encodes small neuroendocrine peptides and can regulate diverse physiologic functions, including growth hormone, insulin release, and adrenal secretion (Ottlecz et al., 1988; McKnight et al., 1992; Murakami et al., 1989; Hooi et al., 1990). A study found that GAL and ACTH were co-expressed in human pituitary and pituitary adenomas, and suggested that GAL may be locally involved in the regulation of the HPA axis (Hsu et al., 1991). We demonstrated that GAL was expressed in the ACTH+&CRH + pheochromocyte and might participate in the regulation ATCH secretion (Figure 3C). Then we examined the known adrenal chromaffin cell markers (CHGA and CHGB) and the markers for catecholamine-synthesizing enzymes (TH and PNMT) (Figure 3C). These known markers and another new candidate marker CARTPT were observed in both ACTH+&CRH + pheochromocyte and pheochromocyte cell subtypes. The CYP17A1 and CYP21A2, the typical markers of the adrenal cortical cell subtype, were also investigated (Figure 3C). They are members of the cytochrome P450 superfamily, encoding key enzymes, and maybe the precursors of cortisol in the adrenal glucocorticoids biosynthesis pathway (Auchus et al., 1998; Petrunak et al., 2014). Finally, a subtype of cells with positive expression of S100B was identified, called sustentacular cells. Sustentacular cells were found near chromaffin cells and nerve terminations. Several studies have shown that sustentacular cells exhibit stem-like characteristics (Pardal et al., 2007; Fitzgerald et al., 2009; Poli et al., 2019; Scriba et al., 2020). Figure 3 Download asset Open asset A unique tumor cell type was revealed by the composition analysis of cell types in each sample. The results validated an ectopic ACTH and CRH secreting pheochromocytoma. (A) Cell clusters shown in UMAP map can be subdivided by different specimens. (B) Frequency distribution of cell types among … see more Our scRNA-seq analysis validated that the mRNA expression of POMC (precursor for ACTH) and CRH in pheochromocyte triggered the pathophysiology of ectopic ACTH and CRH syndromes, thereby stimulating the adrenal glands to release cortisol. The overexpression of TH and PNMT was responsible for the excessive secretion of catecholamines in the ACTH+&CRH + pheochromocyte and pheochromocyte cell subtypes. Tumor samples (esPHEO_T1, esPHEO_T2, and esPHEO_T3) from Case 1 and PHEO_T from Case 2 were demonstrated to have the function of producing catecholamine. These genes related to catecholamine secretion were all negative for adrenocortical cell subtypes because the catecholamine-producing pheochromocytomas originated from chromaffin cells in the adrenal medulla rather than the adrenal cortex. Our laboratory tests were consistent with these results, that is, both Case 1 and Case 2 had a high level of catecholamines in plasma and 24 hr urine while Case 3 had a normal level. We also found CARTPT was similar to PNMT and can be used as a marker for ACTH+&CRH + pheochromocyte and pheochromocyte. Chromaffin cell markers CHGA and CHGB were mainly characterized in PHEO_T and three tumor samples from Case 1. Adrenocortical cell clusters mainly existed in ACA_T1 and ACA_T2, but a few existed in esPHEO_Adj. S100B was specifically identified in PHEO_T. An absence of S100-positive sustentacular cells has been previously confirmed in most malignant adrenal pheochromocytomas, and the locally aggressive or recurrent group usually contains a large number of these cells (Unger et al., 1991). It suggests that PHEO_T from Case 2 might be a locally aggressive case, while Case 1 is the opposite. To validate this finding, we performed additional IHC staining experiments on paraffin-embedded serial slices with similar tissue regions from the tumor specimen esPHEO_T3 using antibodies against CgA, ACTH, POMC, CRH, TH, and GAL. We did find that these markers were all positive in the tumor tissue, which further indicated that the special rare pheochromocytoma exhibited multiple hormone-secreting characteristics, including ACTH, CRH, and catecholamines (Figure 3D, Appendix 1—figure 8). We also prepared two serial slices for immunofluorescence co-staining for POMC&CRH and POMC&TH. The legible co-localization signals were observed, where the green signal was for POMC, and the red signal was for CRH and TH (Figure 3E, Appendix 1—figure 9). This result confirmed the ACTH and CRH secreting pheochromocytoma from Case 1 contained a unique multi-functional chromaffin-like cell type, which was consistent with the analysis result by scRNA-seq. Differential expression genes show adrenal tumor cell-type specificity Next, we analyzed the DEGs between ACTH+&CRH + pheochromocyte and the other two subtypes of adrenal tumor cells (pheochromocyte and adrenocortical cells). It is worth noting that many genes were dramatically upregulated specifically in ACTH+&CRH + pheochromocyte when compared with the other tumor cell types, such as GAL, POMC, PNMT, and CARTPT (Figure 4A). Using these upregulated or downregulated genes, we performed functional enrichment analysis based on gene ontology (GO) annotation to further characterize the molecular characteristics of different tumor cell types. In comparison with adrenocortical cell types, the highly upregulated genes of ACTH+&CRH + pheochromocyte were mainly enriched in the neuropeptide signaling pathway, hormone secretion, and transport, while the downregulated genes were mostly enriched in the pathway of adrenocortical hormones (Figure 4B). Comparing the two types of pheochromocyte, GO functional enrichment analysis for the biology process (BP) revealed that the upregulated genes for ACTH+&CRH + pheochromocyte were also enriched in the neuropeptide signaling pathway, while the enrichment of the downregulated genes from the GO functional result hardly reach statistical significance. Interestingly, compared with adrenocortical cells, a total of 248 upregulated and 198 downregulated genes were detected in ACTH+&CRH + pheochromocyte, while only 95 upregulated and 111 downregulated genes were detected in ACTH+&CRH + pheochromocyte when compared with pheochromocyte (Figure 4C), which suggested that the difference between ACTH+&CRH + pheochromocyte and pheochromocyte was relatively small. The known adrenal chromaffin cell markers (CHGA and CHGB) were differential expressed significantly between ACTH+&CRH + pheochromocyte and adrenocortical cells, but not observed significant difference between two subtypes of pheochromocytes. Besides, the co-upregulated genes, such as CARTPT, PNMT, POMC, GAL, and CRH, were responsible for the production of a variety of hormones and involved in neuropeptide signaling pathways. Of which, the product of PNMT catalyzes the last step of the catecholamine biosynthesis pathway, methylating norepinephrine to form epinephrine. The overexpression of PNMT was responsible for the significantly elevated epinephrine (Appendix 1—table 1) of the rare Case 1 with ectopic ACTH and CRH secretory pheochromocytoma. The elevated plasma ACTH (Appendix 1—table 1) of the rare Case 1 could be explained by specific high expression signals of GAL, POMC, and CRH. In details, POMC is the precursor of ACTH; CRH is the most important regulator of ACTH secretion; and GAL was co-expressed in the ACTH+&CRH + pheochromocyte, which might be locally involved in the regulation of the HPA axis. Therefore, we concluded that the tumor cell type of ACTH+&CRH + pheochromocyte from Case 1 had multiple hormone secretion functions, namely, CRH secretion function, ACTH secretion function, and catecholamine secretion function. Furthermore, we believed that the rare Case 1 harbor the ACTH-dependent CS is due to the presence of the identified novel tumor cell type of ACTH+&CRH + pheochromocyte, which secretes both ACTH and CRH. The secretion of ACTH had a direct effect on the adrenal gland to produce cortisol, while the secretion of CRH can indirectly stimulate the secretion of ACTH from the anterior pituitary (Figure 4D). Figure 4 Download asset Open asset Altered functions in POMC+&CRH + pheochromocyte revealed by differential gene expression analysis. (A) Volcano plot of changes in gene expression between POMC+&CRH + pheochromocytes and other adrenal cell types (pheochromocytes and adrenocortical cells). The x-axis specifies the natural logarithm … see more RNA velocity analysis To investigate dynamic information in individual cells, we performed RNA velocity analysis using velocyto.py for spliced or unspliced transcripts annotation followed by scVelo pipeline for RNA dynamics modeling. RNA velocity is the time derivative of the measured mRNA abundance (spliced/unspliced transcripts) and allows to estimate the future developmental directionality of each cell (La Manno et al., 2018). We observed the ratios of spliced and unspliced mRNA, and sustentacular cell type was ranking first with 36% unspliced proportions among non-immune cell types (Figure 5A and B). The balance of unspliced and spliced mRNA abundance is an indicator of the future state of mature mRNA abundance, and thus the future state of the cell (Bergen et al., 2020). Previously study had observed unspliced transcripts were enriched in genes involved in DNA binding and RNA processing in hematopoietic stem cells (Bowman et al., 2006). For the high proportions of unspliced/spliced transcripts, stem-like characteristics of sustentacular cells were supported. There were more spliced transcripts proportions in POMC+&CRH + pheochromocytes than in pheochromocytes (Figure 5B). Then, we estimated pseudotime grounded on transcriptional dynamics and generated velocity streamlines that account for speed and direction of motion. As observed in the pseudotime of four adrenal cell subtypes, medullary cells are earlier than cortical cells (Figure 5C). From velocity streamlines, we found the four adrenal cell subtypes, that is, POMC+&CRH + pheochromocytes, pheochromocytes adrenocortical cells, and sustentacular cells, were independent respectively and not directed toward other cell types (Figure 5D). Newly transcribed, unspliced pre-mRNAs were distinguished from mature, spliced mRNAs by detecting the presence of introns. Genes, like POMC and CRH, only contain one coding sequence (CDS) region, were all detected as spliced (Appendix 1—figure 5). It indicated that the actual values of RNA velocity for POMC+&CRH + pheochromocytes might be larger than the predicted ones. Furthermore, the spliced versus unspliced phase for CHGA, CHGB, and TH demonstrated a clear more dynamics expression in POMC+&CRH + pheochromocytes than in pheochromocytes (Appendix 1—figure 5). Figure 5 Download asset Open asset RNA velocity analysis supported sustentacular cells as root and indicated four adrenal cell subtypes were independent respectively and not directed toward other cell types. RNA velocity is the time derivative of the measured mRNA abundance (spliced/unspliced transcripts) and allows to estimate the future developmental directionality of each cell. (A) The total ratios … see more Lineage tracing analysis confirms the plasticity of adrenal tumor cell subsets We performed the pseudotime analysis for the adrenal tumor cell subsets to determine the pattern of the dynamic cell transitional states. We used the recommended strategy of Monocle to order cells based on genes that differ between clusters. The sustentacular cells were in an early state in pseudotime analysis (Figure 6A, B and C), which was in accordance with their exhibited stem-like properties and the highest unspliced proportion among non-immune cell types in the RNA velocity analysis. The results also showed a transition from sustentacular cells to pheochromocytes and then to ACTH+&CRH + pheochromocyte, and adrenocortical cells were on another branch (Figure 6A, B and C). To determine whether specific gene modules might be responsible for this cell plasticity, we calculated the expression levels of all the genes in the single-cell transcriptome identified the DEGs on the different paths through the entire trajectory (Figure 6D), which showed the dynamic changes of each gene over pseudotime. Figure 6 Download asset Open asset Pseudotime analysis of adrenal cells inferred by Monocle. We ran reduce dimension with t-SNE for four types of adrenal cells and sorted cells along pseudotime using Monocle. The single-cell pseudotime trajectories by ordering cells were constructed based … see more scRNA-seq reveals distinct immune and endothelial cell type in the tumor microenvironment scRNA-seq allowed us to use an unbiased approach to discover the composition of immune cell populations of the adrenal tumor specimens. Analysis of our transcriptional profiles revealed that from the frequency distribution of cell clusters, immune cells accounted for more than ~50% of total cells (Figure 3B). We identified and annotated the immune cell types based on the expression of conventional markers, such as B cells with MS4A1, NK cells with GNLY, and Neutrophil with S100A8 and S100A9 (Figure 7A). The various frequency distribution of immune cell sub-clusters was observed among different samples (Figure 7B). Due to the identical tumor microenvironment, all three tumor specimens one peritumoral specimen from the rare case had similar immune cell composition. Interestingly, the CD4 T cells, B cells, and macrophages are mainly presented in two adrenal cortical adenomas (ACA_T1 and ACA_T2), while the CD8 T cells mostly resided in the microenvironment of other pheochromocytoma tumor and the peritumoral specimen. We found the heterogeneity of T cells in different adrenal tumor subtypes, that is, compared with CD4 T cells in adrenocortical adenomas, the pheochromocytoma types were mostly manifested by activated CD8+, especially in the anatomic specimens from the ectopic ACTH&CRH secreting pheochromocytoma. Figure 7 Download asset Open asset Diverse immune microenvironments in different adrenal tumor subtypes and tumor-adjacent tissue. (A) The UMAP diagram shows the expression levels of well-known marker genes of immune cell types. (B) Frequency distribution of immune cell sub-clusters in different adrenal tumors and … see more Endothelial cells consisted of four distinct sub-clusters: vascular endothelial cells, lymphatic endothelial cells, cortical endothelial cells, and other endothelial cells, as shown in the cell cluster distribution map highlighted by endothelial cells (Figure 8A, Supplementary file 3). Various adrenal tumor subtypes had different endothelial compositions (Figure 8B). Vascular endothelial cells were mainly identified in pheochromocytoma samples (esPHEO_T1, esPHEO_T2, esPHEO_T3, and PHEO_T), because pheochromocytoma is a tumor arising in the adrenal medulla, and vascular endothelial cells might be detected from the medullary capillary. Cortical endothelial cells were mainly detected in adrenocortical adenomas (ACA_T1 and ACA_T2). Lymphatic endothelial cells were found in the adjacent adrenal specimen of the rare ACTH+&CRH + pheochromocytoma (esPHEO_Adj). Then, by comparing vascular endothelial cells with two other subclusters (lymphatic endothelial cells and cortical endothelial cells), we found the markers across the subclusters of endothelial cells and annotated GO function of differentially expressed genes (Figure 8C and D). Vascular endothelial cells are the barrier between the blood and vascular wall and have the functions of organizing the extracellular matrix and regulating the metabolism of vasoactive substances. Lymphatic endothelial cells are responsible for chemokine-mediated pathways. Cortical endothelial cells express TFF3 and FABP4, which are involved in repairing and maintaining stable functions. Figure 8 Download asset Open asset Differential gene expression analysis shows changes in endothelial cell functions. (A) The UMAP diagram shows four different endothelial cell sub-clusters. (B) Frequency distribution of endothelial cell sub-clusters among different adrenal tumors and tumor-adjacent specimen. (C) … see more Discussion Both CS and pheochromocytoma are serious clinical conditions. In this study, we reported an extremely rare patient (Case 1) with ATCH-dependent CS due to an ectopic ACTH&CRH secreting pheochromocytoma. Surgery is the most common treatment strategy for this type of tumor. After the operation, our clinical manifestations of Case 1 showed the complete remission of CS. The IHC of the dissected tumor confirmed the diagnosis with positive staining for CRH and ACTH. In this study, scRNA-seq was used for the first time to identify the rare ACTH+&CRH + pheochromocyte cell subset. Compared with other subtypes of adrenal tumors, the common pheochromocytoma (from Case 2) and adrenal cortical cells (from Case 3), the DEGs in Case 1 were further characterized. Case 2 was examined to have normal levels of cortisol and ACTH, but Case 3 showed a Cushingoid appearance. The molecular mechanism of CS in Case 3 was different, which was attributed to two cortical adenomas on the left adrenal, showing ACTH-independent hypercortisolemia. In addition, to investigate the genetic driver for Case 1, we supplemented whole-exome sequencing experiments for all rest specimens, that is, tumors (esPHEO_T2 and esPHEO_T3) and controls (esPHEO_Adj and esPHEO_Blood) from the rare case with ectopic ACTH&CRH-secreting pheochromocytoma. Filtered germline and somatic mutations were listed in Supplementary file 4 including detailed annotations. Genetic mutations of phaeochromocytoma and paraganglioma are mainly classified into two major clusters, that is, pseudo hypoxic pathway and kinase signaling pathways (Pillai et al., 2016; Nölting and Grossman, 2012). We did not find any gene mutations that were related to these two major clusters. We only identified one shared somatic variant of ACAN (c.5951T > A:p.L1984Q) comparing variants in tumor samples to controls but Sanger sequencing only confirmed the presence in esPHEO_T3 which was not observed in esPHEO_T2 (Appendix 1—figure 7). ACAN, encoding a major component of the extracellular matrix, is a member of the aggrecan/versican proteoglycan family. Mutations of ACAN were reported related to steroid levels (Yousri et al., 2018). It is well-established that circulating steroid levels are linked to inflammation diseases such as arthritis, because arthritis as well as most autoimmune disorders results from a combination of several predisposing factors including the stress response system such as hypothalamic-pituitary-adrenocortical axis (Cutolo et al., 2003). But no direct evidence related to ACAN to phaeochromocytoma. Therefore, no obvious genetic driver was found to explain the rare case of ACTH/CRH-secreting phaeochromocytoma. Further investigations would be needed to uncover the relation between ACAN and phaeochromocytoma. For many years, the understanding of neurotransmission has been dominated by the concept of ‘one cell, one hormone, and one neuron one transmitter,’ which is known as Dale’s Principle (Dale in 1934; for detailed discussion, see Burnstock, 1976; Burnstock, 1976). Sakuma et al., 2016 reported an ectopic ACTH pheochromocytoma case and proved that ACTH and catecholamine were produced by two functionally distinct chromaffin-like tumor cell types through immunohistochemical analysis Sakuma et al., 2016. However, more and more evidence has emerged that Dale’s principle is incorrect because existing studies have shown that these cells are multi-messenger systems (Hakanson and Sundler, 1983; Apergis-Schoute et al., 2019; Svensson et al., 2018). Based on scRNA-seq results, we concluded that the tumor cells from Case 1 had multiple hormone secretion functions, namely, CRH secretion function, ACTH secretion function, and catecholamine secretion function. CRH is the most important regulator of ACTH secretion. Therefore, we believed that the secretion of both CRH and ACTH of this tumor led to ACTH-dependent CS. Besides, the secretion of ACTH had a direct impact on the adrenal gland to produce cortisol, and the secretion of CRH indirectly stimulated the secretion of ACTH by the anterior pituitary. Jessop et al., 1987 also draw the same conclusion in their report in 1987. However, in the reported case, the histological immunostained result was shown only for the corticotropin-releasing factor (CRF-41), but not for ACTH (Jessop et al., 1987). Adrenal glands are composed of two main tissue types, namely, the cortex and the medulla, which are responsible for producing steroid and catecholamine hormones, respectively. The inner medulla is derived from neuroectodermal cells of neural crest origin, while the outer cortex is derived from the intermediate mesoderm. In the adrenal pheochromocytomas, a third cell type with the positive expression of S100B was identified, called ‘sustentacular’ cells (Suzuki and Kachi, 1995; Lloyd et al., 1985). By evaluating 17 malignant and recurrent or