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  1. Objective Postoperative data on Cushing’s disease (CD) are equivocal in the literature. These discrepancies may be attributed to different series with different criteria for remission and variable follow-up durations. Additional data from experienced centers may address these discrepancies. In this study, we present the results obtained from 96 endoscopic transsphenoidal surgeries (ETSSs) for CD conducted in a well-experienced center. Methods Pre- and postoperative data of 96 ETSS in 87 patients with CD were included. All cases were handled by the same neurosurgical team between 2014 and 2022. We obtained data on remission status 3−6 months postoperatively (medium-term) and during the latest follow-up (long-term). Additionally, magnetic resonance imaging (MRI) and pathology results were obtained for each case. Results The mean follow-up duration was 39.5±3.2 months. Medium and long-term remission rates were 77% and 82%, respectively. When only first-time operations were considered, the medium- and long-term remission rates were 78% and 82%, respectively. The recurrence rate in this series was 2.5%. Patients who showed remission between 3−6 months had higher longterm remission rates than did those without initial remission. Tumors >2 cm and extended tumor invasion of the cavernous sinus (Knosp 4) were associated with lower postoperative remission rates. Conclusion Adenoma size and the presence/absence of cavernous sinus invasion on preopera-tive MRI may predict long-term postoperative remission. A tumor size of 2 cm may be a supporting criterion for predicting remission in Knosp 4 tumors. Further studies with larger patient populations are necessary to support this finding. Key Words: Complete remission · Neuroendoscopy · Pituitary-dependant Cushing syndrome · Treatment outcome. Go to : INTRODUCTION Cushing’s disease (CD) is characterized by excessive secretion of adrenocorticotropic hormone (ACTH) by a corticotropic adenoma in the pituitary gland. In patients with CD whose hypercortisolism is inadequately corrected, morbidity and mortality can increase by up to 4.8 times due to Cushingrelated complications such as osteoporosis, hypertension, dyslipidemia, insulin resistance, and hypercoagulability [11,18]. Endoscopic transsphenoidal surgery (ETSS), the first-line treatment for CD [7], is performed to decrease complications while achieving remission and long-term disease control. Previous studies on CD have reported varying remission rates between 45% and 95% and recurrence rates ranging from 3−66% [2,4,9,16,21,30]. This wide range of differences can be primarily attributed to differences in surgical experience among centers: centers with higher surgical experience have fewer postoperative complications and higher remission rates [4,6]. However, despite initial remission, patients with CD may eventually experience recurrence. The mean recurrence rate at the 5-10-year follow-up is 23% for microadenomas and 33% for macroadenomas [19,23,30]. Since the postoperative rates in the literature are variable, additional data from experienced centers may be necessary to resolve these discrepancies. In this study, we present the medium- and long-term follow-up data from 96 operations for CD that were conducted in a center with a high level of experience for ETSS. Go to : MATERIALS AND METHODS The study was conducted in accordance with the Declaration of Helsinki (as revised in 2013). The study was approved by the Ethics Committee of Basaksehir Cam and Sakura City Hospital (No. 2022185). Informed consent was obtained from all patients. The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. This retrospective study included pre and postoperative data of 96 ETSS performed in 87 patients with CD (Fig. 1). CD was diagnosed based on unsuppressed cortisol levels (>1.8 µg/dL) following the 1-mg dexamethasone suppression test, high levels of urinary free cortisol, or late night salivary cortisol and plasma ACTH levels >20 pg/mL [28]. Between 2014 and 2022, all surgeries were conducted by the experienced neurosurgical team (Ö.G., O.T., B.E., E.A.) responsible for endoscopic transsphenoidal procedures at the Pituitary Research Center. The surgeries were performed under perioperative glucocorticoid coverage. Fig. 1. Number of operations and patients included in the study. Size, cavernous sinus invasion, sellar and suprasellar infiltration of adenoma on preoperative magnetic resonance imaging (MRI) scans, residual tumor on postoperative MRI scans, postoperative complications, pathology results, remission and recurrence status, and additional postoperative management were evaluated in addition to patients’ demographic data. For follow-up assessments, data obtained 3−6 months postoperatively and during the latest follow-up were included. Three different classifications obtained during radiologic evaluation using MRI were used for pituitary adenomas : 1) maximum size of tumor (MST) : 0−5 mm (group 1), 6−10 mm (group 2), 11−20 mm (group 3), and >20 mm (group 4); 2) Knosp classification : for evaluation of cavernous sinus invasion [22]; and 3) modified Hardy classification : for evaluation of sellar and suprasellar infiltrations [20,39]. In cases of CD without a lesion or with a lesion <6 mm on MRI, confirmation of the central origin and lateralization was provided by inferior petrosal sinus sampling (IPSS) with corticotropin-releasing hormone stimulation [25,26,29]. Under neuronavigation guidance, pure ETSS surgical interventions were performed for all patients by a single surgical team using the Medtronic StealthStation™ S7 and S8 systems (Medtronic, Minneapolis, MN, USA) together with 4-mm 0°, 30°, and 45° rigid optical instruments and an endoscope. A nasal decongestant spray was administered 1 hour before the operation. The sphenoid ostium was detected from both nostrils, and a bi-nostril approach was used by resecting the posterior nasal septum. After sphenoidectomy, the standard sellar approach was used for lesions in the sellar region. The details of these surgical procedures are described in previous study [14]. Selective adenectomy with ETSS was performed for preoperatively localized and visible tumors, whereas hemihypophysectomy was performed for non-lesional cases. In cases with cavernous sinus-invading tumors, particularly Knops 3-4, the defect which was created by the tumor on the medial wall of anterior cavernous sinus was identified and, it was expanded for resection of the tumor tissue within the cavernous sinus. If a defect was not visible, blunt-ended hook-shaped dissectors were used to create a defect on the medial wall, allowing access for the tumor to enter the cavernous sinus. Hematoxylin and Eosin (H&E) and immunohistochemistry staining were performed for the specimens obtained during ETSS. Adenomas showing positive immunohistological staining for ACTH were diagnosed histologically as corticotropinomas. CD was considered to be in remission when the cases showed basal cortisol levels <5 µg/dL or suppressed cortisol levels (≤1.8 µg/dL) following the 1-mg dexamethasone suppression test, 3-6 months postoperation, and during the latest follow-up. The study protocol was approved by the ethics committee of our institution. Data were statistically analyzed using the SPSS 15.0 package (IBM Corp., Armonk, NY, USA). The chi-square test was used for categorical variables. Sample distribution was evaluated with the Kolmogorov-Smirnov test. Continuous independent variables with a normal distribution were compared using the Student’s t-test. Continuous variables with non-normal distributions were compared using the Mann-Whitney U test. p<0.05 was considered statistically significant. A Kaplan-Meier survival analysis was conducted to determine probability and time to recurrence in cases with initial remission. Go to : RESULTS Demographic data A total of 96 ETSS were performed for 87 patients with CD. Of the 87 patients, 68 (79%) were female, and 19 (21%) were male. The mean patient age was 42.2±12.9 years, and the mean duration of follow-up was 39.5±3.2 months. Of the 96 surgeries, 79 (82%) were performed for the first time, six (6%) were performed for residual tumors, and 11 (12%) were performed following a recurrence of the disease. Eight of the 17 patients who underwent reoperations had undergone their first operation at another center. Preoperative imaging Table 1 shows the maximum tumor size on preoperative pituitary MRI before each surgical procedure. Preoperative IPSS for lateralization was performed in 42 operations (44%), all of which were first-time cases. Knosp classification based on preoperative pituitary MRI and the modified Hardy classification is presented in Table 1. Table 1. Preoperative pituitary magnetic resonance imaging scans Number of tumors (n=96) Maximum tumor size  Group 1, 0−5 mm 41 (42.7)  Group 2, 6−10 mm 24 (25.0)  Group 3, 11−20 mm 20 (20.8)  Group 4, >20 mm 11 (11.5) Knosp classification  Grade 0 52 (54.2)  Grade 1 22 (22.9)  Grade 2 6 (6.3)  Grade 3 8 (8.3)  Grade 4 8 (8.3) Modified Hardy classification  0   A 41 (42.8)   B -   C -   D -   E -  1   A 14 (14.6)   B -   C -   D -   E 4 (4.2)  2   A 5 (5.2)   B -   C -   D -   E 5 (5.2)  3   A 1 (1.0)   B 2 (2.1)   C -   D -   E 1 (1.0)  4   A 1 (1.0)   B -   C -   D 1 (1.0)   E 3 (3.1)  NA 18 (18.8) Values are presented as number (%). Invasion : 0, sella normal; 1, sella focally expanded and tumor ≤10 mm; 2, sella enlarged and tumor ≥10 mm; 3, localized perforation of the sellar floor; 4, diffuse destruction of the sellar floor. Suprasellar extension : A, no suprasellar extension; B, anterior recesses of the third ventricle obliterated; C, floor of the third ventricle grossly displaced with parasellar extension; D, intracranial (intradural) : anterior, middle or middle fossa; E, into/beneath the cavernous sinus (extradural). NA : not available Postoperative results Remission was achieved between the 3rd and 6th months in 74 (77%) of the 96 operations, and long-term remission in 79 operations (82%). Among all 96 operations, eight (8%) concluded with a residual tumor. Regarding only first-time operations, five (6%) of the 79 concluded with a postoperative residual tumor. Of the 79 first-time operations, there were 62 cases (78%) of remission between 3 and 6 months. Two (2.5%) of these 79 operations involved recurrence during follow-up, while 60 (97%) showed sustained remission. Those with sustained remission had a median disease-free survival time of 31 months (interquartile range, 14-64) during long-term followup, two cases with recurrence had their recurrence 49 and 54 months after their operation. Survival analysis of cases with remisson and recurrence is presented in Fig. 2. CD persisted after 17 (21.5%) of the 79 first operations. Fig. 2. Survival analysis after the first operation in cases with remission at 3-6 months. Dashed line represents cases with recurrence and, straight line represents cases with sustained remission during long-term follow-up. Ten (13%) of the 79 cases underwent reoperation; two were due to recurrence, and eight due to disease persistence. In five cases (29%), the patients were initially unresponsive but showed remission later during the long-term follow-up. Remission was achieved with stereotactic radiosurgery (STRS) and medical treatment in one of these cases, with only STRS in two and only medical treatment in two cases. At the latest follow-up visit, the total number of cases showing remission after the first operation was 65 (82%). Additional details regarding the results of the first operations are provided in Fig. 3. Fig. 3. Results of the cases who had operation for the first time. Of the 18 reoperations, the results for one case were excluded since the patient was operated at another center. After the reoperation (n=17), the medium and long-term remission rates were 71% (n=12) and 77% (n=13), respectively. The 3-6-month remission rate did not differ significantly between first-time and reoperations (p=0.5). Residual tumors were present in three cases (18%) after reoperation. Of the early non-responders, one case showed remission after STRS, and none of the responders showed recurrence during long-term follow-up. Additional details regarding the results of reoperations are provided in Fig. 4. Fig. 4. Results of the reoperations in our center. Remission rates based on tumor size are presented in Table 2. The initial remission rates of the tumors in MST group 4 were significantly lower than those in the other MST groups (MST 1 vs. 4, p=0.01; MST 2 vs. 4, p=0.001; and MST 3 vs. 4, p=0.006). Comparisons of the other MST groups showed no significant differences. When adenomas were stratified using the 10-mm cut-off, the remission rates did not differ significantly (remission rate, 81% for adenomas <10 mm and 68% for adenomas ≥10 mm; p=0.2). Postoperative residual tumors were observed in five of the 11 tumors (46%) >2 cm (MST group 4) and in one tumor in each of MST groups 1-3 (2%, 4%, and 5%, respectively, p<0.001). Reoperation rate was 17% (n=7) for adenomas ≤5 mm, 18% (n=10) for adenomas ≥6 mm (p=0.9), and 27% (n=3) for adenomas >20 mm (among all grades, p=0.3). Table 2. Comparison of remission rates in preoperative pituitary magnetic resonance imaging scans 3−6-month remission Long-term remission Maximum tumor size  Group 1, 0−5 mm (n=41) 31 (75.6) 33 (80.5)  Group 2, 6−10 mm (n=24) 22 (91.7) 22 (91.7)  Group 3, 10−20 mm (n=20) 17 (85.0) 17 (85.0)  Group 4, >20 mm (n=11) 4 (36.4) 7 (63.6)  p-value 0.003* 0.200 Knops classification  0 (n=52) 41 (78.8) 44 (84.6)  1 (n=22) 21 (95.5) 21 (95.5)  2 (n=6) 4 (66.7) 3 (50.0)  3 (n=8) 7 (87.5) 7 (87.5)  4 (n=8) 1 (12.5) 4 (50.0)  p-value <0.001* 0.010* Modified Hardy classification  0   A (n=41) 32 (78.0) 34 (82.9)  1   A (n=14) 12 (85.7) 12 (85.7)  2   E (n=4) 3 (75.0) 3 (75.0)   A (n=5) 5 (100.0) 5 (100.0)  3   E (n=5) 2 (40.0) 2 (40.0)   A (n=1) 1 (100.0) 1 (100.0)   B (n=2) 2 (100.0) 2 (100.0)  4   E (n=1) 0 (0.0) 0 (0.0)   A (n=1) 1 (100.0) 1 (100.0)   D (n=1) 0 (0.0) 0 (0.0)   E (n=3) 1 (33.3) 3 (100.0)  p-value 0.10 0.06 Pathology result  Corticotropinoma (+) (n=71) 58 (81.7) 60 (84.5)  Corticotropinoma (-) (n=25) 16 (64.0) 19 (76.0)  p-value 0.07 0.30 Values are presented as number (%). Invasion : 0, sella normal; 1, sella focally expanded and tumor ≤10 mm; 2, sella enlarged and tumor ≥10 mm; 3, localized perforation of the sellar floor; 4, diffuse destruction of the sellar floor. Suprasellar extension : A, no suprasellar extension; B, anterior recesses of the third ventricle obliterated; D, intracranial (intradural) with anterior, middle, or middle fossa; E, into/beneath the cavernous sinus (extradural). * Statistically significant p-value Remission rates based on Knosp and Hardy classifications are presented in Table 2, respectively. The medium-term remission rates in Knosp group 4 were significantly lower than the rates in the other groups (Knosp 0 vs. 4, p<0.001; Knosp 1 vs. 4, p<0.001; Knosp 2 vs. 4, p=0.04; and Knosp 3 vs. 4, p=0.003). Additionally, the medium-term remission rate of tumors in Knosp group 2 was lower than that in Knosp group 1 (p=0.04). However, remission rates did not differ significantly among the other groups. Comparing invasive (Knosp 3 and 4) and noninvasive (Knosp 0, 1, and 2) tumors, remission rates within 3-6 months were 50% and 83% in the invasive and noninvasive groups, respectively. We further stratified cases with tumor size ≥20 mm (n=11) using Knosp classification; one case (9%) was Knosp 0, one case (9%) was Knosp 1, two cases (18%) were Knosp 3, and seven cases (64%) were Knosp 4 tumors. For ≥20 mm, all cases with Knosp 0, 1, and 3 tumors achieved remission within 3-6 months postoperatively, while none of the cases with Knosp 4 tumors had remission (p=0.01). All the cases with Knosp 0, 1, and 3 tumors sustained remission, and three cases with Knosp 4 tumor later achieved long-term remission (p=0.3). Of the cases that achieved long-term remission, two underwent STRS, and one had medical therapy with additional STRS. Of the 96 tissue specimens obtained during ETSS, 71 (74%) stained positive for ACTH and were histologically identified as corticotropic adenomas, while 25 (26%) were negative. Remission rates based on the pathology results are compared in Table 2. Of the lesions with conclusive findings on MRI (≥6 mm lesions), 89% (n=49) were pathologically confirmed as corticotropinomas, whereas 54% (n=22) of those with inconclusive MRI f indings were pathologically conf irmed (p<0.001). Among the lesions that showed negative results for both conclusive MRI findings (≤5 mm) and pathologic confirmation (negative for a corticotropinoma) (n=19), 12 (63%) showed remission at 3-6 months and 14 (74%) showed remission during long-term follow-up. During the exploration of the cavernous sinus in one patient (1%), postoperative lateral gaze paralysis of the eye developed due to right abducens nerve palsy. The patient was treated with anti-inflammatory doses of steroids, and the symptom completely resolved within 1 month. In three other patients (3%), severe epistaxis was observed in the postoperative period, 1 to 3 weeks after surgery. Nasal packing was applied for 3 days. Additionally, three patients (3%) experienced postoperative rhinorrhea. To address this issue, a reconstruction of the skull base was performed using fat tissue harvested from the leg, fascia lata graft, and tissue adhesive material. These patients were monitored with a lumbar drain for 1 week. Among the patients who developed rhinorrhea, one patient also developed meningitis and received intravenous antibiotic therapy for about 3 weeks and, the situation compeletly resolved during follow-up. The postoperative complications are summarized in Table 3. Comparison of various characteristics of the cases with and without medium and long-term remission are presented in Table 3, respectively. Table 3. Comparison of cases with and without remission, postoperative complications 3−6-month remission Long-term remission Number of cases (n=96) Remission (+) (n=74) Remission (-) (n=22) p-value Remission (+) (n=79) Remission (-) (n=17) p-value Operation 0.500 0.08  First time 62 (83.8) 17 (77.3) 66 (83.5) 13 (76.5)  Re-operation 12 (16.2) 5 (22.7) 13 (16.5) 4 (23.5) Tumor characteristics 0.003* 0.20  MST   Grade 1 31 (42.0) 10 (45.0) 33 (41.8) 8 (47.1)   Grade 2 22 (30.0) 2 (9.0) 22 (27.8) 2 (11.8)   Grade 3 17 (23.0) 3 (14.0) 17 (21.5) 3 (17.6)   Grade 4 4 (5.0) 7 (32.0) 7 (8.9) 4 (23.5)  Knosp classification <0.001* 0.01*   0 41 (56.0) 11 (50.0) 44 (55.5) 9 (53.0)   1 21 (28.0) 1 (4.5) 21 (26.5) 2 (12.0)   2 4 (5.0) 2 (9.0) 3 (4.0) 1 (6.0)   3 7 (10.0) 1 (4.5) 7 (9.0) 1 (6.0)   4 1 (1.0) 7 (32.0) 4 (5.0) 4 (23.0)  Hardy classification 0.09 0.06   0A 32 (43.2) 9 (41.0) 34 (43.0) 7 (41.0)   1A 12 (16.2) 2 (9.0) 12 (15.0) 2 (12.0)   1E 3 (4.0) 1 (4.5) 3 (4.0) 1 (6.0)   2A 5 (6.7) 0 (0.0) 5 (6.0) 0 (0.0)   2E 2 (2.7) 3 (14.0) 2 (3.0) 3 (17.0)   3A 1 (1.4) 0 (0.0) 1 (1.0) 0 (0.0)   3B 2 (2.7) 0 (0.0) 2 (3.0) 0 (0.0)   3E 0 (0.0) 1 (4.5) 0 (0.0) 1 (6.0)   4A 1 (1.4) 0 (0.0) 1 (1.0) 0 (0.0)   4D 0 (0.0) 1 (4.5) 0 (0.0) 1 (6.0)   4E 1 (1.4) 2 (9.0) 3 (4.0) 0 (0.0)   NA 15 (20.3) 3 (13.5) 16 (20.0) 2 (12.0) Postoperative  Complication 0.900 0.30   (+) 10 (13.5) 3 (13.6) 12 (15.2) 1 (5.9)   (-) 64 (86.5) 19 (86.4) 67 (84.8) 16 (94.1)  Pathologic diagnosis 0.070 0.30   Corticotropinoma 58 (78.4) 13 (59.1) 60 (75.9) 11 (64.7)   Negative 16 (21.6) 9 (40.9) 19 (24.1) 6 (35.3)  Remission during long-term F/U <0.001*   (+) 72 (97.3) 7 (31.8)   (-) 2 (2.7) 15 (68.2)  Residual tumor 0.001*   (+) 3 (3.8) 5 (29.4)   (-) 76 (96.2) 12 (70.6)  Remission during long-term F/U <0.001*   (+) 72 (91.1) 2 (11.8)   (-) 7 (8.9) 15 (88.2) Postoperative complication  DI-temporary 4 (4.2)  DI-permanent 4 (4.2)  Meningitis 1 (1.0)  CSF leak 3 (3.1)  Epistaxis 3 (3.1)  Cranial nerve palsy, transient 1 (1.0) Hypopituitarism 4 (4.2)  Hypocortisolism 2 (2.1)  Hypothyroidisim 2 (2.1) Values are presented as number (%). *Statistically significant p-values. MST : maximum size of tumor, NA : not available, F/U : follow up, DI : diabetes insipidus, CSF : cerebrospinal fluid Go to : DISCUSSION This study reported an overall postoperative 3-6 month remission rate of 77% and a long-term remission rate of 82% after 3 years of follow-up. The initial and long-term remission rates after first operations were 78% and 82%, respectively, with a recurrence rate of 2.5% over a follow-up period of 3-3.5 years. Additionally, our findings revealed that tumor size >2 cm and extended tumor invasion of the cavernous sinus (Knosp 4) might be associated with lower postoperative remission rates. Patients who showed remission within 3-6 months showed higher rates of long-term remission than those in patients without initial remission. Pituitary surgery is the first-line treatment modality for CD. ETSS is a safe and less invasive method for treating pituitary adenomas; therefore, it has been increasingly preferred in CD [5,15]. However, the postsurgical outcomes in patients with CD have shown variable remission and recurrence rates [2,4,9,16,17,21,30]. These discrepancies may be attributable to differences in population and number of cases involved in the studies, tumor characteristics, criteria for remission and recurrence used by the centers, laboratory parameters, time of evaluation and followup durations, surgical and imaging techniques used by different centers, and neurosurgical expertise. In this study, we present the medium- and long-term postoperative results of 96 ETSS procedures performed in 87 patients. The medium-term results (obtained 3-6 months postoperation) were preferred to immediate results since a subset of cases may show delayed remission, and immediate postoperative results could be misleading in almost 6% of cases [37]. The overall medium-term remission rate was 77%, consistent with the results published by Serban et al. [34], who reported an overall remission rate of 77% 2 months postoperation. A larger series of 1106 cases reported an immediate remission rate of 72.5% within 7 days postoperation; however, this rate decreased to 67% after delayed remission rates and recurrences 56 months postoperation were considered [12]. The long-term remission rate obtained over a median period of 3 years was 82% in our series. The increased long-term remission rate was attributed to reoperations, additional medical therapies, and the use of STRS in those who did not show remission initially. Of the 96 procedures, 79 were performed for the first time. The medium-term remission rate after first operations was 78%. Recent studies have reported remission rates of 74-82% after first operations [12,34]. The recurrence rates reported previously varied between 3% and 66% [5,12,34]. However, the duration of follow-up differed among the studies. Dai et al. [12] and Brady et al. [5] reported recurrence rates of 12% and 3%, respectively, after a follow-up period of 2 years. In contrast, Serban et al. [34] reported a recurrence rate of 17% after a longer followup duration of 6 years. In this series, after a median follow-up period of 3 years, the overall recurrence rate was 2.5%. Residual tumors were observed in five cases (6%), and the reoperation rate after the first operation was 13%. Including the eight patients admitted for reoperation after having undergone their first surgery in another center, 17 cases involved reoperations in our center. Of these cases, 71% (n=12) showed remission between 3-6 months postoperation, while none showed recurrence; thus, the long-term remission rate was 77%. Residual tumors were detected in three cases (18%), and the disease persisted in four (24%) of these 17 reoperated cases. Previous studies have reported remission rates of 22-75% after repeated surgery in CD [5,12,34,38]. Although the success rates after reoperations were lower than those in first-time operations in some studies [38], the remission rates after the first and reoperations did not differ significantly in our study. Tumor size has been reported to contribute to the success of transsphenoidal surgery [12,34], with microadenomas showing a higher success rate after surgery [5,12,34]. Our remission rates for micro- and macroadenomas were similar to those reported by Dai et al. [12] : 81% for adenomas <10 mm and 68% for adenomas ≥10 mm. However, the statistical significance of our study differed from that in the series presented by Dai et al. [12] (p=0.2 vs. p=0.002). This may be due to the large difference in the number of cases included in the two studies and the differences in size scales for tumors ≥10 mm. In our series, when the tumors were stratified further by the tumor size, the medium-term remission rate further decreased to 36% for tumors ≥20 mm in size, although the remission rates for other groups <20 mm were all above 75% (p=0.003). Sharifi et al. [35] classified pituitary MRI scans in CD showing a tumor size <6 mm as “inconclusive” because incidentalomas are frequent among tumors in this size range, and this size is not indicative of CD. Previously published series reported that the rate of inconclusive MRI scans in CD was 36-64%, and the remission rates varied between 50% and 71% for those with an inconclusive MRI scan [10,24,27,32,33]. In our series, 54% of the preoperative MRI scans were inconclusive. In the series presented by Sharifi et al. [35] and some other series [8,12,32,36], no significant difference was observed between the remission rates of CD cases with and without a conclusive MRI.This finding is controversial since other studies showed decreased remission rates with preoperative inconclusive MRIs [13,40]. Similar to the results reported by Sharifi et al. [35], we did not find a statistically significant difference between the remission rates of tumors <6 mm and those between 6-20 mm. However, a significant difference was observed between tumors <6 mm and those ≥20 mm. Residual tumors were more frequent after operating tumors >20 mm compared to those <20 mm, but the number of reoperations did not differ among the groups. Additionally, tumors >20 mm were primarily Knosp 4 (64%), probably contributing to lower remission rates in this group. Interestingly, two Knosp 3 cases had postoperative remission within 3-6 months without additional intervention. In these two cases, the surgical team explored the cavernous sinus and could resect the tumor. However, complete excision was not feasible with Knosp 4 tumors, where there is a complete encasement of the intracavernous internal carotid artery. Thus, a tumor size of 20 mm may be supportive data in predicting non-remission in the presence of complete cavernous sinus infiltration. Cavernous sinus invasion, determined by the Knosp classification, and sellar invasion and/or suprasellar extension, determined by the Hardy-Wilson classification, indicate the radiologic status of local invasion in cases of pituitary tumors [20,22,39]. Invasion to surrounding structures and tissues may be a limiting factor for postoperative remission of pituitary tumors. In the series presented by Dai et al. [12], remission rates of corticotropinomas with Knosp grade 4 (definitive cavernous sinus invasion) dropped to 53% from a remission rate of 77% for corticotropinomas with less likely or no cavernous sinus invasion (p<0.001). Similarly, our results showed that both medium- and long-term remission rates for Knosp grade 4 tumors decreased to 13% and 50%, respectively, and were lower than the remission rates in other grades (p<0.001 and p=0.01, respectively). While remission rates in Knosp group 3 were not inferior to noninvasive tumors, remission rates in Knosp group 4 were lower than all the other groups. In this regard, the extent of invasion may be more determinative. In contrast, in our series, the modified Hardy classification did not show a significant effect on postoperative remission rates in medium- and long-term follow-up assessments. Araujo-Castro et al. [3] had previously shown that for pituitary adenomas, the Hardy-Wilson classification lacked utility in predicting postoperative remission compared to the Knosp classification. The difference in the utility of these classifications for predicting postoperative remission may be due to differences in accessing tissues during surgery. In the present series, 74% (n=71) of tissues were histologically proven to be corticotropinomas, while 26% (n=25) did not show histologic confirmation. Medium- and long-term remission rates did not differ between histologically proven and unproven CD cases (medium-term remission rates, 82% vs. 64%, p=0.07; long-term remission rates, 85% vs. 76%, p=0.3). A conclusive finding of an adenoma on MRI increased the rate of histologic proof of corticotropinoma in our series, implying that adenomas showing a ≥6-mm lesion on MRI more frequently stained positive for ACTH. In previous studies 12-53% of CD did not have postoperative pathologic identification and the rate increased in those with a preoperative inconclusive MRI [25,31,38]. However, this did not have a significant influence on our remission rates. The remission rates did not decrease even for CD cases that were not conclusively detected on MRI and could not be histologically proven. On