locally aggressive adrenal pheochromocytomas, Unger et al., 1991 found that sustentacular cells were absent in most malignant cases (Unger et al., 1991). Because there are no sustentacular cells in ACTH&CRH secreting pheochromocytoma, ACTH&CRH secreting pheochromocytoma is more serious than the common pheochromocytoma. Furthermore, several studies have demonstrated that sustentacular cells exhibit stem-like characteristics (Pardal et al., 2007; Fitzgerald et al., 2009; Poli et al., 2019; Scriba et al., 2020). A unique case of a tumor originating from S100-positive sustentacular cells was previously reported (Lau et al., 2006). The RNA velocity estimation and pseudo-time analysis of different adrenal cell subtypes supported the sustentacular cells exhibiting stem-like properties. Although pheochromocyte was prior to ACTH&CRH secreting pheochromocyte in pseudotime order, the RNA velocity prediction of POMC+&CRH+ pheochromocytes might be under-estimated because the transcripts of POMC and CRH were all predicted as spliced ones. Based on the spliced versus unspliced phase for CHGA, CHGB, and TH, it showed a clear more dynamics expression in POMC+&CRH+ pheochromocytes than in pheochromocytes. We assumed that ACTH&CRH secreting pheochromocyte have more hormone-producing functions, retain stem- and endocrine-differentiation ability. But further experiments are needed to validate our hypothesis. There are bidirectional communications between the immune system and the neuroendocrine system (Blalock, 1989). Hormones produced in the endocrine system, especially glucocorticoids, affect the immune system to modulate its function (Imura and Fukata, 1994). Other hormones, such as growth hormone (GH) and prolactin (PRL), also modulate the immune system (Blalock, 1989). It has been proved that the exogenous production of cytokines can stimulate and mediate the release of multiple hormones including ACTH, CRH (Rivier et al., 1989; Bernton et al., 1987), and induce the activation of the HPA axis (Gisslinger et al., 1993; Fukata et al., 1994; Kakucska et al., 1993; Murakami N Fukata et al., 1992). Human T cells coordinate the adaptive immunity of different anatomic compartments by producing cytokines and effector molecules (Szabo et al., 2019). The activation of naive T cells through the antigen-specific T cell receptor (TCR) can initiate transcriptional programs that can drive the differentiation of lineage-specific effector functions. CD4+ T cells secrete cytokines to recruit and activate other immune cells, while CD8+ T cells have cytotoxic functions and can directly kill infected or tumor cells. Recent studies have shown that the composition of the T cell subset is related to the specific tissue locations (Carpenter et al., 2018; Thome et al., 2014). scRNA-seq can be used to deconvolve the immune system heterogeneity with high resolution. Compared with adrenocortical adenomas which were in CD4+ (with the expression of cytokine receptors, such as the IL-7R) state, T cells in pheochromocytoma, especially T cells in the ectopic ACTH&CRH secreting pheochromocytoma were inactivated CD8+ state, suggesting different tumor microenvironments between adrenocortical adenomas and pheochromocytoma. Previous studies have shown that signaling through IL-7R is essential in the developmental process and regulation of lymphoid cells (Kondrack et al., 2003; Tan et al., 2001; Tan et al., 2002; Lenz et al., 2004; Li et al., 2003; Seddon et al., 2003), and disruption of the IL-7R signaling pathway may lead to skewed T cell distribution and cause immunodeficiency (Maraskovsky et al., 1996; Kaech et al., 2003; Carini et al., 1994). Our results indicated the heterogeneity of the immune system between different samples, and CD4+ T cells with the high expression level of IL-7R might be related to adrenal tumor progression, apoptosis, or factors influencing progression such as immune activation. Although we have shown the heterogeneity of immune cell types in different adrenal tumor subtypes, it is unclear how T cells influence different markers, including effector states and interferon-response states. In addition to composition differences, a deeper understanding of the complex interactions between adrenal tumor tissues and immune systems is a key issue in neuroendocrine tumor research. Overall, we reported a rare case in which ectopic ACTH&CRH-secreting pheochromocytoma on the left adrenal that infiltrated around the kidney and psoas major tissues. We applied scRNA-seq to identify this rare and special adrenal tumor cell. Thus, the majority of our analysis focused on the validation of novel tumor cell type and their multiple hormones-secreting functions, namely, CRH secretion function, ACTH secretion function, and catecholamine secretion function. Also, GAL could be a candidate marker to detect the rare ectopic ACTH+&CRH + secreting pheochromocytes. For future studies, on one hand, we are very concerned about similar suspicious cases in the clinic. On the other hand, we are going for following research for further downstream experiments to validate the molecular mechanism for secreting multiple hormones. Materials and methods Clinical specimens collection Request a detailed protocol Our study included three adrenal tumor patients, that is, pheochromocytoma with ectopic ACTH and CRH secretion, common pheochromocytoma, and adrenocortical adenoma. All three patients had signed the consent forms at the General Surgery Department of Peking Union Medical College Hospital (PUMCH). The enhanced CT scanning images and laboratory test (ACTH, 24 hr urine-free cortisol, Catecholamines) of relevant patients are listed in Appendix 1. Fresh tumor specimens were collected during surgical resection. For the case of ACTH and CRH secreting pheochromocytoma, we performed the surgical resection of the tumor at left adrenal (esPHEO_T1) and its infiltrating tissues located in the kidney (esPHEO_T3) and masses (esPHEO_T2), and obtained three tumor specimens. The peritumor sample (esPHEO_Adj) was collected from the left adrenal tissue under the supervision of a qualified pathologist. The other two patients underwent left adrenalectomy and provided the other three tumor specimens. In details, one tumor specimen was obtained from the patient with common pheochromocytoma and two tumor specimens were obtained from the patient with adrenocortical adenoma. A total of seven specimens were carefully dissected under the microscope and confirmed by a qualified pathologist. Single-cell transcriptome library preparation and sequencing Request a detailed protocol After the resection, tissue specimens were rapidly processed for single-cell RNA sequencing. Single-cell suspensions were prepared according to the protocol of Chromium Single Cell 3′ Solution (V2 chemistry). All specimens were washed two times with cold 1× phosphate-buffered saline (PBS). Haemocytometer (Thermo Fisher Scientific) was used to evaluate cell viability rates. Then, we used Countess (Thermo Fisher Scientific) to count the concentration of single-cell suspension, and adjust the concentration to 1000 cells/μl. Samples that were lower than the required cell concentration defined in the user guide (i.e., <400 cells/µl) were pelleted and re-suspended in a reduced volume; and then the concentration of the new solution was counted again. Finally, the cells of the sample were loaded, and the libraries were constructed using a Chromium Single-Cell Kit (version 2). Single-cell libraries were submitted to 150 bp paired-end sequencing on the Illumina NavoSeq platform. Single-cell RNA-seq data pre-processing and quality control Request a detailed protocol After obtaining the paired-end raw reads, we used CellRanger (10× Genomics, v3.1.0) to pre-process the single-cell RNA-seq data. Cell barcodes and unique molecular identifiers (UMIs) of the library were extracted from read1. Then, the reads were split according to their cell (barcode) IDs, and the UMI sequences from read2 were simultaneously recorded for each cell. Quality control on these raw readings was subsequently performed to eliminate adapter contamination, duplicates, and low-quality bases. After filtering barcodes and low-quality readings that were not related to cells, we used STAR (version 2.5.1b) to map the cleaned readings to the human genome (hg19) and retained the uniquely mapped readings for UMIs counts. Next, we estimated the accurate molecular counts and generated a UMI count matrix for each cell by counting UMIs for each sample. Finally, we generated a gene-barcode matrix that showed the barcoded cells and gene expression counts. Based on the number of total reads, the number of detected gene features, and the percentage of mitochondrial genes, we performed quality control filtering through Seurat (v3.1.5) (Butler et al., 2018; Stuart et al., 2019) to discard low-quality cells. Briefly, mitochondrial genes inside one cell were calculated lower than 20%, and total reads in one cell were below 40,000. Also, the cells were further filtered according to the following criteria: PHEO, ACA, and esPHEO samples with no more than 5000, 3000, and 2500 genes were detected, respectively, and at least 200 genes were detected per cell in any sample. Low-quality cells and outliers were discarded, and the single cells that passed the QC criteria were used for downstream analyses. Doublets were predicted by DoubletFinder (v2.0) (McGinnis et al., 2019) and DoubletDecon (v1.1.6) (DePasquale et al., 2019; Appendix 1—figure 2). Clustering analysis and cell phenotype recognition Request a detailed protocol Seurat (Butler et al., 2018; Stuart et al., 2019) software package was used to perform cell clustering analysis to identify major cell types. All Seurat objects constructed from the filtered UMI-based gene expression matrixes of given samples were merged. We first applied ‘SCTransform’ function to implement normalization, variance stabilization, and feature selection through a regularized negative binomial model. Then, we reduced dimensionality through PCA. According to standard steps implemented in Seurat, highly variable numbers of principal components (PCs) 1–20 were selected and used for clustering using the t-distributed stochastic neighbor embedding method (t-SNE). We identified cell types of these groups based on the expression of canonic cell type markers or inferred by CellMarker database (Zhang et al., 2019). Finally, the four groups of endothelial cells were combined to a larger endothelial cell cluster for downstream analysis. Cellular cluster statistics were added in Supplementary file 2, which presented cell counts for each cellular cluster in different samples and top 10 gene markers. Endothelial cell cluster statistics were added in Supplementary file 3, which presented cell counts for each endothelial cell cluster in different samples and top 10 gene markers. DEG analysis Request a detailed protocol The cell-type-specific genes were identified by running Seurat (Butler et al., 2018; Stuart et al., 2019) containing the function of ‘FindAllMarkers’ on a log-transformed expression matrix with the following parameter settings: min.pct=0.25, logfc.threshold=0.25 (i.e., there is at least 0.25 log-scale fold change between the cells inside and outside a cluster), and only.pos=TRUE (i.e., only positive markers are returned). For heatmap and violin plots, the SCT-transformed data from Seurat pipeline were used. Using the Seurat ‘FindMarkers’ function, we found the DEGs between two cell types. We also used R package of clusterProfiler with default parameters to identify gene sets that exhibited significant and consistent differences between two given biological states. RNA velocity estimation Request a detailed protocol We used the velocyto python package (v0.17.17) (La Manno et al., 2018) for distinguishing transcripts as spliced or unspliced mRNAs based on the presence or absence of intronic regions in the transcript. We took aligned reads of BAM file for each sample as input. After per sample abundance estimation, it generated a LOOM file with the loompy package. Then, we used the scVelo (v0.2.3; Bergen et al., 2020) to combine each sample abundance data as well as cell cluster information from the Seurat object. We showed the proportions of abundances for each sample using scvelo.pl.proportions function. The RNA velocity was estimated for each cell for an individual gene at a given time point based on the ratio of its spliced and unspliced transcript. RNA velocity graph was visualized on a UMAP plot, with vector fields representing the averaged velocity of nearby cells. We also visualized some marker genes dynamics portraits with scv.pl.velocity to examine their spliced versus unspliced phase in different cell types. Pseudotime analysis Request a detailed protocol The Monocle2 packages (v2.14.0) (Trapnell et al., 2014) for R were used to determine the pseudotimes of the differentiation of four different cell subtypes, that is, POMC+/CRH + pheochromocytoma, pheochromocytoma, adrenocortical, and sustentacular cells. We converted a Seurat3 integrated object into a Monocle cds object and distributed the composed cell clusters to the Monocle cds partitions. Then, we used Monocle2 to perform trajectory graph learning and pseudotemporal sorting analysis by specifying the sustentacular cells as the root nodes. To identify genes that are significantly regulated as the cells differentiate along the cell-to-cell distance trajectory, we used the differentialGeneTest() function implemented in Monocle2 (Trapnell et al., 2014). Finally, we selected the genes that were differentially expressed on different paths through the trajectory and plotted the pseudotime_heatmap. Gene regulatory network (regulon) analysis Request a detailed protocol We used R package SCENIC (v1.1.2) (Aibar et al., 2017) for gene regulatory network inference. Normalized log counts were used as input to identify co-expression modules by the GRNBoost2 algorithm. Following which, regulons were derived by identifying the direct-binding TF target genes while pruning others based on motif enrichment around transcription start site (TSS) with cisTarget databases. Using aucell, the regulon activity score was measured as the area under the recovery curve (AUC). Additionally, regulon specificity score (RSS) was used for the detection of the cell-type-specific regulons. Cell-cell communication analysis Request a detailed protocol Given the diverse immune and endothelial cell types in the tumor microenvironment, we performed cell-cell communication analysis using CellPhoneDB Python package (2.1.7) (Efremova et al., 2020). We visualized the potential cell-cell interactions among various immune cells, endothelial cells, and other cell types in the different tumor microenvironment (esPHEO, esPHEO_Adj, PHEO, and ACA) (Appendix 1—figure 6). Whole-exome sequencing Request a detailed protocol Genomic DNA extracted from whole blood (esPHEO_Blood), esPHEO_T2, esPHEO_T3, and esPHEO_Adj of the rare Case 1 were sent for whole-exome sequencing. The exomes were captured using the Agilent SureSelect Human All Exon V6 Kit and the enriched exome libraries were constructed and sequenced on the Illumina NovaSeq 6000 platform to generate WES data (150 bp paired-end reads, >100×) according to standard manufacturer protocols. The cleaned reads were aligned to the human reference genome sequence NCBI Build 38 (hg38) using Burrows-Wheeler Aligner (BWA) (v0.7.17) (Li and Durbin, 2009). All aligned BAM were then performed through the same bioinformatics pipeline according to GATK Best Practices (v4.2) (McKenna et al., 2010). We obtained germline variants shared by all tumors and control samples based on variant calling from GATK-HaplotypeCaller. We then used GATK-MuTect2 to call somatic variants in tumors and obtained a high-confidence mutation set after rigorous filtering by GATK-FilterMutectCalls. All variants were annotated using ANNOVAR (v2018Apr16) (Wang et al., 2010). The criteria for filtering variants were as follows: (1) only retained variants located on exon or splice site, and excluded synonymous variants; (2) retained rare variants with minor allele frequencies <5% in any ancestry population groups from public databases (1000 Genomes, ESP6500, ExAC, or the GnomAD); (3) For germline variants, excluded common variants in dbSNP (Build 138) and predicted benign missense variants by SIFT, Polyphen2, and Mutation Taster. Immunocytochemistry and Immunofluorescence Request a detailed protocol Immunocytochemical and immunofluorescent staining experiments were conducted according to standard protocols using antibodies against malinfixed paraffin-embedded (FFPE) tissue specimens. The antibodies and reagents used in the experiments are listed as follows: ACTH (Abcam, ab199007), POMC (ProteinTech, 66358-1-Ig), TH (Abcam, ab112), CRH (ProteinTech, 10944-1-AP), CgA (ProteinTech, 60135-1-Ig), and Human Galanin Antibody (R&D, MAB5854). Appendix 1 Clinical samples description Case 1: A 39-year-old lady underwent laparoscopic left adrenal tumor resection in July 2012 at a local hospital. She had a 2-year history of headache, generalized swelling, and palpitations. She was noted to have hypertensive (BP 240/120 mmHg) and typical Cushingoid characteristics, including asthenia, supraclavicular fat deposits, bruises, purple striae, proximal myopathy, and hyperpigmentation. Histopathology confirmed an adrenomedullary chromaffin tumor. During tumor immunostaining, the tumor stained positively for ACTH. After the adrenal surgery, her Cushingoid characteristics, hypokalemia, and hypertension were all relieved. However, the patient experienced recurrence of symptoms and signs in January 2019 and was admitted to our hospital. It was found that urine and plasma metanephrine were significantly elevated, and plasma ACTH was also high. Enhanced CT scanning of the abdomen revealed bilateral adrenocortical hyperplasia and multiple masses in the left adrenal and around the left kidney. The largest mass lesion was 2.3×1.6 cm2, which invaded upper pole of left kidney. But the I123-MIBG scintigraphy was negative. We performed a surgery to remove left adrenal, kidney, and masses. After the surgery, the patient’s clinical features and symptoms were improved, and the excessive hypercortisolemia and catecholamine eventually returned to normal. IHC revealed positive staining for chromogranin A, ACTH, and CRH, confirming the diagnosis of pheochromocytoma secreting both ACTH and CRH. Case 2: A 42-year-old male with a 3-year history of headache and palpitations, and a 6-month history of hypertension was admitted to our hospital. Laboratory tests showed that the plasma and urine catecholamines and their metabolites were elevated, and cortisol and ACTH were at the normal level. Enhanced CT showed a 67×70 mm2 left adrenal tumor, and I123-MIBG scintigraphy exhibited positive. We performed a surgery to remove the left adrenal gland. After the surgery, the patient’s clinical features and symptoms were relieved. IHC confirmed the diagnosis of pheochromocytoma. Case 3: A 50-year-old female came to our hospital with hypertension, hyperkalemia, and Cushingoid symptoms (moon face and central obesity). Enhanced CT scanning revealed a 19×36 mm2 irregular mass in left adrenal gland. The laboratory tests showed ACTH-independent hypercortisolemia. The left adrenal gland was removed, and Cushing’s syndrome was relieved. Resected specimen revealed two tumors in the left adrenal gland, and IHC confirmed the diagnosis of adrenal adenoma. Appendix 1—table 1 Summary of laboratory test for three cases. Laboratory test Case 1 Case 2 Case 3 Reference range ACTH 519.0 24.0 <5 0–46.0 pg/ml 24 hr urine-free cortisol 2024.4 332.4 12.3–103.5 μg/24 hr Catecholamines Plasma metanephrines Normetanephrine 3.28 10.81 0.4 <0.9 nmol/L Metanephrine 3.44 11.55 0.2 <0.5 nmol/L 24 hr urine Epinephrine 397.63 56.23 1.92 1.74–6.42 μg/24 hr Norepinephrine 475.43 82.29 26.17 16.69–40.65 μg/24 hr Dopamine 432.21 301.71 240.5 120.93–330.5 μg/24 hr Appendix 1—figure 1 Download asset Open asset Enhanced CT scanning image for three cases. (A) Enhanced CT scanning for Case 1 with pheochromocytoma secreting both ACTH and CRH. The abdomen revealed bilateral adrenocortical hyperplasia and multiple masses in the left adrenal and around … see more Appendix 1—figure 2 Download asset Open asset Quality control plots and doublet detection for this scRNA-seq study. Violin plots showing number of total RNAs (A), number of genes (B), and percentage of mitochondrial (mito) genes (C) for cells in seven samples. Doublets were predicted by DoubletFinder (D) and … see more Appendix 1—figure 3 Download asset Open asset Four adrenal cell types and their highly expressed genes through single-cell transcriptomic analysis. Heatmap shows the scaled expression patterns of top 10 marker genes in each cell type. The color keys from white to red indicate relative expression levels from low to high. Appendix 1—figure 4 Download asset Open asset Transcription factors detection using SCENIC pipeline. (A) Binarized heatmap showing the AUC score (area under the recovery curve, scoring the activity of regulons) of the identified regulons plotted for each cell. (B) For each cellular cluster, dot … see more Appendix 1—figure 5 Download asset Open asset The spliced versus unspliced phase for marker genes in four types of adrenal cells. Transcripts were marked as either spliced or unspliced based on the presence or absence of intronic regions in the transcript. For each gene, the scatter plot shows spliced and unspliced ratios in a … see more Appendix 1—figure 6 Download asset Open asset Ligand-receptor interaction analysis for CD4+ T cells, CD8+ T cells, and endothelial cells in different tumor microenvironments. Overview of ligand-receptor interactions between the CD4+ T cells (A), CD8+ T cells (B), endothelial (C), and the other cell types in the different tumor microenvironments. p-values are represented … see more Appendix 1—figure 7 Download asset Open asset Whole-exome sequencing identified one shared somatic variant of ACAN comparing variants in tumor samples to controls and Sanger sequencing only confirmed the presence in esPHEO_T3 but not observed in esPHEO_T2. (A) Distribution of somatic mutations for the rare case with ectopic ACTH&CRH-secreting pheochromocytoma. OncoPrint plots were generated using the R package Maftools for somatic mutations from five … see more Appendix 1—figure 8 Download asset Open asset Immunohistochemistry of CgA, ACTH, POMC, CRH, TH, or GAL on serial biopsies from tumor specimen infiltrating tissues located in the kidney (esPHEO_T3). We observed positive staining signal at tumor left in each slice, while the adjacent kidney was un-stained could be negative controls. The magnification is 0.5×, 2.5×, 10×, and 40× from left to … see more Appendix 1—figure 9 Download asset Open asset Immunofluorescence co-staining for POMC&CRH and POMC&TH on two serial biopsies from tumor specimen esPHEO_T3. The magnification is 10× (top) and 40× (bottom). Red rectangular indicates the magnified area of the location, as shown in Figure 3E. Data availability The raw data of scRNA-seq sequencing reads generated in this study were deposited in The National Genomics Data Center (NGDC, https://bigd.big.ac.cn/) under the accession number: PRJCA003766. 