the other hand, in previous studies, ACTH positivity was higher, and the lack of proof for a corticotropinoma decreased the remission rates [1,12,31,32,34]. The higher remission rates despite reduced localization with MRI and/or lower rates of histologic confirmation in our series may be explained by the successful preoperative IPSS lateralization, surgical exploration, and hemi-hypophysectomy procedure. Furthermore, tumor tissues might have been aspirated along with blood and other materials through the suction tube, potentially resulting in less histological confirmation despite postoperative remission of CD. Additionally, tumor tissues might have been aspirated along with blood and other materials through the suction tube, potentially resulting in less histological confirmation despite postoperative remission of CD. The total rate of complications in this series was 20%, and the most frequent complication was diabetes insipidus (DI; 8%, both permanent and temporary). The incidence of hypopituitarism was relatively lower (4%), mainly involving hypocortisolism and hypothyroidism. Recent studies have shown postoperative DI rates of 25-66% and hypothyroidism rates of 11-23% [5,34]. Although our neurosurgical team was experienced in conducting pituitary surgeries, other factors may have resulted in these differences. Since not all the cases were postoperatively followed in our center, with some patients lost to follow-up, there may be missing data. Comparing cases with and without remission in the medium term, cases of remission frequently involved adenomas >20 mm and less cavernous sinus invasion. Additionally, cases that achieved medium-term remission showed long-term remission more frequently. In the long term, those showing remission had less cavernous sinus invasion and residual tumors compared to those without remission. Therefore, we may conclude that a tumor size of 20 mm may predict medium-term remission, while the absence of/less cavernous sinus invasion, early remission, and absence of residual tumor may predict long-term remission. This study had limitations. First, the retrospective nature of the study and the limited number of cases, which was inevitable due to the low incidence of CD, may have distorted our results. Although the same neurosurgical team operated on all patients, they were followed up pre and postoperatively at different endocrinology centers, causing difficulty in obtaining the full postoperative data of certain cases. Lastly, some patients recently underwent ETSS; thus, they had a shorter follow-up period. However, we intend to present the longer-term outcomes of all patients in a separate study. Although ETSS is the first-line treatment for CD, previous studies on the use of ETSS for CD have reported a wide range of remission and recurrence rates, which can be primarily attributed to differences in the surgical experience levels among centers. This trend highlights the need for additional data from experienced centers to resolve the discrepancies in the existing data. Therefore, we present medium- and long-term follow-up data from 96 operations for CD conducted in a center with a high level of experience for ETSS. We believe our study makes a significant contribution to the literature because the findings reconfirm the usefulness of ETSS for the treatment of CD and highlight the importance of the size of the adenoma and presence/absence of cavernous sinus invasion on preoperative MRI in predicting long-term remission postoperatively. Go to : CONCLUSION ETSS is a safe and effective method for the treatment of CD. Some characteristics of adenomas, such as size, cavernous sinus invasion, and postoperative residue, may predict long-term remission. A tumor size of 2 cm may be a supporting criterion for predicting remission status in the presence of complete cavernous sinus infiltration. Further studies with larger patient populations are necessary to support this finding. Go to : Notes Conflicts of interest No potential conflicts of interest relevant to this study exist. Informed consent Informed consent was obtained from all individual participants included in this study. Author contributions Conceptualization : BE, MB, EH; Data curation : EA, OH, DT, MM; Formal analysis : LŞP, DAB, DT, İÇ; Funding acquisition : OT, ÖG, DAB; Methodology : LŞP, İÇ, MM, ÖG; Project administration : BE, SÇ, EH; Visualization : EA, OT, OH; Writing - original draft : BE, MB, SÇ; Writing - review & editing : BE, EH Data sharing None Preprint None Go to : Acknowledgements This manuscript was edited by a certified English Proofreading Service (Editage). Go to : References 1. Acebes JJ, Martino J, Masuet C, Montanya E, Soler J : Early post-operative ACTH and cortisol as predictors of remission in Cushing’s disease. Acta Neurochir (Wien) 149 : 471-477; discussion 477-479, 2007 2. Aranda G, Enseñat J, Mora M, Puig-Domingo M, Martínez de Osaba MJ, Casals G, et al : Long-term remission and recurrence rate in a cohort of Cushing’s disease: the need for long-term follow-up. Pituitary 18 : 142-149, 2015 3. 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World Neurosurg 77 : 525-532, 2012 From https://jkns.or.kr/journal/view.php?doi=10.3340/jkns.2023.0100
  2. YOU’RE INVITED! Webinar on Dr. Theodore Friedman’s update on medical treatment for Cushing’s disease In this informative webinar, Dr. Friedman will discuss What medicines to use to treat Cushing’s disease Side effects and timing of the medicines The use of ketoconazole for a medication trial before surgery Longer-term treatment for Cushing’s How to determine when a patient should go to surgery Sunday • March 31 • 6 PM PDT here to join the meeting or https://us02web.zoom.us/j/4209687343?pwd=amw4UzJLRDhBRXk1cS9ITU02V1pEQT09&omn=88672684111 OR +16699006833,,4209687343#,,,,*111116# US (San Jose) OR Join on Facebook Live https://www.facebook.com/goodhormonehealth Slides will be available on the day of the talk here. There will be plenty of time for questions using the chat button. For more information, email us at mail@goodhormonehealth.com
  3. YOU’RE INVITED! Webinar on Dr. Theodore Friedman’s update on medical treatment for Cushing’s disease In this informative webinar, Dr. Friedman will discuss What medicines to use to treat Cushing’s disease Side effects and timing of the medicines The use of ketoconazole for a medication trial before surgery Longer-term treatment for Cushing’s How to determine when a patient should go to surgery Sunday • March 31 • 6 PM PDT here to join the meeting or https://us02web.zoom.us/j/4209687343?pwd=amw4UzJLRDhBRXk1cS9ITU02V1pEQT09&omn=88672684111 OR +16699006833,,4209687343#,,,,*111116# US (San Jose) OR Join on Facebook Live https://www.facebook.com/goodhormonehealth Slides will be available on the day of the talk here. There will be plenty of time for questions using the chat button. For more information, email us at mail@goodhormonehealth.com
  4. Cushing’s syndrome (CS) secondary to adrenocorticotropic hormone (ACTH) producing tumours is a severe condition with a challenging diagnosis. Ectopic ACTH-secretion often involves neuroendocrine tumours (NET) in the respiratory tract. ACTH-secreting small intestine neuro-endocrine tumours (siNET) are extremely rare entities barely reported in literature. This review is illustrated by the case of a 75-year old woman with fulminant ectopic CS caused by a ACTH-secreting metastatic siNET. Severe hypokalemia, fluid retention and refractory hypertension were the presenting symptoms. Basal and dynamic laboratory studies were diagnostic for ACTH-dependent CS. Extensive imaging studies of the pituitary and thorax-abdomen areas were normal, while [68Ga]Ga-DOTATATE PET-CT revealed increased small intestine uptake in the left iliac fossa. The hypercortisolism was well controlled with somatostatin analogues, after which a debulking resection of the tumour was performed. Pathological investigation confirmed a well-differentiated NET with sporadic ACTH immunostaining and post-operative treatment with somatostatin analogues was continued with favourable disease control. © Acta Gastro-Enterologica Belgica. ABOUT THE CONTRIBUTORS B alliet, c severi, t veekmans, j cuypers, h topal, c m deroose, t roskams, m bex, j dekervel B Alliet Department of Gastroenterology, UZ Leuven, Leuven, Belgium. C Severi Department of Gastroenterology, ZOL, Genk, Belgium. T Veekmans Department of Pathology, UZ Leuven, Leuven, Belgium. J Cuypers Department of Endocrinology, AZ Turnhout, Turnhout, Belgium. H Topal Department of Abdominal Surgery, UZ Leuven, Leuven, Belgium. C M Deroose Department of Nuclear Medicine, UZ Leuven, Leuven, Belgium. T Roskams Department of Pathology, UZ Leuven, Leuven, Belgium. M Bex Department of Endocrinology, UZ Leuven, Leuven, Belgium. J Dekervel Department of Gastroenterology – Digestive Oncology, UZ Leuven, Leuven, Belgium. From https://www.physiciansweekly.com/fulminant-ectopic-cushings-syndrome-caused-by-metastatic-small-intestine-neuroendocrine-tumour-a-case-report-and-review-of-the-literature/
  5. Highlights Phaeochromocytoma with ectopic ACTH secretion. Its clinical presentation is varied, and diagnosis is challenging. Ectopic ACTH secretion from a phaeochromocytoma can rapidly progress to severe Cushing’s syndrome. Removal of the primary tumour often leads to full recovery. Abstract Introduction The occurrence of hypercortisolism resulting from adrenocorticotropic hormone (ACTH)-secreting pheochromocytoma is exceedingly uncommon, with limited documented instances thus far. Presentation of case We present a case of ectopic ACTH-secreting pheochromocytoma in a patient who suffered from severe metabolic disorders. Our clinical case outlines the diagnostic history, preoperative correction of the patient's metabolic disturbances and surgical strategy for management of a rare ectopic ACTH producing pheochromocytoma. Discussion Ectopic adrenocorticotropic hormone-secreting pheochromocytoma displays multifaceted clinical features and requires prompt diagnosis and multidisciplinary management in order to overcome the related severe clinical derangements. Conclusion The combination of biochemical and hormonal testing and imaging procedures is mandatory for the diagnosis of ectopic ACTH secretion, and in the presence of an adrenal mass, the possibility of an ACTH-secreting pheochromocytoma should be taken into account. Keywords Hypokalemia Adrenal gland Pheochromocytoma Ectopic cushing's syndrome Cushing's syndrome 1. Introduction Neuroendocrine tumors such as Pheochromocytoma and paraganglioma (PPGL) are an uncommon occurrence. The prevalence of PPGL has been estimated to be between (2–8)/1 million, with a population rate of 1:2500–1:6500 [1], and it is associated with symptoms such as headache, irregular heartbeats, profuse sweating, high blood pressure, nausea, vomiting, nervousness, irritability, and a sense of imminent mortality [2]. Hypercortisolism is also a rare disorder with an incidence of 5/1 million, <10 % of patients with hypercortisolism are caused by ectopic secretion of ACTH [3], and these are most commonly seen in APUD tumors such as small cell bronchopulmonary carcinoma, pancreatic islet carcinoma, medullary thyroid carcinoma, pheochromocytoma, and melanoma [4]. Tumors that secrete both ACTH and catecholamines are much rarer. Here, we present a case of ectopic ACTH-secreting pheochromocytoma with severe metabolic disorders. The case report is compliant with SCARE Guidelines [5]. 2. Case report The patient is a 46-year-old male who presented to our hospital with recurrent symptoms of pheochromocytoma. He reported that he experienced unexplained symptoms such as panic attacks, headache, sweating, nausea, vomiting, and a feeling of imminent death, which could be alleviated by rest. His blood pressure was around 160–220/110–120 mmHg, and he was taking oral antihypertensive drugs regularly, with poor control of his blood pressure. The patient was admitted with a body temperature of 36.7 °C, heart rate of 130 beats/min, respiratory rate of 20 cycles per minute, blood pressure of 138/88 mmHg, height of 175 cm, weight of 67 kg, Body Mass Index (BMI): 21.88, normal physical examination, emaciated body type, thin subcutaneous fat, self-reported weight loss of 20 kg within 10 months, and history of diabetes mellitus of >1 year. Laboratory tests showed that the blood potassium levels were within the normal range, while the blood sugar and beta-hydroxybutyrate levels were elevated (Table 1). Hormonal analysis showed plasma levels of free catecholamine and its metabolites were much higher than normal, in addition to a severe excess of cortisol secretion with circadian rhythm disorders and elevated serum ACTH (Table 2). Small dose dexamethasone suppression test (1 mg) yielded cortisol levels of over 1750 nmol/L (negative: no decrease in blood cortisol), thus confirming the presence of ACTH-dependent hypercortisolism. The results of electrocardiogram, chest computerized tomography (CT), cardiac ultrasound and thyroid ultrasound showed no obvious abnormality. Enhanced CT of the adrenal glands (Fig. 1) revealed the presence of a right adrenal tumor measuring approximately 5.3 ∗ 4.7 cm. Despite undergoing cranial MRI, no pituitary lesion was detected, thereby ruling out the possibility of Cushing's disease. The patient was further considered for possible ectopic ACTH syndrome and suspected ectopic ACTH-secreting pheochromocytoma. Table 1. Laboratory test results. Laboratory test Result Reference value Unit White blood cell 17.03 3.5–9.5 109/L Red blood cell 5.06 3.8–5.1 1012/L Hemoglobin 147 115–150 g/L Platelets 206 125–350 109/L Glucose 12.13 3.9–6.1 mmol/L β-Hydroxybutyric acid 8.680 0–0.30 mmol/L Creatinine 55.30 40–105 umol/L Calcium 2.47 2.2–2.7 mmol/L Phosphate 1.26 0.85–1.51 mmol/L Potassium 3.66 3.5–5.5 mmol/L Sodium 147.1 137–147 mmol/L Table 2. The patient's adrenal hormone results Empty Cell Preoperative Postoperative Reference value Unit Norepinephrine, free 11,900 118 217–1109 pg/ml Adrenaline, free 3940 <24 <95 pg/ml Dopamine 207 <18 <20 pg/ml Methoxy norepinephrine 4130 87.80 <145 pg/ml Methoxy adrenaline 1850 <12 <62 pg/ml Adrenocorticotropic hormone (8:00) 544 10.60 7.2–63.3 pg/ml Cortisol (8:00) >1750 246.00 166–507 nmol/L Adrenocorticotropic hormone (16:00) 647 33.50 – pg/ml Cortisol (16:00) >1750 536.00 73.8–291 nmol/L Adrenocorticotropic hormone (00:00) 566 – – pg/ml Cortisol (00:00) >1750 – nmol/L Renin 2.82 3.10 2.4–32.8 pg/ml Aldosterone 81.51 73.56 16–160 pg/ml Aldosterone/renin concentration ratio 28.90 23.73 0–25 Download : Download high-res image (184KB) Download : Download full-size image Fig. 1. Adrenal CT showed a 53 ∗ 47 mm mass in the right adrenal gland. In response to the patient's pheochromocytoma symptoms and improve preoperative preparation, we used α-blocker (Phenoxybenzamine 20 mg q8h) to lower blood pressure and increase blood volume, antihypertensive medication (nifedipine 30 mg q12h, olmesartan tablets 20 mg q12h) to assist in lowering blood pressure, and β-blocker (metoprolol 47.5 mg q12h) to control the heart rate. On the 4th day in hospital, the patient was lethargic and had weak limbs. Urgent blood workup showed severe hypokalemia (2.85 mmol/L) as well as hyperglycemia (10.26 mmol/L). Patient was transferred to intensive care to correct intractable hypokalemia and diabetic ketoacidosis. After the patient was transferred to ICU, a deep vein cannulation was performed with intravenous potassium chloride supplementation, and the patient's blood potassium was maintained at normal levels prior to surgery through a large amount of potassium supplementation (Fig. 2A). For diabetic ketoacidosis, insulin administration, rehydration, ketone elimination and other treatments were given and the amount of access was recorded, and it was found that the patient was polyuric, with the highest urine volume of 21,800 ml in a single day (Fig. 2B), and the amount of urine did not decrease by taking oral desmopressin tablets 0.1 mg bid. Download : Download high-res image (255KB) Download : Download full-size image Fig. 2. Changes in blood potassium and urine volume during the patient's hospitalization. A: Blood potassium level. B: Daily urine vlume. Eventually, the patient underwent laproscopic right adrenal tumor resection. Intraoperative changes in blood pressure and heart rate are shown in Fig. 3. On day 1 after surgery, the morning (8:00) ACTH level was 10.60 pg/ml, antihypertensive medications were discontinued, and his blood pressure was 100–120/60–90 mmHg. The patient's daily urine output and blood glucose gradually returned to normal levels after surgery. Pathology (Fig. 4😞 Adrenal pheochromocytoma with ACTH immunopositive staining, cellular heterogeneity was unremarkable, nuclear schizophrenic images were rare, no pericytes, choroidal invasion and necrosis were seen. The patient was discharged from the clinic in a satisfactory condition with adrenal insufficiency compensated by daily intake of Prednisone Acetate Tablets (20 mg), discontinued 6 months after surgery. No signs of recurrence were noted upon frequent follow-up examinations. Download : Download high-res image (295KB) Download : Download full-size image Fig. 3. Changes in patient's intraoperative blood pressure and heart rate. Download : Download high-res image (313KB) Download : Download full-size image Fig. 4. Immunohistochemistry. A: hematoxylin and eosin staining B: ACTH. 3. Discussion We share the management of a patient with ectopic ACTH-secreting pheochromocytoma with severe metabolic disturbances, where, in addition to the rare etiology, perioperative management of the clinical complications of catecholamines and hypercortisolism is very challenging [6]. Patients suffering from ectopic ACTH syndrome caused by pheochromocytoma commonly exhibit severe Cushing's syndrome (CS), significant diabetes mellitus, hypertension, and hypokalemia [7]. Additionally, a retrospective study revealed that the majority of patients presented with Cushing's syndrome [8], whereas another report indicated that only 30 % of patients presented with typical Cushing's syndrome, but weight loss was frequently observed [9]. Our patient's recent weight loss may be attributed to the body's hypermetabolic condition caused by catecholamines. Recent reports claim that catecholamines directly reduce subcutaneous and visceral fat [10]. Rapid onset of cortisolism appears to be a feature of ACTH-secreting pheochromocytomas, because of the rapid onset of severe hypercortisolism, and our patient did not exhibit typical Cushing's symptoms [8]. Despite the absence of typical Cushing-like symptoms, this patient displayed persistent hypokalemia, a prevalent metabolic manifestation of Cushing's syndrome, particularly in ectopic ACTH syndrome, where hypokalemia is observed in 74 %–95 % of patients, in contrast to 10 % of patients with Cushing's disease [11]. Glucocorticoids have the ability to interact with aldosterone receptors, resulting in specific aldosterone-like reactions, while ectopic ACTH syndrome typically generates a higher amount of cortisol compared to Cushing's disease, ultimately causing more pronounced hypokalemia [7]. The perioperative management of patients with ACTH-secreting pheochromocytomas poses a significant challenge due to severe hypokalemia, and our patient's potassium levels remained within the normal range through extensive central venous potassium supplementation, without the need for cortisol secretion inhibition medications. The severity of hypertension in patients with ACTH-secreting pheochromocytomas seems to surpass that of patients with pheochromocytomas alone [12]. Hypercortisolism amplifies catecholamine-induced hypertension [13]. In the case of hypertension in patients with pheochromocytomas, alpha-blockers are favored for reducing blood pressure and enlarging blood volume, while for individuals whose blood pressure is not adequately managed with alpha-blockers alone, a combination of medications is recommended. Proper preoperative readiness for expanding the volume is crucial for a successful surgical procedure. Patients with ACTH-secreting pheochromocytoma have a greater prevalence and intensity of diabetes mellitus compared to those with pheochromocytoma alone [14], and our patient displayed a combination of severe diabetes mellitus and ketoacidosis. Insulin exhibits swift action and adaptable dosage, effectively averting hypoglycemia and effectively addressing hyperglycemia, rendering it the preferred medication for regulating blood glucose levels in individuals with ectopic CS [6]. Managing the water-electrolyte balance in this patient proved to be an arduous task, and the diabetes insipidus may have been one of the complications, with a maximum urine output of 21,800 ml in a single day (Fig. 2), and we hold the belief that the patient's diabetes insipidus is caused by a range of factors, such as hypokalemia, hypercortisolism, and severe diabetes mellitus. Indeed, hypokalemia may cause renal impairment, which reduces the ability to concentrate urine and lack of response to antidiuretic hormone (ADH), leading to nephrogenic diabetes insipidus [15]. Cortisol increases renal plasma flow and glomerular filtration rate, and also inhibits the secretion of antidiuretic hormone, leading to neurogenic diabetes insipidus [16]. For hypercortisolism, surgery to target the cause is the first-line treatment, and surgical removal of primary tumor may lead to 40 % radical treatment and 80 % complete remission of ectopic ACTH syndrome [17]. 4. Conclusion Preoperative diagnosis and management of pheochromocytoma, an extremely rare cause of ectopic ACTH syndrome, is challenging. Proper preoperative recognition of complications of both hypercortisolism and catecholamines excess is the key to prevent the morbidity and mortality of an ACTH-producing pheochromocytoma. If diagnosed successfully and managed intensively, they are curable. Consent Written informed consent was obtained from the patient for publication of this case report and accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal on request. Ethical approval Shandong Provincial Hospital Affiliated to Shandong First Medical University does not require ethical approval for publication of case reports. Signed consent from the patient has been received. Funding No funding was received for this research. Author contribution Shangjian Li: study concept or design, data collection, data analysis or interpretation, writing the paper Xudong Guo: study concept or design, data collection, data analysis or interpretation, writing the paper Hanbo Wang: study concept or design, data analysis or interpretation Ni Suo: study concept or design, data analysis or interpretation Xiuqing Mi: study concept,data collection Shaobo Jiang: study concept or design, data analysis or interpretation, writing the paper Guarantor Shangjian Li Xudong Guo Shaobo Jiang Conflict of interest statement All authors declare no conflict of interest. Acknowledgements None. References [1] A. Jain, R. Baracco, G. Kapur Pheochromocytoma and paraganglioma-an update on diagnosis, evaluation, and management Pediatr. Nephrol., 35 (2020), pp. 581-594 View article CrossRefView in ScopusGoogle Scholar [2] F.A. Farrugia, A. Charalampopoulos Pheochromocytoma Endocr. Regul., 53 (2019), pp. 191-212 View article CrossRefView in ScopusGoogle Scholar [3] M. Gadelha, F. Gatto, L.E. Wildemberg, et al. Cushing’s syndrome Lancet, 402 (2023), pp. 2237-2252 View PDFView articleView in ScopusGoogle Scholar [4] O. Ragnarsson, C.C. Juhlin, D.J. Torpy, et al. A clinical perspective on ectopic Cushing’s syndrome Trends Endocrinol. Metab. (2023) Google Scholar [5] C. Sohrabi, G. Mathew, N. Maria, et al. The SCARE 2023 guideline: updating consensus Surgical CAse REport (SCARE) guidelines Int. J. Surg., 109 (2023), pp. 1136-1140 View article CrossRefView in ScopusGoogle Scholar [6] M.F. Birtolo, E.M. Grossrubatscher, S. Antonini, et al. Preoperative management of patients with ectopic Cushing’s syndrome caused by ACTH-secreting pheochromocytoma: a case series and review of the literature J. Endocrinol. Investig., 46 (2023), pp. 1983-1994 View article CrossRefView in ScopusGoogle Scholar [7] J.N. Gabi, M.M. Milhem, Y.E. Tovar, et al. Severe Cushing syndrome due to an ACTH-producing pheochromocytoma: a case presentation and review of the literature J Endocr Soc, 2 (2018), pp. 621-630 View article CrossRefView in ScopusGoogle Scholar [8] P.F. Elliott, T. Berhane, O. Ragnarsson, et al. Ectopic ACTH- and/or CRH-producing pheochromocytomas J. Clin. Endocrinol. Metab., 106 (2021), pp. 598-608 View article CrossRefView in ScopusGoogle Scholar [9] J.E. Paleń-Tytko, E.M. Przybylik-Mazurek, E.J. Rzepka, et al. Ectopic ACTH syndrome of different origin-diagnostic approach and clinical outcome. Experience of one clinical centre PLoS One, 15 (2020), Article e0242679 View article CrossRefView in ScopusGoogle Scholar [10] L.N. Krumeich, A.J. Cucchiara, K.L. Nathanson, et al. Correlation between plasma catecholamines, weight, and diabetes in pheochromocytoma and paraganglioma J. Clin. Endocrinol. Metab., 106 (2021), pp. e4028-e4038 View article CrossRefGoogle Scholar [11] J. Young, M. Haissaguerre, O. Viera-Pinto, et al. Management of endocrine disease: Cushing’s syndrome due to ectopic ACTH secretion: an expert operational opinion Eur. J. Endocrinol., 182 (2020), pp. R29-r58 View article CrossRefView in ScopusGoogle Scholar [12] H. Falhammar, M. Kjellman, J. Calissendorff Initial clinical presentation and spectrum of pheochromocytoma: a study of 94 cases from a single center Endocr. Connect., 7 (2018), pp. 186-192 View article CrossRefView in ScopusGoogle Scholar [13] E.L. Alba, E.A. Japp, G. Fernandez-Ranvier, et al. The Mount Sinai clinical pathway for the diagnosis and management of hypercortisolism due to ectopic ACTH syndrome J Endocr Soc, 6 (2022), Article bvac073 View in ScopusGoogle Scholar [14] L. Foppiani, M.G. Poeta, M. Rutigliani, et al. Catastrophic ACTH-secreting pheochromocytoma: an uncommon and challenging entity with multifaceted presentation Endocrinol. Diabetes Metab. Case Rep., 2023 (2023) Google Scholar [15] S. Khositseth, P. Uawithya, P. Somparn, et al. Autophagic degradation of aquaporin-2 is an early event in hypokalemia-induced nephrogenic diabetes insipidus Sci. Rep., 5 (2015), Article 18311 View PDF This article is free to access. View in ScopusGoogle Scholar [16] M.M. Hammami, N. Duaiji, G. Mutairi, et al. Case report of severe Cushing’s syndrome in medullary thyroid cancer complicated by functional diabetes insipidus, aortic dissection, jejunal intussusception, and paraneoplastic dysautonomia: remission with sorafenib without reduction in cortisol concentration BMC Cancer, 15 (2015), p. 624 View PDF This article is free to access. View in ScopusGoogle Scholar [17] A. Ferriere, A. Tabarin Cushing’s syndrome: treatment and new therapeutic approaches Best Pract. Res. Clin. Endocrinol. Metab., 34 (2020), Article 101381 View PDFView articleView in ScopusGoogle Scholar From https://www.sciencedirect.com/science/article/pii/S2210261224001226