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Decision letter after peer review: Thank you for submitting your work entitled "Single-cell transcriptome analysis identifies a unique tumor cell type producing multiple hormones in ectopic ACTH and CRH secreting pheochromocytoma" for further consideration by eLife. Your article has been reviewed by 3 peer reviewers, one of whom is a member of our Board of Reviewing Editors, and the evaluation has been overseen by Mone Zaidi as the Senior Editor. Reviewer #1: The authors identified an extremely rare case of ATCH-dependent Cushing syndrome due to ACTH&CRH secreting pheochromocytoma. They retrieved sugically resected samples from the tumor and subjected them to scRNA-seq, which led them to identify a group of cells that are double-positive for ACTH&CRH. They then performed a series of expriments to confirm that the cells are indeed present in the tissue, and attempted to identify genes that may lie upstream of the process. Perhaps the most important point of the study is the identification of the double-positive (DP) cells from the patient. However, evidence supporting this observation is relatively scarce other than showing a cell cluster that express POMC, CRH etc (as displayed in Figure 3A, C). Gene expression pattern shown in Figure 3C supports that the DP cells share molecular characteristics with those of pheochromocytes. But in the t-SNE plot, these cells are located far from pheochromocytes in PHEO_T. Rather, the DP cell cluster seems to be branched out from immune cells. If I didn't read the t-SNP plot wrong, I wonder why the identity of DP cells is closer to the immune cells. Also, it needs to be clarified if the DP cells could be doublets? The authors did not show basic statistics and QA/QC data of the scRNA-seq experiment (as supplementary data for example). They should show that the DP cells are not technical doublet cells. Another critical question would be what is the genetic driver that induces expression of both hormones in the DP cells? They propose GAL, but the evidence supporting its direct role is not strong and remains speculative. Comments for the authors: Overall, this study requires more carefully designed expriments and interpretation. Otherwise, it remains as a descriptive study with vague conclusions, leaving the uniqueness of the sample being the only strength of the study. 1. Colors in Figure 3A are confusing. 2. Figure 5 does not add much to the molecular mechanism. Rather it merely describes physiological consequences by the presence of DP cells. Please consider strengthen or remove it. 3. Isn't Figure 7B a duplication of Figure 3B? 4. IHC data in Figure 3E, F lack negative controls. And the readers need additional markers to be guided of its anatomical location. 5. Figure 4 compared DEGs between DP cells and other tumor cells. Since the cell groups that were being compared are too different, observing such dramatic differences is not unexpected and hard to coin physiological relevance. Wouldn't it be more meaningful to compare them to pheochromocytes? 6. The pseudotime analysis in Figure 6 does not answer the question of how the DP cells originated. It should be performed in a such way to suggest genes that marks critical points during the pseudotime branching or proceeding. Reviewer #2: In this manuscript Zhang et al. generated single cell RNA sequencing data for the adrenal gland tumors including extremely rare type of tumor, ACTH & CRH-secreting pheochromocytoma. Unbiased clustering analysis discovered a unique tumor cell type that expresses multiple hormones unlike normal adrenal gland cells and other tumor cell types that produce a single hormone. By comparing with other type of tumor cells, they identified specific marker genes of the novel tumor cell type. They also revealed the distinct immune and endothelial cell populations in the microenvironment of different tumor samples. Although the gene expression profiles of novel cell type can be utilized to reveal the molecular mechanism of this rare tumor associated with Cushing's syndrome, the data was generated from only a single patient and have not validated in other samples. In addition, the results only provide the list of genes that were specifically expressed in the novel tumor cell type and their potentially related biological pathways, but not detail molecular and cellular characters of the cells. The single cell gene expression profiling data are definitely useful for the researches. Comments for the authors: I have several concerns and suggestions, which if addressed would improve the manuscript. 1. The major finding of this manuscript is the presence of multi-functional tumor cell type which produce multiple hormones such as POMC, the precursor of ACTH and CRH. But, this finding was only derived from a single sample and experimentally validated using the same tissue. I understand the sample is very rare, but could the authors validate the result in different tumor samples at least using IHC or IF? If sample is not available, the limitation of the study should be mentioned. 2. Please consider providing full list of marker genes that were used for cell type annotation. 3. Figure 3C does not seem to support the statement "We demonstrated that GAL was expressed in the ACTH+&CRH+ pheochromocyte and 'regulated the secretion of ACTH'". 4. The authors identified a unique and important multi-functional cell type but current analyses (differentially expressed genes identification and gene ontology analysis) seem insufficient to characterize molecular feature of ACTH+&CRH+ pheochromocyte. The authors could perform additional comprehensive analysis such as SCENIC analysis in order to identify the master transcription regulator of the cell type. 5. The pseudo-time analysis indicated that sustentacular cells transform to ACTH+&CRH+ pehochromocytes and then to pheochromocyte. The authors utilized Monocle3 in which user has to define the starting points. The authors can validate the result using RNA velocity analysis which also predicts cell transition without the need of prior knowledge about starting point cell type. 6. Given the diverse immune and endothelial cell type in the tumor microenvironment, it would be interesting to perform the cell-cell interaction analysis using the programs such as CellPhoneDB to see if they have distinct regulatory role in different tumor microenvironment. 7. How did the authors define the four subclusters of endothelial cells? Please consider providing list of marker genes. 8. In the method part, how did the authors determine different criteria for the maximum number of genes (no more than 5000, 3000, and 2500 genes for PHEO, ACA, and esPHEO samples, respectively)? Reviewer #3: Zhang et al. perform single cell RNA sequencing (scRNA-Seq) of one rare ACTH+CRH-secreting phenochromocytoma (3 anatomically distinct sites from the tumor and one peritumoral site), one typical pheochromocytoma, and two typical adrenocortical adenomas. Their main findings are as follows: (1) They identify a unique cell type, which they term ACTH+CRH+ pheochromocyte, which appears to be the tumor cell present in the rare ACTH+CRH+ tumor (2) Marker gene analysis reveals that while known adrenal chromaffin markers (CHGA, PNMT) are present in both pheochromocytes and ACTH+CRH+ pheochromocyte, the latter has some unique markers such as GAL and POMC. They validate the marker genes with IHC. (3) Profiling of the non-tumor populations reveals distinct immune microenvironment profile and endothelial cell profile to the rare tumor compared with classical pheochromocytoma and adrenalocortical adenoma. The main strength of this manuscript is that it involves single-cell profiling of an exceptionally rare tumor type and a distinction from the more common adrenal tumors (pheochromocytoma and adrenocortical adenoma). The broader implication of the authors' findings is with respect to Dale's principle, which states that a given neuron releases only one type of neurotransmitter. However, in the case of this tumor, single cell analysis clearly shows that ACTH, CRH, and chatacholemines are being released from the same cell. This is quite interesting and significant. The data will also potentially be valuable to others in the field for analysis in future studies. There remain some unanswered questions – namely: (1) What is the cell in normal physiology that gives rise to this ACTH+CRH+ pheochromocytoma? (2) Do conventional phenochromocytomas differ from the ACTH+CRH+ pheochromocytoma in terms of the cell of origin that is transformed, or in the spectrum of genetic alterations that result in transformation? Comments for the authors: Overall, I think this study is of broad interest given the rarity of this tumor type. My comments to the authors to improve the manuscript are as follows: 1. Given how rare the ACTH+CRH+ pheochromocytoma is, I think the study would be substantially strengthened if the authors could perform DNA sequencing (WGS or WES) and describe how, if at all, the genomic landscape differs from conventional pheochromocytoma. 2. Can the authors comment on whether the hypothesis is whether the ACTH+CRH+ pheochromocytoma originates from a rare progenitor cell that is distinct from the chromaffin cell giving rise to pheochromocytoma? If so, can the authors stain a panel of normal adrenal glands with some of their marker genes to try and identify this cell in normal tissues? 3. While the tumor type is interesting for its rarity, the analysis performed is quite standard and comes across as a bit superficial in parts. Although it is understandable that the authors have only one ACTH+CRH+ sample I think they can do more with the data and this would significantly strengthen the manuscript. For example, it would be interesting if the authors can point to specific master regulatory factors that drive the distinct programs in pheochromocytes vs. ACTH+CRH+ pheochromocytes. The immune microenvironment analysis, while inherently descriptive, is also somewhat superficial. [Editors' note: further revisions were suggested prior to acceptance, as described below.] Thank you for submitting your revised article "Single-cell transcriptome analysis identifies a unique tumor cell type producing multiple hormones in ectopic ACTH and CRH secreting pheochromocytoma" for consideration by eLife. Your article has been reviewed by 3 peer reviewers, including Murim Choi as the Reviewing Editor and Reviewer #1, and the evaluation has been overseen by Mone Zaidi as the Senior Editor. The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission. Essential revisions: Although the reviewers thought that many issues were addressed, they still concerned on the superficial analysis results. Nonetheless, they agreed that the manuscript contains a common interest for publication in eLife as the tumor is an extremely rare case. Please address reviewers' concerns below. Reviewer #1: Although the authors could not address all the questions, especially regarding the origin of DP cells and genetic driver for DP cells, it appears reasonable that they are hard to address as the tumor sample was extremely rare. Reviewer #2: Although the authors have satisfactorily addressed most of my points, there are remaining concerns about RNA velocity data. Please cite any reference for the statement "For the high proportions of unspliced/spliced transcripts, stem-like characteristics of sustentacular cells were supported." Can global ratio of unspliced/spliced transcripts support stem-like characteristics? Please elaborate Figure 5 C-F. Currently, they don't seem to add any information. Reviewer #3: In the revised manuscript Zhang et al. have included additional data and analyses including more exhaustive QC, RNA velocity analysis, regulome analysis, and have performed WES of the ACTH/CRH-secreting pheochromocytoma. They have generally addressed my technical concerns from the prior review. I maintain that the analysis remains somewhat superficial and descriptive in parts and this may be somewhat of a missed opportunity to more deeply explore the underlying biology of this unique case, understanding the caveats of its rarity. Nonetheless, I think a description of this tumor at single-cell resolution and availability of the dataset is of value to the scientific community. However, I would like to see a more careful analysis of the WES data prior to publication. I do not see any basic metrics (mutation rate etc.), description of pathogenicity filtering/annotation, or copy number analysis. The mutations shown are primarily missense and I do not really see any obvious driver genes – how many of these are putative driver vs. passenger mutations? ACAN is mentioned, but what is its significance, if any? The somatic landscape should be discussed in comparison to typical phenochromocytomas and adrenocortical carcinomas, which have been more extensively sequenced. If there is no obvious genetic driver of this ACTH/CRH-secreting phenochromocytoma, that should be stated. If the claim is that ACAN alterations are somehow related to this tumor type, that needs to be substantiated. Or if the implication is that ACAN is a passenger alteration, that needs to be stated explicitly also. https://doi.org/10.7554/eLife.68436.sa1 Author response Reviewer #1: The authors identified an extremely rare case of ATCH-dependent Cushing syndrome due to ACTH&CRH secreting pheochromocytoma. They retrieved surgically resected samples from the tumor and subjected them to scRNA-seq, which led them to identify a group of cells that are double-positive for ACTH&CRH. They then performed a series of experiments to confirm that the cells are indeed present in the tissue, and attempted to identify genes that may lie upstream of the process. We thank the reviewer for carefully reviewing the manuscript. We updated graphs, added supplementary files of raw data QC and cell cluster statistics, and performed RNA velocity analysis, scenic analysis for the single cell RNA sequencing experiments to response the reviewer’s critiques and strengthen the manuscript. In addition, to investigate the genetic driver for Case 1, we supplemented whole-exome sequencing experiments for all rest specimens, that is, tumors (esPHEO_T2, esPHEO_T3) and controls (esPHEO_Adj, esPHEO_Blood) from the rare case with ectopic ACTH&CRH-secreting pheochromocytoma. Perhaps the most important point of the study is the identification of the double-positive (DP) cells from the patient. However, evidence supporting this observation is relatively scarce other than showing a cell cluster that express POMC, CRH etc (as displayed in Figure 3A, C). Gene expression pattern shown in Figure 3C supports that the DP cells share molecular characteristics with those of pheochromocytes. But in the t-SNE plot, these cells are located far from pheochromocytes in PHEO_T. Rather, the DP cell cluster seems to be branched out from immune cells. If I didn't read the t-SNP plot wrong, I wonder why the identity of DP cells is closer to the immune cells. Also, it needs to be clarified if the DP cells could be doublets? The authors did not show basic statistics and QA/QC data of the scRNA-seq experiment (as supplementary data for example). They should show that the DP cells are not technical doublet cells. We thank the reviewer for raising the concerns and providing these helpful suggestions. First, we updated the colors mapped to 16 cellular clusters in Figure 2A and Figure 3A to enhance the color difference between doublet-positive (DP) cells and immune cells. Then, the new analysis based on RNA velocity was performed in the revision, and the results showed that DP cluster was isolated and not branched out from other cell types (including immune cells) from velocity streamlines (Figure 5F). In addition, we added the raw data QC and doublet prediction results of the scRNA-seq experiment as shown in Appendix 1—figure 2 and Supplementary File 1. From the doublets predicted by DoubletFinder and DoubletDecon, it is clarified that almost noDP cells were defined as doublets. Cellular cluster statistics were shown in Supplementary File 2, which presented cell counts for each cellular cluster in different samples and top10 gene markers. Another critical question would be what is the genetic driver that induces expression of both hormones in the DP cells? They propose GAL, but the evidence supporting its direct role is not strong and remains speculative. We thank the reviewer for raising these important concerns, and we agree with the reviewer that the presentation about the genetic driver in the previous version of the manuscript is not sufficient enough. We changed the conclusion statement "We demonstrated that GAL was expressed in the ACTH+&CRH+ pheochromocyte and regulated the secretion of ACTH" to "We demonstrated that GAL was expressed in the ACTH+&CRH+ pheochromocyte and might participate in the regulation of ACTH secretion". (Page 7 line 175-182) We provided more description and additional analysis about putative genetic driver in the DP cells, as follows: First, we found GAL co-expressed with POMC and CRH, could be a candidate marker to detect the rare ectopic ACTH+&CRH+ secreting pheochromocytes. It might be involved in the regulation of the hypothalamic-pituitary-adrenal axis. (Page 7 line 175-182, Figure 3, Figure 4). Second, we also found an additional weak signal of transcription regulons for the DP cells (Page 6 line 153-157, Appendix 1—figure 4). It showed XPBP1 as the specific regulons for ACTH+&CRH+ pheochromocyte and adrenocortical cell type. Third, to investigate the genetic driver, we supplemented whole-exome sequencing experiments for tumors (esPHEO_T2, esPHEO_T3) and controls (esPHEO_Adj, esPHEO_Blood) from the rare case with ectopic ACTH&CRH-secreting pheochromocytoma. We identified 1 shared somatic variant of ACAN (c.5951T>A:p.L1984Q) comparing variants in tumor samples to controls but Sanger sequencing only confirmed the presence in esPHEO_T3 which was not observed in esPHEO_T2 (Page 13 line 352-358, Appendix 1—figure 7). Comments for the authors: Overall, this study requires more carefully designed experiments and interpretation. Otherwise, it remains as a descriptive study with vague conclusions, leaving the uniqueness of the sample being the only strength of the study. We thank the reviewer for carefully reviewing and helpful suggestions. We updated graphs and tables, implemented supplementary analysis for the single-cell RNA sequencing data. Because this case is particularly rare, fresh tissue samples are lacking, currently, frozen tissue samples cannot be assayed by flow cytometry. For all rest of the samples, we can only supplement the whole-exome sequencing experiments for tumors (esPHEO_T2, esPHEO_T3) and controls (esPHEO_Adj, esPHEO_Blood) from the rare case with ectopic ACTH&CRH-secreting pheochromocytoma to make our results more comprehensive. Lastly, on one hand, we are very concerned about similar suspicious cases in the clinic. On the other hand, we are going for the following research for further downstream experiments to validate the molecular mechanism for secreting multiple hormones. 