  6. Abstract Context Patients with Cushing’s disease (CD) face challenges living with and receiving appropriate care for this rare, chronic condition. Even with successful treatment, many patients experience ongoing symptoms and impaired quality of life (QoL). Different perspectives and expectations between patients and healthcare providers (HCPs) may also impair well-being. Objective To examine differences in perspectives on living with CD between patients and HCPs, and to compare care goals and unmet needs. Design Memorial Sloan Kettering Pituitary Center established an annual pituitary symposium for pituitary patients and HCPs. Through anonymous pre-program surveys distributed at the 2020 and 2022 symposia, patients and HCPs answered questions related to their own sense, or perception of their patients’ sense, of hope, choice, and loneliness in the context of living with CD. Participants From 655 participants over two educational events, 46 patients with CD and 116 HCPs were included. Median age of both groups was 51 years. 78.3% of the patients were female vs. 53.0% of the HCPs. Results More patients than HCPs reported they had no choices in their treatment (21.7% vs. 0.9%, P < 0.001). More patients reported feeling alone living with CD than HCPs’ perception of such (60.9% vs. 45.5%, P = 0.08). The most common personal care goal concern for patients was ‘QoL/mental health,’ vs. ‘medical therapies/tumor control’ for HCPs. The most common CD unmet need reported by patients was ‘education/awareness’ vs. ‘medical therapies/tumor control’ for HCPs. Conclusions CD patients experience long term symptoms and impaired QoL which may in part be due to a perception of lack of effective treatment options and little hope for improvement. Communicating experiences and care goals may improve long term outcomes for CD patients. Introduction Patients with rare diseases face challenges receiving appropriate care. Cushing’s disease (CD), a condition associated with excess endogenous glucocorticoids due to an ACTH-secreting pituitary tumor, is a rare disease, occurring in 0.7 to 2.4 per million per year [1]. Patients with CD are at high risk for metabolic, cardiovascular, and psychiatric disease, in addition to long-term symptom burden and impaired quality of life (QoL), despite adequate treatment [1,2,3]. A critical aspect of effective patient care is communication and mutual understanding between healthcare provider (HCP) and patient. Patients with pituitary tumors experience significant anxiety associated with their diagnosis, in large part due to difficulties interacting with healthcare systems and limited communication of information [4]. Many pituitary patients express concern regarding the complexity of their care, and satisfaction improves with the delivery of more information by the HCP [4]. Patients with pituitary tumors, and CD specifically, require multidisciplinary care which necessitates effective communication in order to provide the best possible outcomes [5]. Similar to acromegaly patients [6], CD patients’ long-term well-being may be adversely affected by different perspectives and expectations between patients and HCPs, especially after treatment [7]. While HCPs primarily use biochemical data to define successful treatment, patients rely more on their symptoms and ability to regain normal functioning [7]. Despite achieving biochemical remission, CD patient perception of having persistent disease negatively impacts QoL [8]. In addition, 67.5% of Cushing’s syndrome patients report receiving insufficient information from their HCPs regarding the recovery experience after surgery despite the fact that all HCPs report providing this information [9]. Improved communication between HCPs and CD patients is vital to optimizing patients’ QoL and long term outcomes. Recently there has been a growing emphasis on the use of internet-based platforms for healthcare delivery and education [10]. With the goals of offering HCP and patient education and assessing pituitary patients’ needs, since 2019 the pituitary center at Memorial Sloan Kettering (MSK) has offered annual virtual educational programs for pituitary patients, caregivers, HCPs, and members of the pharmaceutical industry. For the current study, we gathered deidentified information from 2020 to 2022 MSK program participants on CD patients’ and HCPs’ attitudes about CD, related to their sense of hope, choice, and loneliness, through anonymous pre-program surveys. Our specific aims were to: (1) Assess differences in perspectives between patients’ and HCPs’ responses in the pre-program survey; (2) Compare patients’ and HCPs’ perceived care goals and unmet needs. Methods Educational program enrollment The MSK program was offered to patients with any type of pituitary tumor as well as HCPs, family members, caregivers, and members of industry. The role of the registrant as a patient, caregiver/family member, HCP, and/or member of industry was determined for all registrants of the virtual programs. Any patient with a pituitary tumor treated at our center and outside institutions, inclusive of patients at all points along their treatment journey, were invited to register for the virtual education program. HCPs, including endocrinologists, neurosurgeons, otolaryngologists, radiation oncologists, neurologists, ophthalmologists, neuro-oncologists, family medicine and internal medicine physicians, physicians in training and other allied health professionals who treat and manage patients with pituitary diseases were also invited to register. Invitations were sent through email to neuroendocrine experts and endocrinologists, patient support groups on social media, direct messaging to patients with pituitary tumors by their treating physicians and via patient databases, advertisements through endocrine societies, brochure/postcard mailing, and Eventbrite, a virtual platform for live events. Study participants Registrants from MSK virtual programs held on December 5, 2020, (n = 328) and April 9, 2022, (n = 327) were included in the pool of subjects, among which the qualifying participants were determined. Of the 655 total registrants from the 2020 and 2022 programs, 320 (48.9%) were patients or caregivers and 309 (47.2%) were HCPs (Fig. 1). Of the 147 providers (88 in 2020 and 59 in 2022) that attended and filled out a pre-program survey 31 were excluded from our analysis. Eight filled out surveys in both 2020 and 2022, 4 were members of industry, 3 did not fill out any responses, and 1 was not in the healthcare field. In addition, 12 providers had at least three fields missing in the survey and 3 had filled out two surveys for the same year, so they were also excluded. A total of 116 providers (72 from 2020 to 44 from 2022) were included in the analysis. Fig. 1 Enrollment flowchart Full size image Among the 320 pituitary patients who attended the programs (157 from 2020 to 163 from 2022), 53 identified as ‘patients with Cushing’s’ and submitted surveys (34 participants from 2020 to 19 from 2022). Seven patients were excluded from the 2022 surveys as they had also filled out surveys in 2020, leaving a final group of 46 patients who were included in the analysis. Virtual education programs For each program, there was a single day of live interactive programming, meaning that all participants attended at the same time. The programs were recorded and made available for several weeks as enduring material for registrants on an online website. After joint sessions in the morning, both programs consisted of two tracks in the afternoon: the ‘provider/clinical track’ and the ‘patient/caregiver track’. During the programs, an ongoing chat reeled through the virtual program which allowed patients to continually ask questions. Faculty experts answered these questions in written responses directly within the chat and/or in spoken responses during one of the live broadcasted Q&A sessions. Additionally, the programs both included panel discussions answering patient questions and moderated patient discussions with invited patient speakers. Study procedures Through anonymous pre-program surveys distributed at the 2020 and 2022 symposia, patients and HCPs answered questions related to their own sense, or perception of their patients’ sense, of hope, choice, and loneliness in the context of living with CD. This survey was developed by a multidisciplinary team and has been reported previously [11]. Demographic and clinical information was also assessed including year of diagnosis, prior treatments, and current medications (for patients) and specialty and practice type (for providers), as shown in Tables 1 and 2. Multiple-choice questions assessing patients’ attitudes toward their disease included possible answers of ‘I have no hope for improvement,’ ‘I have some hope for improvement,’ and ‘I have a lot of hope for improvement;’ and ‘I have no choice in my treatment,’ ‘I have some choices in my treatment,’ and ‘I have many choices in my treatment.’ Patients were also asked to respond ‘TRUE’ or ‘FALSE’ to the following statements: ‘I feel alone living with my Cushing’s,’ ‘Hearing the journeys of other patients helps me better understand my own,’ and ‘I feel anxious about my Cushing’s diagnosis.’ Table 1 Patient demographic data Full size table Table 2 Provider demographic data Full size table Multiple-choice questions assessing providers’ attitudes about their patients' Cushing’s included possible answers of ‘I have no hope for their improvement,’ ‘I have some hope for their improvement,’ and ‘I have a lot of hope for their improvement;’ and ‘my patients have no choice in their treatment,’ ‘my patients have some choices in their treatment,’ and ‘my patients have many choices in their treatment.’ Providers were also asked to respond ‘TRUE’ or ‘FALSE’ to the following statements: ‘my patients feel alone living with their Cushing’s,’ ‘Hearing the journeys of other patients helps will help my patients better understand their own,’ and ‘my patients feel anxious about their Cushing’s diagnosis.’ Additionally, patients were surveyed on care goals and unmet needs related to their treatment. Specifically, patients were asked, ‘What are the healthcare outcomes/goals that matter to you the most?’ and ‘What do you think are unmet needs for the diagnosis or treatment of your condition?’ The first question was intended to refer to the patient specifically, while the second question was meant to examine how the condition is treated in general. Survey responses were submitted as free text and subsequently grouped by the authors (AH and EBG) into nine different categories: (a) Quality of life (QoL)/Mental Health; (b) Medical Therapies/Tumor Control; (c) Education/Awareness; (d) Communications/Multidisciplinary Care; (e) Insurance/Access; (f) Fertility; (g) Controlling Comorbidities; (h) Support System and (i) none. Responses could receive multiple designations if applicable. AH coded the free text themes independently, then EBG reviewed each answer and corresponding grouping to confirm accuracy. If there was disagreement or confusion, coding from our prior work [11] was reviewed. HCPs were also surveyed on care goals and unmet needs related to their patient’s treatment. Providers were asked, ‘What are the healthcare outcomes/goals that matter to you the most?’ and ‘what do you think are unmet needs for the diagnosis or treatment of your patient’s condition?’ The first question was intended to refer to the provider and their goals related to Cushing’s, while the second question was meant to examine how the condition is treated in general. Survey responses were submitted as free text and subsequently grouped by the authors (AH and EBG) into nine different categories: (a) Quality of life (QoL)/Mental Health; (b) Medical Therapies/Tumor Control; (c) Education/Awareness; (d) Communications/Multidisciplinary Care; (e) Insurance/Access; (f) Fertility; (g) Controlling Comorbidities; (h) Support System and (i) none. Responses could receive multiple designations if applicable. Statistical analysis Descriptive statistics were presented as counts and percentages for categorical variables and as medians and interquartile range (IQR) for continuous variables. The Chi-square test or Fisher’s exact test was used to compare gender and survey responses between the CD patient group and the HCP group. All statistical tests were two-tailed, and a P-value of < 0.05 was considered statistically significant. SAS Software® (version 9.4; SAS Institute Inc., Cary, NC) was used for all analyses. Results Between the 2020 and 2022 events, there was combined representation from 25 different countries. A map and a full list of the countries is shown in Fig. 2. Fig. 2 Map of registrant locations. Locations (listed alphabetically): Argentina, Australia, Belgium, Brazil, Canada, Chile, China, Greece, Hong Kong, India, Israel, Jamaica, Latvia, Malaysia, Netherlands, New, Zealand, Oman, Peru, Philippines, Qatar, Romania, Saudi Arabia, Singapore, UK, US Full size image From a total of 655 participants over two educational events, 46 patients with CD and 116 HCP caring for CD patients were included in the analysis. The demographic data of the patients and HCPs are outlined in Tables 1 and 2, respectively. Median age of the patients and HCPs was 51 years. 78.3% of the CD group was female vs. 53.0% of the HCP group (P = 0.003). CD patients ranged from newly diagnosed to being diagnosed 33 years prior. The HCPs who filled out the pre-program surveys were in practice for a mean duration of 18.5 years, with a range from 1 to 54 years. As shown in Table 1, CD patients had a mean duration of suspected active disease prior to diagnosis of 5.26 years, as defined by onset of CD symptoms until diagnosis, and a mean duration of disease since diagnosis of 5.9 years. 42 (91%) had undergone surgical treatment of their Cushing’s. For those who underwent surgery, the mean number of surgeries was 1.17 (range 0–4). 20% had received pituitary radiation. Overall, 31% of patients were on medical therapy for Cushing’s. Metyrapone was the most used CD therapy (in 11%), followed by ketoconazole (in 9%). Of those requiring pituitary hormone replacement, 34.8% had one pituitary hormone deficiency and 21.7% had multiple hormone deficiencies. Thyroid hormone replacement (37%) and adrenal replacement (30%) were the most common. As shown in Table 2, the majority of the HCPs were endocrinologists (72%) followed by neurosurgeons (9%) and nurses (8%). There was a total of 9 different specialties represented by the provider group. 16% of the providers worked in private practice, 16% were hospital based, and 16% worked in ‘unspecified clinical care.’ 38% of the providers practice type was ‘unspecified.’ Based on the pre-program survey responses, we identified different attitudes between patients and HCPs in several domains. Table 3 depicts pre-program survey responses from CD patients and HCPs assessing their attitudes about CD. 21.7% of patients reported they had no choices in their treatment, compared to 0.9% of HCPs (P < 0.001). Almost all HCPs (99.1%) reported that CD patients had least some choice in their management. In addition, less than half (45.7%) of patients reported they had a lot of hope for improvement whereas 71.3% of HCPs had a lot of hope for their patients’ improvement. Surprisingly, fewer CD patients reported feeling anxious about their diagnosis compared to HCPs’ perceived patient anxiety (65.2% vs 94.6%, P < 0.001). However, more patients tended to feel more alone living with CD than HCPs’ perception of such (60.9% vs. 45.5%, P = 0.08). Both CD patients and HCPs agreed that hearing the journeys of other CD patients would help patients better understand their own disease (97.8% vs 100%). Table 3 Patient and provider attitudes by pre-program survey Full size table CD patients and HCPs were also surveyed on their personal care goals and unmet needs, results of which are shown in Figs. 3A, B and 4A, B. The most common personal care goal concern for patients was ‘QoL/mental health’ which was reported by 70%, followed by ‘controlling comorbidities’ (39%) and ‘medical therapies/tumor control’ (24%). HCPs prioritized the same three care goals as patients but ‘medical therapies/tumor control’ was the most common (44%). ‘Controlling comorbidities’ and ‘QoL/mental health’ were the second and third most often HCP reported care goals (31 and 22% respectively). ‘Education/awareness’ was the most common perceived CD unmet need by patients (59%). HCPs reported both ‘medical therapies/tumor control’ and ‘education/awareness’ to be the most common unmet needs (35 and 26%, respectively). Examples of patient and provider responses, and how they were coded, are shown in Supplemental Table 1. Fig. 3 A Care goals according to participants with Cushing’s who completed pre-program survey. This pie graph represents the free-text survey response from patients regarding their personal care goals as categorized by topic. B Care goals according to providers who completed pre-program survey. This pie graph represents the free-text survey response from providers regarding their personal care goals as categorized by topic Full size image Fig. 4 A Unmet needs for the field of Cushing’s disease according to participants with Cushing’s who completed pre-program survey. This pie graph represents the free-text survey response from patients regarding unmet needs in Cushing’s as categorized by topic. B Unmet needs for the field of Cushing’s disease according to providers who completed pre-program survey. This pie graph represents the free-text survey response from providers regarding unmet needs in Cushing’s as categorized by topic Full size image Discussion This study examined the differences between patients and HCP-reported perceptions of living with CD. We identified several differences in disease outlook between CD patients and HCPs. We found that more patients than HCPs reported they had no choices in their treatment. Furthermore, less than half of patients reported they had a lot of hope for improvement whereas most (71.3%) of HCPs had a lot of hope for their patients’ improvement. Interestingly, fewer CD patients reported feeling anxious about their diagnosis compared to HCPs’ perceived patient anxiety, although a higher percentage of patients reported feeling alone living with CD compared to the HCPs’ perception of patient loneliness. We also identified HCP and patient differences in reported personal care goals and perceived unmet needs in the field. The most common personal care goal concern for patients was ‘QoL/mental health,’ whereas it was ‘medical therapies/tumor control’ for HCPs. ‘Education/awareness’ was the most commonly perceived unmet need by patients, whereas it was ‘medical therapies/tumor control’ for HCPs. Our findings support prior work demonstrating a discrepancy between patients and HCPs regarding the need for improved multidisciplinary care [12]. 43% of patients listed ‘communication/multidisciplinary care’ as an unmet need in the field, compared to 3% of providers. Pituitary centers of excellence provide expert multidisciplinary care in the neuroendocrine, neurosurgical, and radiation oncology domains, but often lack expertise in mental and physical health domains salient for CD patients, who suffer from depression, anxiety, myopathy and joint pain. In order to offer comprehensive care, psychiatrists, psychologists, social workers, pain medicine experts, physical therapists, and nutritionists with expertise in CD should be included in the pituitary center multidisciplinary team [13]. Our findings suggest that pituitary centers of excellence should take into account the most important personal care goal reported by CD patients, which is Qol/mental health, and provide expert treatment in this domain. It is not surprising that Qol/mental health is the personal care goal most reported by CD patients. Prior assessment of acromegaly patients demonstrated the same finding: QoL/mental health was the most common personal care goal concern [11]. While surgical [14] and medical [15,16,17,18] treatment of Cushing’s improves QoL, QoL has been shown to remain impaired over time after treatment [19]. Several factors may contribute to long-term Qol impairments, including the presence of persistent disease, imperfect treatment modalities which themselves may be associated with burden and adverse side effects, and persistent comorbidities including depression, anxiety, fatigue, and overweight. Perception of disease status may also play a role in QoL. In surgically remitted CD patients, there may be discordance between biochemical remission and perceived disease status [8]. Specifically, this study found that of those with self-identified persistence of disease, 65% were in fact biochemically remitted. This group had lower QoL scores than the concordant group who self-identified as in remission with biochemical evidence of remission. CD patients’ outlook on their condition, including their perception of choices and hope for change, has not been previously well described, despite the fact that these perceptions likely inform long term Qol. Patient outlook may be a modifiable target that if addressed, could improve long term patient well-being and outcomes. Aside from continuing progress in the development of new therapies for CD patients which can offer patients more objective choices in their treatment, other modalities should be considered. Prior work has shown that virtual educational programs improve acromegaly patients’ hope for improvement, perception of having choices in their treatment, and sense of loneliness [11]. Educational programs have also been shown to result in improved physical activity and sleep, and reduced pain levels in CS patients [20]. More work is needed to develop effective education programming tailored for CD patients to provide the appropriate support that these patients need. Difference in HCP and patient disease perceptions may also play a role in Cushing’s patients’ quality of life and outcomes. Among a cohort of patients who underwent surgical resection for Cushing’s, 32.4% reported not receiving information from their doctors about the recovery experience, despite the fact that all physicians surveyed reported giving information about the recovery process [9]. Furthermore, 16.1% of patients in this cohort reported that not enough medical professionals were familiar with the symptoms of Cushing’s. Recovery time was also reported to be longer by patients than providers [9]. Similarly, discordance was found between acromegaly patients and HCPs regarding reported severity of symptoms, with patients more frequently reporting symptoms as severe compared to HCPs, and many patients reporting symptoms which were not reported by HCPs [6]. Improving communication between HCP and patients may positively affect CD patient outlook and QoL. We identified a similar disparity between CD patients and HCP regarding care goals and unmet needs. 70% of patients surveyed considered QoL/mental health to be a top care goal, but only 22% of provider shared this goal. 59% of patients reported education/awareness as an unmet need, compared to 26% of HCPs. These findings support data shown by Acre et al. in which Cushing’s patients report a lack of symptom recognition by their providers [9]. HCPs should be aware that their patients may have different treatment priorities. Our finding that more HCPs reported patient anxiety living with CD compared to patients themselves needs further exploration. This could reflect inadequate communication between HCP and patient, or skewed HCP perceptions of CD. This, and other findings in our study should be viewed in light of the small cohort, and as such, needs confirmation in larger cohorts and more in-depth symptom assessments. Additional limitations of our study include lack of paired patient-HCP responses as the HCPs included were not providing care for this specific CD cohort. Since this was a pituitary educational forum, likely most or all patients who identified as having Cushing’s had CD. However, our survey did not specify the type of surgery patients underwent or the etiology of their Cushing’s. Additionally, we used multidisciplinary team agreed upon measures and not validated assessments. Further work should consider validating a tool to assess patient-provider discordances. Our findings may also be confounded by selection bias, given that the patients participating in our virtual education programs are more likely to be under the care of experts in the field and may not represent the attitudes of all patients living with CD. Finally, the included HCPs were representatives from a range of specialties with different levels of experience taking care of patients with CD which may also affect their responses. Our findings highlight the importance of understanding CD patients’ outlook and perspective in their condition, and that they may differ from their HCP. More than half of CD patients did not have a lot of hope for improvement and reported feeling alone, and many patients felt they had no choices in their treatment. QOL/mental health was the most commonly reported care goal for patients, which was not the case for HCPs. Comprehensive multidisciplinary care for CD patients should include mental health professionals with expertise in CD. Regular open communication between HCPs and CD patients will help bridge perception differences and facilitate personalized care, which will ultimately improve long-term outcomes for CD patients. Data availability The data that support the findings of this study are available from the authors upon request. References Newell-Price J, Bertagna X, Grossman AB, Nieman LK (2006) Cushing’s syndrome. 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Endocrine 53:199–209. https://doi.org/10.1007/s12020-015-0737-0 Article CAS PubMed Google Scholar Download references Acknowledgements The authors would like to thank the HCP and patient participants who attended the events, the MSK faculty, invited speakers, Leslie Edwin of Cushing’s Support and Research Foundation, Amy Edouard and the MSK CME team, and Recordati Rare Diseases, Inc., Amryt Pharma (previously Chiasma, Inc.), Crinetics, Sparrow Pharmaceuticals, Corcept Therapeutics, and Xeris Biopharma (previously Strongbridge Biopharma) for providing educational grants for these educational activities. Funding This research was funded in part through the NIH/NCI Cancer Center Support Grant P30 CA008748. Author information Authors and Affiliations Division of Endocrinology, Department of Medicine, Weill Cornell Medicine, New York, NY, USA Amanda Halstrom Department of Epidemiology and Biostatistics, Memorial Sloan Kettering Cancer Center, New York, NY, USA I.-Hsin Lin Multidisciplinary Pituitary & Skull Base Tumor Center, Memorial Sloan Kettering Cancer Center, New York, NY, USA Andrew Lin, Marc Cohen, Viviane Tabar & Eliza B. Geer Department of Neurology, Memorial Sloan Kettering Cancer Center, New York, NY, USA Andrew Lin Department of Neurosurgery, Memorial Sloan Kettering Cancer Center, New York, NY, USA Andrew Lin, Marc Cohen & Eliza B. Geer Head and Neck Service, Department of Surgery, Memorial Sloan Kettering Cancer Center, New York, NY, USA Marc Cohen & Viviane Tabar Department of Medicine, Memorial Sloan Kettering Cancer Center, New York, NY, USA Eliza B. Geer Contributions A.H. and E.B.G. wrote the manuscript text and prepared the figures. All authors reviewed the manuscript. Corresponding author Correspondence to Eliza B. Geer. Ethics declarations Competing interests The authors declare no competing interests. Ethical approval As an educational quality initiative project using de-identified data, it was determined that our project did not constitute human subjects research and thus did not require IRB oversight. Additional information Publisher's Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Supplementary Information Below is the link to the electronic supplementary material. Supplementary file1 (DOCX 15 kb) Rights and permissions Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. Reprints and permissions From https://link.springer.com/article/10.1007/s11102-024-01381-4
  7. Abstract Pituitary surgery, a critical intervention for various pituitary disorders, has sparked ongoing debates regarding the preference between endoscopic and microscopic transsphenoidal approaches. This systematic review delves into the outcomes associated with these techniques, taking into account the recent advancements in neurosurgery. The minimally invasive nature of endoscopy, providing improved visualization and reduced morbidity, stands in contrast to the well-established track record of the conventional microscopic method. Examining outcomes for disorders such as Cushing's disease and acromegaly, the review synthesizes evidence from Denmark, Bulgaria, and China. Noteworthy advantages of endoscopy encompass higher resection rates, shorter surgery durations, and fewer complications, endorsing its effectiveness in pituitary surgery. While emphasizing the necessity for prospective trials, the review concludes that endoscopic approaches consistently showcase favorable outcomes, influencing the ongoing discourse on the optimal surgical strategies for pituitary disorders. Introduction & Background Pituitary surgery is a critical intervention for various pituitary disorders, and the choice between endoscopic and microscopic transsphenoidal approaches has been a subject of ongoing debate within the medical community. This systematic review aims to explore and analyze the outcomes associated with endoscopic and microscopic transsphenoidal pituitary surgery. As advancements in surgical techniques continue to shape the field of neurosurgery, understanding the comparative effectiveness of these two approaches becomes imperative. The endoscopic approach, characterized by its minimally invasive nature, has gained popularity for pituitary surgery in recent years [1]. Proponents argue that it provides enhanced visualization, improved maneuverability, and reduced patient morbidity. On the other hand, traditional microscopic transsphenoidal surgery has been the conventional method for decades, known for its familiarity among surgeons and established track record [2]. Several studies have investigated the outcomes of these approaches in treating pituitary disorders, including but not limited to Cushing's disease, pituitary adenomas, and other tumors. For instance, a systematic review and meta-analysis by Chen et al. compared endoscopic and microscopic transsphenoidal surgery specifically for Cushing's disease, shedding light on the effectiveness of these approaches in managing this specific condition [3]. Moreover, Møller et al. reported promising results for endoscopic pituitary surgery based on the experiences of experienced microscopic pituitary surgeons, indicating a potential shift towards the adoption of the endoscopic technique [1]. Guo et al. conducted a meta-analysis comparing the effectiveness of microscopic and endoscopic surgery for treating pituitary disorders, contributing valuable insights into the overall efficacy of these approaches [4]. This review aims to contribute to the ongoing discourse on pituitary surgery by providing a comprehensive analysis of the outcomes associated with endoscopic versus microscopic transsphenoidal approaches. By synthesizing the existing evidence, we strive to offer valuable insights that can guide both clinicians and researchers in making informed decisions regarding the optimal surgical approach for pituitary disorders. Review Materials and methods This systematic review strictly adheres to the established Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines, employing a comprehensive approach to investigate the outcomes of endoscopic versus microscopic transsphenoidal pituitary surgery. The subsequent sections delineate the criteria for study inclusion, the search strategy utilized, and the methodology employed for data synthesis. Search Strategy We conducted a meticulous search across prominent electronic databases, including PubMed, Embase, and the Cochrane Library, to identify pertinent articles. Our search strategy comprised a combination of Medical Subject Headings (MeSH) terms and keywords related to pituitary surgery, encompassing both endoscopic and microscopic approaches. Boolean operators (AND, OR) were strategically employed to refine the search and identify studies meeting our predetermined inclusion criteria. The search string used for PubMed was ("Outcomes" OR "Treatment Outcome" OR "Surgical Outcome") AND ("Endoscopic Transsphenoidal Pituitary Surgery" OR "Endoscopic Pituitary Surgery" OR "Endoscopic Hypophysectomy") AND ("Microscopic Transsphenoidal Pituitary Surgery" OR "Microscopic Pituitary Surgery" OR "Microscopic Hypophysectomy" OR "Endoscopy"[Mesh] OR "Endoscopy, Surgical"[Mesh] OR "Transsphenoidal Hypophysectomy"[Mesh] OR "Microsurgery"[Mesh] OR "Microscopic Hypophysectomy"[Mesh]). Eligibility Criteria Stringent inclusion criteria were predefined to ensure the selection of high-quality and relevant studies. The included studies focused on investigating the outcomes of endoscopic versus microscopic transsphenoidal pituitary surgery. Only articles published in peer-reviewed journals within the timeframe from the inception of relevant databases until October 2023 were considered. Exclusion criteria encompassed studies on other interventions, those lacking sufficient data on surgical outcomes, and studies solely involving animal cells. Additionally, only studies in the English language with full-text availability were included, and gray literature was not considered eligible. Data Extraction and Synthesis Two independent reviewers meticulously screened titles and abstracts to identify potentially eligible studies. Subsequently, full-text articles were retrieved and evaluated for adherence to inclusion criteria. Discrepancies between reviewers were resolved through discussion and consultation with a third reviewer. Relevant data, including study design, patient characteristics, interventions, and surgical outcomes, were systematically extracted using a predefined data extraction form. Data Analysis A narrative synthesis approach was employed to summarize findings from included studies due to anticipated heterogeneity in study designs and outcome measures. Key themes and patterns related to the outcomes of endoscopic versus microscopic transsphenoidal pituitary surgery were identified and presented. Results Study Selection Process Following four database searches, 97 articles were initially identified. After eliminating eight duplicates, the titles and abstracts of the remaining 89 publications were evaluated. Subsequently, 17 potential studies underwent eligibility verification through a thorough examination of their full texts. Ultimately, three articles satisfied the inclusion criteria. No additional studies meeting the eligibility criteria were found during the examination of references in the selected articles. The entire process is visually depicted in the PRISMA flowchart (Figure 1). Figure 1: PRISMA flow diagram of the selection of studies for inclusion in the systematic review. PRISMA: Preferred Reporting Items for Systematic Reviews and Meta-Analyses Characteristics of Selected Studies Overall, three papers met the inclusion criteria. Two studies were randomized controlled trials (RCTs), one each from Bulgaria and China. One study was an observational study from Denmark. The main findings and characteristics of the included studies are mentioned in the following tables (Table 1 and Table 2). Author Year Country Study type Sample size No. of participants in the endoscopic group No. of participants in the microscopic group Main findings Møller et al. [1] 2020 Denmark Observational study 240 45 195 The study comparing endoscopic and microscopic transsphenoidal pituitary surgery revealed that the endoscopic technique exhibited advantages, achieving a higher rate of gross total resection (39% vs. 22%) and shorter surgery duration (86 minutes vs. 106 minutes). Complications within 30 days were lower with the endoscope (17% vs. 27%), and grade II complications or higher were significantly reduced (4% vs. 20%) compared to the microscopic approach. Pituitary function outcomes favored the endoscope, with fewer new deficiencies in the HPA axis (3% vs. 34%) and TSH-dependent deficiencies (15% vs. 38%). The HPG axis also showed better normalization in the endoscopic group (32% vs. 19%). Visual field impairment and postoperative improvement did not significantly differ between the two techniques. Overall, the findings suggest that endoscopic transsphenoidal pituitary surgery may offer superior outcomes compared to the microscopic approach, particularly in terms of resection rates and complication profiles. Vassilyeva et al. [5] 2023 Bulgaria RCT 83 43 40 The study compared endoscopic and microscopic transsphenoidal pituitary surgery in acromegaly patients, revealing comparable demographic profiles between the groups. Endoscopic surgery demonstrated advantages with shorter anesthesia and surgery times, as well as a reduced postoperative hospital stay. Complete tumor removal was more frequent with endoscopic adenomectomy, while microscopic surgery showed a higher rate of sub-total removal. Both techniques led to a tendency for somatic improvement, with more pronounced visual function improvement in the endoscopic group. Complications, such as liquorrhea and endocrine disorders, were generally low, with endoscopic surgery showing mainly mild complications. Disease remission rates were similar between the groups at various follow-up intervals. In conclusion, while both techniques proved effective in achieving remission, endoscopic surgery exhibited favorable outcomes in terms of efficiency and some aspects of complication profiles. Zhang et al. [6] 2021 China RCT 46 23 23 Endoscopic transsphenoidal pituitary surgery for the treatment of Cushing's disease showed comparable efficacy to microscopic transseptal pituitary surgery but with the added benefits of shorter operative time, reduced estimated blood loss, shorter hospital stays, and fewer complications. Table 1: Summary of the studies included in this systematic review. RCT: randomized controlled trial; HPA: hypothalamic-pituitary-adrenal; TSH: thyroid-stimulating hormone; HPG: hypothalamic-pituitary-gonadal Technique Møller et al. [1] Vassilyeva et al. [5] Zhang et al. [6] Male-to-female ratio (endoscopic) 25:20 17:26 13:10 Male-to-female ratio (microscopic) 107:88 16:24 12:11 Mean age in years (endoscopic) 61 43.26 55.6 Mean age in years (microscopic) 58 44.12 53.2 Functional tumors (endoscopic) 15 All functional All functional Non-functional tumors (endoscopic) 29 - - Functional tumors (microscopic) 69 All functional All functional Non-functional tumors (microscopic) 115 - - Microadenoma size (mm) (endoscopic) - 4 19 Macroadenoma size (mm) (endoscopic) - 39 4 Microadenoma size (mm) (microscopic) - 3 18 Macroadenoma size (mm) (microscopic) - 37 5 Mean operative time (min) (endoscopic) 86 142 108 Mean operative time (min) (microscopic) 106 176 174 Mean hospital stay (days) (endoscopic) - 5 2.8 Mean hospital stay (days) (microscopic) - 7 5.1 Postoperative complications (endoscopic) 2 15 3 Postoperative complications (microscopic) 39 10 8 Table 2: Summary of demographics, tumor characteristics, and postoperative outcomes of the studies included in this systematic review. The quality assessment of the selected studies was done using the Newcastle-Ottawa Quality Assessment Scale. All three studies included in this study turned out to be of high quality with a rating of 9/9 stars (Table 3). Author Selection Comparability Outcome Total stars Møller et al. [1] ★★★★ ★★ ★★★ ★★★★★★★★★ Vassilyeva et al. [5] ★★★★ ★★ ★★★ ★★★★★★★★★ Zhang et al. [6] ★★★★ ★★ ★★★ ★★★★★★★★★ Table 3: Quality assessment of the included studies using the Newcastle-Ottawa Quality Assessment Scale. Discussion This systematic review thoroughly assesses the effectiveness and results of endoscopic transsphenoidal pituitary surgery in comparison to microscopic transsphenoidal surgery, with a specific focus on pituitary adenomas, including Cushing's disease and acromegaly. The results contribute significant insights into the evolving landscape of pituitary surgery, highlighting the benefits and limitations of both surgical techniques. The selected studies offer valuable insights into the comparative outcomes. Møller et al.'s observational study in Denmark suggests that endoscopic surgery exhibits superior outcomes with higher gross total resection rates, shorter surgery duration, and fewer complications [1]. Vassilyeva et al.'s RCT in Bulgaria, focusing on acromegaly patients, indicates endoscopic advantages such as shorter anesthesia and surgery times, reduced postoperative stay, and comparable remission rates [5]. Zhang et al.'s RCT in China, specifically for Cushing's disease, suggests comparable efficacy with added benefits favoring endoscopy [6]. The endoscopic approach has been advocated for its panoramic visualization and superior mobility, which are crucial in resecting tumors while preserving normal structures [7,8]. Studies have shown a higher remission rate in endoscopic procedures for endocrine-active tumors, like growth hormone or adrenocorticotropic hormone (ACTH)-secreting adenomas, compared to the microscopic approach [9,10]. Patient comfort and recovery play a significant role in evaluating surgical methods. The endoscopic technique, by avoiding submucosal excision of nasal tissues, typically results in less postoperative pain and rhinological dysfunction. Studies, including ours, have reported shorter operative times and hospital stays with endoscopic surgery, attributed to fewer intraoperative and postoperative complications and a reduced need for wound management [11-13]. Safety is paramount to any surgical intervention. The endoscopic method has shown a decrease in common complications such as cerebrospinal fluid (CSF) leak, pituitary hormone dysfunction, and diabetes insipidus. Additionally, the endoscopic procedure exhibited fewer complications, which could be attributed to the enhanced visualization allowing for more precise tumor excision and preservation of vital structures [14-16]. In the context of acromegaly patients, the endoscopic technique has demonstrated increased radicality in tumor removal. Our review aligns with these findings, showing a higher rate of total tumor resection in endoscopic patients compared to those undergoing microscopic surgery. This improved outcome is likely due to better illumination and a wider angle of vision provided by endoscopic operations [5,17]. The endoscopic technique has shown a statistically significant improvement in visual function post surgery compared to the microscopic method. However, the frequency of certain postoperative complications, such as intraoperative liquorrhea, was higher in microscopic surgery. These differences underline the importance of the surgical technique in influencing the outcomes and complications of pituitary surgery [5,18]. Despite these findings, it is important to recognize the limitations inherent in these studies. Factors such as tumor size, density, and localization significantly influence surgical outcomes and procedure times. Additionally, the retrospective nature of many studies introduces potential biases, underscoring the need for more prospective, randomized trials for a comprehensive understanding of the long-term outcomes of these techniques. Conclusions This systematic review comparing endoscopic and microscopic transsphenoidal pituitary surgery outcomes indicates consistent evidence favoring the endoscopic approach. Notable studies from Denmark, Bulgaria, and China reveal superior results with endoscopic surgery, demonstrating higher resection rates, shorter surgery duration, and fewer complications. Endoscopy's benefits extend to patient comfort, as evidenced by shorter operative times and hospital stays. Safety considerations also support endoscopy, showing a decrease in common complications such as CSF leaks and hormonal dysfunction. Despite these strengths, the review underscores the need for prospective, randomized trials to address limitations and provide a comprehensive understanding of long-term outcomes. References Møller MW, Andersen MS, Glintborg D, Pedersen CB, Halle B, Kristensen BW, Poulsen FR: Endoscopic vs. microscopic transsphenoidal pituitary surgery: a single centre study. Sci Rep. 2020, 10:21942. 10.1038/s41598-020-78823-z Gao Y, Zhong C, Wang Y, et al.: Endoscopic versus microscopic transsphenoidal pituitary adenoma surgery: a meta-analysis. World J Surg Oncol. 2014, 12:94. 10.1186/1477-7819-12-94 Chen J, Liu H, Man S, et al.: Endoscopic vs. microscopic transsphenoidal surgery for the treatment of pituitary adenoma: a meta-analysis. Front Surg. 2022, 8:806855. 10.3389/fsurg.2021.806855 Guo S, Wang Z, Kang X, Xin W, Li X: A meta-analysis of endoscopic vs. microscopic transsphenoidal surgery for non-functioning and functioning pituitary adenomas: comparisons of efficacy and safety. Front Neurol. 2021, 12:614382. 10.3389/fneur.2021.614382 Vassilyeva N, Mena N, Kirov K, Diatlova E: Comparative effectiveness of endoscopic and microscopic adenoma removal in acromegaly. Front Endocrinol (Lausanne). 2023, 14:1128345. 10.3389/fendo.2023.1128345 Zhang T, Zhang B, Yuan L, Song Y, Wang F: Superiority of endoscopic transsphenoidal pituitary surgery to microscopic transseptal pituitary surgery for treatment of Cushing's disease. Rev Assoc Med Bras (1992). 2021, 67:1687-91. 10.1590/1806-9282.20210732 Yadav Y, Sachdev S, Parihar V, Namdev H, Bhatele P: Endoscopic endonasal trans-sphenoid surgery of pituitary adenoma. J Neurosci Rural Pract. 2012, 3:328-37. 10.4103/0976-3147.102615 Louis RG, Eisenberg A, Barkhoudarian G, Griffiths C, Kelly DF: Evolution of minimally invasive approaches to the sella and parasellar region. Int Arch Otorhinolaryngol. 2014, 18:S136-48. 10.1055/s-0034-1395265 Broersen LH, Biermasz NR, van Furth WR, de Vries F, Verstegen MJ, Dekkers OM, Pereira AM: Endoscopic vs. microscopic transsphenoidal surgery for Cushing's disease: a systematic review and meta-analysis. Pituitary. 2018, 21:524-34. 10.1007/s11102-018-0893-3 Torales J, Halperin I, Hanzu F, et al.: Endoscopic endonasal surgery for pituitary tumors. Results in a series of 121 patients operated at the same center and by the same neurosurgeon. Endocrinol Nutr. 2014, 61:410-6. 10.1016/j.endoen.2014.07.002 Zubair A, M Das J: Transsphenoidal hypophysectomy. StatPearls [Internet]. StatPearls Publishing, Treasure Island (FL); 2023. Pan X, Ma Y, Fang M, Jiang J, Shen J, Zhan R: Improvement in the quality of early postoperative course after endoscopic transsphenoidal pituitary surgery: description of surgical technique and outcome. Front Neurol. 2020, 11:527323. 10.3389/fneur.2020.527323 Aiyer RG, Upreti G: Endoscopic endo-nasal trans-sphenoidal approach for pituitary adenomas: a prospective study. Indian J Otolaryngol Head Neck Surg. 2020, 72:36-43. 10.1007/s12070-019-01725-8 Oertel J, Gaab MR, Linsler S: The endoscopic endonasal transsphenoidal approach to sellar lesions allows a high radicality: the benefit of angled optics. Clin Neurol Neurosurg. 2016, 146:29-34. 10.1016/j.clineuro.2016.04.016 Hanson M, Li H, Geer E, Karimi S, Tabar V, Cohen MA: Perioperative management of endoscopic transsphenoidal pituitary surgery. World J Otorhinolaryngol Head Neck Surg. 2020, 6:84-93. 10.1016/j.wjorl.2020.01.005 Qiao N: Endocrine outcomes of endoscopic versus transcranial resection of craniopharyngiomas: a system review and meta-analysis. Clin Neurol Neurosurg. 2018, 169:107-15. 10.1016/j.clineuro.2018.04.009 Nie D, Fang Q, Wong W, Gui S, Zhao P, Li C, Zhang Y: The effect of endoscopic transsphenoidal somatotroph tumors resection on pituitary hormones: systematic review and meta-analysis. World J Surg Oncol. 2023, 21:71. 10.1186/s12957-023-02958-2 Butenschoen VM, Schwendinger N, von Werder A, Bette S, Wienke M, Meyer B, Gempt J: Visual acuity and its postoperative outcome after transsphenoidal adenoma resection. Neurosurg Rev. 2021, 44:2245-51. 10.1007/s10143-020-01408-x From https://www.cureus.com/articles/213241-navigating-the-surgical-landscape-a-comprehensive-analysis-of-endoscopic-vs-microscopic-transsphenoidal-pituitary-surgery-outcomes#!/
  8. Abstract We investigated the impact of metformin on ACTH secretion and tumorigenesis in pituitary corticotroph tumors. The mouse pituitary tumor AtT20 cell line was treated with varying concentrations of metformin. Cell viability was assessed using the CCK-8 assay, ACTH secretion was measured using an ELISA kit, changes in the cell cycle were analyzed using flow cytometry, and the expression of related proteins was evaluated using western blotting. RNA sequencing was performed on metformin-treated cells. Additionally, an in vivo BALB/c nude xenograft tumor model was established in nude mice, and immunohistochemical staining was conducted for further verification. Following metformin treatment, cell proliferation was inhibited, ACTH secretion decreased, and G1/S phase arrest occurred. Analysis of differentially expressed genes revealed cancer-related pathways, including the MAPK pathway. Western blotting confirmed a decrease in phosphorylated ERK1/2 and phosphorylated JNK. Combining metformin with the ERK1/2 inhibitor Ulixertinib resulted in a stronger inhibitory effect on cell proliferation and POMC (Precursors of ACTH) expression. In vivo studies confirmed that metformin inhibited tumor growth and reduced ACTH secretion. In conclusion, metformin inhibits tumor progression and ACTH secretion, potentially through suppression of the MAPK pathway in AtT20 cell lines. These findings suggest metformin as a potential drug for the treatment of Cushing's disease. Introduction Pituitary neuroendocrine tumors (PitNETs) are common intracranial tumors with an incidence of 1/1000, and pituitary corticotroph tumors (corticotroph PitNETs) account for approximately 15% of all PitNETs. Most corticotroph PitNETs are functional tumors with clinical manifestations of Cushing's disease characterized by central obesity, hypertension, diabetes mellitus, and psychosis (Cui et al., 2021). The increased cortisol due to the overproduction of adrenocorticotropic hormone (ACTH) significantly reduces the overall quality of survival and life expectancy of patients (Sharma et al., 2015; Barbot et al., 2018). Currently, treatment of corticotroph PitNETs mainly relies on surgery resection, pharmacologic therapy or radiotherapy may be considered for patients with residual tumors or those who are unable to undergo surgery. While several agents, such as cabergoline and pasireotide, are clinically approved, the effect is unsatisfactory, and potentially serious side effects exist. Therefore, there is an urgent need to develop novel therapeutic drugs for corticotroph PitNETs. Metformin is a biguanide hypoglycemic agent for the treatment of type 2 diabetes. In addition to its hypoglycemic effect, numerous studies identified the therapeutic role of metformin in the prevention and treatment of various tumors including small cell lung cancer, colorectal cancer, breast cancer, ovarian cancer, and neuroendocrine tumors (Lu et al., 2022; Kamarudin et al., 2019; Wang et al., 2019; Thakur et al., 2019), making metformin a promising adjuvant drug in the therapy of cancers. Besides, it has been reported that metformin improves metabolic and clinical outcomes in patients treated with glucocorticoids. However, to date, limited studies explore the potential anti-cancer effect of metformin in corticotroph PitNETs. Recent studies report the use of metformin for blood glucose and body weight control in patients with Cushing's disease (Ceccato et al., 2015), while the role of metformin on ACTH secretion and tumor growth in corticotroph PitNETs remains to be elucidated. In the current study, we investigated the effect of metformin in corticotroph PitNETs and performed RNA-sequencing to identify the potential mechanisms of metformin. We found that metformin inhibited cell proliferation and ACTH secretion of AtT20 cells in a dose-dependent manner. Besides, metformin induced cell cycle arrest via decreased ERK1/2 phosphorylation and increased P38 phosphorylation. Our results revealed that metformin is a potential drug for corticotroph PitNET therapy. Section snippets Cell culture The ACTH-secreting mouse pituitary tumor cell line AtT-20 was purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA). Cells were cultured in F-12K medium (ATCC; Catalog No. 30-2004), supplemented with 15% fetal bovine serum (FBS; Gibco), and 2.5% horse serum (Gibco) as suggested. AtT20 cells were cultured in a humidified incubator at 37 °C in 5% CO2. Reagents and drugs Metformin and Ulixertinib were purchased from MedChemExpress (MCE), Metformin was dissolved in sterile H2O and prepared as a Results Metformin inhibits cell proliferation and ACTH secretion, and leads to cell cycle arrest in AtT20 cells. We used CCK-8 assay to detect the cell viability of AtT20 cells after treatment with different concentrations of metformin at 24 h, 48 h, and 72 h. The results showed that metformin significantly inhibited the proliferation of AtT20 cells in a dose-dependent manner (Fig. 1A). Similarly, prolonged (6 days) treatment of AtT20 cells with a lower concentration (400 μM) of metformin also inhibited Discussion Metformin, acting by binding to PEN2 and initiating the subsequent AMPK signaling pathway in lysosomes, is the most commonly used oral hypoglycemic agent (Hundal et al., 2000; Ma et al., 2022). Previous reports demonstrated metformin as a potential anti-tumor agent in cancer therapy (Evans et al., 2005). Metformin, either alone or in combination with other drugs, has been shown to reduce cancer risk in a variety of tumors including pituitary neuroendocrine tumors (PitNETs) (Thakur et al., 2019; Conclusion Our study demonstrated that metformin suppressed cell proliferation and decreased ACTH secretion in AtT20 cells via the MAPK pathway. Our results revealed that metformin is a potential anti-tumor drug for the therapy of corticotroph PitNETs, which deserves further study. Funding This study was supported by the National Natural Science Foundation of China (82072804, 82071559). CRediT authorship contribution statement Yingxuan Sun: Conceptualization, Formal analysis, Investigation, Writing – original draft, Writing – review & editing. Jianhua Cheng: Data curation, Formal analysis, Visualization, Writing – original draft, Writing – review & editing. Ding Nie: Formal analysis, Writing – review & editing. Qiuyue Fang: Data curation, Formal analysis, Writing – review & editing. Chuzhong Li: Conceptualization, Supervision, Writing – original draft, Writing – review & editing, Funding acquisition. Yazhuo Zhang: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgement We thank Mr. Hua Gao (Cell Biology Laboratory, Beijing Neurosurgical Institute, China) for support with the techniques. References (30) K. Jin et al. Metformin suppresses growth and adrenocorticotrophic hormone secretion in mouse pituitary corticotroph tumor AtT20 cells Mol. Cell. Endocrinol. (2018) R. Krysiak et al. The effect of metformin on prolactin levels in patients with drug-induced hyperprolactinemia Eur. J. Intern. Med. (2016) X. Liu et al. Combination treatment with bromocriptine and metformin in patients with bromocriptine-resistant prolactinomas: pilot study World neurosurgery (2018) J. Sinnett-Smith et al. Metformin inhibition of mTORC1 activation, DNA synthesis and proliferation in pancreatic cancer cells: dependence on glucose concentration and role of AMPK Biochem. Biophys. Res. Commun. (2013) C.R. Triggle et al. Metformin: is it a drug for all reasons and diseases? Metab., Clin. Exp. (2022) J.C. Wang et al. Metformin inhibits metastatic breast cancer progression and improves chemosensitivity by inducing vessel normalization via PDGF-B downregulation J. Exp. Clin. Cancer Res. : CR (2019) J. An et al. Metformin inhibits proliferation and growth hormone secretion of GH3 pituitary adenoma cells Oncotarget (2017) M. Barbot et al. Diabetes mellitus secondary to Cushing's disease Front. Endocrinol. (2018) F. Ceccato et al. Clinical use of pasireotide for Cushing's disease in adults Therapeut. Clin. Risk Manag. (2015) M. Cejuela et al. Metformin and breast cancer: where are we now? Int. J. Mol. Sci. (2022) From https://www.sciencedirect.com/science/article/abs/pii/S0303720723002915