1. Colors in Figure 3A are confusing. We have updated the colors mapped to 16 cellular clusters in Figure 2 and Figure 3 to enhance the color difference between doublet-positive (DP) cells and immune cells. 2. Figure 5 does not add much to the molecular mechanism. Rather it merely describes physiological consequences by the presence of DP cells. Please consider strengthen or remove it. Due to the previous Figure 5 mainly describe the physiological consequences by the presence of DP cells as the reviewer commented. We have moved it to Figure 4D, because the differential expressed genes between DP cells and other adrenal cell types were shown in Figure 4A and Figure 4C. Combining these figures into a group could complement each other and clarify the secreting functions of the DP cells. 3. Isn't Figure 7B a duplication of Figure 3B? Figure 3B presents the frequency distribution of all cell types among different samples, while in Figure 7B we specifically focused on the immune microenvironments and showed statistics of immune cell types. To some extent, they are repetitive since both describe the percentage of immune cells. But the denominators are different for percentage calculation, that is, one is the total number of cells in Figure 3B, the other is the total number of immune cells in Figure 7B. 4. IHC data in Figure 3E, F lack negative controls. And the readers need additional markers to be guided of its anatomical location. We supplemented IHC figures of CgA, ACTH, POMC, CRH, TH or GAL with magnification (0.5x, 2.5x, 10x, 40x) from tumor specimen infiltrating tissues located in the kidney (esPHEO_T3) in Appendix 1—figure 8. We observed positive staining signal at tumor left in each slice, while the adjacent kidney was un-stained could be negative controls. Red rectangular indicates the magnified area of the location as shown in Figure 3D. The. We supplemented the immunofluorescence (IF) co-staining figures with magnification (10x, 40x) for POMC&CRH and POMC&TH from tumor specimen esPHEO_T3 in Appendix 1—figure 9, where red rectangular indicates the magnified area of the location in Figure 3E. 5. Figure 4 compared DEGs between DP cells and other tumor cells. Since the cell groups that were being compared are too different, observing such dramatic differences is not unexpected and hard to coin physiological relevance. Wouldn't it be more meaningful to compare them to pheochromocytes? We analyzed the differentially expressed genes (DEGs) between ACTH+&CRH+ pheochromocyte and the other two subtypes of adrenal tumor cells (pheochromocyte and adrenocortical cells) (Page 9 line 241-245). Such dramatic differences were observed because we set the statistically significant differences as a cut-off p-value < 0.05 and a fold change ≥ 1.5 ( which means a log2 fold change |logFC| ≥ 0.585 ) (Figure 4A). It could more strict such as a cut-off p-value <0.01 and a fold change ≥ 2 ( which means a log2 fold change |logFC| ≥ 1 ). But the top significantly differentially expressed genes were POMC, CRH, GAL etc, as marked in Figure 4A. There is a relatively larger difference in gene expression between DP cells and adrenocortical cells than that between DP cells and pheochromocytes (Figure 4C). Since we didn’t identify any pheochromocytes in esPHEO_adj, we could not compare the DP cells to their adjacent pheochromocytes (Supplementary File 2). Reviewer #2: In this manuscript Zhang et al. generated single cell RNA sequencing data for the adrenal gland tumors including extremely rare type of tumor, ACTH & CRH-secreting pheochromocytoma. Unbiased clustering analysis discovered a unique tumor cell type that expresses multiple hormones unlike normal adrenal gland cells and other tumor cell types that produce a single hormone. By comparing with other type of tumor cells, they identified specific marker genes of the novel tumor cell type. They also revealed the distinct immune and endothelial cell populations in the microenvironment of different tumor samples. Although the gene expression profiles of novel cell type can be utilized to reveal the molecular mechanism of this rare tumor associated with Cushing's syndrome, the data was generated from only a single patient and have not validated in other samples. In addition, the results only provide the list of genes that were specifically expressed in the novel tumor cell type and their potentially related biological pathways, but not detail molecular and cellular characters of the cells. The single cell gene expression profiling data are definitely useful for the researches. We thank the reviewer for carefully reviewing and raising insightful critiques. In this study, we reported a rare case in which ectopic ACTH&CRH-secreting pheochromocytoma in the left adrenal. To identify the hormones-secreting cells, we sent specimens for single-cell transcriptome sequencing immediately after the resection. Thus, the majority of our analysis focused on the validation of novel tumor cell type and their multiple hormones-secreting functions. For future studies, on one hand, we are very concerned about similar suspicious cases in the clinic. On the other hand, we are going for following research for further downstream experiments to validate the molecular mechanism for secreting multiple hormones. Comments for the authors:I have several concerns and suggestions, which if addressed would improve the manuscript. 1. The major finding of this manuscript is the presence of multi-functional tumor cell type which produce multiple hormones such as POMC, the precursor of ACTH and CRH. But, this finding was only derived from a single sample and experimentally validated using the same tissue. I understand the sample is very rare, but could the authors validate the result in different tumor samples at least using IHC or IF? If sample is not available, the limitation of the study should be mentioned. For the case of ACTH and CRH secreting pheochromocytoma, we performed the surgical resection of the tumor at left adrenal (esPHEO_T1) and its infiltrating tissues located in the kidney (esPHEO_T3) and masses (esPHEO_T2), and obtained 3 tumor specimens. The peritumor sample (esPHEO_Adj) was collected from the left adrenal tissue under the supervision of a qualified pathologist. At first, we performed immunohistochemistry (IHC) staining with chromogranin A (CgA) and ACTH markers for esPHEO_T1 and adjacent specimen (esPHEO_Adj) (Figure 1B). To validate our discovery from scRNA-seq data we implemented IHC of CgA, ACTH, POMC, CRH or TH (Figure 3D) on serial biopsies from another tumor specimen (esPHEO_T3) and added immunofluorescence co-staining for POMC&CRH and POMC&TH on two serial biopsies from esPHEO_T3 (Figure 3E). The frozen tissue of esPHEO_T1 is unavailable and a few remaining for esPHEO_T2. For all rest of tissue samples, we supplemented with the whole-exome sequencing experiments for tumors (esPHEO_T2, esPHEO_T3) and controls (esPHEO_Adj) from the rare case with ectopic ACTH&CRH-secreting pheochromocytoma. 2. Please consider providing full list of marker genes that were used for cell type annotation. We add row annotations for top10 marker genes at the heatmap showing different cellular clusters and their highly expressed genes (Figure 2B). Cellular cluster statistics were supplemented in Supplementary File 2, which presented cell counts for each cellular cluster in different samples and top10 gene markers. 3. Figure 3C does not seem to support the statement "We demonstrated that GAL was expressed in the ACTH+&CRH+ pheochromocyte and 'regulated the secretion of ACTH'". We changed the conclusion sentence to "We demonstrated that GAL was expressed in the ACTH+&CRH+ pheochromocyte and might participate in the regulation of ACTH secretion". We’re trying to express that: [We found GAL co-expressed with POMC and CRH, could be a candidate marker to detect the rare ectopic ACTH+&CRH+ secreting pheochromocytes. As previous research reported, it might be involved in the regulation of the hypothalamic-pituitary-adrenal axis.] 4. The authors identified a unique and important multi-functional cell type but current analyses (differentially expressed genes identification and gene ontology analysis) seem insufficient to characterize molecular feature of ACTH+&CRH+ pheochromocyte. The authors could perform additional comprehensive analysis such as SCENIC analysis in order to identify the master transcription regulator of the cell type. We have performed additional analysis (Page 18 line 519-570), including RNA velocity analysis, SCENIC analysis etc. In addition, whole-exome sequencing experiments for tumors (esPHEO_T2, esPHEO_T3) and controls (esPHEO_Adj, esPHEO_Blood) from the rare case with ectopic ACTH&CRH-secreting pheochromocytoma were performed to make our results more comprehensive. First, based on differentially expressed genes identification, we mainly found GAL co-expressed with POMC and CRH, could be a candidate marker to detect the rare ectopic ACTH+&CRH+ secreting pheochromocytes. It might be involved in the regulation of the hypothalamic-pituitary-adrenal axis. (Page 7 line 175-182, Figure 3, Figure 4). Second, applied the SCENIC pipeline, we found an additional weak signal of transcription regulons for the DP cells (Page 6 line 153-157, Appendix 1—figure 4). It showed XPBP1 as the specific regulons for ACTH+&CRH+ pheochromocyte and adrenocortical cell type. Third, the spliced vs. unspliced phase for CHGA, CHGB, and TH from RNA velocity analysis demonstrated a clear more dynamics expression in POMC+&CRH+ pheochromocytes than in pheochromocytes (Appendix 1—figure 5). Lastly, to investigate the genetic driver, the whole exome sequencing identified 1 shared somatic variant of ACAN (c.5951T>A:p.L1984Q) comparing variants in tumor samples to controls but Sanger sequencing only confirmed the presence in esPHEO_T3 which not observed in esPHEO_T2 (Page 13 line 352-358, Appendix 1—figure 7). 5. The pseudo-time analysis indicated that sustentacular cells transform to ACTH+&CRH+ pehochromocytes and then to pheochromocyte. The authors utilized Monocle3 in which user has to define the starting points. The authors can validate the result using RNA velocity analysis which also predicts cell transition without the need of prior knowledge about starting point cell type. At first, we have added RNA velocity analysis (Figure 5B, Page 10 line 268-286). For the high proportions of unspliced/spliced transcripts in Figure 5B, stem-like characteristics of sustentacular cells were supported. We performed the pseudo-time analysis for the adrenal tumor cell subsets to determine the pattern of the dynamic cell transitional states. Then, we re-run the pseudo-time analysis and used the recommended strategy of Monocel to order cells based on genes that differ between clusters. The sustentacular cells were also in an early stage (Figure 6). 6. Given the diverse immune and endothelial cell type in the tumor microenvironment, it would be interesting to perform the cell-cell interaction analysis using the programs such as CellPhoneDB to see if they have distinct regulatory role in different tumor microenvironment. To investigate the potential cell-cell interactions among various immune cells, endothelial cells, and other cell types in the different tumor microenvironment (esPHEO, esPHEO_Adj, PHEO, and ACA), we performed additional analysis using the CellPhoneDB Python package in the revised version of our manuscript. As shown in the new Appendix 1—figure 6, we observed very distinct patterns of ligand-receptor pairs for cell-cell interactions in the different tumor microenvironments. Notably, the diverse cell clusters within PHEO tumors exhibited a relatively high abundance of cell-cell connections between different cell types, while the cell-cell interactions within esPHEO_Adj samples were totally different. For example, MIF, one of the most enigmatic regulators of innate and adaptive immune responses, was shown as a specific regulator in esPHEO and PHEO, in contrast to ACA. 7. How did the authors define the four subclusters of endothelial cells? Please consider providing list of marker genes. The four groups of endothelial cells were combined to a larger endothelial cell cluster for downstream analysis. Endothelial cell cluster statistics were added in Supplementary File 3, which presented cell counts for each endothelial cell cluster in different samples and top10 gene markers. 8. In the method part, how did the authors determine different criteria for the maximum number of genes (no more than 5000, 3000, and 2500 genes for PHEO, ACA, and esPHEO samples, respectively)? We set the different criteria for the maximum number of genes (no more than 5000, 3000, and 2500 genes for PHEO, ACA and esPHEO samples respectively) based on QC violin plot showing the number of detected genes (Appendix 1—figure 2B). Reviewer #3: Zhang et al. perform single cell RNA sequencing (scRNA-Seq) of one rare ACTH+CRH-secreting phenochromocytoma (3 anatomically distinct sites from the tumor and one peritumoral site), one typical pheochromocytoma, and two typical adrenocortical adenomas. Their main findings are as follows: (1) They identify a unique cell type, which they term ACTH+CRH+ pheochromocyte, which appears to be the tumor cell present in the rare ACTH+CRH+ tumor (2) Marker gene analysis reveals that while known adrenal chromaffin markers (CHGA, PNMT) are present in both pheochromocytes and ACTH+CRH+ pheochromocyte, the latter has some unique markers such as GAL and POMC. They validate the marker genes with IHC. (3) Profiling of the non-tumor populations reveals distinct immune microenvironment profile and endothelial cell profile to the rare tumor compared with classical pheochromocytoma and adrenalocortical adenoma. The main strength of this manuscript is that it involves single-cell profiling of an exceptionally rare tumor type and a distinction from the more common adrenal tumors (pheochromocytoma and adrenocortical adenoma). The broader implication of the authors' findings is with respect to Dale's principle, which states that a given neuron releases only one type of neurotransmitter. However, in the case of this tumor, single cell analysis clearly shows that ACTH, CRH, and chatacholemines are being released from the same cell. This is quite interesting and significant. The data will also potentially be valuable to others in the field for analysis in future studies. There remain some unanswered questions – namely: (1) What is the cell in normal physiology that gives rise to this ACTH+CRH+ pheochromocytoma? (2) Do conventional phenochromocytomas differ from the ACTH+CRH+ pheochromocytoma in terms of the cell of origin that is transformed, or in the spectrum of genetic alterations that result in transformation? We thank the reviewer for carefully reviewing the manuscript and raising insightful questions. To response the reviewer’s questions and strengthen the manuscript, we supplemented analysis and experiments as much as possible. First, we performed RNA velocity analysis (Figure 5, Page 10 line 268-286) to investigate dynamic information in individual cells. For the high proportions of unspliced/spliced transcripts in Figure 5B, stem-like characteristics of sustentacular cells were supported. Also, the spliced vs. unspliced phase for CHGA, CHGB, and TH from RNA velocity analysis demonstrated a clear more dynamics expression in POMC+&CRH+ pheochromocytes than in pheochromocytes (Appendix 1—figure 5). Second, we re-run the pseudo-time analysis (Page 10 line 288-300) and used the recommended strategy of Monocel to order cells based on genes that differ between clusters. The sustentacular cells were also in an early state (Figure 6), which was in accordance with their exhibited stem-like properties and the highest unspliced proportion among non-immune cell types in the RNA velocity analysis (Figure 5B). The results also showed a transition from sustentacular cells to pheochromocytes and then to ACTH+&CRH+ pheochromocyte, and adrenocortical cells were on another branch (Figure 6). As we discussed in manuscript (Page 14 line 391-398), although pheochromocyte was prior to ACTH&CRH secreting pheochromocyte in pseudotime order, we assumed that ACTH&CRH secreting pheochromocyte have more hormone-producing functions, retain stem- and endocrine-differentiation ability. But further experiments are needed to validate our hypothesis. Third, we applied SCENIC analysis pipeline (Page 6 line 153-157, Appendix 1—figure 4) to detect the transcription factors (which are jointly called regulons) alongside their candidate target genes, and yield specific regulons for each cellular cluster. We observed an additional weak signal of transcription regulons (XPBP1) for the ACTH+CRH+ pheochromocytoma and adrenocortical cell type. Furthermore, to investigate the genetic driver, we supplemented with the whole-exome sequencing (WES) experiments for all rest of tissue samples (esPHEO_T2, esPHEO_T3 and esPHEO_Adj) from the rare case with ectopic ACTH&CRH-secreting pheochromocytoma and the blood sample (esPHEO_Blood). Based on WES data, we identified 1 shared somatic variant of ACAN (c.5951T>A:p.L1984Q) comparing variants in tumor samples to controls but Sanger sequencing only confirmed the presence in esPHEO_T3 which not observed in esPHEO_T2 (Page 13 line 352-358, Appendix 1—figure 7). Overall, additional analyses and experiments have presented more comprehensive results which appropriately address the questions raised by the reviewer. But they also provide new hypothesis remaining unanswered questions. For future studies, on one hand, we are very concerned about similar suspicious cases in the clinic. On the other hand, we are going for following research for further downstream experiments to validate the molecular mechanism for secreting multiple hormones. Comments for the authors: Overall, I think this study is of broad interest given the rarity of this tumor type. My comments to the authors to improve the manuscript are as follows: 1. Given how rare the ACTH+CRH+ pheochromocytoma is, I think the study would be substantially strengthened if the authors could perform DNA sequencing (WGS or WES) and describe how, if at all, the genomic landscape differs from conventional pheochromocytoma. The frozen tissue of esPHEO_T1 and PHEO_T is unavailable and a few remaining for esPHEO_T2. For all rest of tissue samples, we supplemented with the whole-exome sequencing experiments for tumors (esPHEO_T2, esPHEO_T3) and controls (esPHEO_Adj) from the rare case with ectopic ACTH&CRH-secreting pheochromocytoma. (Page 13 line 352-358, Appendix 1—figure 7) 2. Can the authors comment on whether the hypothesis is whether the ACTH+CRH+ pheochromocytoma originates from a rare progenitor cell that is distinct from the chromaffin cell giving rise to pheochromocytoma? If so, can the authors stain a panel of normal adrenal glands with some of their marker genes to try and identify this cell in normal tissues? (Page 14 line 389-398) The RNA velocity estimation and pseudo-time analysis of different adrenal cell subtypes supported the sustentacular cells exhibiting stem-like properties. Although pheochromocyte was prior to ACTH&CRH secreting pheochromocyte in pseudotime order, the RNA velocity prediction of POMC+&CRH+ pheochromocytes might be under-estimated because the transcripts of POMC and CRH were all predicted as spliced ones. Based on the spliced vs. unspliced phase for CHGA, CHGB and TH it showed a clear more dynamics expression in POMC+&CRH+ pheochromocytes than in pheochromocytes. We assumed that ACTH&CRH secreting pheochromocyte have more hormone-producing functions, retain stem- and endocrine-differentiation ability. But further experiments are needed to validate our hypothesis. We thank the reviewer for raising good recommendations. We would like to test marker genes in normal tissues. But it is difficult to obtain normal adrenal glands in clinic. We searched POMC, CRH and GAL in Genotype-Tissue Expression Project (GTEx), which launched by the National Institutes of Health (NIH). GTEx has established a database (https://www.gtexportal.org/home/) to study genes in different normal tissues. The results, as shown in Author response images 1-3: POMC is over-expressed in pituitary, but expressed at a very low level in adrenal gland. CRH is overexpressed in brain-hypothalamus, but almost not expressed in adrenal gland. GAL is overexpressed in pituitary and brain-hypothalamus, but almost not expressed in adrenal gland. Author response image 1 Download asset Open asset Author response image 2 Download asset Open asset Author response image 3 Download asset Open asset 3. While the tumor type is interesting for its rarity, the analysis performed is quite standard and comes across as a bit superficial in parts. Although it is understandable that the authors have only one ACTH+CRH+ sample I think they can do more with the data and this would significantly strengthen the manuscript. For example, it would be interesting if the authors can point to specific master regulatory factors that drive the distinct programs in pheochromocytes vs. ACTH+CRH+ pheochromocytes. The immune microenvironment analysis, while inherently descriptive, is also somewhat superficial. Based on the routine differentially expressed genes analysis, we mainly found GAL co-expressed with POMC and CRH, could be a candidate marker to detect the rare ectopic ACTH+&CRH+ secreting pheochromocytes. As previous research reported, it might be involved in the regulation of the hypothalamic-pituitary-adrenal axis. (Page 7 line 175-182, Figure 3, Figure 4). Second, applied the SCENIC pipeline, we found an additional weak signal of transcription regulons for the DP cells (Page 6 line 153-157, Appendix 1—figure 4). It showed XPBP1 as the specific regulons for ACTH+&CRH+ pheochromocyte and adrenocortical cell type. Furthermore, RNA velocity analysis (Appendix 1—figure 5) demonstrated a clear more dynamics expression in POMC+&CRH+ pheochromocytes than in pheochromocytes. [Editors' note: further revisions were suggested prior to acceptance, as described below.] Reviewer #2: Although the authors have satisfactorily addressed most of my points, there are remaining concerns about RNA velocity data. Please cite any reference for the statement "For the high proportions of unspliced/spliced transcripts, stem-like characteristics of sustentacular cells were supported." Can global ratio of unspliced/spliced transcripts support stem-like characteristics? Please elaborate Figure 5 C-F. Currently, they don't seem to add any information. (Page 10 line 269-286, Figure 5 and its legend) We thank the reviewer for carefully reviewing and raising this concern about RNA velocity. We have revised our manuscript to add a paragraph and cite the appropriate references in the updated revision. Previously study had observed that the unspliced transcripts were enriched in genes involved in DNA binding and RNA processing in hematopoietic stem cells [1]. And Schwann cell precursors, which can differentiate into chromaffin cells, also had positive unspliced-spliced phase portrait [2]. Therefore, we claimed that, as for the high proportions of unspliced/spliced transcripts, stem-like characteristics of sustentacular cells were supported. We remove Figure 5 C-D, as the reviewer mentioned, because they don't seem to add any valuable information. Besides, we added more description about the results for new Figure 5 C-D (old Figure 5 E-F) in Page 10 line 282-288, which showed estimated pseudo-time grounded on transcriptional dynamics and velocity streamlines accounting for speed and direction of motion. These results indicated that medullary cells are earlier than cortical cells (new Figure 5C). From velocity streamlines (new Figure 5D), we found the four adrenal cell subtypes, that is, POMC+&CRH+ pheochromocytes, pheochromocytes adrenocortical cells, and sustentacular cells, were independent respectively and not directed toward other cell types. Reviewer #3: In the revised manuscript Zhang et al. have included additional data and analyses including more exhaustive QC, RNA velocity analysis, regulome analysis, and have performed WES of the ACTH/CRH-secreting pheochromocytoma. They have generally addressed my technical concerns from the prior review. I maintain that the analysis remains somewhat superficial and descriptive in parts and this may be somewhat of a missed opportunity to more deeply explore the underlying biology of this unique case, understanding the caveats of its rarity. Nonetheless, I think a description of this tumor at single-cell resolution and availability of the dataset is of value to the scientific community. However, I would like to see a more careful analysis of the WES data prior to publication. I do not see any basic metrics (mutation rate etc.), description of pathogenicity filtering/annotation, or copy number analysis. The mutations shown are primarily missense and I do not really see any obvious driver genes – how many of these are putative driver vs. passenger mutations? ACAN is mentioned, but what is its significance, if any? The somatic landscape should be discussed in comparison to typical phenochromocytomas and adrenocortical carcinomas, which have been more extensively sequenced. If there is no obvious genetic driver of this ACTH/CRH-secreting phenochromocytoma, that should be stated. If the claim is that ACAN alterations are somehow related to this tumor type, that needs to be substantiated. Or if the implication is that ACAN is a passenger alteration, that needs to be stated explicitly also. (Page 13 line 359-378; Page 21 line 587-597; Supplementary File 4) We thank the reviewer for carefully reviewing and raising concerns about our WES analysis. We supplemented the variants filtering criteria in Page 21 line 587-597, and further discussed the WES results in Page 13 line 359-378. Besides, the germline and somatic mutations were listed in Supplementary File 4 including detailed annotations. Genetic mutations of phaeochromocytoma and paraganglioma are mainly classified into two major clusters, that is, pseudo hypoxic pathway and kinase signaling pathways [3-4]. We did not find any gene mutations or copy number variations that were related to these two major clusters. We only identified 1 shared somatic variant of ACAN mutation (c.5951T>A:p.L1984Q) comparing variants in tumor samples to controls. ACAN, encoding a major component of the extracellular matrix, is a member of the aggrecan/versican proteoglycan family. Mutations of ACAN were reported related to steroid levels [5]. It is well-established that circulating steroid levels are linked to inflammatory diseases such as arthritis, because arthritis as well as most autoimmune disorders result from a combination of several predisposing factors including the stress response system such as the hypothalamic-pituitary-adrenocortical axis [6]. But no direct evidence related to ACAN for phaeochromocytoma. Therefore, no obvious genetic driver was found to explain the rare case of ACTH/CRH-secreting phaeochromocytoma. Further investigations would be needed to uncover the relation between ACAN to phaeochromocytoma. References: [1]. Bowman TV, McCooey AJ, Merchant AA, Ramos CA, Fonseca P, Poindexter A, Bradfute SB, Oliveira DM, Green R, Zheng Y, Jackson KA, Chambers SM, McKinney-Freeman SL, Norwood KG, Darlington G, Gunaratne PH, Steffen D, Goodell MA. Differential mRNA processing in hematopoietic stem cells. Stem Cells. 2006. Mar;24(3):662-70. [2]. La Manno G., Soldatov R., Zeisel A., Braun E., Hochgerner H., Petukhov V., Lidschreiber K., Kastriti M.E., Lönnerberg P., Furlan A. RNA velocity of single cells. Nature. 2018 560:494-498. [3] Pillai S, Gopalan V, Smith RA, Lam AK. Updates on the genetics and the clinical impacts on phaeochromocytoma and paraganglioma in the new era. Crit Rev Oncol Hematol. 2016. Apr;100:190-208. [4] Nölting S, Grossman AB. Signaling pathways in pheochromocytomas and paragangliomas: prospects for future therapies. Endocr Pathol. 2012. Mar;23(1):21-33. [5] Yousri NA, Fakhro KA, Robay A, Rodriguez-Flores JL, Mohney RP, Zeriri H, Odeh T, Kader SA, Aldous EK, Thareja G, Kumar M, Al-Shakaki A, Chidiac OM, Mohamoud YA, Mezey JG, Malek JA, Crystal RG, Suhre K. Whole-exome sequencing identifies common and rare variant metabolic QTLs in a Middle Eastern population. Nat Commun. 2018 Jan 23;9(1):333. [6]. Cutolo M, Sulli A, Pizzorni C, Craviotto C, Straub RH. Hypothalamic-pituitary-adrenocortical and gonadal functions in rheumatoid arthritis. Ann N Y Acad Sci. 2003 May;992:107-17. https://doi.org/10.7554/eLife.68436.sa2 Article and author information Author details Xuebin Zhang Department of Urology, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, China Contribution Data curation, Formal analysis, Investigation, Methodology, Resources, Software, Writing – original draft, Writing – review and editing Contributed equally with Penghu Lian and Mingming Su Competing interests No competing interests declared Penghu Lian Department of Urology, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, China Contribution Formal analysis, Investigation, Methodology, Project administration, Resources, Software, Validation, Visualization, Writing – original draft, Writing – review and editing Contributed equally with Xuebin Zhang and Mingming Su Competing interests No competing interests declared Mingming Su Institute of Basic Medical Sciences Chinese Academy of Medical Sciences, School of Basic Medicine Peking Union Medical College, Beijing, China Contribution Data curation, Formal analysis, Investigation, Methodology, Resources, Software, Validation, Visualization, Writing – original draft, Writing – review and editing Contributed equally with Xuebin Zhang and Penghu Lian Competing interests No competing interests declared "This ORCID iD identifies the author of this article:"0000-0002-1393-0800 Zhigang Ji Department of Urology, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, China Contribution Data curation, Investigation, Methodology, Visualization, Writing – review and editing Competing interests No competing interests declared Jianhua Deng Department of Urology, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, China Contribution Data curation, Investigation, Methodology, Writing – review and editing Competing interests No competing interests declared Guoyang Zheng Department of Urology, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, China Contribution Data curation, Investigation, Writing – review and editing Competing interests No competing interests declared Wenda Wang Department of Urology, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, China Contribution Data curation, Investigation, Writing – review and editing Competing interests No competing interests declared Xinyu Ren Department of Pathology, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, China Contribution Data curation, Visualization Competing interests No competing interests declared Taijiao Jiang Institute of Basic Medical Sciences Chinese Academy of Medical Sciences, School of Basic Medicine Peking Union Medical College, Beijing, China Suzhou Institute of Systems Medicine, Jiangsu, China Contribution Conceptualization, Funding acquisition, Project administration, Supervision, Writing – review and editing Competing interests No competing interests declared Peng Zhang Beijing Key Laboratory for Genetics of Birth Defects, Beijing Pediatric Research Institute, Beijing Children's Hospital, Capital Medical University, National Center for Children's Health, Beijing, China Contribution Investigation, Methodology, Supervision, Validation, Writing – original draft, Writing – review and editing For correspondence zhangpengdyx@163.com Competing interests No competing interests declared "This ORCID iD identifies the author of this article:"0000-0002-6218-1885 Hanzhong Li Department of Urology, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, China Contribution Conceptualization, Funding acquisition, Project administration, Supervision, Writing – review and editing For correspondence lihzh@pumch.cn Competing interests No competing interests declared Funding Chinese Academy of Medical Sciences (2017-I2M-1-001) Hanzhong Li Chinese Academy of Medical Sciences (2021-I2M-1-051) Taijiao Jiang Chinese Academy of Medical Sciences (2021-I2M-1-001) Taijiao Jiang The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication. Acknowledgements This work was supported by CAMS Innovation Funds for Medical Sciences (CIFMS), which were 2017-I2M-1-001, 2021-I2M-1-051 and 2021-I2M-1-001. Ethics Specimen collection was obtained after appropriate research consents (and assents when applicable) and was approved (protocol number: S-K431) by the Institutional Review Board, Peking Union Medical College Hospital. All information obtained was protected and de-identified. Senior Editor Mone Zaidi, Icahn School of Medicine at Mount Sinai, United States Reviewing Editor Murim Choi, Seoul National University, Republic of Korea Reviewer Murim Choi, Seoul National University, Republic of Korea Publication history Received: March 16, 2021 Accepted: December 13, 2021 Accepted Manuscript published: December 14, 2021 (version 1) Accepted Manuscript updated: December 15, 2021 (version 2) Version of Record published: December 31, 2021 (version 3) Copyright © 2021, Zhang et al. This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited. from https://elifesciences.org/articles/68436

Millions of people are at increased risk of type 2 diabetes and high blood pressure and don't even know it, due to a hidden hormone problem in their bodies. As many as 1 in 10 people have a non-cancerous tumor on one or both of their adrenal glands that could cause the gland to produce excess amounts of the stress hormone cortisol. Up to now, doctors have thought that these tumors had little impact on your health. But a new study out of Britain has found that up to half of people with these adrenal tumors are secreting enough excess cortisol to raise their risk of diabetes and high blood pressure. Nearly 1.3 million adults in the United Kingdom alone could suffer from this disorder, which is called Mild Autonomous Cortisol Secretion (MACS), the researchers said. Anyone found with one of these adrenal tumors should be screened to see if their health is at risk, said senior researcher Dr. Wiebke Arlt, director of the University of Birmingham Institute of Metabolism and Systems Research in England. "People who are found to have an adrenal tumor should undergo assessment for cortisol excess and if they are found to suffer from cortisol overproduction they should be regularly screened for type 2 diabetes and hypertension and receive treatment if appropriate," Arlt said. These tumors are usually discovered during imaging scans of the abdomen to treat other illnesses, said Dr. André Lacroix, an endocrinologist at the University of Montreal Hospital Center, who wrote an editorial accompanying the study. Both were published Jan. 4 in the Annals of Internal Medicine. Adrenal glands primarily produce the hormone adrenaline, but they are also responsible for the production of a number of other hormones, including cortisol, Lacroix said. Cortisol is called the "fight-or-flight" hormone, and can cause blood sugar levels to rise and blood pressure to surge -- usually in response to some perceived bodily threat. Previous studies had indicated that about 1 in 3 adrenal tumors secrete excess cortisol, and an even lower number caused cortisol levels to rise so high that they affected health, researchers said in background notes. But this new study of more than 1,300 people with adrenal tumors found that previous estimates were wrong. About half of these patients had excess cortisol due to their adrenal tumors. Further, more than 15% had levels high enough to impact their health, compared to those with truly benign tumors. MACS patients were more likely to be diagnosed with high blood pressure, and were as much as twice as likely to be on three or more blood pressure medications. They also were more likely to have type 2 diabetes, and were twice as likely to require insulin to manage their blood sugar, the study found. "This study clearly shows that mild cortisol production is more frequent than we thought before, and that the more cortisol you produce, the more likely to you are to have consequences such as diabetes and hypertension," Lacroix said. About 70% of people with MACS were women, and most were of postmenopausal age, the researchers said. "Adrenal tumor-related cortisol excess is an important previously overlooked health issue that particularly affects women after the menopause," Arlt said. Lacroix agreed that guidelines should be changed so that people with adrenal tumors are regularly screened. "Everybody who is found to have an adrenal nodule larger than 1 centimeter needs to be screened to see if they're producing excess hormone or not," he said. "That's very clear." A number of medications can reduce cortisol overproduction or block cortisol action, if an adrenal tumor is found to be causing an excess of hormone. People with severe cortisol excess can even have one of their two adrenal glands removed if necessary, Lacroix said. "It is quite possible to live completely normally with one adrenal gland," he said. More information The Cleveland Clinic has more about adrenal tumors. SOURCES: Wiebke Arlt, MD, DSc, director, Institute of Metabolism and Systems Research, University of Birmingham, U.K.; André Lacroix, MD, endocrinologist, University of Montreal Hospital Center; Annals of Internal Medicine, Jan. 4, 2022 From https://consumer.healthday.com/1-4-benign-adrenal-gland-tumors-might-cause-harm-to-millions-2656172346.html

Researchers in Europe say they have shown for the first time that the SARS-CoV-2 virus attacks the human stress system by limiting how our adrenal glands can respond to the threat of Covid-19. According to a study, the coronavirus targets the adrenal glands, thereby weakening the body’s ability to produce the stress hormones cortisol and adrenaline needed to help battle a serious infection. Part of the body’s defence mechanism, these glands are indispensable for our survival of stressful situations, particularly with a coronavirus infection. The research was published by a group of scientists in London, United Kingdom; Zurich, Switzerland; and Dresden and Regensburg in Germany, in the journal The Lancet Diabetes and Endocrinology last month (November 2021). “The results of our latest work now show for the first time that the virus directly affects the human stress system to a relevant extent,” says Dr Stefan Bornstein, director of the Medical Clinic and Polyclinic III and the Centre for Internal Medicine at the University Hospital in Dresden. Whether these changes directly contribute to adrenal insufficiency, or even lead to long Covid is still unclear, he says. This question must be investigated in further clinical studies. Pointing to recent research showing the effect of inhaling steroids to prevent clinical deterioration in patients with Covid-19, the researchers say certain drugs may be able to help limit this effect of the SARS-CoV-2 virus. “This evidence underlines the potentially important role for adrenal steroids in coping with Covid-19,” scientists at the University of Zurich say. The researchers analysed the data of 40 deceased Covid-19 patients in Dresden and found that their tissue samples showed clear signs of adrenal gland inflammation. From https://www.thestar.com.my/lifestyle/health/2021/12/22/how-the-sars-cov-2-virus-undermines-our-bodys-039fight039-response

Any condition that causes the adrenal gland to produce excessive cortisol results in the disorder Cushing's syndrome. Cushing syndrome is characterized by facial and torso obesity, high blood pressure, stretch marks on the belly, weakness, osteoporosis, and facial hair growth in females. Cushing's syndrome has many possible causes including tumors within the adrenal gland, adrenal gland stimulating hormone (ACTH) produced from cancer such as lung cancer, and ACTH excessively produced from a pituitary tumors within the brain. ACTH is normally produced by the pituitary gland (located in the center of the brain) to stimulate the adrenal glands' natural production of cortisol, especially in times of stress. When a pituitary tumor secretes excessive ACTH, the disorder resulting from this specific form of Cushing's syndrome is referred to as Cushing's disease. As an aside, it should be noted that doctors will sometimes describe certain patients with features identical to Cushing's syndrome as having 'Cushingoid' features. Typically, these features are occurring as side effects of cortisone-related medications, such as prednisone and prednisolone.