  9. Here, we report the first adult case of pancreatic yolk sac tumor with ectopic adrenocorticotropic hormone (ACTH) syndrome. The patient was a 27-year-old woman presenting with abdominal distension, Cushingoid features, and hyperpigmentation. Endogenous Cushing’s syndrome was biochemically confirmed. The ACTH level was in the normal range, which raised the suspicion of ACTH precursor-dependent disease. Elevated ACTH precursors were detected, supporting the diagnosis of ectopic ACTH syndrome. Functional imaging followed by tissue sampling revealed a pancreatic yolk sac tumor. The final diagnosis was Cushing’s syndrome due to a yolk sac tumor. The patient received a steroidogenesis inhibitor and subsequent bilateral adrenalectomy for control of hypercortisolism. Her yolk sac tumor was treated with chemotherapy and targeted therapy. Cushing’s syndrome secondary to a yolk sac tumor is extremely rare. This case illustrated the utility of ACTH precursor measurement in confirming an ACTH-related pathology and distinguishing an ectopic from a pituitary source for Cushing’s syndrome. Introduction Ectopic adrenocorticotrophic hormone (ACTH) syndrome, also termed paraneoplastic Cushing’s syndrome, can be caused by the secretion of ACTH and/or ACTH precursors from ectopic tumors. The tumors concerned secrete ACTH precursors, including unprocessed proopiomelanocortin (POMC) and POMC-derived peptides, owing to the altered post-translational processing of POMC (1). These tumors are associated with intense hypercortisolism and various complications, such as hypertension, hyperglycemia, osteoporosis, infection risks, and thrombotic tendencies (2). Distinguishing ectopic from pituitary-dependent Cushing’s syndrome is often challenging. The two conditions are classically distinguished by their variable responses to dynamic endocrine tests, including the high-dose dexamethasone suppression test, the corticotrophin-releasing-factor (CRF) test, and the desmopressin test (3). Pituitary imaging may sometimes provide a diagnosis if a pituitary macroadenoma is identified at this juncture. The gold standard for diagnosing pituitary Cushing’s is a positive inferior petrosal sinus sampling (IPSS) result. The measurement of ACTH precursors is reported to have diagnostic value in this scenario (4). The most common source of ectopic ACTH is intrathoracic tumors, including bronchial carcinoid and small cell lung cancers. Other possible sources include gut neuroendocrine tumors and medullary thyroid cancer. Recognizing the potential causes of ectopic ACTH syndrome is essential as this provides guidance in locating the causative tumor and allows tumor-directed therapies. A yolk sac tumor as a cause of ectopic ACTH syndrome has only been reported in a 2-year-old child but not in adults (5). Here, we present a case of a 27-year-old Chinese woman who had Cushing’s syndrome due to ectopic ACTH precursor production from a pancreatic yolk sac tumor. Case description A 27-year-old Chinese woman, who had unremarkable past health and family history, presented with right upper quadrant abdominal pain and nausea in early 2020. Abdominal ultrasonography was unrevealing. A few months later, she developed Cushingoid features and oligomenorrhea. At presentation, her blood pressure was 160/95 mmHg, body weight was 65.6 kg, and body mass index was 23.2 kg/m2. She had a moon face, hirsutism, proximal myopathy, bruising, thinning of the skin, and acne. She also had hyperpigmentation on the nails and knuckles of both hands (Figure 1). Figure 1 Figure 1. Cushingoid features at presentation include moon face, acne, thin skin, and easy bruising. Hyperpigmentation on the nails and knuckles was also noted. Diagnostic assessments Her 9 am and 9 pm cortisol were both >1,700 nmol/L. Her 24-h urine-free cortisol was beyond the upper measurable limit at >1,500 nmol/L. Her serum cortisol was 759 nmol/L after a 1 mg overnight-dexamethasone suppression test, confirming endogenous Cushing’s syndrome. The morning ACTH was 35 pg/mL (upper limit of normal is 46 pg/mL). After excluding a high dose-hook effect, her blood sample was concomitantly sent for ACTH measurement using two different platforms to eliminate possible interference, which might cause a falsely low ACTH reading. ACTH was 19 pg/mL (upper limit of normal is 46 pg/mL) using an IMMULITE 2000 XPI, Siemens Healthineers, Erlangen, Germany, and 17 pg/mL (reference range: 7–63 pg/mL) using a Cobas e-801, Roche Diagnostics, Indianapolis, IN, United States, therefore verifying the ACTH measurement. In view of this being ACTH-dependent Cushing’s syndrome, a high-dose-dexamethasone suppression test (HDDST) was performed, and her cortisol was not suppressed at 890 nmol/L, with ACTH 42 pg/mL. The serum cortisol day profile showed a mean cortisol level of >1,700 nmol/L (i.e., higher than the upper measurable limit of the assay) and an ACTH of 17 pg/mL. A CRF test using 100 μg of corticorelin showed less than a 50% rise in ACTH and no rise in cortisol levels (Supplementary Table S1). She suffered from multiple complications of hypercortisolism, including thoracic vertebral collapse with back pain, diabetes mellitus (HbA1c 6.7% and fasting glucose 7.6 mmol/L), and hypokalemic hypertension, with a lowest potassium level of 2.3 mmol/L. The rapid onset of intense hypercortisolism and refractory hypokalemia, as well as the responses in the HDDST and CRF tests raised the suspicion of ectopic ACTH syndrome. Tumor markers were measured. Alpha-fetoprotein (AFP) was markedly raised at 33,357 ng/mL (reference range: <9 ng/mL). Beta-human chorionic gonadotropin (beta-hCG) was not elevated. Carcinoembryonic antigen (CEA) was 4.0 ng/mL (reference range: <3 ng/mL) and CA 19–9 was 57 U/mL (reference range: <37 U/mL). The marked hyperpigmentation in the context of normal ACTH levels pointed to the presence of an underlying tumor producing circulating ACTH precursors. Hence, magnetic resonance imaging (MRI) of the pituitary gland was not performed at this juncture. ACTH precursors were measured using a specialized immunoenzymatic assay (IEMA) employing in-house monoclonal antibodies against the ACTH region and the gamma MSH region. Both monoclonal antibodies have to bind to these regions in POMC and pro-ACTH to create a signal. The patient had a level of 4,855 pmol/L (upper limit of normal is 40 pmol/L) (6). This supported Cushing’s syndrome from an ectopic source secondary to an excess in ACTH precursors. Localization studies were arranged to identify the source of ectopic ACTH precursors. Computed tomography (CT) of the thorax did not show any significant intrathoracic lesion but incidentally revealed a pancreatic mass. Dedicated CT of the abdomen confirmed the presence of a 7.9 × 5.6 cm lobulated mass in the pancreatic body; the adrenal glands were unremarkable. 18-FDG and 68Ga-DOTATATE dual-tracer positron-emission tomography-computed tomography (PET-CT) showed that the pancreatic mass was moderately FDG-avid and non-avid for DOTATATE (Supplementary Figure S1). Multiple FDG-avid nodal metastases were also present, including left supraclavicular fossa lymph nodes. Fine needle aspiration of the left supraclavicular fossa lymph node yielded tumor cells featuring occasional conspicuous nucleoli, granular coarse chromatin, irregular nuclei, and a high nuclear-to-cytoplasmic ratio. Mitotic figures were infrequent. On immunostaining, the tumor cells were positive for cytokeratin 7 and negative for cytokeratin 20. Focal expression of CDX-2, chromogranin, and synaptophysin was noted. They were negative for TTF-1, GCDPF, Gata 3, Pax-8, CD56, ACTH, inhibin, and S-100 protein. Further immunostaining was performed in view of highly elevated AFP. The tumor cells expressed AFP, Sall4, and MNF-116. They were negative for c-kit, calretinin, Melan A and SF-1. Placental ALP (PLAP) was weak and equivocal. The features were in keeping with a yolk sac tumor. Therapeutic intervention and outcome The patient had significant hypokalemic hypertension requiring losartan 100 mg daily, spironolactone 100 mg daily, and a potassium supplement of 129 mmol/day. Co-trimoxazole was given for prophylaxis against Pneumocystis jirovecii pneumonia. Metyrapone was started and up-titrated to 1 gram three times per day. However, in view of persistent hypercortisolism, with urinary free cortisol persistently above the upper measurable limit of the assay, bilateral adrenalectomy was performed. The tumor was mainly in the periadrenal soft tissue, with vascular invasion. The tumor formed cords, nests, and ill-defined lumen (Figure 2). The tumor cells were polygonal and contained pale to eosinophilic cytoplasm and pleomorphic nuclei, some with large nucleoli. Mitosis was present while tumor necrosis was not obvious. The stroma was composed of vascular fibrous tissue, with minimal inflammatory reaction. Immunohistochemical study showed that the tumor was positive for cytokeratin 7, MNF-116, AFP, and glypican-3, and also positive for Sall4 and HNF1β. The tumor cells were negative for cytokeratin 20, PLAP, CD30, negative for neuroendocrine markers including S100 protein, synaptophysin, chromogranin, and also negative for Melan-A, inhibin, and ACTH. Histochemical study for Periodic acid–Schiff–diastase (PAS/D) showed no cytoplasmic zymogen granules like those of acinar cell tumor. The features were compatible with yolk sac tumor. She was put on glucocorticoid and mineralocorticoid replacements post-operatively. Figure 2 Figure 2. Histology and immunohistochemical staining pattern of tumor specimen. (A) HE stain x 40 showing tumor cells in the soft tissue and peritoneum. (B) HE × 400 showing that the tumor forms cords, nests, and ill-formed lumen in the vascular stroma. The tumor cells are polygonal with pale cytoplasm and pleomorphic nuclei. (C) PAS/D stain showing no cytoplasmic zymogen granules. (D) Tumor is diffusely positive for cytokeratin 7. (E) Tumor is positive for AFP. (F) Tumor is positive for glypican-3. (G) Tumor is diffusely positive for HNF1β. (H) Tumor is diffusely positive for SALL4. Regarding her oncological management, she received multiple lines of chemotherapy, but the response was poor. Due to limited access to the ACTH precursor assay, serial measurement was unavailable. Treatment response was monitored by repeated imaging and monitoring of AFP. Figure 3 shows a timeline indicating the key events of the disease, showing the trends of the AFP and cortisol levels. Apart from (i) bleomycin, etoposide, and platinum, she was sequentially treated with (ii) etoposide, ifosfamide with cisplatin, and (iii) palliative gemcitabine with oxaliplatin. Next-generation sequencing showed a BRAF V600E mutation, for which (iv) dabrafenib and trametinib were given. Unfortunately, the disease progressed, and the patient succumbed approximately one year after the disease was diagnosed. Figure 3 Figure 3. Timeline with serial cortisol and alpha-fetoprotein levels from diagnosis to patient death. Discussion This case demonstrates the diagnostic value of ACTH precursor measurement in the diagnosis of ectopic Cushing’s syndrome. ACTH precursors are raised in all ectopic tumors responsible for Cushing’s syndrome and could be useful in distinguishing ectopic from pituitary Cushing’s syndrome (4). Moreover, Cushing’s syndrome due to a yolk sac tumor has been reported only once in a pediatric case, and this is the first adult case reported in the literature (5). POMC is sequentially cleaved in the anterior pituitary into pro-ACTH and then into ACTH, which is released into the circulation and binds to ACTH receptors in the adrenal cortex, leading to glucocorticoid synthesis (5, 7). Due to incomplete processing, ACTH precursors are found in normal subjects at a concentration of 5–40 pmol/L (6). Pituitary tumors are traditionally well-differentiated and can also relatively efficiently process ACTH precursors. However, this processing is less efficient in ectopic tumors that cause Cushing’s syndrome (8). Some less differentiated pituitary macroadenomas can secrete ACTH precursors into the circulation; however, these tumors are diagnosed by imaging and so do not, in general, cause problems with differential diagnosis (9). Measurement of ACTH precursors by immunoradiometric assay (IRMA) was first described by Crosby et al. (10). The assay utilized monoclonal antibodies specific for ACTH and the other binding gamma-MSH. The assay only detects peptides expressing both epitopes and therefore measures POMC and pro-ACTH. The assay does not cross-react with other POMC-derived peptides such as beta-lipotropin, ACTH, and N-POMC. Oliver et al. demonstrated that, compared to the pituitary adenomas in Cushing’s disease, all ectopic tumors responsible for Cushing’s syndrome in their study produce excessive POMC and pro-ACTH (4). The excessive production of ACTH precursors may reflect neoplasm-induced modification and amplification of POMC production. It is suggested that POMC binds to and activates the ACTH receptor because it contains the ACTH amino-acid sequence, or it is cleaved to ACTH in the adrenal glands to cause hypercortisolism (5) (Figure 4). Moreover, cleavage of POMC may produce peptides that exert mitogenic actions on adrenal cells and lead to adrenocortical growth. Outside the adrenal tissue, excessive ACTH precursors in Cushing’s syndrome caused by ectopic tumors can lead to marked hyperpigmentation. Both hypercortisolism and hyperpigmentation were observed in the reported case. Figure 4 Figure 4. Postulated pathological mechanism of ectopic ACTH precursors. In patients with ACTH-dependent Cushing’s syndrome, ectopic tumors should be distinguished from pituitary tumors. The HDDST, at a cut-off of 50% cortisol suppression, gives a sensitivity of 81% and a specificity of 67% for pituitary dependent Cushing’s syndrome (11). The CRF test provides 82% sensitivity and 75% specificity for pituitary disease (8). IPSS is the gold standard in distinguishing pituitary from ectopic tumors in Cushing’s syndrome. Utilization of CRF-stimulated IPSS provides 93% sensitivity and 100% specificity for pituitary disease. It also allows correct lateralization in 78% of patients with pituitary tumors. However, it is only available in specialized centers. In a retrospective cohort, the ACTH precursor level distinguished well between Cushing’s disease and ectopic ACTH syndrome (4). With a cut-off of 100 pmol/L, the test achieved 100% sensitivity and specificity for ectopic ACTH syndrome. More recently, this assay has been used to diagnose patients with occult ectopic ACTH syndrome, with ACTH precursors above 36 pmol/L (8). Unfortunately, the immunoassay for ACTH precursor measurement utilizes in-house monoclonal antibodies, which are not widely available. Cross-reactivity of POMC in commercially available ACTH assays ranges from 1.6% to 4.7% (12). In cases of ectopic tumors causing Cushing’s syndrome with markedly raised ACTH-precursors and intense hypercortisolism, the cross-reactivity would give significantly high ‘ACTH’ measurements to suggest an ACTH-related pathology. The degree of cross-reactivity, which is variable, should ideally be provided by the assay manufacturer as it affects result interpretation. Lower levels of ACTH precursor production might not be detected, especially by assays with low precursor cross-reactivity. Clinical vigilance is crucial in reaching the correct diagnosis. In patients with marked hypercortisolism and a normal ACTH concentration, like in this case, the measurement of ACTH precursors would allow the accurate diagnosis of Cushing’s syndrome caused by ACTH precursors. Ectopic tumors causing Cushing’s syndrome are associated with more intense hypercortisolism than Cushing’s disease (11). However, due to variable cross-reactivity, commercial ACTH assays might not accurately detect the excessive ACTH precursors responsible for the clinical syndrome. For this reason, ACTH measurements in these two conditions can significantly overlap and may not differentiate between ectopic and pituitary diseases (4). On the other hand, the more specific POMC assay described in 1996, which does not cross-react with pro-ACTH, has a low sensitivity of 80% for ectopic Cushing’s syndrome and is not now available (13). Hence, the ACTH precursor assay used in this reported case, which detects POMC and pro-ACTH, appears to provide the best diagnostic accuracy from the available literature. Serial measurement of ACTH precursors may play a role in monitoring the treatment response in an ACTH precursor secreting tumor. In the case of ectopic ACTH secretion, the corticotropic axis is slowed down and ACTH is almost exclusively of paraneoplastic origin. Immunotherapy is known to alter the functioning of the hypothalamic–pituitary corticotropic axis; however, its effect on ectopic secretions is not known. More data is required before the role of ACTH precursor measurement for disease monitoring in these scenarios can be ascertained. The incidence of endogenous Cushing’s syndrome is reported to be 2 to 4 per million people per year (14). Ectopic sources of Cushing’s syndrome are responsible for 9 to 18% of these cases. Typical sources of these ectopic tumors include bronchial carcinoid tumors, small-cell lung cancer, and gut neuroendocrine tumors. Notably, germ cell tumors, including teratomas, ovarian epithelial tumors, and ovarian endometrial tumors, are also possible ectopic sources of Cushing’s syndrome. The histological diagnosis of germ cell tumor in a non-genital site is challenging, especially for the poorly differentiated, or with somatic differentiation. Immunostaining, chromosomal, or genetic study are very important in confirming the diagnosis. AFP elevation in our case limited the differential diagnoses to germ cell tumors/yolk sac tumors, hepatocellular carcinoma, and rare pancreatic tumors. The specimen was biopsied from the retroperitoneum, and the morphology was a dominant trabecular pattern or a hepatoid pattern. It showed diffuse positive immunostaining for cytokeratin, AFP, and glypican-3. It was also diffusely and strongly positive for HNF1β and SALL4, supporting the diagnosis of yolk sac tumor. Both HNF1β and SALL4, being related with the expression of genes associated with stem cells or progenitor cells, are used as sensitive and specific markers for germ cell tumors/yolk sac tumors (15, 16). Staining related to pancreatic acinar cell carcinoma and neuroendocrine tumor were performed. PAS/D staining showed a lack of zymogen granules. A lack of nuclear β-catenin positivity was shown. Staining for neuroendocrine markers, including chromogranin and synaptophysin, was negative. Bcl-10 and trypsin were not available in the local setting. Cushing’s syndrome due to a yolk sac tumor was reported only once, in a 2-year-old child (5). The abdominal yolk sac tumor was resistant to cisplatin, with rapid disease progression, and the patient succumbed 1.5 years after initial presentation. Yolk sac tumor in the pancreas is also rare, with only 4 cases reported so far. The first case was reported in a 57-year-old woman with an incidentally detected abdominal mass (17). The tumor stained positive for AFP, PLAP, and CEA. The second case was a 70-year-old asymptomatic woman with histology showing a group of tumor cells with features of a yolk sac tumor, and another group showing features of pancreatic ductal adenocarcinoma with mucin production, suggesting a yolk sac tumor derived from pancreatic ductal adenocarcinoma (18). The tumor showed partial positivity for AFP, Sall4, glypican-3, and cytokeratin 7, as found in our case, while MNF-116 and PLAP staining results were not described. The third was in a 33-year-old man with a solitary pancreatic head mass with obstructive jaundice (19). The patient had undergone Whipple’s procedure followed by cisplatin-based chemotherapy, resulting in at least 5 years of disease remission. The latest reported case was in a 32-year-old man presenting with abdominal pain (20). Notably, initial imaging showed diffuse enlargement of the pancreas and increased FDG uptake without a distinct mass. Reassessment imaging 11 months later showed a 13 cm pancreatic mass. The initial imaging findings suggested initial intraductal growth of the tumor, as reported in some subtypes of pancreatic carcinoma. None of the reported cases of adult pancreatic yolk sac tumors were associated with abnormal hormone secretion. We reported the first adult case of pancreatic yolk sac tumor with ectopic ACTH syndrome. The case represents an overlap of two rarities. It demonstrates that pancreatic yolk sac tumor is a possible cause of ectopic ACTH syndrome. Conclusion ACTH precursor measurement helps to distinguish ectopic ACTH syndrome from Cushing’s disease. The test has superior diagnostic performance and is less invasive than IPSS. Nonetheless, the limited availability of the assay may restrict its broader use in patient management. We describe the first adult case of pancreatic yolk sac tumor with ACTH precursor secretion resulting in Cushing’s syndrome. This adds to the list of origins of ectopic ACTH syndrome in adults. Data availability statement The original contributions presented in the study are included in the article/Supplementary material, further inquiries can be directed to the corresponding author. Ethics statement Written informed consent was obtained from the individual to publish any potentially identifiable images or data in this article. Author contributions JC wrote the manuscript. JC, CW, WC, AW, KW, and PT researched the data. WC, AL, EL, YW, KT, KL, and CL critically reviewed and edited the manuscript. DL initiated and conceptualized this case report and is the guarantor of this work. All authors contributed to the article and approved the submitted version. Funding The author(s) declare that no financial support was received for the research, authorship, and/or publication of this article. Conflict of interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Publisher’s note All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher. Supplementary material The Supplementary material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fmed.2023.1246796/full#supplementary-material References 1. Stewar, PM, Gibson, S, Crosby, SR, Pennt, R, Holder, R, Ferry, D, et al. ACTH precursors characterize the ectopic ACTH syndrome. Clin Endocrinol. (1994) 40:199–204. doi: 10.1111/j.1365-2265.1994.tb02468.x PubMed Abstract | CrossRef Full Text | Google Scholar 2. Young, J, Haissaguerre, M, Viera-Pinto, O, Chabre, O, Baudin, E, and Tabarin, A. 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SALL4 expression in germ cell and non-germ cell tumors: a systematic immunohistochemical study of 3215 cases. Am J Surg Pathol. (2014) 38:410–20. doi: 10.1097/PAS.0000000000000116 PubMed Abstract | CrossRef Full Text | Google Scholar 17. Zhang, B, Gao, S, Chen, Y, and Wu, Y. Primary yolk sac tumor arising in the pancreas with hepatic metastasis: a case report. Korean J Radiol. (2010) 11:472–5. doi: 10.3348/kjr.2010.11.4.472 PubMed Abstract | CrossRef Full Text | Google Scholar 18. Yonemaru, J, Takahashi, M, Nara, S, Ichikawa, H, Ishigamori, R, Imai, T, et al. A yolk sac tumor of the pancreas and derived xenograft model effectively responded to VIP chemotherapy. Pancreatology. (2020) 20:551–7. doi: 10.1016/j.pan.2019.12.021 PubMed Abstract | CrossRef Full Text | Google Scholar 19. Galanis, I, Floros, G, Simou, M, Kyriakopoulos, G, and Stylianidis, G. An extremely rare case of a primary pancreatic yolk sac tumor. Cureus. (2022) 14:e26007. doi: 10.7759/cureus.26007 PubMed Abstract | CrossRef Full Text | Google Scholar 20. Sui, H, Zhu, Z, Li, Z, and Luo, Y. Primary pancreatic yolk sac tumor presenting as diffusely enlarged pancreas in initial 18F-FDG PET/CT. Clin Nucl Med. (2020) 45:483–6. doi: 10.1097/RLU.0000000000003038 PubMed Abstract | CrossRef Full Text | Google Scholar Keywords: Cushing’s syndrome, ectopic ACTH syndrome, yolk sac tumor, pancreatic tumor, ACTH precursor Citation: Chang JYC, Woo CSL, Chow WS, White A, Wong KC, Tsui P, Lee ACH, Leung EKH, Woo YC, Tan KCB, Lam KSL, Lee CH and Lui DTW (2023) Cushing’s syndrome caused by ACTH precursors secreted from a pancreatic yolk sac tumor in an adult—a case report and literature review. Front. Med. 10:1246796. doi: 10.3389/fmed.2023.1246796 Received: 18 July 2023; Accepted: 20 November 2023; Published: 05 December 2023. Edited by: Alessandro Vanoli, University of Pavia, Italy Reviewed by: Petar Brlek, St. Catherine Specialty Hospital, Croatia Wafa Alaya, Hospital University Tahar Sfar, Tunisia Copyright © 2023 Chang, Woo, Chow, White, Wong, Tsui, Lee, Leung, Woo, Tan, Lam, Lee and Lui. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. *Correspondence: David Tak Wai Lui, dtwlui@hku.hk Disclaimer: All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article or claim that may be made by its manufacturer is not guaranteed or endorsed by the publisher. From https://www.frontiersin.org/articles/10.3389/fmed.2023.1246796/full
  10. Lydia, a 28-year-old Florida resident, wife, and mother of two, first noticed a drastic increase in her weight around Easter of 2022 in a family photo. She was shocked by how different she looked despite not making any drastic changes to her diet. “While those I loved would say ‘you look beautiful’, to me I looked like a completely different person,” recalled Lydia. When Lydia asked her mother, Jeanne, if she had noticed her weight gain, her mother observed that some days Lydia’s face looked swollen. They both recognized that this was not normal, and decided, like many pituitary patients, to make an appointment with a primary care provider. “I remember her saying to me ‘something is wrong with me’ and ‘something is not right’”, recalled Jeanne. Lydia’s weight gain was most noticeable in her face and around her abdomen. “She was exercising all the time and trying to watch what she ate and cut down on sugars,” said Jeanne. “But she kept putting on more weight. We knew something was not right.” Lydia scheduled the first of what would be many doctor appointments hoping for answers. Her primary care provider recognized that her rapid weight gain was abnormal and ordered standard blood work. When that blood work came back normal, her doctor referred her to an endocrinologist and to her OBGYN. In addition to her weight gain, Lydia had begun developing other symptoms including excessive sweating day and night, severe acne, hair loss, hair gain on her face, insomnia, thin skin, and brittle nails. “The worst symptom was the constant feeling of fight or flight,” recalled Lydia. “I always felt on edge and was letting things bother me.” Lydia would later learn that this feeling was caused by the drastic increase of cortisol in her body. When Lydia first met with her OBGYN to address her weight gain and the overall feeling that something was wrong with her body, her concerns were quickly dismissed. “He told me ‘You’re almost 30 and you’ve had two kids, no wonder you feel the way that you do,’” said Lydia. “He blew me off and told me that I needed more diet and exercise. He didn’t order other tests.” Figure illustrates the drastic physical changes and symptoms caused by a pituitary tumor and Cushing’s disease. (Medical illustration by Mark Schornak, MS, CMI) A couple of months later, Lydia went to see an endocrinologist. Despite watching her calories and exercising almost every day of the week, she had gained more weight and felt more miserable. When her labs came back, Lydia’s cortisol levels were so high that the endocrinologist thought there had been a lab error. A 24-hour urine test confirmed that Lydia’s cortisol levels were off the charts. “I was in full panic mode at this point,” said Lydia. Lydia could not get back in to see her endocrinologist in a timely manner, so she ended up back at her primary care provider’s office. Her primary care provider suggested that it could be a tumor on her adrenal glands and that it was probably not in her brain since she was not experiencing headaches. A CT scan of the adrenal glands came back clean. “I remember telling my primary care doctor ‘I just don’t feel normal’”, recalls Lydia. “His response was ‘everyone’s normal is different’ and I told him ‘I’m not normal for me.’” At this point, Lydia was desperate for answers. “All these doctors were telling me it could be in my head or because I was almost 30,” said Lydia. “I kept getting shut down. I told friends and family that there was something seriously wrong with me and no one was believing me.” Finally, a friend sent Lydia information on another endocrinologist in Florida. “He was the first doctor to care about me,” said Lydia. “He said, ‘I’m so sorry you’ve been treated like this. Everyone you have seen before me is an idiot.’” More specific bloodwork and an MRI confirmed that Lydia had a macroadenoma, a benign tumor in the pituitary gland, and Cushing’s disease. After the diagnosis, Lydia was told that she would need to have the tumor removed. “He told me, ‘Find where you want to go and I’ll refer you,’” said Lydia. Lydia and her mother Jeanne began searching online for the right pituitary tumor surgeon. “Once I realized how serious it was, we started researching different doctors,” recalled Jeanne. Both Lydia and Jeanne spent time researching different doctors, but could not find a doctor that had experience treating Cushing’s disease. “We researched all kinds of surgeons to find the best one,” said Jeanne. “Then we found Dr. Oyesiku. He understood Cushing’s disease. That was important to me.” Jeanne is the one who found world-renowned pituitary tumor surgeon, Dr. Nelson Oyesiku. “I called him and said, ‘I have a 28-year-old daughter with a pituitary tumor and Cushing’s disease and I need you to operate on her,’” said Jeanne. Dr. Oyesiku has performed over 4,000 pituitary tumor operations and is currently the Chair of the Department of Neurosurgery at UNC Health. “Cushing’s is a rare disease so not many surgeons have a lot of experience with the various technical nuances required to achieve a high likelihood of cure and reduce the incidence of re-operations and complications,” said Dr. Oyesiku. Since Lydia lives in Florida, her initial consultation with Dr. Oyesiku was over Zoom. “I Zoomed with another local neurosurgeon and I was going back and forth,” said Lydia. “Dr. Oyesiku told me that he looks at the whole picture and what the tumor is doing to you. He said that he wanted to get the tumor out and then cure the Cushing’s disease.” Jeanne was also with her daughter during the initial Zoom appointment with Dr. Oyesiku. “I couldn’t find anyone else that had that background knowledge for Cushing’s disease,” said Jeanne. Dr. Oyesiku ordered more labs. “He told me ‘I want to measure twice and cut once,’” recalled Lydia. “That phrase is something my dad always said growing up and that felt like fate. So that made my decision for me and made me want to see him.” After her initial consultation with Dr. Oyesiku, both Lydia and her mother felt confident that they had found the right surgeon. Lydia met with Dr.Oyesiku in December of 2022, then had her surgery on January 23, 2023. “I called UNC and made sure that I could go in with her and stay while she was recovering,” said Jeanne. “We had contacted a different hospital early on, and I would have had to drop her off and not see her until after her surgery and only during visiting hours.” Patient coordinator David Baker, who also played an important role in Lydia’s care, helped Lydia and Jeanne find a local hotel for them to stay in before surgery at a discounted rate. After surgery, UNC Health endocrinologist Dr. Atil Kargi spoke with Lydia and her mom to help them understand the severity of Cushing’s disease and the importance of monitoring Lydia closely. “Dr. Kargi and David Baker really helped us to truly understand Cushing’s disease,” said Jeanne. Jeanne was impressed with the level of patient care that Lydia experienced during her surgery at UNC Health. “Lydia had her own nurse that would text me or call me to let me know how things were progressing.” Jeanne said. Jeanne explained that the same nurse was with her daughter going into the surgery and when she woke up after the surgery. She was also able to stay with Lydia in the hospital while she recovered from the surgery. “UNC was such an uplifting place. All these residents, they all love what they’re doing,” said Jeanne. Lydia stayed in the hospital for six days so Dr. Oyesiku and the endocrine team could monitor her levels. “I was in the normal range, and then I started to tank,” said Lydia. “I had read that a lot of patients are sent home right after surgery. If they would have sent me, I would have been adrenally insufficient.” Lydia also expressed gratitude for ENT surgeon, Dr. Brian Thorp. “During my surgery, Dr. Thorp also repaired my deviated septum,” said Lydia. “Even after surgery when I was home miles away in Florida, he was always available to me. I appreciate Dr. Oyesiku and everyone at UNC,” said Lydia. “I can’t imagine going anywhere else for this. Dr. Oyesiku truly saved my life.” After her discharge, Jeanne drove Lydia back to Florida. “Dr. Oyesiku followed-up after surgery with the Cushing’s disease treatment,” said Jeanne. “Our local endocrinologist could not believe how fast she recovered.” Jeanne also noted that she was always able to get ahold of Dr. Oyesiku, Dr. Thorp, or David Baker to answer her questions. “You feel like you’re their only patient,” said Jeanne. “We are 8-9 hours away, and it didn’t feel like it.” After Lydia weaned off of her medication, she started to lose weight, her face changed, and her body started to feel “normal” again. “My biggest symptom that I am thankful went away was my literally going crazy feeling,” said Lydia. “I am very thankful that I was able to catch it early enough so that this awful disease didn’t leave me with any lifelong complications.” Lydia, like many pituitary tumor patients, still has lingering feelings of anxiety and frustration with the long road from initial symptoms to diagnosis. It takes the average pituitary tumor patient 5-8 years to be properly diagnosed. Lydia and her mother were extremely proactive and still spent 18 months looking for answers. Lydia went to her primary care provider, her OB, a second OB, and two endocrinologists before she had a proper diagnosis. “Cushing’s disease can mimic many other vastly common medical disorders and is often misdiagnosed or mistaken for something else such as diabetes, hypertension, obesity, infertility, depression, or autoimmune disorders,” said Dr. Oyesiku. “Making the diagnosis requires expert clinical acumen supported by sophisticated medical tests, and many of these tests have to be repeated to confirm the diagnosis.” Because Cushing’s disease is so rare, many of the providers that initially saw Lydia dismissed it. After her surgery, Lydia returned to her OB office in Florida for her annual exam and was seen by the OB that told her that her symptoms were “all in her head”. “I told him, ‘Remember that you blew me off? I had a brain tumor that caused Cushing’s disease,’” said Lydia. “He told me that in all his years practicing, he had never had a patient with an endocrine disorder caused by a pituitary tumor.” Lydia’s story and other pituitary tumor patient stories serve as a reminder that while Cushing’s disease is rare, it is worth ruling out when a patient complains of these symptoms. “Part of the problem is that people just do not have access to good doctors,” said Jeanne. “If we had not had that endocrinologist, I don’t know how much longer it would have taken. It makes me sad that other women and even men can have it for so long because they cannot figure out what is going on.” Both Jeanne and Lydia are thankful that the surgery was a success, but the symptoms and long road to a diagnosis left Lydia with a few emotional scars. “I’m fine and healthy on paper, but still battling the mental aspects and the toll it took on me,” said Lydia. “Sometimes I feel resentful because it took away a year and a half of my life. I feel very blessed to be on the other side of this disease, but I’m ready to not be a patient anymore.” From https://www.med.unc.edu/neurosurgery/i-dont-feel-normal-the-diagnosis-of-a-pituitary-tumor-cushings-disease/?fbclid=IwAR1I12ND084Ato5lloDalTEcIFycV5HpLiR7S1brNxr7Lux1BZ6g_vySHOA