An updated guideline for the treatment of Cushing’s disease focuses on new therapeutic options and an algorithm for screening and diagnosis, along with best practices for managing disease recurrence. Despite the recent approval of novel therapies, management of Cushing’s disease remains challenging. The disorder is associated with significant comorbidities and has high mortality if left uncontrolled. Source: Adobe Stock “As the disease is inexorable and chronic, patients often experience recurrence after surgery or are not responsive to medications,” Shlomo Melmed, MB, ChB, MACP, dean, executive vice president and professor of medicine at Cedars-Sinai Medical Center in Los Angeles, and an Endocrine Today Editorial Board Member, told Healio. “These guidelines enable navigation of optimal therapeutic options now available for physicians and patients. Especially helpful are the evidence-based patient flow charts [that] guide the physician along a complex management path, which usually entails years or decades of follow-up.” Shlomo Melmed The Pituitary Society convened a consensus workshop with more than 50 academic researchers and clinical experts across five continents to discuss the application of recent evidence to clinical practice. In advance of the virtual meeting, participants reviewed data from January 2015 to April 2021 on screening and diagnosis; surgery, medical and radiation therapy; and disease-related and treatment-related complications of Cushing’s disease, all summarized in recorded lectures. The guideline includes recommendations regarding use of laboratory tests, imaging and treatment options, along with algorithms for diagnosis of Cushing’s syndrome and management of Cushing’s disease. Updates in laboratory, testing guidance If Cushing’s syndrome is suspected, any of the available diagnostic tests could be useful, according to the guideline. The authors recommend starting with urinary free cortisol, late-night salivary cortisol, overnight 1 mg dexamethasone suppression, or a combination, depending on local availability. If an adrenal tumor is suspected, the guideline recommends overnight dexamethasone suppression and using late-night salivary cortisol only if cortisone concentrations can also be reported. The guideline includes several new recommendations in the diagnosis arena, particularly on the role of salivary cortisol assays, according to Maria Fleseriu, MD, FACE, a Healio | Endocrine Today Co-editor, professor of medicine and neurological surgery and director of the Pituitary Center at Oregon Health & Science University in Portland. Maria Fleseriu “Salivary cortisol assays are not available in all countries, thus other screening tests can also be used,” Fleseriu told Healio. “We also highlighted the sequence of testing for recurrence, as many patients’ urinary free cortisol becomes abnormal later in the course, sometimes up to 1 year later.” The guideline states combined biochemical and imaging for select patients could potentially replace petrosal sinus sampling, a very specialized procedure that cannot be performed in all hospitals, but more data are needed. “With the corticotropin-releasing hormone stimulation test becoming unavailable in the U.S. and other countries, the focus is now on desmopressin to replace corticotropin-releasing hormone in some of the dynamic testing, both for diagnosis of pseudo-Cushing’s as well as localization of adrenocorticotropic hormone excess,” Fleseriu said. The guideline also has a new recommendation for anticoagulation for high-risk patients; however, the exact duration and which patients are at higher risk remains unknown. “We always have to balance risk for clotting with risk for bleeding postop,” Fleseriu said. “Similarly, recommended workups for bone disease and growth hormone deficiency have been further structured based on pitfalls specifically related to hypercortisolemia influencing these complications, as well as improvement after Cushing’s remission in some patients, but not all.” New treatment options The guideline authors recommended individualizing medical therapy for all patients with Cushing’s disease based on the clinical scenario, including severity of hypercortisolism. “Regulatory approvals, treatment availability and drug costs vary between countries and often influence treatment selection,” the authors wrote. “However, where possible, it is important to consider balancing cost of treatment with the cost and the adverse consequences of ineffective or insufficient treatment. In patients with severe disease, the primary goal is to treat aggressively to normalize cortisol concentrations.” Fleseriu said the authors reviewed outcomes data as well as pros and cons of surgery, repeat surgery, medical treatments, radiation and bilateral adrenalectomy, highlighting the importance of individualized treatment in Cushing’s disease. “As shown over the last few years, recurrence rates are much higher than previously thought and patients need to be followed lifelong,” Fleseriu said. “The role of adjuvant therapy after either failed pituitary surgery or recurrence is becoming more important, but preoperative or even primary medical treatment has been also used more, too, especially in the COVID-19 era.” The guideline summarized data on all medical treatments available, either approved by regulatory agencies or used off-label, as well as drugs studied in phase 3 clinical trials. “Based on great discussions at the meeting and subsequent emails to reach consensus, we highlighted and graded recommendations on several practical points,” Fleseriu said. “These include which factors are helpful in selection of a medical therapy, which factors are used in selecting an adrenal steroidogenesis inhibitor, how is tumor growth monitored when using an adrenal steroidogenesis inhibitor or glucocorticoid receptor blocker, and how treatment response is monitored for each therapy. We also outline which factors are considered in deciding whether to use combination therapy or to switch to another therapy and which agents are used for optimal combination therapy.” Future research needed The guideline authors noted more research is needed regarding screening and diagnosis of Cushing’s syndrome; researchers must optimize pituitary MRI and PET imaging using improved data acquisition and processing to improve microadenoma detection. New diagnostic algorithms are also needed for the differential diagnosis using invasive vs. noninvasive strategies. Additionally, the researchers said the use of anticoagulant prophylaxis and therapy in different populations and settings must be further studied, as well as determining the clinical benefit of restoring the circadian rhythm, potentially with a higher nighttime medication dose, as well as identifying better markers of disease activity and control. “Hopefully, our patients will now experience a higher quality of life and fewer comorbidities if their endocrinologist and care teams are equipped with this informative roadmap for integrated management, employing a consolidation of surgery, radiation and medical treatments,” Melmed told Healio. From https://www.healio.com/news/endocrinology/20211029/updated-cushings-disease-guideline-highlights-new-diagnosis-treatment-roadmap

Jessica Rotham, National Center for Health Research What is it? Cushing’s syndrome is a condition you probably have never heard of, but for those who have it, the symptoms can be quite scary. Worse still, getting it diagnosed can take a while. Cushing’s syndrome occurs when the tissues of the body are exposed to high levels of cortisol for an extended amount of time. Cortisol is the hormone the body produces to help you in times of stress. It is good to have cortisol at normal levels, but when those levels get too high it causes health problems. Although cortisol is related to stress, there is no evidence that Cushing’s syndrome is directly or indirectly caused by stress. Cushing’s syndrome is considered rare, but that may be because it is under-reported. As a result, we don’t have good estimates for how many people have it, which is why the estimates for the actual number of cases vary so much–from 5 to 28 million people.[1] The most common age group that Cushing’s affects are those 20 to 50 years old. It is thought that obesity, type 2 diabetes, and high blood pressure may increase your risk of developing this syndrome.[2] What causes Cushing’s Syndrome? Cushing’s syndrome is caused by high cortisol levels. Cushing’s disease is a specific form of Cushing’s syndrome. People with Cushing’s disease have high levels of cortisol because they have a non-cancerous (benign) tumor in the pituitary gland. The tumor releases adrenocorticotropin hormone (ACTH), which causes the adrenal glands to produce excessive cortisol. Cushing’s syndrome that is not Cushing’s disease can be also caused by high cortisol levels that result from tumors in other parts of the body. One of the causes is “ectopic ACTH syndrome.” This means that the hormone-releasing tumor is growing in an abnormal place, such as the lungs or elsewhere. The tumors can be benign, but most frequently they are cancerous. Other causes of Cushing’s syndrome are benign tumors on the adrenal gland (adrenal adenomas) and less commonly, cancerous adrenal tumors (adrenocortical carcinomas). Both secrete cortisol, causing cortisol levels to get too high. In some cases, a person can develop Cushing’s syndrome from taking steroid medications, such as prednisone. These drugs, known as corticosteroids, mimic the cortisol produced by the body. People who have Cushing’s syndrome from steroid medications do not develop a tumor.[3] What are the signs and symptoms of Cushing’s Syndrome? The appearance of people with Cushing’s syndrome starts to change as cortisol levels build up. Regardless of what kind of tumor they have or where the tumor is located, people tend to put on weight in the upper body and abdomen, with their arms and legs remaining thin; their face grows rounder (“moon face”); they develop fat around the neck; and purple or pink stretch marks appear on the abdomen, thighs, buttocks or arms. Individuals with the syndrome usually experience one or more of the following symptoms: fatigue, muscle weakness, high glucose levels, anxiety, depression, and high blood pressure. Women are more likely than men to develop Cushing’s syndrome, and when they do they may have excess hair growth, irregular or absent periods, and decreased fertility.[4] Why is Cushing’s Syndrome so frequently misdiagnosed? These symptoms seem distinctive, yet it is often difficult for those with Cushing’s syndrome to get an accurate diagnosis. Why? While Cushing’s is relatively rare, the signs and symptoms are common to many other diseases. For instance, females with excess hair growth, irregular or absent periods, decreased fertility, and high glucose levels could have polycystic ovarian syndrome, a disease that affects many more women than Cushing’s. Also, people with metabolism problems (metabolic syndrome), who are at higher than average risk for diabetes and heart disease, also tend to have abdominal fat, high glucose levels and high blood pressure.[5] Problems in testing for Cushing’s When Cushing’s syndrome is suspected, a test is given to measure cortisol in the urine. This test measures the amount of free or unbound cortisol filtered by the kidneys and then released over a 24 hour period through the urine. Since the amount of urinary free cortisol (UFC) can vary a lot from one test to another—even in people who don’t have Cushing’s—experts recommend that the test be repeated 3 times. A diagnosis of Cushing’s is given when a person’s UFC level is 4 times the upper limit of normal. One study found this test to be highly accurate, with a sensitivity of 95% (meaning that 95% of people who have the disease will be correctly diagnosed by this test) and a specificity of 98% (meaning that 98% of people who do not have the disease will have a test score confirming that).[6] However, a more 2010 study estimated the sensitivity as only between 45%-71%, but with 100% specificity.[7] This means that the test is very accurate at telling people who don’t have Cushing’s that they don’t have it, but not so good at identifying the people who really do have Cushing’s. The authors that have analyzed these studies advise that patients use the UFC test together with other tests to confirm the diagnosis, but not as the initial screening test.[8] Other common tests that may be used to diagnose Cushing’s syndrome are: 1) the midnight plasma cortisol and late-night salivary cortisol measurements, and 2) the low-dose dexamethasone suppression test (LDDST). The first test measures the amount of cortisol levels in the blood and saliva at night. For most people, their cortisol levels drop at night, but people with Cushing’s syndrome have cortisol levels that remain high all night. In the LDDST, dexamethasone is given to stop the production of ACTH. Since ACTH produces cortisol, people who don’t have Cushing’s syndrome will get lower cortisol levels in the blood and urine. If after giving dexamethasone, the person’s cortisol levels remain high, then they are diagnosed with Cushing’s.[9] Even when these tests, alone or in combination, are used to diagnose Cushing’s, they don’t explain the cause. They also don’t distinguish between Cushing’s syndrome, and something called pseudo-Cushing state. Pseudo-Cushing state Some people have an abnormal amount of cortisol that is caused by something unrelated to Cushing’s syndrome such as polycystic ovarian syndrome, depression, pregnancy, and obesity. This is called pseudo-Cushing state. Their high levels of cortisol and resulting Cushing-like symptoms can be reversed by treating whatever disease is causing the abnormal cortisol levels. In their study, Dr. Giacomo Tirabassi and colleagues recommend using the desmopressin (DDAVP) test to differentiate between pseudo-Cushing state and Cushing’s. The DDAVP test is especially helpful in people who, after being given dexamethasone to stop cortisol production, continue to have moderate levels of urinary free cortisol (UFC) and midnight serum cortisol.[10] An additional test that is often used to determine if one has pseudo-Cushing state or Cushing’s syndrome is the dexamethasone-corticotropin-releasing hormone (CRH) test. Patients are injected with a hormone that causes cortisol to be produced while also being given another hormone to stop cortisol from being produced. This combination of hormones should make the patient have low cortisol levels, and this is what happens in people with pseudo-Cushing state. People with Cushing’s syndrome, however, will still have high levels of cortisol after being given this combination of hormones.[11] How can Cushing’s be treated? Perhaps because Cushing’s is rare or under-diagnosed, few treatments are available. There are several medications that are typically the first line of treatment. None of the medications can cure Cushing’s, so they are usually taken until other treatments are given to cure Cushing’s, and only after that if the other treatment fails. The most common treatment for Cushing’s disease is transsphenoidal surgery, which requires the surgeon to reach the pituitary gland through the nostril or upper lip and remove the tumor. Radiation may also be used instead of surgery to shrink the tumor. In patients whose Cushing’s is caused by ectopic ACTH syndrome, all cancerous cells need to be wiped out through surgery, chemotherapy, radiation or a variety of other methods, depending on the location of the tumor. Surgery is also recommended for adrenal tumors. If Cushing’s syndrome is being caused by corticosteroid (steroid medications) usage, the treatment is to stop or lower your dosage.[12] Medications to control Cushing’s (before treatment or if treatment fails) According to a 2014 study in the Journal of Clinical Endocrinology and Metabolism, almost no new treatment options have been introduced in the last decade. Researchers and doctors have focused most of their efforts on improving existing treatments aimed at curing Cushing’s. Unfortunately, medications used to control Cushing’s prior to treatment and when treatment fails are not very effective. Many of the medications approved by the FDA for Cushing’s syndrome and Cushing’s disease, such as pasireotide, metyrapone, and mitotane, have not been extensively studied. The research presented to the FDA by the makers of these three drugs did not even make clear what an optimal dose was.[13] In another 2014 study, published in Clinical Epidemiology, researchers examined these three same drugs, along with ten others, and found that only pasireotide had moderate evidence to support its approval. The other drugs, many of which are not FDA approved for Cushing’s patients, had little or no available evidence to show that they work.[14] They can be sold, however, because the FDA has approved them for other diseases. Unfortunately, that means that neither the FDA nor anyone else has proven the drugs are safe or effective for Cushing patients. Pasireotide, the one medication with moderate evidence supporting its approval, caused hyperglycemia (high blood sugar) in 75% of patients who participated in the main study for the medication’s approval for Cushing’s. As a result of developing hyperglycemia, almost half (46%) of the participants had to go on blood-sugar lowering medications. The drug was approved by the FDA for Cushing’s anyway because of the lack of other effective treatments. Other treatments used for Cushing’s have other risks. Ketoconazole, believed to be the most commonly prescribed medications for Cushing’s syndrome, has a black box warning due to its effect on the liver that can lead to a liver transplant or death. Other side effects include: headache, nausea, irregular periods, impotence, and decreased libido. Metyrapone can cause acne, hirsutism, and hypertension. Mitotane can cause neurological and gastrointestinal symptoms such as dizziness, nausea, and diarrhea and can cause an abortion in pregnant women.[15] So, what should you do if you suspect you have Cushing’s Syndrome? Cushing’s syndrome is a serious disease that needs to be treated, but there are treatment options available for you if you are diagnosed with the disease. If the symptoms in this article sound familiar, it’s time for you to go see your doctor. Make an appointment with your general practitioner, and explain your symptoms to him or her. You will most likely be referred to an endocrinologist, who will be able to better understand your symptoms and recommend an appropriate course of action. All articles are reviewed and approved by Dr. Diana Zuckerman and other senior staff. Nieman, Lynette K. Epidemiology and clinical manifestations of Cushing’s syndrome, 2014. UpToDate: Wolters Kluwer Health Cushing’s syndrome/ disease, 2013. American Association of Neurological Surgeons. http://www.aans.org/Patient Information/Conditions and Treatments/Cushings Disease.aspx Cushing’s syndrome, 2012. National Endocrine and Metabolic Diseases: National Institutes of Health. http://endocrine.niddk.nih.gov/pubs/cushings/cushings.aspx#treatment Cushing’s syndrome, 2012. National Endocrine and Metabolic Diseases: National Institutes of Health. http://endocrine.niddk.nih.gov/pubs/cushings/cushings.aspx#treatment Cushing’s syndrome, 2012. National Endocrine and Metabolic Diseases: National Institutes of Health. http://endocrine.niddk.nih.gov/pubs/cushings/cushings.aspx#treatment Newell-Price, John, Peter Trainer, Michael Besser and Ashley Grossman. The diagnosis and differential diagnosis of Cushing’s syndrome and pseudo-Cushing’s states, 1998. Endocrine Reviews: Endocrine Society Carroll, TB and JW Findling. The diagnosis of Cushing’s syndrome, 2010. Reviews in Endocrinology and Metabolic Disorders: Springer Ifedayo, AO and AF Olufemi. Urinary free cortisol in the diagnosis of Cushing’s syndrome: How useful?, 2013. Nigerian Journal of Clinical Practice: Medknow. Cushing’s syndrome, 2012. National Endocrine and Metabolic Diseases: National Institutes of Health. http://endocrine.niddk.nih.gov/pubs/cushings/cushings.aspx#treatment Tirabassi, Giacomo, Emanuela Faloia, Roberta Papa, Giorgio Furlani, Marco Boscaro, and Giorgio Arnaldi. Use of the Desmopressin test in the differential diagnosis of pseudo-Cushing state from Cushing’s disease, 2013. The Journal of Clinical Endocrinology & Metabolism: Endocrine Society. Cushing’s syndrome, 2012. National Endocrine and Metabolic Diseases: National Institutes of Health. http://endocrine.niddk.nih.gov/pubs/cushings/cushings.aspx#treatment Cushing’s syndrome, 2012. National Endocrine and Metabolic Diseases: National Institutes of Health. http://endocrine.niddk.nih.gov/pubs/cushings/cushings.aspx#treatment Tirabassi, Giacomo, Emanuela Faloia, Roberta Papa, Giorgio Furlani, Marco Boscaro, and Giorgio Arnaldi. Use of the Desmopressin test in the differential diagnosis of pseudo-Cushing state from Cushing’s disease, 2013. The Journal of Clinical Endocrinology & Metabolism: Endocrine Society. Galdelha, Monica R. and Leonardo Vieira Neto. Efficacy of medical treatment in Cushing’s disease: a systematic review, 2014. Clinical Endocrinology: John Wiley & Sons. Adler, Gail. Cushing syndrome treatment & management, 2014. MedScape: WebMD. Adapted from https://www.center4research.org/cushings-syndrome-frequent-misdiagnosis/?fbclid=IwAR1lfJPilmaTl1BhR-Esi69eU7Xjm3RlO4f8lmFBIviCtHHXmVoyRxOlJqE