  11. ABSTRACT Objective To determine whether accurate inferior petrosal sinus sampling (IPSS) tumor lateralization is associated with improved clinical outcomes following the surgical treatment of Cushing’s disease. Methods The presented study was performed in accordance with PRISMA guidelines. Data regarding patient demographics, IPSS tumor lateralization, and postoperative endocrinologic outcomes were abstracted and pooled with random effects meta-analysis models. Additional meta-regression models were used to examine the association between the accuracy of IPSS tumor lateralization and postoperative outcomes (recurrence/persistence or remission/cure). Statistical analyses were performed using the Comprehensive Meta-Analysis software (significance of P<0.05). Results Seventeen eligible articles were identified, yielding data on 461 patients. Within average follow-up duration (∼59 months), the rate of correct IPSS tumor lateralization was 69% [95% Confidence Interval: 61%, 76%], and the rate of postoperative remission/cure was 78% [67%, 86%]. Preoperative IPSS tumor lateralization was concordant with MRI lateralization for 53% of patients [40%, 66%]. There was no significant association between the rate of correct IPSS tumor lateralization and postoperative remission/cure among study-level data (P=0.735). Additionally, there was no association among subgroup analyses for studies using stimulatory agents during IPSS (corticotropin-releasing hormone or desmopressin, P=0.635), nor among subgroup analyses for adult (P=0.363) and pediatric (P=0.931) patients. Conclusions Limited data suggest that the rate of correct IPSS tumor lateralization may not be positively associated with postoperative remission or cure in patients with Cushing’s disease. These findings bring into question the utility of IPSS tumor lateralization in the context of preoperative planning and surgical approach rather than confirming a pituitary source. From https://www.sciencedirect.com/science/article/abs/pii/S187887502301745X
  12. BACKGROUND Double pituitary adenomas are rare presentations of two distinct adenohypophyseal lesions seen in <1% of surgical cases. Increased rates of recurrence or persistence are reported in the resection of Cushing microadenomas and are attributed to the small tumor size and localization difficulties. The authors report a case of surgical treatment failure of Cushing disease because of the presence of a secondary pituitary adenoma. OBSERVATIONS A 32-year-old woman with a history of prolactin excess and pituitary lesion presented with oligomenorrhea, weight gain, facial fullness, and hirsutism. Urinary and nighttime salivary cortisol elevation were elevated. Magnetic resonance imaging confirmed a 4-mm3 pituitary lesion. Inferior petrosal sinus sampling was diagnostic for Cushing disease. Primary endoscopic endonasal transsphenoidal resection was performed to remove what was determined to be a lactotroph-secreting tumor on immunohistochemistry with persistent hypercortisolism. Repeat resection yielded a corticotroph-secreting tumor and postoperative hypoadrenalism followed by long-term normalization of the hypothalamic-pituitary-adrenal axis. LESSONS This case demonstrates the importance of multidisciplinary management and postoperative hormonal follow-up in patients with Cushing disease. Improved strategies for localization of the active tumor in double pituitary adenomas are essential for primary surgical success and resolution of endocrinopathies. Keywords: pituitary neuroendocrine tumor; PitNET; pituitary adenoma; Cushing disease; prolactinoma; transsphenoidal ABBREVIATIONS ACTH = adrenocorticotrophic hormone; BMI = body mass index; DHEA-S = dehydroepiandrosterone sulfate; FSH = follicle-stimulating hormone; GH = growth hormone; IHC = immunohistochemical; IPSS = inferior petrosal sinus sampling; LH = luteinizing hormone; MRI = magnetic resonance imaging; POD = postoperative day; T4 = thyroxine; TF = transcription factor; TSH = thyroid-stimulating hormone; UFC = urinary free cortisol Pituitary adenomas are adenohypophyseal tumors that can cause endocrinopathies, such as pituitary hormone hypersecretion or anterior hypopituitarism. Cell lineages are used to classify these tumors on the basis of immunohistochemical (IHC) staining of transcription factors, hormones, and other biomarkers.1 Pituitary adenomas differentiate from pluripotent stem cells along one of three lineage pathways, depending on the following active transcription factors (TFs): pituitary transcription factor 1 (PIT-1), T-box transcription factor (TPIT), or steroidogenic factor-1 (SF-1). Rarely, two or more discrete pituitary adenomas from different lineages are identified in patients; however, the etiology remains unclear.2 The incidence of multiple pituitary adenomas has been reported to be 1%–2% of all resected pituitary adenomas but is likely underestimated based on data from large autopsy series.1–4 Pluri-hormonal adenomas are also rare entities in which a single tumor contains multiple TF lineages with one or more hormonal excesses.1–3 Preoperative recognition of multiple or pluri-hormonal pituitary adenomas is rare, and most tumors are discovered incidentally upon autopsy, intraoperatively, or on histological analysis.2,3,5 In cases of multiple synchronous pituitary adenomas, only one hormone excess syndrome is most frequently evident on clinical presentation and endocrine workup. Silent pituitary tumors positive for prolactin on immunohistochemistry are the most prevalent additional, incidentally found tumor in cases of multiple pituitary adenomas.5 This is particularly true in Cushing disease.6,7 It is important to recognize the presence of multiple pituitary adenomas especially in the setting of hormonally active pituitary adenomas to provide optimal management for this subset of patients. Complete resection is curative for Cushing disease with the standard of care achieved through a transsphenoidal approach. Localization of the tumor presents a challenge because of suboptimal sensitivity of magnetic resonance imaging (MRI) in demonstrating microadenomas, the inconsistency of lateralization with inferior petrosal sinus sampling (IPSS), and delays in pathological analysis.1,8,9 Additionally, the presence of an additional pituitary adenoma can obscure the microtumor through its large size and mass effect and can act as a “decoy lesion” during MRI, IPSS, and resection.6 Consideration of multiple pituitary tumors is necessary for successful resection. In a patient with a biochemical picture of Cushing disease, the demonstration of an adenoma with negative adrenocorticotrophic hormone (ACTH) immunostaining and the absence of postoperative hypoadrenalism may indicate the existence of a double adenoma. Few cases have described a lack of remission of an endocrinopathy after transsphenoidal resection due to the presence of an additional adenoma,2,6,10 and even less so in the instance of the persistence of Cushing disease.6 We present a rare case of double pituitary adenomas in a patient presenting with Cushing disease who underwent two endoscopic endonasal transsphenoidal resections and immunostaining for prolactin and ACTH, respectively, with long-term normalization of her hypothalamic-pituitary-adrenal (HPA) axis. Illustrative Case History and Presentation A 32-year-old female, gravida 0 para 0, with a history of a pituitary lesion and hyperprolactinemia presented to our institution for the evaluation for Cushing disease. Ten years earlier, the patient had presented to a gynecologist with hirsutism, galactorrhea, and oligomenorrhea. Her endocrine workup was remarkable for an elevated prolactin at 33.8 ng/mL (2.3–23.3 ng/mL), while follicle-stimulating hormone (FSH), luteinizing hormone (LH), and thyroid-stimulating hormone (TSH) levels were normal. No ACTH or cortisol levels were available. MRI demonstrated a 5 × 6 × 5–mm T1-weighted isointense pituitary lesion protruding into the suprasellar cistern due to a small sella size. She was treated with bromocriptine 2.5 mg daily for 5 years, with normalization of her prolactin level. Subsequent MRI demonstrated a stable lesion size and T1 and T2 hyperintensity in the region of the known pituitary lesion, considered to be posttreatment cystic change with proteinaceous contents and blood. After the normalization of her prolactin levels, she continued to have oligomenorrhea and abnormal hair growth. Polycystic ovaries were not visualized on ultrasound. She was started on oral contraceptives and then switched to the etonorgestrel implant. A decade after initial presentation, she presented to endocrinology at our institution with 3 years of weight gain, hirsutism, and potential oligomenorrhea. Vital signs were stable (blood pressure: 122/86; heart rate: 72 beats/min), and facial fullness and striae on her bilateral breasts were appreciated on physical examination. She was taking isoniazid and pyridoxine for a recent diagnosis of latent tuberculosis and had discontinued bromocriptine 5 years earlier. Her weight was 66.3 kg and body mass index (BMI) was 23.9 kg/m2. She reported that her maternal uncle had a pituitary tumor. Laboratory analysis was positive for elevated urinary free cortisol (UFC) of 109 µg per 24 hours (2.5–45 µg/24 h; Table 1) and nighttime salivary cortisol of 142 ng/mL (<100 ng/dL) with high-normal prolactin of 22.8 ng/mL (2.3–23.3 ng/dL) and normal FSH, LH, TSH, and thyroxine (T4). Dehydroepiandrosterone sulfate (DHEA-S) was 128 µg/dL (98.8–340.0 µg/dL). Imaging demonstrated a 4 × 4 × 4–mm pituitary lesion with decreased T1-weighted and increased central T2-weighted signal intensity in the left lateral pituitary (Fig. 1A–C). Desmopressin (Ferring Pharmaceuticals DDAVP) stimulation increased a basal ACTH of 49.9 pg/mL to ACTH of 91.2 pg/mL, and cortisol increased from 13.7 µg/dL to 21.2 µg/dL, consistent with neoplastic hypercortisolism. IPSS was performed, which showed a right-sided, central-to-peripheral ACTH gradient (Table 2). The patient elected to undergo endoscopic endonasal resection with the initial target as the left-lateral pituitary mass to achieve a cure for Cushing disease. TABLE 1 Urinary free cortisol at baseline and 3, 5, and 7 months after the primary resection Variable Baseline 3 Mos 5 Mos 7 Mos on Osilodrostat Urinary free cortisol (4–50 µg/24 hrs) 109 134.2 125.4 40.3 Urinary creatinine (0.5–2.5 g/24 hrs) 0.995 1.17 1.42 1.11 Urinary vol (mL) 1950 2300 2100 2125 FIG. 1 Preoperative coronal precontrast (A) and postcontrast (B) T1-weighted magnetic resonance imaging (MRI) and T2-weighted MRI (C) demonstrated a 4-mm3 lesion (arrows) with decreased T1 and increased central T2 signal intensity in the left lateral pituitary. Two days after surgery, coronal precontrast (D) and postcontrast T1-weighted (E) and T2-weighted (F) MRI demonstrated the unchanged adenoma. TABLE 2 Preoperative inferior petrosal sinus sampling with corticorelin ovine triflutate 68 µg Time (mins) ACTH (pg/mL) Prolactin (ng/mL) Peripheral Petrosal Sinus ACTH Ratio Peripheral Petrosal Sinus Prolactin Ratio Rt Lt Rt Lt Rt Lt Rt Lt −5 50.6 225 1586 4.45 31.34 21 124 295 5.90 14.05 0 48.8 389 1376 7.97 28.20 22.2 185 198 8.33 8.92 3 69.8 4680 1333 67.05 19.1 22.1 396 32.5 17.92 1.47 5 80.9 4590 1623 56.74 20.06 22.1 436 32.2 19.73 1.46 10 112 4160 1660 37.14 14.82 20.2 367 42 17.90 2.05 ACTH or prolactin ratio = inferior petrosal sinus ACTH or prolactin/peripheral blood ACTH or prolactin. Primary Resection and Outcomes During the primary resection, abnormal tissue was immediately visible after a linear incision along the bottom of the dura, with an excellent plane of dissection. The inferomedial adenoma was distinct from the known left lateral lesion, and the resection was considered complete by the primary neurosurgeon. Subsequently, the left-sided adenoma was not pursued because of the historical prolactinoma diagnosis and an assumption that the newly discovered adenoma was the cause of ACTH hypersecretion. However, pathology of the inferomedial tumor was strongly and diffusely positive for prolactin (Fig. 2B), synaptophysin, and cytokeratin, with an Mindbomb Homolog-1 (MIB-1) proliferative index of 2.4%. ACTH, growth hormone (GH), FSH, LH, and TSH immunostaining were negative. TF immunohistochemistry was not available. On postoperative day (POD) 1, pituitary MRI was performed and demonstrated the unchanged 4-mm3 T1-weighted hypointense lesion with small central T2-weighted hyperintensity in the left lateral gland (Fig. 1D–F). Cortisol levels ranged from 9.7 to 76.2 µg/dL (4.8–19.5 µg/dL), and ACTH was 19.5 pg/mL (7.2–63.3 pg/mL) on POD 1. FIG. 2 Histological examination of surgical specimens from the inferomedial (A–C) and left lateral (D–F) lesions. The initial resection (hematoxylin and eosin [H&E], A) was strongly and diffusely positive for prolactin (B) with normal reticulin levels (C) indicating a lactotrophic pituitary adenoma. The second operation (H&E, D) was diagnostic for a corticotropic pituitary adenoma with diffusely positive adrenocorticotrophic hormone (ACTH) (E) and decreased reticulin (F). Original magnification ×100. Early reoperation was discussed with the patient based on the pathology and persistent hypercortisolism; however, she elected to pursue conservative management with close follow-up. Postoperative cortisol nadir was 4.8 µg/dL (4.8–19.5 µg/dL) on POD 2 during her 4-day hospital stay. DHEA-S was significantly decreased from baseline at 22.3 µg/dL (98.8–340.0 µg/dL) and a prolactin level of 3.4 ng/mL (2.3–23.3 ng/dL) was low-normal. No glucocorticoids were administered during her hospital course. There was no clinical evidence of vasopressin deficiency while she was an inpatient. Three months postoperatively, the patient reported insomnia, poor hair quality, fatigue, nocturnal sweating, and continued increasing weight gain with fat accumulation in the supraclavicular and dorsal cervical area. She had one spontaneous menstrual period despite the use of etonogestrel implant. UFC was increased at 134.2 µg/24 hours (4–50 µg/24 h; Table 1). The 8:00 am serum cortisol was 10.2 µg/dL (5.0–25.0 µg/dL). She was started on osilodrostat 2 mg twice daily for her persistent hypercortisolism, and she reported some clinical improvement; however, she had continued elevation in her late-night salivary cortisol levels up to 7.0 nmol/L. Other endocrine lab work was normal, with a prolactin of 13.5 ng/mL (2.8–23.3 ng/mL) and TSH of 3.67 µIU/mL (0.4–4.0 µIU/mL). Her weight had increased by 4.9 kg to 71.2 kg with a BMI of 25.3 kg/m2. Approximately 6 months postoperatively, she was amenable to a secondary resection targeting the remaining left lateral pituitary adenoma. Secondary Resection and Outcomes After obtaining adequate exposure for the secondary resection, the lesion in the left lateral aspect of the pituitary was targeted. The tumor was clearly identified and completely resected without intraoperative complication. IHC staining was diffusely positive for ACTH (Fig. 2E), synaptophysin, and cytokeratin with decreased reticulin and an MIB-1 index of 3.3%. Prolactin, GH, TSH, LH, and FSH immunostaining were negative. Postoperative cortisol monitoring demonstrated decreased levels, with a nadir of 2.0 µg/dL on POD 0. Levels of ACTH and DHEA-S were decreased at 4.4 pg/mL (7.2–63.3 pg/mL) and 13.3 µg/dL (98.8–340 µg/dL), respectively, on POD 1. Prolactin remained within the normal range at 8.2 ng/mL (2.8–23.3 ng/mL). The patient was started on intravenous hydrocortisone 50 mg every 8 hours for adrenal insufficiency. Postoperative symptoms of nausea, headache, and muscle weakness resolved with hydrocortisone administration. She was discharged on hydrocortisone 60 mg daily in divided doses for adrenal insufficiency and had no signs of vasopressin deficiency during her 2-day hospital course. By 3 months, the patient reported decreased fatigue, myalgia, and insomnia and improved overall well-being and physical appearance. She was weaned down to a total daily dose of 20 mg of hydrocortisone and had lost 5.2 kg. Her menstruation returned while having an etonogestrel implant. Rapid ACTH stimulation was abnormal, with decreased cortisol at 30 minutes of 4.1 µg/dL (7.2–63.3 pg/mL) demonstrating continued adrenal insufficiency. Follow-up MRI demonstrated miniscule remaining left pituitary adenoma (Fig. 3). Seven months after her second surgery, she was started on 50 µg levothyroxine for primary hypothyroidism in the setting of slightly elevated TSH of 4.1 µIU/mL (0.4–4.0 µIU/mL) and a low-normal T4 of 0.8 ng/dL (0.7–1.5 ng/dL). FIG. 3 Postoperative imaging 3 months after the second operation demonstrates near gross-total resection (yellow arrows: surgical cavity) of the left lateral pituitary adenoma on coronal precontrast (A) and postcontrast T1-weighted (B) and T2-weighted (C) MRI. Two years after the second resection, the patient lost 10.1 kg (weight, 61.1 kg; BMI, 21.76 kg/m2). Her ACTH stimulation test became normal, and hydrocortisone therapy was discontinued. At the 2-year time point, the patient and her husband successfully conceived a child. Patient Informed Consent The necessary patient informed consent was obtained in this study. Discussion Double or multiple pituitary adenomas are discovered in 0.37%–2.6% of resected pituitary lesions.3,4,6,11,12 A majority of multiple pituitary adenomas are not suspected before surgery with an inconclusive clinical presentation or endocrine laboratory workup.6 The presentation of multiple synchronous neoplasms is thought to be more common than having a single neoplasm with multiple lineages.1 Studies have shown that additional pituitary adenomas are seen at a rate of 1.6%–3.3% in Cushing disease in studies including both contiguous and noncontiguous double pituitary adenomas.6 Additional pituitary adenomas that are hormonally active make up 40% of resected double pituitary adenomas, with most staining for gonadotroph adenoma.13 Overall, the most common incidental pituitary adenoma is prolactinoma,6 which occurs most frequently with GH or ACTH adenomas.5 In very rare instances, Cushing cases can present with hyperprolactinemia and Cushing synchronously.6 Hormonal secretion and clinical presentation are variable, with the pathology most often attributed to only one component of double pituitary adenoma.3,14 The multiple-hit theory is the most common hypothesis for double pituitary adenoma etiology with coincidental monoclonal expansion of two or more lineages, which present with separate pseudo-capsules for each lesion.15 Observations On presenting with Cushing disease, the differential diagnosis before the initial operation considered that the known left lateral pituitary adenoma could be a mixed tumor with both prolactin and ACTH lineages. Therefore, it was the initial target of the resection until discovering the second adenoma intraoperatively. With two distinct adenomas, the inferomedial adenoma was presumed to be the source of the ACTH hypersecretion and was subsequently resected. The left lesion was thought to be a prolactinoma and hormonally inactive after historical dopaminergic therapy and thus was not pursued during the initial surgery. However, pathology confirmed that the opposite was true. Few cases have also involved incidental pituitary tumors that look like the hormonally active adenoma and encourage resection of it, leaving the primary pituitary adenoma behind.6,7 It has been reported that these “decoy lesions” can cause surgical failure and require secondary operations.6,7,10,16 Intraoperative localization and confirmation of the adenoma classification may have also been helpful during the case, including tissue-based ACTH antibody assay,9 plasma ACTH measurements with a immunochemiluminometric method,17 or intraoperative ultrasound.5,6 The inferomedial second tumor was not appreciated or reported throughout her serial MRI studies from 2010 to 2020. Interestingly, imaging did demonstrate the left pituitary adenoma that was medically treated as a prolactinoma, although it was later diagnosed as an ACTH-secreting lesion on IHC staining. Preoperative visualization of a pituitary adenoma in Cushing disease is reported to be limited, with a reported 50% incidence with negative MRI with standard 1.5 T.1,18,19 MRI technical refinements in magnet strength, slice thickness, or enhanced spin sequences have increased sensitivity, but one-third of patients with Cushing disease still have negative scans.20 Small prolactinomas, especially those near the cavernous sinus, are also notoriously difficult to visualize on MRI, although recent advances using co-registration of 11C-methionine positron emission tomography–computed tomography with MRI (Met-PET/MRICR) may prove useful.21 Difficulty with preoperative visualization complicates a diagnosis of multiple adenomas, with or without multiple endocrinopathies, and negatively affects surgical planning. In a single-institution retrospective review of MRI in all cases of double pituitary tumors, only one of eight patients (12.5%) over 16 years of age had a positive MRI for double pituitary tumors and was diagnosed preoperatively.2 The patient’s preoperative IPSS demonstrated a right central-to-peripheral gradient. This was incongruent with the MRI demonstrating the single left-sided tumor. While IPSS is useful in confirming Cushing disease, its sensitivity for lateralization has been reported at only 59%–71%.9 With this in mind and a known left-sided adenoma on MRI, exploration of the right side of the pituitary was not originally planned. Ultimately, the left-sided adenoma was the source of ACTH hypersecretion, which remains incongruent with preoperative IPSS. It has been suggested that multiple pituitary adenomas in Cushing disease could further decrease its accuracy.1,6 The patient’s initial historical prolactin levels (33.8 ng/dL) were lower than reported levels of 100–250 ng/dL for microadenoma and >250 ng/dL in cases of macroadenoma. Normally, in active single prolactinoma, prolactin secretion is correlated to size. We do not suspect that the presence of more than one pituitary adenoma would affect the level of prolactin hypersecretion.6 Slight elevations in prolactin can be attributed to causes such as pituitary stalk effect, medications, and physiological stimulation. During the 5 years of bromocriptine therapy, the effect on the inferomedial prolactinoma was unknown, as it was not appreciated on MRI. There are reports of prolactinomas being less responsive to dopaminergic agonist therapy in cases of double adenomas.14,22 Upon resection of the inferomedial prolactinoma during the initial operation, there was no further change in the patient’s prolactin levels, which could most likely be attributed to prior dopaminergic therapy. Unfortunately, the initial endocrine laboratory workup did not include levels of ACTH or cortisol. In addition to hyperprolactinemia, Cushing disease can also present with changes in menstruation. After the secondary resection and removal of the ACTH-secreting pituitary adenoma, the patient’s oligomenorrhea resolved and she achieved pregnancy. Retrospectively, it remains unclear if the prolactinoma was once truly active hormonally. Lessons The rare presence of two pituitary adenomas can complicate the diagnosis, medical and surgical management, and long-term outcomes for patients. A complete endocrine workup is essential when a pituitary adenoma is suspected and can help screen for pluri-hormonal and multiple pituitary adenomas. In our patient, it is unknown when the onset of hypercortisolism was with the limited initial hormonal workup. Currently, localizing and resecting the hormonally active adenoma in double or multiple pituitary adenomas remain a challenge, with limitations in preoperative imaging and intraoperative measures. After encountering the additional inferomedial lesion during surgery, resection of both adenomas during the initial surgery may have been prudent to ensure the resolution of Cushing disease. Although exploration for additional pituitary adenomas is not usually recommended, it could be considered in cases of multiple pituitary adenomas and uncertainty of the culprit of Cushing disease. The current characterization of pituitary tumors by the World Health Organization includes immunohistochemistry for both transcription factors and pituitary hormones, with clinical usefulness to be determined by future studies. Multiple lineages can occur mixed in a single pituitary adenoma or across different noncontiguous adenomas and can only be determined by TF immunostaining. The left ACTH-staining lesion in our patient had some shrinkage and MRI changes, which may have been a response to dopaminergic therapy. Full characterization of the tumor cell lineages in this case remains undetermined without staining for TFs. In conclusion, we report a rare case of Cushing disease concurrent with a prolactinoma leading to the need for repeat resection. This is one of the few reported cases of a double pituitary adenoma leading to a lack of biochemical remission of hypercortisolism after the initial surgery. Strategies for localization of the active tumor in double pituitary adenomas are essential for primary surgical success and the resolution of endocrinopathies. Author Contributions Conception and design: Zwagerman, Tavakoli, Shah, Findling. Acquisition of data: Zwagerman, Armstrong, Tavakoli, Shah, Ioachimescu, Findling. Analysis and interpretation of data: Zwagerman, Armstrong, Tavakoli, Shah, Coss, Ioachimescu, Findling. Drafting of the article: Zwagerman, Armstrong, Shah. Critically revising the article: Zwagerman, Armstrong, Tavakoli, Shah, Ioachimescu, Findling. Reviewed submitted version of the manuscript: Zwagerman, Armstrong, Tavakoli, Shah, Laing, Ioachimescu, Findling. Approved the final version of the manuscript on behalf of all authors: Zwagerman. Statistical analysis: Armstrong, Shah. Administrative/technical/material support: Zwagerman, Armstrong, Shah. Study supervision: Zwagerman, Tavakoli, Shah, Laing. References 1↑ Asa SL, Mete O, Perry A, Osamura RY. Overview of the 2022 WHO Classification of Pituitary Tumors. Endocr Pathol. 