TAMPA, Fla., Nov. 3, 2021 /PRNewswire/ -- The Carling Adrenal Center, a worldwide destination for the surgical treatment of adrenal tumors, becomes the first center to offer adrenal vein sampling and curative surgery in one visit. The novel protocol and diagnostic method for adrenal tumors will condense a 2–4-week process of localization of hyper-secreting adrenal tumors and subsequent curative surgery down to just one day. The innovative approach combines highly specialized adrenal vein sampling with rapid adrenal hormone lab testing and then consultation with the world's highest volume adrenal surgeon. If appropriate, a patient may even complete their mini-surgery during that same visit. Established by Dr. Tobias Carling in 2020, the Carling Adrenal Center located at the Hospital for Endocrine Surgery in Tampa FL, is the highest volume adrenal surgical center in the world. The Center now averages nearly 20 adrenal tumor patients every week that could benefit from this novel diagnostic and treatment approach to address a decades-long problem for patients with adrenal tumors. The Endocrine Society Clinical Practice Guideline recommends adrenal vein sampling (AVS) as the preferred method to select patients with primary hyperaldosteronism for an adrenalectomy. "The difficulty and complexity of testing and diagnosing adrenal tumors secreting excess aldosterone is the primary reason why less than 5% of these adrenal tumors are diagnosed and treated," says Dr. Carling. "By combining expertise in interventional radiology for adrenal vein sampling and rapid laboratory measurements of adrenal hormones with our unique international consulting capability, we can determine which adrenal gland is bad and whether or not the patient needs that adrenal gland removed." Adrenal vein sampling is performed through small catheters placed in very specific veins where blood samples are obtained from both adrenal veins and the inferior vena cava. In experienced centers, the bilateral adrenal veins are catheterized and sampled with a success rate exceeding 90%. Technical success is directly associated with operator experience, leading to the recommendation that the procedure only be performed by true experts or the test will very likely be of no help. Dr. Carling's very high volume of adrenal surgery for many years has allowed him to publish scientific studies demonstrating that in aldosterone-producing adenomas, there is a strong correlation between the imaging phenotype (i.e., what the tumor looks like on a CT scan), histology (what the tumor looks like under the microscope) and genotype (what gene is mutated in the tumor). This knowledge allows Dr. Carling and his team at the Hospital for Endocrine Surgery to predict who can go straight to surgery with an excellent outcome, and who may first need adrenal vein sampling to determine which adrenal gland is over-producing the hormone causing significant morbidity and mortality. With adrenal vein sampling proving lateralization, the next step is surgical removal of the adrenal tumor. Dr. Carling has more experience with all types of adrenal surgery than any surgeon in the United States, but especially with advanced, minimally invasive adrenal operations which are the best options for aldosterone-secreting adrenal tumors. A fellow of the American College of Surgeons, Dr. Carling is a member of both the American Association of Endocrine Surgeons (AAES) and the International Association of Endocrine Surgeons (IAES). Dr. Carling moved his world-renowned adrenal surgery program from Yale University to Tampa, Florida in early 2020 to start the Carling Adrenal Center. Here, patients needing adrenal surgery have access to the best practices and best techniques the world has to offer. In January 2022, the Carling Adrenal Center will unite with the Norman Parathyroid Center, the Clayman Thyroid Center and the Scarless Thyroid Surgery Center at the brand-new Hospital for Endocrine Surgery located in Tampa, Florida. About the Carling Adrenal Center: Founded by Dr. Tobias Carling, one of the world's leading experts in adrenal gland surgery, the Carling Adrenal Center is a worldwide destination for the surgical treatment of adrenal tumors. Dr. Carling spent nearly 20 years at Yale University, including 7 as the Chief of Endocrine Surgery before leaving in 2020 to open to Carling Adrenal Center, which performs more adrenal operations than any other hospital in the world. More about adrenal vein sampling for adrenal tumors can be found at the Center's website www.adrenal.com and here. (813) 972-0000. Contact: Julie Canan, Director of Marketing Carling Adrenal Center juliec@parathyroid.com SOURCE Carling Adrenal Center From https://www.prnewswire.com/news-releases/innovative-one-visit-adrenal-tumor-diagnosis-and-treatment-program-begins-in-tampa-301414465.html

Cortisol is a hormone which produced by the adrenal gland (cortex) to control blood sugar. The production of cortisol is triggered by the pituitary hormone ACTH. Cortisol is a glucocorticoid which stimulates an increase in blood glucose. Cortisol will also stimulate the release of amino acids from muscle tissue and fatty acids from adipose tissue. The amino acids are then converted in the liver to glucose (for use by the brain). The fatty acids can be used by skeletal muscles for energy (rather than glucose) thereby freeing up glucose for selective utilization by the brain. Cortisol levels are often measured to evaluate the function of the pituitary or adrenal glands. Some of the cortisol is metabolized by the liver to produce 17 hydroxycorticosteroids, which is then excreted in the urine. The primary stress hormone. Cortisol is the major natural GLUCOCORTICOID (GC) in humans. Synthetic cortisol, also known as hydrocortisone, is used as a drug mainly to fight allergies and inflammation. A certain amount of cortisol is necessary for life. Without cortisol even a small amount of stress will kill you. Addison's disease is a disease which causes low cortisol levels, and which is treated by cortisol replacement therapy. Cortisol...

Abstract Cushing’s disease is an abnormal secretion of ACTH from the pituitary that causes an increase in cortisol production from the adrenal glands. Resultant manifestations from this excess in cortisol include multiple metabolic as well as psychiatric disturbances which can lead to significant morbidity and mortality. In this report, 23-year-old woman presented to mental health facility with history of severe depression and suicidal ideations. During evaluation, she found to have Cushing’s disease, which is unusual presentation. She had significant improvement in her symptoms with reduction of antidepressant medications after achieving eucortisolism. Cushing syndrome can present with wide range of neuropsychiatric manifestations including major depression. Although presentation with suicidal depression is unusual. Early diagnosis and prompt management of hypercortisolsim may aid in preventing or lessening of psychiatric symptoms The psychiatric and neurocognitive disorders improve after disease remission (the normalization of cortisol secretion), but some studies showed that these disorders can partially improve, persist, or exacerbate, even long-term after the resolution of hypercortisolism. The variable response of neuropsychiatric disorders after Cushing syndrome remission necessitate long term follow up. Keywords cushing syndrome, cushing disease, hypercortisolism Introduction Endogenous Cushing syndrome is a complex disorder caused by chronic exposure to excess circulating glucocorticoids. It has a wide range of clinical signs and symptoms as a result of the multisystem effects caused by excess cortisol.1 The hypercortisolism results in several complications that include glucose intolerance, diabetes, hypertension, dyslipidemia, thromboembolism, osteoporosis, impaired immunity with increased susceptibility to infection as well as neuropsychiatric disorders.2,3 Cushing syndrome presents with a wide variety of neuro-psychiatric manifestations like anxiety, major depression, mania, impairments of memory, sleep disturbance, and rarely, suicide attempt as seen in this case.2,4 The mechanism of neuropsychiatric symptoms in Cushing’s syndrome is not fully understood, but multiple proposed theories have been reported, one of which is the direct brain damage secondary to excess of glucocorticoids.5 Case Report A 23-year-old female presented to Al-Amal complex of mental health in Riyadh, Saudi Arabia with history of suicidal tendencies and 1 episode of suicidal attempt which was aborted because of religious reasons. She reported history of low mood, having disturbed sleep, loss of interest, and persistent feeling of sadness for 4 months. She also reported history of weight gain, facial swelling, hirsutism, and irregular menstrual cycle with amenorrhea for 3 months. She was prescribed fluoxetine 40 mg and quetiapine 100 mg. She was referred to endocrinology clinic at King Fahad Medical City, Riyadh for evaluation and management of possible Cushing syndrome as the cause of her abnormal mental health. She was seen in the endocrinology clinic where she reported symptoms as mentioned above in addition to headache, acne, and proximal muscle weakness. On examination her vital signs were normal. She had depressed affect, rounded face with acne and hirsutism, striae in the upper limb, and abdomen with proximal muscle weakness (4/5). Initial investigations showed that 24 hour urinary free cortisol was more than 633 µg which is more than 3 times upper limit of normal (this result was confirmed on second sample with level more than 633 µg/24 hour), cortisol level of 469 nmol/L after low dose 1 mg-dexamethasone suppression test and ACTH level of 9.8 pmol/L. Levels of other anterior pituitary hormones tested were within normal range. She also had prediabetes with HbA1c of 6.1 and dyslipidemia. Serum electrolytes, renal function and thyroid function tests were normal. MRI pituitary showed left anterior microadenoma with a size of 6 mm × 5 mm. MRI pituitary (Figure 1). Figure 1. (A-1) Coronal T2, (B-1) post contrast coronal T1 demonstrate small iso intense T1, heterogeneous mixed high, and low T2 signal intensity lesion in the left side of anterior pituitary gland which showed micro adenoma with a size of 6 mm × 5 mm. (A-2) Post-operative coronal T2 and (B-2) post-operative coronal T1. Demonstrates interval resection of the pituitary micro adenoma with no recurrence or residual lesion and minimal post-operative changes. There is no abnormal signal intensity or abnormal enhancing lesion seen. No further hormonal work up or inferior petrosal sinus sampling were done as the tumor size is 6 mm and ACTH level consistent with Cushing’s disease (pituitary source). She was referred to neurosurgery and underwent trans-sphenoidal resection of the tumor. Histopathology was consistent with pituitary adenoma and positive for ACTH. Her repeated cortisol level after tumor resection was less than 27 and ACTH 2.2 with indicated excellent response to surgery. She was started on hydrocortisone until recovery of her hypothalamic pituitary adrenal axis documented by normal morning cortisol 3 months after surgery (Table 1). Table 1. Labs. Table 1. Labs. View larger version During follow up with psychiatry her depressive symptoms improved but not resolved and she was able to stop fluoxetine 5 months post-surgery. Currently she is maintained on quetiapine 100 mg with significant improvement in her psychiatric symptoms. Currently she is in remission from Cushing’s disease based on the normal level of repeated 24 hour urinary free cortisol and with an over-all improvement in her metabolic profile. Discussion Cushing syndrome is a state of chronic hypercortisolism due to either endogenous or exogenous sources. Glucocorticoid overproduction by adrenal gland can be adrenocorticotropic (ACTH) hormone dependent which represent most of the cases and ACTH independent.6 To the best of our knowledge this is the first case documented in Saudi Arabia. There are multiple theories behind the neuropsychiatric manifestations in Cushing syndrome. These include increased stress response leading to behavioral changes, prolonged cortisol exposure leading to decreased brain volume especially in the hippocampus, reduced dendritic mass, decreased glial development, trans-cellular shift of water and synaptic loss, and excess glucocorticoid levels inhibiting neurogenesis and promoting neuronal tendency to toxic insult.3,7 In this report, the patient presented with severe depression with suicidal attempt. She had significant improvement in her symptoms with reduction of antidepressant medications but her depression persisted despite remission of Cushing disease. A similar case has been reported by Mokta et al,1 about a young male who presented with suicidal depression as initial manifestation of Cushing disease. As opposed to the present case he had complete remission of depression within 1 month of resolution of hypercortisolism. In general, psychiatric and neurocognitive disorders secondary to Cushing syndrome improves after normalization of cortisol secretion, but some studies showed that these disorders can partially improve, persist, or exacerbate, even long-term after the resolution of hypercortisolism. This may be due to persistence hypercortisolism creating toxic brain effects that occur during active disease.2,8 Similar patients need to be followed up for mental health long after Cushing syndrome has been resolved. Conclusion Depression is a primary psychiatric illness, that is, usually not examined for secondary causes. Symptoms of depression and Cushing syndrome overlap, so diagnosis and treatment of Cushing disease can be delayed. Early diagnosis and prompt management of hypercortisolsim may aid in preventing or lessening psychiatric symptoms. The variable neuropsychiatric disorders associated with Cushing syndrome post-remission necessitates long term follow up. Declaration of Conflicting Interests: The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article. Funding: The author(s) received no financial support for the research, authorship, and/or publication of this article. Informed Consent Written informed consent was obtained from the patient for the publication of this case and accompanying images. ORCID iD Sultan Dheafallah Al-Harbi https://orcid.org/0000-0001-9877-9371 References 1. Mokta, J, Sharma, R, Mokta, K, Ranjan, A, Panda, P, Joshi, I. Cushing’s disease presenting as suicidal depression. J Assoc Physicians India. 2016;64:82-83. Google Scholar | Medline 2. Pivonello, R, Simeoli, C, De Martino, MC, et al. Neuropsychiatric disorders in cushing’s syndrome. Front Neurosci. 2015;9:1-6. Google Scholar | Crossref | Medline 3. Pereira, AM, Tiemensma, J, Romijn, JA. Neuropsychiatric disorders in Cushing’s syndrome. Neuroendocrinology. 2010;92:65-70. Google Scholar | Crossref | Medline | ISI 4. Tang, A, O’Sullivan, AJ, Diamond, T, Gerard, A, Campbell, P. Psychiatric symptoms as a clinical presentation of Cushing’s syndrome. Ann Gen Psychiatry. 2013;12:1. Google Scholar | Crossref | Medline 5. Sonino, N, Fava, GA, Raffi, AR, Boscaro, M, Fallo, F. Clinical correlates of major depression in Cushing’s disease. Psychopathology. 1998;31:302-306. Google Scholar | Crossref | Medline 6. Wu, Y, Chen, J, Ma, Y, Chen, Z. Case report of Cushing’s syndrome with an acute psychotic presentation. Shanghai Arch Psychiatry. 2016;28:169-172. Google Scholar | Medline 7. Rasmussen, SA, Rosebush, PI, Smyth, HS, Mazurek, MF. Cushing disease presenting as primary psychiatric illness: a case report and literature review. J Psychiatr Pract. 2015;21:449-457. Google Scholar | Crossref | Medline 8. Sonino, N, Fava, GA. Psychiatric disorders associated with Cushing’s syndrome. Epidemiology, pathophysiology and treatment. CNS Drugs. 2001;15:361-373. Google Scholar | Crossref | Medline From https://journals.sagepub.com/doi/10.1177/11795476211027668

Dr. Friedman will host Tobias Carling, MD, PhD, FACS Surgeon-in-Chief & Founder Carling Adrenal Center Hospital for Endocrine Surgery www.adrenal.com Who will talk on: The 20-minute Mini Back Scope Adrenalectomy (MBSA) The Carling Adrenal Center is the world's busiest adrenal surgery center, operating on patients from all 50 states and all over the world. Dr. Carling is the most experienced adrenal surgeon in the United States, and by far the world's most knowledgeable surgeon-scientist when it comes to adrenal gland function and disease, adrenal tumors and cancer, and all forms of adrenal gland surgery. Dr. Carling has more experience with advanced minimally invasive adrenal and endocrine operations than any surgeon in the United States. A fellow of the American College of Surgeons, Dr. Carling is a significant member of both the American Association of Endocrine Surgeons (AAES) and the International Association of Endocrine Surgeons (IAES). Dr. Carling spent 17.5 years at Yale University and the Yale University School of Medicine where he served as the Chief of Endocrine Surgery, Associate Professor of Surgery, Program Director of the Yale Endocrine Surgery Fellowship and the Founder & Director of the Yale Endocrine Neoplasia Laboratory, a supreme scientific program focused on the molecular pathogenesis of tumors arising in the adrenal, thyroid and parathyroid glands. Dr. Carling moved his world-renowned adrenal surgery program to Tampa, Florida in early 2020 to start the Carling Adrenal Center. Here, patients needing adrenal surgery have access to the best practices and best techniques the world has to offer. Dr. Carling works closely with Dr. Friedman and will be able to perform a Mini Back Scope Adrenalectomy with a referral from Dr. Friedman. Sunday • November 7• 6 PM PST Via Zoom Click here to join the meeting or https://us02web.zoom.us/j/4209687343?pwd=amw4UzJLRDhBRXk1cS9ITU02V1pEQT09 OR +16699006833,,4209687343#,,,,*111116# Slides will be available before the webinar and recording after the meeting at slides Your phone/computer will be muted on entry. There will be plenty of time for questions using the chat button. For more information, email us at mail@goodhormonehealth.com