2022;33(1😞6–26. PubMed Search Google Scholar Export Citation 2↑ Roberts S, Borges MT, Lillehei KO, Kleinschmidt-DeMasters BK. Double separate versus contiguous pituitary adenomas: MRI features and endocrinological follow up. Pituitary. 2016;19(5😞472–481. PubMed Search Google Scholar Export Citation 3↑ Mete O, Alshaikh OM, Cintosun A, Ezzat S, Asa SL. Synchronous multiple pituitary neuroendocrine tumors of different cell lineages. Endocr Pathol. 2018;29(4😞332–338. PubMed Search Google Scholar Export Citation 4↑ Kontogeorgos G, Kovacs K, Horvath E, Scheithauer BW. Multiple adenomas of the human pituitary. A retrospective autopsy study with clinical implications. J Neurosurg. 1991;74(2😞243–247. PubMed Search Google Scholar Export Citation 5↑ Budan RM, Georgescu CE. Multiple pituitary adenomas: a systematic review. Front Endocrinol (Lausanne). 2016;7:1. 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  13. Abstract The occurrence of a second neoplasm possibly constitutes an adverse and uncommon complication after radiotherapy. The incidence of a second pituitary tumor in patients irradiated for adrenocorticotropic hormone secreting pituitary adenoma is rare. We report a case of a 40-year-old female with Cushing disease who underwent surgical management followed by radiotherapy. After 5 years of initial treatment, an increase in tumor size was evident at the same location, with a significant interval growth of the parasellar component of the lesion. Histology revealed an undifferentiated highly malignant sarcoma. In the span of next 2 years, the patient was followed with 2 repeat decompression surgeries and radiotherapy because of significant recurrent compressive symptoms by locally invasive malignant tumor. Despite the best efforts, the patient remained unresponsive to multiple treatment strategies (eg, surgical resections and radiotherapy) and succumbed to death. radiotherapy, second malignancy, Cushing disease Issue Section: Case Report Introduction Radiation therapy is a commonly used modality for primary or adjuvant treatment of pituitary adenoma. It is also used as an adjuvant therapy for Cushing disease with persistent or aggressive tumor growth or recurrent disease after surgery. The immediate sequelae of radiotherapy for pituitary tumors include nausea, fatigue, diminished taste and olfaction, and hair loss [1]. One frequent long-term side effect is hypopituitarism. The incidence rate of new-onset hypopituitarism after conventional radiotherapy is approximately 30% to 100% after a follow-up of 10 years, whereas after stereotactic radiosurgery or fractionated radiotherapy, the incidence is approximately 10% to 40% at 5 years [2]. The occurrence of a second neoplasm after cranial radiotherapy constitutes possibly one of the most adverse complications. Tumors such as meningioma, glioma, and sarcoma are the most frequently reported secondary neoplasms after pituitary irradiation [3]. The cumulative probability of a second brain tumor in patients irradiated for pituitary adenoma and craniopharyngioma is approximately 4% [4]. We report 1 such case with detailed clinical, histopathological, and radiological characteristics because of its rarity and associated high mortality of radiation-induced sarcoma. Case Presentation The patient first presented at 40 years of age with complaints of weight gain, new-onset diabetes mellitus, hypertension, and cushingoid features in 2014. She was diagnosed with Cushing disease (24-hour urinary cortisol 1384 mcg/24 hours [3819 nmol/24 hours; reference >2 upper limit of normal], low-dose dexamethasone suppression test serum cortisol 16.6 mcg/dL [457.9 nmol/L], ACTH 85 pg/mL [18.7 pmol/L; reference range, <46 pg/mL, <10 pmol/L]) caused by invasive adrenocorticotropic hormone-secreting giant adenoma. The initial imaging revealed a homogenously enhanced pituitary macroadenoma with a size of 42 × 37 × 35 mm with suprasellar extension and encasing both the internal carotid arteries with mass effect on optic chiasma and sellar erosion. The patient underwent tumor excision by endoscopic transsphenoidal transnasal approach. Partial excision of the tumor was achieved because of cavernous sinus invasion. Histopathology and immunohistochemical stains demonstrated a corticotrophin-secreting (ACTH-staining positive) pituitary adenoma with MIB labeling index of 1% to 2%. Because biochemical remission was not achieved (urinary cortisol 794 mcg/24 hours [2191 nmol/24 hours]; ACTH 66 pg/mL [14.5 pmol/L; reference range, <46 pg/mL, <10 pmol/L]), the patient was started on ketoconazole and was received fractionated radiotherapy with a dose of 5040 cGy in 28 fractions. Diagnostic Assessment For the next 5 years, at yearly follow-up, 400 mg ketoconazole was continued in view of insufficient control of ACTH secretion. During follow-up, the size of the tumor was stable at approximately 23 × 16 × 33 mm after radiotherapy with no significant clinical and biochemical changes. Five years after surgery and radiotherapy, the patient developed cerebrospinal fluid rhinorrhea; imaging revealed a cystic transformation of the suprasellar component and increase in the size of the tumor to 39 × 22 × 26 mm, which included visualization of a parasellar component of size 29 × 19 × 15 mm. The patient continued on ketoconazole. The patient was also advised to undergo hypofractionated radiotherapy but did not return for follow-up. Treatment In 2021, 1.5 years after the last visit, the patient developed severe headache, altered sensorium, ptosis, focal seizures, and left-sided hemiparesis. During this episode, the patient had an ACTH of 66 pg/mL (14.53 pmol/L; reference range, <46 pg/mL [<10 pmol/L]) and baseline cortisol of 25 mcg/dL (689 nmol/L; reference range, 4-18 mcg/dL [110-496 nmol/L]). Repeat imaging revealed a significant decrease in the suprasellar cystic component but an increase in the size of the parasellar component to 38 × 21 × 25 mm from 29 × 19 × 15 mm, which was isointense on T1 and T2 with heterogeneous enhancement. Significant brain stem compression and perilesional edema was also visible. The patient underwent urgent frontotemporal craniotomy and decompression of the tumor. On pathological examination, the tumor tissue was composed of small pleomorphic round cells arranged in sheets and cords separated by delicate fibrocollagenous stroma. Cells had a round to oval hyperchromatic nucleus with scanty cytoplasm. Areas of hemorrhage, necrosis, and a few apoptotic bodies were seen. The tumor tissue had very high mitotic activity of >10/10 hpf and MIB labeling index of 70%. Immunohistochemistry demonstrated positivity for vimentin, CD99, and TLE-1. Dot-like positivity was present for HMB 45, synaptophysin. INI-1 loss was present in some cells. Ten percent patchy positivity was present for p53. The tumor cells, however, consistently failed to express smooth muscle actin, CD34, Myf-4, epithelial membrane antigen, desmin, LCA, SADD4, CD138, and S-100 protein. ACTH and staining for other hormones was negative. Based on the immunological and histochemical patterns, a diagnosis of high-grade poorly differentiated malignant tumor with a probability of undifferentiated sarcoma was made. Because of the invasion of surrounding structures and surgical inaccessibility, repeat fractionated radiotherapy was given with a dose of 4500 cGy over 25 fractions at 1.8 Gy daily to the planned target volume via image-guided fractionated radiotherapy. During the next 1.5 years, patient improved clinically with no significant increase in the size of tumor (Fig. 1). The patient was gradually tapered from ketoconazole and developed hypopituitarism requiring levothyroxine and glucocorticoid replacement. There was a significant improvement in the power of the left side and ptosis. Figure 1. Open in new tabDownload slide Contrast-enhanced T1 magnetic resonance imaging dynamic pituitary scan (A, sagittal; B, axial; C, coronal sections) reveals postoperative changes with residual enhancing tumor in the right lateral sella cavity with extension into the right cavernous sinus and parasellar region encasing the cavernous and inferiorly extends through the foramen ovale below the skull base up to approximately 1.5 cm. Anteriorly, it extends up to the right orbital apex and posteriorly extends along the right dorsal surface of clivus. Outcome and Follow-up After 1.5 years of reradiation in 2022, the patient again developed palsies of the abducens, trigeminal, oculomotor, and trochlear cranial nerve on the right side and left-sided hemiparesis. A significant increase in tumor size to 50 × 54 × 45 mm with anterior, parasellar, and infratentorial extension was seen (Fig. 2). Again, repeat decompression surgery was done. Two months after surgery, there was no improvement in clinical features and repeat imaging suggested an increased size of the tumor by 30%, to approximately 86 × 68 × 75 mm. Nine years after initial presentation, the patient had an episode of aspiration pneumonia and died. Figure 2. Open in new tabDownload slide Contrast-enhanced T1 magnetic resonance imaging dynamic pituitary images (A, sagittal; B, axial; C, coronal sections) after 1.5 years of a second session of radiotherapy reveal a significant interval increase in size of heterogeneously enhancing irregular soft tissue in sellar cavity with extension into the right cavernous sinus and parasellar region when compared with previous imaging. Superiorly, it extends in the suprasellar region, causing mass effect on the optic chiasma with encasement of the right prechiasmatic optic nerve and right-sided optic chiasma. Inferiorly, the lesion extends into the sphenoid sinus. Posteriorly, there is interval increase in the lesion involving the clivus and extending into the prepontine and interpeduncular cistern. Anteriorly, mass has reached up to the right orbital apex optic nerve canal, which shows mild interval increase. Discussion Radiation-induced tumors were initially described by Cahan et al in 1948. They also described the prerequisites for a tumor to be classified as a radiation-induced sarcoma [5]. The modified Cahan criteria state that (1) the presence of nonmalignancy or malignancy of a different histological type before irradiation, (2) development of sarcoma within or adjacent to the area of the radiation beam, (3) a latent period of at least 3 years between irradiation and diagnosis of secondary tumor, and (4) histological diagnosis of sarcoma, can be classified as radiation-induced sarcoma [5]. Our patient fulfilled the criteria for a radiation-induced sarcoma with a highly malignant tumor on histopathology. Radiation-induced sarcomas after functional pituitary tumors, especially Cushing disease, are rarely reported. One of the case reports revealed a high-grade osteoblastic osteosarcoma 30 years after treatment for Cushing disease with transsphenoidal resection and external beam radiotherapy [6]. In our case, there was a lag period of approximately 5 years before the appearance of a second highly undifferentiated, malignant, histologically distinct tumor. The cellular origin of this relatively undifferentiated tumor cannot be determined with certainty. However, the interlacing sarcomatous and adenomatous components resulting from distinct positive immunohistochemistry may indicate that the sarcomatous component may be derived from the preexisting pituitary adenoma. A hormonally functional pituitary tumor is not itself expected to be associated with an increased risk of secondary malignancy, except in the case of GH-secreting tumors and those with a hereditary cancer syndrome. Although not proven, immunosuppression from hypercortisolism in Cushing disease has been proposed as a contributor to secondary tumor development [7]. Other mechanisms causing increased risk of secondary malignancy can be double-stranded DNA damage and genomic instability caused by ionizing radiation and germline mutations in tumor suppressor genes such as TP53 and Rb [7]. Radiation-induced intracranial tumors were studied in a multicenter, retrospective cohort of 4292 patients with pituitary adenoma or craniopharyngioma. Radiotherapy exposure was associated with an increased risk of a second brain tumor with a rate ratio of 2.18 (95% CI, 1.31-3.62, P < .0001). The cumulative probability of a second brain tumor was 4% for the irradiated patients and 2.1% for the controls at 20 years [7]. In another study including 426 patients irradiated for pituitary adenoma between 1962 and 1994, the cumulative risk of second brain tumors was 2.0% (CI, 0.9-4.4) at 10 years and 2.4% (95% CI, 1.2-5.0) at 20 years. The relative risk of a second brain tumor compared with the incidence in the normal population is 10.5 (95% CI, 4.3-16.7) [8]. The incidence of radiation-induced sarcomas has been estimated at 0.03% to 0.3% of patients who have undergone radiation therapy. The risk of radiation-induced sarcomas increases with field size and dose. In a systemic review and analysis of 180 cases of radiation-induced intracranial sarcomas, the average dose of radiation delivered was 51.4 ± 18.6 Gy and latent period of sarcoma onset was 12.4 ± 8.6 years. A total of 49 cases were developed after radiation treatment of pituitary adenomas (27.2%). The median overall survival time for all patients with sarcoma was 11 months, with a 5-year survival rate of 14.3% [9]. Our patient received approximately 50 Gy twice through fractionated radiotherapy, resulting in larger field size and significantly higher dose than one would expect with a modern stereotactic treatment. Such a high dose of radiation is indeed a risk factor for secondary malignancy. In our patient, in a period of 2 months, there was already >30% tumor growth after recent repeat decompression surgery. The risk of secondary malignancy is thought to be much lower with stereotactic radiosurgery than conventional external beam radiation therapy, with an estimated cumulative incidence of 0.045% over 10 years (95% CI, 0.00-0.34) [10]. However, long-term follow-up data for patients receiving stereotactic radiation therapy are shorter and thus definitive conclusions cannot be made at this stage. Our case highlights a rare but devastating long-term complication of pituitary tumor irradiation after Cushing disease. The limited response to various available treatment options defines the aggressive nature of radiation-induced malignancy. Learning Points The occurrence of a second neoplasm constitutes possibly one of the most adverse and rare complication after radiotherapy. The incidence of radiation-induced sarcomas has been estimated at 0.03% to 0.3% of patients, but cases after Cushing disease are rarely reported. Patients often present with advanced disease unresponsive to various treatment modalities because of aggressive clinical course. New modalities with stereotactic radiosurgery and proton beam therapy are to be reviewed closely for risk assessment of secondary tumor. Acknowledgments The authors acknowledge Dr. Ishani Mohapatra for her support with histopathology and interpretation. Contributors All authors made individual contributions to authorship. G.B., S.K.M., and V.A.R. were involved in diagnosis and management of the patient. G.B. was involved in the writing of this manuscript and submission. V.P.S. was responsible for patient surgeries. All authors reviewed and approved the final draft. Funding The authors received no financial support for the research, authorship, and/or publication of this article. Disclosures The authors have nothing to disclose. Informed Patient Consent for Publication Signed informed consent could not be obtained from the patient or a proxy but was approved by the treating institute. Data Availability Statement Data sharing is not applicable to this article as no data sets were generated or analyzed during the current study. © The Author(s) 2023. Published by Oxford University Press on behalf of the Endocrine Society. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. From https://academic.oup.com/jcemcr/article/1/6/luad119/7343968?login=false
  14. Abstract Introduction: Chronic exposure to excessive endogenous cortisol leads to brain changes in Cushing’s disease (CD). However, it remains unclear how CD affects large-scale functional networks (FNs) and whether these effects are reversible after treatment. This study aimed to investigate functional network changes of CD patients and their reversibility in a longitudinal cohort. Methods: Active CD patients (N = 37) were treated by transsphenoidal pituitary surgery and reexamined 3 months later. FNs were computed from resting-state fMRI data of the CD patients and matched normal controls (NCs, N = 37). A pattern classifier was built on the FNs to distinguish active CD patients from controls and applied to FNs of the CD patients at the 3-month follow-up. Two subgroups of endocrine-remitted CD patients were identified according to their classification scores, referred to as image-based phenotypically (IBP) recovered and unrecovered CD patients, respectively. The informative FNs identified by the classification model were compared between NCs, active CD patients, and endocrine-remitted patients as well as between IBP recovered and unrecovered CD patients to explore their functional network reversibility. Results: All 37 CD patients reached endocrine remission after treatment. The classification model identified three informative FNs, including cerebellar network (CerebN), fronto-parietal network (FPN), and default mode network. Among them, CerebN and FPN partially recovered toward normal at 3 months after treatment. Moreover, the informative FNs were correlated with 24-h urinary-free cortisol and emotion scales in CD patients. Conclusion: These findings suggest that CD patients have aberrant FNs that are partially reversible toward normal after treatment. Journal Section: Research Article Keywords: Cushing’s disease, Reversibility, Functional networks, Cortisol, Emotion Introduction Cushing’s disease (CD) is characterized by chronic exposure to excessive endogenous glucocorticoid most commonly caused by an adrenocorticotropic hormone (ACTH) pituitary adenoma [1, 2]. The CD is accompanied by multiple physical manifestations such as hypertension and osteoporosis, as well as various neuropsychiatric symptoms including memory lapses, attention deficits, executive function decline, emotional dysfunction, visual-spatial disability, and language defects [3‒14]. These neuropsychiatric symptoms are indicative of the effects of CD on the brain anatomy and function. Therefore, CD provides a unique and naturalistic model for investigating both the effects of hypercortisolism on the human brain and the reversibility of these effects after resolution of hypercortisolism. Recent studies have documented brain structural and metabolic abnormalities in CD patients with a variety of neuroimaging techniques, including structural magnetic resonance imaging (sMRI) [11, 12, 15‒24], diffusion tensor imaging [10, 25‒27], proton magnetic resonance spectroscopy [21, 28‒30], positron emission topography [21, 31], and arterial spin labeling [32]. These studies have shown that brain structural and metabolic abnormalities in CD patients can be partially restored after resolution of hypercortisolism [16, 18, 20‒22, 24, 32‒34], typically after transsphenoidal pituitary surgery (TSS), a safe and effective first-line treatment with a high endocrine remission rate [35, 36]. Several functional magnetic resonance imaging (fMRI) studies have also documented brain functional abnormalities in CD patients [37‒42]. Particularly, aberrant functional connectivity between the anterior cingulate cortex and the limbic network, as well as the lateral occipital cortex and the default mode network (DMN) was observed in endocrine-remitted CD patients after TSS treatment in a cross-sectional resting-state fMRI (rs-fMRI) study [40]. However, the causal effects of hypercortisolism on brain functional connectivity cannot be well investigated in CD patients only through the cross-sectional study. Additionally, the large-scale functional networks (FNs) of CD patients were not well investigated through univariate analyses in previous studies, which only examined one or few FNs in CD patients independently [37‒42]. The present study aims to jointly investigate a number of whole-brain large-scale intrinsic FNs and their reversibility due to hypercortisolism in CD patients based on rs-fMRI with a longitudinal design through multivariate analysis. Particularly, intrinsic FNs altered by CD were identified using a multivariate pattern classification model optimized by selecting intrinsic FNs informative for distinguishing CD patients from matched normal controls (NCs). The changes in these informative FNs of endocrine-remitted CD patients after TSS treatment were quantified at the 3-month follow-up with the established pattern classification model. Furthermore, changes in clinical measures, including serum cortisol, 24-h urinary-free cortisol (24hUFC), ACTH, self-rating depression scale (SDS), and self-rating anxiety scale (SAS), were detected between active and endocrine-remitted CD patients using pseudo paired t tests. Finally, the association between aberrant FNs and clinical measures was investigated in CD patients. Materials and Methods Participants In this study, 50 CD patients undergoing TSS, and 38 NCs with no history of glucocorticoid treatment were recruited at the Department of Neurosurgery, Peking Union Medical College Hospital. All these participants were assessed for depression and anxiety measured by the SDS and SAS, respectively [43]. The inclusion criteria for NCs were no past or present heart history of disease, atherosclerosis, hyperlipidemia, diabetes, neurological/psychiatric disorders, and claustrophobia. The exclusion criteria for both CD patients and NCs were past or present brain trauma, other neurological disorders, history of radiotherapy, or contraindications to MRI. Besides the inclusion and exclusion criteria, the quality of the imaging data was controlled as follows. No participant had head motion exceeding 2.0 mm translation in any of the three directions or exceeding 2.0o maximum rotation around any of the axes during rs-fMRI scanning [44]. Additionally, no participant had root-mean-square value of maximum frame-wise displacement greater than 0.3 mm [45]. After quality control of the imaging data, 37 CD patients and 37 sex-, age-, and education level-matched NCs were included in the study. The diagnosis of active CD was confirmed by experienced endocrinologists along with dynamic enhanced pituitary MRI, low- and high-dose dexamethasone suppression tests, and/or inferior petrosal sinus sampling in accordance with the latest clinical practice guidelines [46]. The 37 active CD patients were treated with TSS rather than radiotherapy. All of the 37 CD patients reached endocrine remission after treatment, which was confirmed by their normal serum cortisol (<5 µg/dL within 7 days of surgery) [46]. These patients were asked to revisit the hospital for reexamination 3 months after surgery, and all of them had no recurrence at the 3-month follow-up. Serum cortisol, 24hUFC, and ACTH were measured by direct chemiluminescence immunoassays in CD patients before surgery and at the 3-month follow-up (Siemens Healthcare Diagnostics Inc., USA). This study was approved by the Medical Ethics Committee of Peking Union Medical College Hospital, and written informed consent was obtained from all participants after explaining to them the nature of the study. Imaging Data Acquisition The MRI data were scanned by using an 8-channel phase-array head coil with a 3.0-Tesla MR scanner (Discovery MR750, General Electric) for all participants, including NCs, active CD patients, and endocrine-remitted CD patients without recurrence at the 3-month follow-up. The rs-fMRI data were acquired axially by using a gradient echo-planar imaging sequence, and the scanning parameters were 200 whole-brain volumes, 36 transverse slices with a thickness of 4 mm, in-plane resolution = 3.75 × 3.75 mm2, field of view = 240 × 240 mm2, flip angle = 90°, repetition time = 2,000 ms, and echo time = 30 ms. The extra high-resolution sagittal 3D T1-weighted data were acquired by using a brain volume sequence, and the scanning parameters were 172 slices with a thickness of 1.0 mm, in-place matrix = 512 × 512, field of view = 256 × 256 mm2, voxel size = 0.5 × 0.5 × 1.0 mm3, flip angle = 12°, repetition time = 7.2 ms, echo time = 3.2 ms, and inversion time = 400 ms. Imaging Data Preprocessing The rs-fMRI data were preprocessed as follows: (1) discarding the first four volumes of the fMRI data; (2) correction for slice timing; (3) 3D rigid-body correction for head motion to the middle frame of the data; (4) global 4D intensity scaling of the fMRI data to yield a mean value of 10,000; (5) nonlinear registration of the fMRI data to the MNI template with the deformation field obtained from its co-registered T1-weighted data using DARTEL within statistical parametric mapping (SPM12) software, with a resampled resolution of 3×3×3 mm3; (6) spatial smoothing with a 6-mm full-width at half maximum Gaussian kernel; (7) motion artifacts removal from fMRI data with ICA-AROMA; (8) regressing out averaged signals of white matter, cerebrospinal fluid, and whole brain; (9) temporal band-pass filtering (0.009–0.08 Hz). The preprocessing procedures were performed by using SPM12 software (https://www.fil.ion.ucl.ac.uk/spm/software/spm12/). Identification of Informative FNs in Active CD Patients The flowchart for identifying informative FNs in active CD patients is shown in Figure 1. First, group information-guided independent component analysis was applied to rs-fMRI data of each participant from NCs, active CD patients, and endocrine-remitted CD patients at 3 months after treatment to extract subject-specific independent components (ICs), referred to as intrinsic FNs [47] (Fig. 1a). Specifically, group-level ICs were computed based on all participants from NC, active CD, and endocrine-remitted CD groups, by using the multivariate exploratory linear optimized decomposition into independent components (MELODIC) toolbox in FSL software (https://fsl.fmrib.ox.ac.uk/fsl/fslwiki/melodic). These group-level ICs were used as guidance information to compute subject-specific ICs of all individuals [47]. The number of ICs was empirically set to be 25, and therefore each individual was characterized by 25 FNs. Particularly, these FNs were restricted to gray matter in order to minimize the partial volume effects of cerebrospinal fluid and confounding effects on the estimated components, and to improve the sensitivity to the changes of blood-oxygen-level-dependent signals. Fig. 1. VIEW LARGEDOWNLOAD SLIDE Flowchart of the multivariate pattern classification method for distinguishing active CD patients from NCs, including data preparation (a), classification modeling (b), as well as identifying CD-associated ICs (c). CD, Cushing’s disease; active CD patients, CD patients before treatment; NCs, normal controls; rs-fMRI, resting-state functional magnetic resonance imaging; ICs, independent components; GIG-ICA, group information-guided ICA; SVM, support vector machine; LOOCV, leave-one-out cross-validation. Subsequently, a multivariate pattern classification method based on support vector machine (SVM) was applied to identify cross-sectional informative FNs, which were most discriminative in distinguishing active patients from NCs [48] (Fig. 1b). Specifically, sigmoid kernel SVM classifiers were built upon a subset of 25 FNs obtained via a forward selection technique to optimize the classification performance for differentiating active patients from NCs. The similarity between subjects in SVM classification was defined as the Riemannian distance of the subset of FNs on the Grassmann manifold [48, 49]. Initially, the forward component selection procedure built a classifier on each individual FN, and the performance of the classifier was estimated using leave-one-out cross-validation (LOOCV) so that each FN could be evaluated for its classification performance. The accuracy rate was chosen as the main metric for evaluating the classification performance. The FN with the best performance was selected to be included in the subsequent classification. Through combining the first selected FN and any one of the remaining FNs, classifiers were built upon all paired FNs which were evaluated based on the training data during the current outer round using an inner LOOCV procedure. The paired FNs with the best performance were selected to be included in the classification. The procedure was repeated to add more FNs in the classification one by one until a single classifier was built upon all available FNs. Accordingly, a subset of FNs with the best performance was deemed to be the final selected components in the classification, hereafter referred to as informative FNs. To avoid potential classification biases, a nested LOOCV procedure was applied to optimize the parameters of the sigmoid kernel SVM classifiers to improve the classification performance during the forward component selection procedure [48, 49]. Since different FNs might be selected in each training runs or each testing run during the nested LOOCV procedure, the informative FNs were selected as the best performing ones with higher frequency (selection