Kate** on the Cushing’s support board (Cushing’s Help and Support) wrote this letter after having pituitary surgery… Dear friends and family: I am writing this letter to share with you some basic facts about Cushing’s Disease/Syndrome and the recovery process so that you will have sufficient information to form realistic expectations about me and my ability to engage in certain activities in light of this disease and its aftermath. As you know, Cushing’s is a rarely diagnosed endocrine disorder characterized by hypercortisolism. Cortisol is a hormone produced by the adrenal glands and is vital to regulate the body’s cardivoascular functions and metabolism, to boost the immune system and to fight inflammation. But its most important job is to help the body to respond to stress. The adrenal glands release cortisol in response to stress, so atheletes, women experiencing pregnancy, and those suffering from alcoholism, panic disorders and malnutrition naturally have higher-than-normal levels of cortisol. People with Cushing’s Syndrome live life with too much cortisol for their bodies as a result of a hormone-secreting tumor. Mine is located in the pituitary gland. Endogenous hypercortisolism leaves the body in a constant state of “fight or flight,” which ravages the body and tears down the body’s major systems including cardivascular, musculo-skeletal, endocrine, etc. Symptoms vary, but the most common symptoms include rapid, unexplained weight gain in the upper body with increased fat around the neck and face (“moon facies”); buffalo hump; facial flushing/plethora; muscle wasting in the arms and legs; purplish striae (stretch marks) on the abdomen, thighs, buttocks, arms and breasts; poor wound healing and bruising; severe fatigue; depression, anxiety disorders and emotional lability; cognitive difficulties; sleep disorders due to abnormally high nighttime cortisol production; high blood pressure and high blood sugar/diabetes; edema; vision problems; premature osteoperosis; and, in women, signs of hyperandrogenism such as menstrual irregularities, infertility, hirsutism, male-patterned balding and steroid-induced acne. Cushing's Symptoms http://www.cushings-info.com/images/1/12/Lady.gif A sketch of a typical Cushing’s patient. As you can see, the effects of the disease on the body are dramatic. Worse, the psychological and emotional effects of having a chronic, debilitating and disfiguring disease range from distressing to demoralizing. Imagine that, in the space of a year, you became unrecognizable to those around you and to yourself. You look in the mirror, but the person staring back a tyou is a stranger. You endure the stares and looks of pity from those who knew you before Cushing’s, fully aware that they believe you have “let yourself go” or otherwise allowed this to happen to your body. Nothing you can say or do will persuade them otherwise, so at some point, you stop trying and resolve to live your life in a stranger’s body. You feel increasingly sick, but when you explain your array of symptoms to your doctor, you are dismissed as a depressed hypochondriac who needs to diet and exercise more. Worse, your family members think the same thing — and are often quick to tell you how you need to “change your lifestyle” to overcome the effects of what you eventually will discover, once properly diagnosed, is a serious and rare disease. If only it were so simple! No one would choose to have Cushing’s. Those of us who have it would not wish it even on our worst enemy. Most people with Cushing’s long for the ability to do simple things, like walk a flight of stairs without having to sit for half an hour afterwards, or vacuum the house or even unload a dishwasher. One of the worst parts about this disease is the crushing fatigue and muscle wasting/weakness, which accompanies hypercortisolism. Not only do we become socially isolated because of the virilzing effects of an endocrine tumor, which drastically alters our appearance, but we no longer feel like ourselves with regard to energy. We would love to take a long bike ride, run three miles or go shopping like we used to — activities, which we took for granted before the disease struck. Those activities are sadly impossible at times for those with advanced stages of the disease. Sometimes, as with any serious illness, performing even basic tasks of daily care such as showering and dressing can exhaust the limited reserves of energy available to a Cushing’s patient. How do we explain to you what it’s like to watch our lives slip away? What response is sufficient to express the grief and frustration over losing so much of ourselves? It is often difficult to find the strength to explain how your well-meaning words of prompting and encouragement (to diet or exercise) only serve to leave us more isolated and feeling alone. Though we wouldn’t want it, we wish our disease were as well-understood as cancer so that those who love us would have a frame of reference for what we go through. With Cushing’s, there is such limited public awareness that we are left to describe the effects of the disease from a void, often with limited understanding from those who love us most, which is disheartening. The most frustrating misconception about this disease is that we somehow are “doing this to ourselves,” or delaying recovery because we need to continue steroid replacement or lack the energy to excercise often, which is sadly false. Trust me that we would love to have that much control over such a terrible disease. Fortunately, there is a good likelihood of remission from Cushing’s in the hands of a skilled pituitary surgeon. Unfortunately, the long-term remission rate is only 56%, meaning that 44% of people with Cushing’s will require a second (sometimes third) pituitary surgery, radiation or bilateraly adrenalectomy to resolve the hypercortisolism. Without successful treatment, Cushing’s leads to death. Even with successful treatment, I will have to be monitored for possible recurrence for the rest of my life. After surgery or other treatment, the recovery period can last months or even years. Because the tumor takes over control of the body’s production of cortisol, the adrenal glands, which had lain dormant prior to surgery, require time to start functioning properly again. Until this happens, we must take synthetic steroids or else risk adrenal insufficiency or adrenal crisis, which can be quickly life-threatening. Careful monitoring of our cortisol levels is critical during the weaning period. It is a rare but sad fact that some people’s adrenal glands never return to normal, and those people must continue to take hydrocortisone or prednisone — sometimes for life — simply in order for the body to perform correctly its basic systemic functions. The physical recovery from surgery can be quick, but the withdrawal from hydrocortisone can be a lengthy and extremely painful process. As I described above, Cushing’s causes a tearing-down of muscles and bone. While there is an over-abundance of cortisol in our bodies (as a result of the tumor), we often can’t feel the effects of the muscle-wasting and bone deterioration because of the anti-inflammatory action of cortisol. Upon weaning, however, these become painfully (literally!) evident. The physical pain experienced while weaning from cortisol has been described as worse than weaning from heroin. When cortisol levels are low, one experiences the symptoms akin to a really bad flu, including severe fatigue (”like a wet cement blanket laid on top of me”); weakness and exhaustion; nausea; headache; vomiting; mental confusion. It is imperative for people who are on replacement steroids after Cushing’s surgery to carry extra Cortef (or injectable Solu-Cortef) with them at all times in addition to wearing a medic alert bracelet so that medical professionals will be alerted to the possiblity of adrenal insufficiency in the event of an adrenal crisis. People who have struggled with Cushing’s Syndrome all hope to return to “normal” at some point. Though none of us want to have Cushing’s, it is often a relief finally to have a correct diagnosis and treatment plan. For many, there is a gradual resolution of many Cushing’s symptoms within a few years of surgery or other successful treatment, and a good quality of life can be achieved. But regrettably, this is not possible in every case. Depending on the severity of the disease and the length of time before diagnosis and treatment, the prognosis can be poor and lead to shortened life expectancy and diminished quality of life. This is not a choice or something we can control, but it is the reality for some people who have suffered the consequences of long-term hypercortisolism. The best support you can give someone who is suffering from Cushing’s or its aftermath is to BELIEVE them and to understand that they are not manufacturing their illness or prolonging recovery. Ask them what they are able (and not able) to do, and then be prepared to help them in ways that matter — whether that be to bring them a meal or help them to run errands, pick up prescriptions from the pharmacy or clean their house. Because it’s these little everyday tasks, which can fall by the wayside when someone has (or has had) Cushing’s, and these are the things we miss the most: doing for ourselves. Ask us questions about the disease, and then actively listen to what we say. We know you don’t know much about Cushing’s — even our doctors sometimes lack information about this rare disease. But know we appreciate the interest and will tell you everything you want to know, because those of us who have it necessarily become experts in it just in order to survive. Thank you for caring about me and for hearing what I am saying in this letter. I know you love me and are concerned about me, and I appreciate that so much. Thank you also for taking the time to read this letter. I look forward to discussing further any questions you might have. In the meantime, I am attaching a brief article written by a woman who recently was diagnosed with Cushing’s. I hope hearing another person’s experiences will help you to understand what I’m going through so that when we talk, we will be coming from a similar starting place. Endocrinologists (doctors who specialize in Cushing's Syndrome and its related issues) realize the medical aspect and know the damaging effects that Cushing's has on the body. Family and friends see their Cushie suffering and know they are hurting physically and often times mentally and emotionally. However, understanding the debilitation of Cushing's and how it can affect every aspect of a person's life can only be truly realized by those who have experienced the syndrome. Cushings Help Organization, Inc., a non-profit family of websites maintained by MaryO, a pituitary Cushing's survivor, provides this letter for patients to provide to their family and friends in hopes of providing a better understanding Cushing's and it's many aspects. We're sorry to hear that your family member or friend has Cushing's Syndrome or suspected Cushing's. A person may feel better at times then at other times. It's common for a Cushing's patient to have burst of energy and then all of a sudden they become lethargic and don't feel like moving a muscle. There are many symptoms that are associated with Cushing's. They include weight gain, fatigue, muscle weakness, shortness of breath, feeling achy all over, headaches, blurred vision, mood swings, high blood pressure, stretch marks (straie), buffalo hump, diabetes, edema and the list goes on. Hormones affect every area of the body. It is important to note that not all patients have every symptom. Even some hallmark symptoms, such as straie or the "buffalo hump", may not be noticable on every patient. Not everyone who has Cushing's will experience the same symptoms, treatment, or recovery. Because not all "Cushies" have these symptoms, it makes diagnosis even more difficult. Cushing's can cause the physical appearance change due to weight gain, hair loss, rosacea, acne, etc. This can be very disturbing when looking in the mirror. Changes in appearance can often cause the Cushing's patient to withdraw from family and friends making it a very lonely illness. Patients often feel alone or withdrawn because few others understand. Cushing's can affect affect anyone of any age although it is more commen in women. Cushing's patients need to be able to take one day at time and learn to listen to their bodies. There will most likely be times when naps are needed during the day and often times may not be able to sleep at night due to surges of cortisol. Your Cushie doesn't expect you to understand Cushing's Syndrome completely. They do need you to be there for them and try to understand to the best of your ability what they feel and not give up on them. Often a Cushing's patient may be moody and say things that they don't mean. If this should happen with your Cushie try not to take it personally and know that it's most likely caused by the elevated cortisol and disturbances in other hormone levels caused by the Cushing's and not from the heart or true feelings of your Cushie. It can be very depressing and frustrating having so many limitations and experience things in life being taken from you. Cushing's patients are sick, not lazy, not hypochondriacs or even the newer term "Cyberchondriacs". If a Cushing's patient says they don't feel like doing something or they express how bad they feel let them know that you believe them. One of the most frustrating things to someone who is sick is to have those you love not believe you or support you. Telling a Cushie to think positive thoughts will not make him/her well and will just be aggrivating. Testing procedures can be lengthy and this can become frustrating for the patient and family. Often, it takes a while for results to come back and this can be stressful. Don't look to far ahead just take one day at a time and deal with the situation that is at hand at the present time. After a diagnosis is made then it's time for treatment. Surgery is usually the best treatment option for Cushing's that is caused by tumors. Don't be surprised if the surgeon's facility wants to run even more tests or redo some of those that have already been done. Your Cushie may have to travel a ways to find a surgeon who is trained in these delicate surgeries and who has performed many of them. Once the diagnosis has been made and treatment has finished then it's time for the recovery process. Not all patients who have surgery are cured and they have to make a choice along with the advice of their doctor as to what their next treatment option will be. The recovery from the surgery itself is similar to any other surgery and will take a while to recover. The recovery process obtained from getting a cure from Cushing's is quiet different from other surgeries. A Cushing's patients body has been exposed to excess cortisol, usually for quite a long time, and has become accustomed it. When the tumor is removed that has been responsible for the excessive cortisol and the body is no longer getting it this causes the body to have withdrawal symptoms. Withdrawal can be very hard causing an array of symptoms muscle aches, weakness, bone and joint pain, emotional disturbances etc. Thank you for reading this and we hope it will help you to understand a little more about Cushing's and the dibilating affect it can have on a person. Thank you for being there and supporting your Cushie during this time in their life. We realize that when a family member has Cushing's it not only affects the individual but other family members and those around them as well. Showing your love and support will encourage a speedy recovery for your Cushie. **Note: Kate died on on June 23, 2014. Read her In Memory page here: http://cushingsbios.com/2014/06/25/in-memory-kate-meyers/

The occurrence of different subtypes of endogenous Cushing’s syndrome (CS) in single individuals is extremely rare. We here present the case of a female patient who was successfully cured from adrenal CS 4 years before being diagnosed with Cushing’s disease (CD). The patient was diagnosed at the age of 50 with ACTH-independent CS and a left-sided adrenal adenoma, in January 2015. After adrenalectomy and histopathological confirmation of a cortisol-producing adrenocortical adenoma, biochemical hypercortisolism and clinical symptoms significantly improved. However, starting from 2018, the patient again developed signs and symptoms of recurrent CS. Subsequent biochemical and radiological workup suggested the presence of ACTH-dependent CS along with a pituitary microadenoma. The patient underwent successful transsphenoidal adenomectomy, and both postoperative adrenal insufficiency and histopathological workup confirmed the diagnosis of CD. Exome sequencing excluded a causative germline mutation but showed somatic mutations of the β-catenin protein gene (CTNNB1) in the adrenal adenoma, and of both the ubiquitin specific peptidase 8 (USP8) and the glucocorticoid receptor (NR3C1) genes in the pituitary adenoma. In conclusion, our case illustrates that both ACTH-independent and ACTH-dependent CS may develop in a single individual even without evidence for a common genetic background. Introduction Endogenous Cushing´s syndrome (CS) is a rare disorder with an incidence of 0.2–5.0 per million people per year (1, 2). The predominant subtype (accounting for about 80%) is adrenocorticotropic hormone (ACTH)-dependent CS. The vast majority of this subtype is due to an ACTH-secreting pituitary adenoma [so called Cushing´s disease (CD)], whereas ectopic ACTH-secretion (e.g. through pulmonary carcinoids) is much less common. In contrast, ACTH-independent CS can mainly be attributed to cortisol-producing adrenal adenomas. Adrenocortical carcinomas, uni-/bilateral adrenal hyperplasia, and primary pigmented nodular adrenocortical disease (PPNAD) may account for some of these cases as well (3, 4). Coexistence of different subtypes of endogenous CS in single individuals is even rarer but has been described in few reports. These cases were usually observed in the context of prolonged ACTH stimulation on the adrenal glands, resulting in micronodular or macronodular hyperplasia (5–9). A sequence of CD and PPNAD was also described in presence of Carney complex, a genetic syndrome characterized by the loss of function of the gene encoding for the regulatory subunit type 1α of protein kinase A (PRKAR1A) (10). Moreover, another group reported the case of a patient with Cushing's disease followed by ectopic Cushing's syndrome more than 30 years later (8). To our knowledge, however, we here describe the first case report on a single patient with a cortisol-producing adrenocortical adenoma and subsequent CD. Read the rest of the article at https://www.frontiersin.org/articles/10.3389/fendo.2021.731579/full

This article was originally published here J Clin Endocrinol Metab. 2021 Jul 29:dgab557. doi: 10.1210/clinem/dgab557. Online ahead of print. ABSTRACT CONTEXT: Coronavirus disease 2019 (COVID-19) is a proinflammatory and prothrombotic condition, but its impact on adrenal function has not been adequately evaluated. CASE REPORT: A 46-year-old woman presented with abdominal pain, hypotension, and skin hyperpigmentation after COVID-19 infection. The patient had hyponatremia, serum cortisol <1.0 µg/dL, adrenocorticotropin (ACTH) of 807 pg/mL, and aldosterone ❤️ ng/dL. Computed tomography (CT) findings of adrenal enlargement with no parenchymal and minimal peripheral capsular enhancement after contrast were consistent with bilateral adrenal infarction. The patient had autoimmune hepatitis and positive antiphospholipid antibodies, but no previous thrombotic events. The patient was treated with intravenous hydrocortisone, followed by oral hydrocortisone and fludrocortisone. DISCUSSION: We identified 9 articles, including case reports, of new-onset adrenal insufficiency and/or adrenal hemorrhage/infarction on CT in COVID-19. Adrenal insufficiency was hormonally diagnosed in 5 cases, but ACTH levels were measured in only 3 cases (high in 1 case and normal/low in other 2 cases). Bilateral adrenal nonhemorrhagic or hemorrhagic infarction was identified in 5 reports (2 had adrenal insufficiency, 2 had normal cortisol levels, and 1 case had no data). Interestingly, the only case with well-characterized new-onset acute primary adrenal insufficiency after COVID-19 had a previous diagnosis of antiphospholipid syndrome. In our case, antiphospholipid syndrome diagnosis was established only after the adrenal infarction triggered by COVID-19. CONCLUSION: Our findings support the association between bilateral adrenal infarction and antiphospholipid syndrome triggered by COVID-19. Therefore, patients with positive antiphospholipid antibodies should be closely monitored for symptoms or signs of acute adrenal insufficiency during COVID-19. PMID:34463766 | DOI:10.1210/clinem/dgab557