frequency>0.5). Based on these informative FNs of 74 subjects (including 37 NCs and 37 active CD patients), the LOOCV classification model yielded 74 aggregated SVM classifiers with the nested LOOCV classifiers, respectively. Each aggregated classifier generated a classification score from its corresponding nested classifiers with a positive value indicating CD state and a negative value indicating healthy state. Finally, the classification performance was evaluated with metrics including classification accuracy, specificity, sensitivity, and the area under the receiver operating characteristic curve (AUROC) (Fig. 1c). Non-parametric permutation tests were adopted to examine the statistical significance of the classification performance. The classification rate for the null distribution was estimated by building sigmoid kernel SVM models upon cross-sectional informative FNs of all active CD patients and NCs with subject labels randomly permuted by using the LOOCV strategy. This procedure was repeated for 10,000 times. Finally, the null distribution of the classification rate based on permuted samples was obtained. Longitudinal Analyses of Informative FNs and Emotion Scales from Active to Endocrine-Remitted CD Patients To investigate the longitudinal functional connectivity changes, pseudo paired t tests between active and endocrine-remitted CD patients (10,000 permutations) were applied voxel-wisely to each of the informative FNs using statistical non-parametric mapping (SnPM) software (http://warwick.ac.uk/snpm). Brain regions with statistical significance within each informative FN were identified at a voxel-wise threshold of p < 0.01 and an extent threshold of 40 adjacent voxels (AlphaSim-corrected p < 0.01). Additionally, statistical analyses were performed to compare the IC’s z scores of FNs as well as emotion scales between any pair of NC, active CD, and endocrine-remitted CD groups to further examine the longitudinal brain functional connectivity changes. Particularly, a pseudo paired t test was applied to all IC’s z scores within each informative FN as well as SDS scores and SAS scores between active and endocrine-remitted CD patients (10,000 permutations). While a pseudo two-sample t test with age, sex, and years of school education as covariates was applied to all IC’s z scores within each informative FN as well as SDS scores and SAS scores between NCs and active CD patients and endocrine-remitted CD patients. Significant differences were determined at a false discovery rate (FDR) threshold of p < 0.05 after adjusting for multiple comparisons. Statistical Analyses of Informative FNs in Endocrine-Remitted CD Patients The established pattern classification model was applied to the FNs of the follow-up endocrine-remitted CD patients. Thus, each endocrine-remitted CD patient had a classification score that reflected the likelihood of the endocrine-remitted CD patient to be active CD or healthy state (a positive value indicating active CD state and a negative value indicating healthy state). Based on the follow-up classification scores, endocrine-remitted CD patients who were correctly classified as active CD patients before treatment were further stratified into two subgroups: subjects with negative classification scores, referred to as image-based phenotypically (IBP) recovered CD patients, and those with positive classification scores, referred to as IBP unrecovered CD patients. Additionally, statistical differences in the IC’s z scores within each of the informative FNs between the IBP recovered and unrecovered CD patients, were assessed to elucidate these endocrine-remitted CD patients’ brain recoveries in these informative FNs at 3 months after treatment. Specifically, a pseudo two-sample t test with age, sex, years of school education, and years of disease duration as covariates was applied to all IC’s z scores of each FN between IBP recovered and unrecovered CD patients, and significant differences were determined at an FDR threshold of p < 0.05 (10,000 permutations) after adjusting for multiple comparisons. Correlation Analyses between Informative FNs and Clinical Measures Correlation analyses were performed to investigate the relationship between informative FNs and clinical measures in all 37 CD patients. The clinical measures of interest were serum cortisol, 24hUFC, ACTH, SDS, and SAS. Specifically, the correlation between each clinical measure and the averaged IC’s z score of each informative FN of CD patients before treatment was computed using a general linear model with age, sex, years of school education, and years of disease duration as covariates. Significant correlations were determined at a threshold of p < 0.05 using FDR corrected for multiple comparisons. Additionally, the correlation between the changes of each clinical measure and the averaged IC’s z score of each informative FN for endocrine-remitted CD patients before and after treatment was computed by using a general linear model with age, sex, years of school education, years of disease duration, and this clinical measure before treatment as covariates. The change of the averaged IC’s z score of each informative FN for each CD patient was calculated as the value after treatment minus the value before treatment divided by the value before treatment, and the change of each clinical measure for each CD patient was calculated similarly. Significant correlations were identified at a threshold of p < 0.05 using FDR corrected for multiple comparisons. Results Demographics and Clinical Characteristics The demographic and clinical data, including age, sex, years of school education, hormones, and emotion scales, are summarized in Table 1. There were no significant differences in age, sex, and years of school education between NCs and CD patients before treatment or at the 3-month follow-up (p > 0.05). The hormone levels, including ACTH, 24hUFC, and serum cortisol, were significantly restored (lower to be precise) in endocrine-remitted CD patients at the 3-month follow-up compared to their pre-treatment levels (FDR-corrected p < 0.05). These CD patients reached endocrine remission confirmed by their normal serum cortisol (<5 µg/dL) within 7 days of surgery. The emotion scales, including SDS scores and SAS scores, were significantly improved (smaller to be precise) in endocrine-remitted CD patients at 3 months after treatment compared to their rating scales in active phase (FDR-corrected p < 0.05), and the SDS scores and SAS scores for these endocrine-remitted CD patients were comparable to those of NCs. There was also significant difference in SDS scores between endocrine-remitted CD patients and NCs (FDR-corrected p < 0.05), while no significant difference was found in SAS scores between endocrine-remitted CD patients and NCs (p = 0.70). These psychometric comparison results suggest that depressive symptoms were partially recovered in endocrine-remitted CD patients, while their anxiety symptoms were also not completely recovered. Table 1. Demographic and clinical data of the participants Characteristics NCs (N = 37) Active CDs (N = 37) Endocrine-remitted CDs (N = 37) p value Age, years 38.46±11.85 33.92±8.57 33.92±8.57 0.062a Sex (M/F) 10/27 8/29 8/29 0.83a Years of school education 12.84±3.53 13.27±3.11 13.27±3.11 0.55a ACTH, pg/mL - 75.70 (45.55, 103.25) 23 (10.33, 30.70) <0.01**b 24hUFC, μg/day - 582.34 (351.30, 991.56) 47.77 (14.41, 186.54) <0.01**b Serum cortisol, μg/dL - 26.58 (20.98, 31.84) 5.49 (1.75, 13.69) <0.01**b Depression (SDS) 38.72±7.45 53.99±9.20 45.54±10.24 <0.01**c <0.01**d Anxiety (SAS) 33.34±5.46 45.27±11.92 34.46±9.78 <0.01**c 0.70d Values for characteristics are presented as mean ± SD or median (25th percentiles, 75th percentiles) unless otherwise indicated. Group differences in age, years of school education, SDS, and SAS between NCs and CD patients before or at the 3-month follow-up were examined using pseudo two-sample t tests. Group differences in sex between NCs and the CD patients before treatment or at the 3-month follow-up were examined using a χ2 test. Group differences in ACTH, 24hUFC, serum cortisol, SDS, and SAS between CD patients before treatment and at the 3-month follow-up were examined using pseudo paired t tests. NCs, normal controls; CDs, patients with Cushing’s disease; ACTH, adrenocorticotropic hormone; 24hUFC, 24-h urinary-free cortisol; SDS, self-rating depression scale; SAS, self-rating anxiety scale; M, male; F, female; SD, standard deviations. **p < 0.01. aNCs versus active or endocrine-remitted CDs. bActive CDs versus endocrine-remitted CDs. cActive CDs versus NCs or endocrine-remitted CDs. dNCs versus endocrine-remitted CDs. Informative FNs in Active CD Patients Active CD patients were mostly different from the NCs in 3 out of 25 FNs (selection frequency>0.5), including cerebellar network (CerebN), fronto-parietal network (FPN), and DMN, as shown in Figure 2a and b. The classification models built upon these three informative FNs yielded an accuracy of 72% (sensitivity: 68%, specificity: 76%, AUROC: 0.81), as shown in Figure 2c. Non-parametric permutation tests demonstrated that the classification accuracy was promising and significant (p < 1.0e−04), as suggested by the histogram of permuted classification rates shown in Figure 2d. Particularly, 25 out of 37 (67%) CD patients were correctly classified as active CD patients before treatment. Fig. 2. VIEW LARGEDOWNLOAD SLIDE Three informative functional brain networks identified by the multivariate pattern classification method and the classification performance. a Three highly selected functional brain networks, including CerebN, FPN, and DMN, for differentiating active CD patients from NCs. b The frequency of the functional brain networks selected in the nested LOOCV experiments. c The receiver operating characteristic (ROC) curve (area under the ROC curve [AUROC] = 0.81) of the classification model built upon the selected most discriminative FNs. d The histogram of the classification rates of the permutation tests and the real classification rate. In panel (a), brain regions with significant functional connectivity were obtained by applying voxel-wise one-sample t tests to the IC’s z scores for each of the FNs across all active CD patients and NCs (p < 0.05, FWE corrected for multiple comparisons, and cluster size >400 voxels). CerebN, cerebellar network; FPN, fronto-parietal network; DMN, default mode network; CD, Cushing’s disease; Pres, CD patients before treatment (i.e., active CD patients); NCs, normal controls; FNs, functional networks; ICs, independent components; FWE, family-wise error; L, left; R, right. Changes in Informative FNs from Active to Endocrine-Remitted CD Patients Two out of the three informative FNs, i.e., CerebN and FPN other than DMN, exhibited significant functional connectivity changes in CD patients between active and endocrine-remitted states (Fig. 3a). Compared with their active state, the endocrine-remitted CD patients had significantly improved (increased to be precise) functional connectivity measured by IC’s z scores in both CerebN and FPN circuits at 3 months after treatment. These results indicate that the FNs of the endocrine-remitted CD patients partially recovered toward the NCs at 3 months after treatment (Fig. 3b). Fig. 3. VIEW LARGEDOWNLOAD SLIDE Two informative functional brain networks as well as emotion scales with significant longitudinal changes in CD patients before treatment and at the 3-month follow-up. a Brain regions with significant longitudinal changes in functional connectivity within circuits of CerebN and FPN for CD patients, identified using non-parametric permutation tests (AlphaSim-corrected p < 0.01). b, c Significantly different functional connectivity measured by IC’s z scores across voxels within circuits of CerebN and FPN as well as emotion scales measured by the self-rating depression scale (SDS) and self-rating anxiety scale (SAS) between any two of NCs, CD patients before the treatment (i.e., active CD patients), and endocrine-remitted CD patients at 3-month follow-up (FDR-corrected p < 0.05). A pseudo paired t test with age, sex, and years of school education as covariates was conducted to compare all IC’s z scores within each functional network as well as the SDS scores and SAS scores between CD patients before treatment and at the 3-month follow-up. While a pseudo two-sample t test with age, sex, and years of school education as covariates was conducted to compare IC’s z scores within each functional network as well as SDS scores and SAS scores between NCs and CD patients before treatment, and endocrine-remitted CD patients at 3-month follow-up. CerebN, cerebellar network; FPN, fronto-parietal network; CD, Cushing’s disease; Pres, CD patients before treatment; Posts, endocrine-remitted CD patients at 3-month follow-up; NCs, normal controls; ICs, independent components; FDR, false discovery rate. Changes in Informative FNs of Endocrine-Remitted CD Patients Among the endocrine-remitted CD patients who were correctly classified as active CD patient before treatment, 14 participants were classified as IBP-recovered patients, while 11 participants were classified as IBP-unrecovered patients. The IBP-recovered and -unrecovered CD patients were determined by using the established pattern classification model according to the opposite signs in their classification scores based on their follow-up rs-fMRI data at 3 months after treatment (Fig. 4b). The IBP recovered patients had better recovery of the impaired functional connectivity within the circuits of CerebN and FPN than the IBP-unrecovered patients, as shown in Figure 4a. Fig. 4. VIEW LARGEDOWNLOAD SLIDE Differences in functional connectivity measured by IC’s z scores across voxels within circuits of CerebN and FPN as well as classification scores between image-based phenotypically (IBP)-recovered and -unrecovered CD patients after treatment. In panel (a), statistical comparisons were performed using pseudo two-sample t tests with age, sex, years of school education, and years of disease duration as covariates (FDR-corrected p < 0.05). In panel (b), violin plots showed opposite signs in classification scores between IBP-recovered and -unrecovered CD patients. CerebN, cerebellar network; FPN, fronto-parietal network; CD, Cushing’s disease; CDs, patients with Cushing’s disease; ICs, independent components; FDR, false discovery rate. Relationship between Informative FNs and Clinical Measures Changes of 24hUFC for endocrine-remitted CD patients before and after treatment were negatively correlated with their changes of averaged IC’s z scores within the FPN circuits with statistical significance (r = −0.37, p = 0.020), as shown in Figure 5a. The emotion scales, including SDS and SAS, were significantly negatively correlated with the averaged IC’s z scores within the CerebN circuits in the active CD patients (r = −0.31, p < 0.042), as shown in Figure 5c and d. There was no significant correlation for other clinical measures. Fig. 5. VIEW LARGEDOWNLOAD SLIDE Correlations between clinical measures and averaged IC’s z scores of informative FNs in CD patients (FDR-corrected p < 0.05). a Scatter plot for the significantly negative correlation between changes in the averaged IC’s z scores of the FPN circuits and 24hUFC of these 37 endocrine-remitted CD patients. b Multi-slice view of the FPN circuits whose changes in the averaged z scores were significantly correlated with changes in 24hUFC for all 37 endocrine-remitted CD patients before and after treatment. c, d Scatter plots for the significantly negative correlations between the averaged IC’s z scores of the CerebN circuits, and the SDS scores and SAS scores in these 37 endocrine-remitted CD patients before treatment. In panel (a), the changes in the averaged IC’s z scores of the FPN circuits were adjusted by regressing out covariates including age, sex, years of school education, years of disease duration, and the pre-treatment 24hUFC. In panels (c) and (d), the averaged IC’s z scores of the CerebN circuits were adjusted by regressing out covariates including age, sex, years of school education, and years of disease duration. 24hUFC, 24-h urinary-free cortisol; SDS, self-rating depression scale; SAS, self-rating anxiety scale; FPN, fronto-parietal network; CerebN, cerebellar network; CD, Cushing’s disease; Pres, CD patients before treatment; Posts, endocrine-remitted CD patients at 3-month follow-up; ICs, independent components; FDR, false discovery rate. Discussion The present study investigated the large-scale FNs of CD patients before and after treatment based on longitudinal rs-fMRI data. To the best of our knowledge, this is the first study to characterize longitudinal large-scale functional brain network changes due to hypercortisolism in CD patients using multivariate analysis. Particularly, the active CD patients had aberrant functional connectivity within circuits of CerebN, FPN, and DMN, respectively. More importantly, the impaired functional connectivity within the circuits of the CerebN and FPN was partially recovered in the endocrine-remitted CD patients, respectively. The changes in 24hUFC of CD patients before and after treatment were correlated with their changes in the functional connectivity of the FPN circuits. In addition, the emotion scales, including SDS and SAS, were also correlated with the functional connectivity of the CerebN circuits in CD patients before treatment. Aberrant FNs in Active CD Patients The informative FNs identified by the multivariate method were able to distinguish active CD patients from NCs with an accuracy of 72% (sensitivity: 68%, specificity: 76%, AUROC: 0.81). The non-parametric permutation tests also suggested that the multivariate method performed well in differentiating active CD patients from NCs. The most frequently selected FNs (Fig. 2b), i.e., informative FNs, were CerebN, FPN, and DMN. The cross-sectional multivariate analyses have revealed that the active CD patients were mostly different from the NCs in the functional connectivity within 3 FNs out of 25 FNs, as shown in Figure 2a. The aforementioned cross-sectional results provided new insights into large-scale functional brain network abnormalities due to hypercortisolism in CD patients. Particularly, our study revealed that active CD patients had significantly disrupted functional connectivity within the cerebellum (Fig. 2a, 3b), and their emotional dysfunctions observed by the SDS and SAS were associated with the impaired functional connectivity within the cerebellum (Fig. 5c, d). Therefore, it was reasonable to speculate that the cognitive or emotional dysfunctions for active CD patients, documented in this study as well as numerous previous studies [3‒5, 7‒9, 11‒14, 50], might be closely related to the observed functional connectivity abnormalities in the cerebellum. Additionally, our study found that the functional connectivity within the FPN circuits was significantly reduced in active CD patients (Fig. 2a, 3b). It was postulated that cognitive impairments in active CD patients, reported in several early studies [6, 14, 51], might be associated with the observed functional connectivity abnormalities in the FPN circuits. While several recent studies reported that active CD patients had structural or metabolic abnormalities in two brain regions of the FPN, namely, the middle frontal gyrus and inferior parietal lobule [17, 21, 32] These local morphological or metabolic abnormalities might exacerbate the observed functional network (FPN) alterations in active CD patients. Moreover, our study found that the functional connectivity within the DMN circuits was vulnerable to the detrimental effects of hypercortisolism in active CD patients. Besides our finding, recent studies reported that active CD patients showed structural, metabolic, or spontaneous activity abnormalities in several brain regions of DMN, including the posterior cingulate cortex, precuneus, parahippocampal gyrus, ventral medial prefrontal cortex, superior frontal gyrus, inferior temporal gyrus, and lateral parietal cortex [21, 27, 30‒32, 38, 52] These local morphological, metabolic or activity abnormalities might exacerbate the newly discovered DMN impairments in active CD patients. Essentially, the functional, morphological, and metabolic abnormalities in regions of the DMN might be directly related to the adverse expressions of glucocorticoid receptor genes within these brain regions caused by excessive exposure to endogenous cortisol [53]. Reversible Impaired FNs in Endocrine-Remitted CD Patients after Treatment The longitudinal statistical analysis has revealed that the endocrine-remitted CD patients’ hormones, including ACTH, 24hUFC, and serum cortisol, maintained near-normal levels at 3 months after treatment, suggesting that these patients did not relapse according to the endocrine hormone levels (Table 1). Meanwhile, their functional connectivity within circuits of the FPN and CerebN was partially restored at the 3-month follow-up after resolution of hypercortisolism (Fig. 3b). Particularly, our combined longitudinal and cross-sectional study found that the functional connectivity within the FPN circuits in endocrine-remitted CD patients was partially restored after treatment. While a cross-sectional sMRI study reported that endocrine-remitted CD patients still had structural abnormalities in the FPN-related region, namely, the middle frontal gyrus [17]. Our study also found that the functional connectivity of the cerebellum in endocrine-remitted CD patients was partially restored after treatment (Fig. 3b). Besides this finding, two other cross-sectional sMRI studies reported that the structural abnormalities of the cerebellum in endocrine-remitted patients were present as well [16, 20]. Taken together, it was postulated that the reversibility of the observed functional connectivity impairments within circuits of the FPN and CerebN might be directly influenced by their local morphological abnormalities in endocrine-remitted CD patients. Moreover, our study uncovered that the IBP-recovered patients exhibited better recovery of the functional connectivity within circuits of the FPN and CerebN than the IBP-unrecovered ones, as shown in Figure 4a. This result demonstrated that different endocrine-remitted CD patients had different recovery levels for the impaired functional connectivity within circuits of these brain FNs. More importantly, our study further found that the recovered 24hUFC was associated with the improved functional connectivity within FPN circuits in endocrine-remitted CD patients at the 3-month follow-up after treatment (Fig. 5a). This finding indicated that chronic endogenous hypercortisolism in CD patients might be directly related to their FPN impairments. Strengths of This Study The combined longitudinal and cross-sectional analyses have confirmed that the brain functional network abnormalities in CD patients were partially reversible at 3 months after resolution of the hypercortisolism. Since the brain structural abnormalities in endocrine-remitted CD patients were not completely recovered [16], it merits further investigation how the brain structural and functional network recoveries couple with each other in a longitudinal design. The present study provided complementary information to existing neuroimaging studies of CD patients. The existing neuroimaging studies have reported that CD patients had brain volume loss in cortical and cerebellar regions, hippocampus, and amygdala, as well as enlarged ventricles. These structural abnormalities were partially recovered for endocrine-remitted CD patients after treatment [11, 15, 16, 18‒22, 24] or after resolution of the hypercortisolism [12, 18, 24]. CD patients also had reduced cortical thickness in many brain regions including superior frontal cortex, caudal middle frontal cortex, precentral gyrus, insula, precuneus, cuneus, caudal/rostral anterior cingulate gyrus, and posterior cingulate gyrus [17, 54]. In addition, disrupted white matter integrity was observed in CD patients throughout the brain including frontal lobe, temporal lobe, hippocampus, parahippocampal gyrus, cingulate cingulum, corpus callosum, uncinate fasciculus, and cerebellum [10, 25‒27]. Furthermore, metabolic abnormalities in CD patients have been reported in widely distributed brain regions [21, 28‒32], which could be almost completely restored after resolution of hypercortisolism. Besides aforementioned structural and metabolic abnormalities, functional abnormalities have also been reported in CD patients using fMRI [37‒42]. Particularly, abnormal functional activations in CD patients have been observed in the prefrontal cortex, superior/middle/inferior frontal gyrus, superior parietal lobule, superior/middle temporal gyrus, inferior occipital gyrus, rostral/dorsal anterior cingulate gyrus, anterior/middle/posterior hippocampus, amygdala, precuneus, cuneus, lingual gyrus, caudate body, pulvinar/lateral posterior nuclei of the thalamus, and substantia nigra using task fMRI [37‒39, 41]. Abnormal spontaneous functional activities measured by both the amplitude of low-frequency fluctuation and regional homogeneity for CD patients have been observed in the prefrontal cortex, occipital lobe, postcentral gyrus, posterior cingulate gyrus, precuneus, thalamus, and cerebellum [20]. The dysregulation of functional connectivity density of CD patients has been found primarily in the prefrontal cortex, lateral parietal cortex, anterior/posterior cingulate gyrus, and parahippocampal gyrus [55]. The abnormal functional connectivity for CD patients has also been observed between the prefrontal cortex and medial temporal lobe, ventromedial prefrontal cortex and posterior cingulate cortex, anterior cingulate gyrus and limbic network, and lateral occipital cortex and DMN using task fMRI or rs-fMRI [39, 40]. Limitations and Future Work This study has several limitations. First, the longitudinal sample size is not large enough due to the rarity of CD, which might lead to relatively low statistical power and potential biases. Second, our study mainly investigated the brain functional network reversibility of the CD. Studies of the CD’s structural reversibility may provide complementary information to the current study. Third, our study investigated the short-term (3 months) effects of hypercortisolism on large-scale functional brain networks in CD patients. Nevertheless, the long-term effects of hypercortisolism on large-scale functional brain networks remain unclear and merit further investigation. In future work, long-term follow-up data of the CD patients recruited in the current study will be collected to investigate the long-term dynamic changes of their impaired large-scale functional brain networks. Conclusion This is the first study to investigate large-scale functional brain networks and their reversibility in a longitudinal CD cohort by using multivariate analysis. The large-scale functional brain networks, including the CerebN, FPN, and DMN, were impaired due to elevated cortisol levels in active CD patients. More importantly, the impaired functional brain networks of these CD patients were partially restored when their hormone levels returned to normal at 3 months after treatment. The changes of the functional connectivity within the impaired FPN were correlated with changes of the 24hUFC in endocrine-remitted CD patients, while the functional connectivity within the impaired CerebN was closely associated with emotion dysfunctions in active CD patients. These findings suggest that pattern recognition techniques could help identify informative functional brain networks in CD patients, which may help open up novel avenues for their postoperative interventions and assessments after endocrine remission. Statement of Ethics This study confirmed to the Declaration of Helsinki and was approved by the Medical Ethics Committee of Peking Union Medical College Hospital (approval number S-424). Written informed consent was obtained from all participants. Conflict of Interest Statement All authors reported no financial interests or potential conflicts of interest. Funding Sources This study was supported in part by the China Postdoctoral Science Foundation (2020T130070, 2019M650567), and the Clinical Application Research of Capital Characteristic Fund from the Beijing Municipal Science and Technology Commission (Z151100004015099). Author Contributions Bing Xing, Feng Feng, and Yong Fan were involved in study conception and design. Bo Hou, Xiaopeng Guo, Yong Yao, and Ming Feng collected clinical and imaging data. Hewei Cheng, Lu Gao, and Rixing Jing performed data preparation and statistical analysis. Hewei Cheng, Lu Gao, Rixing Jing, Bing Xing, Feng Feng, and Yong Fan were involved in data interpretation. Hewei Cheng, Lu Gao, and Rixing Jing wrote the first draft of the manuscript. Hewei Cheng, Lu Gao, Rixing Jing, Bo Hou, Xiaopeng Guo, Zihao Wang, Ming Feng, Bing Xing, Feng Feng, and Yong Fan provided critical editing and revision of the manuscript for important intellectual content. All authors approved the final version of the manuscript. Additional Information Hewei Cheng and Lu Gao contributed equally to this work. Data Availability Statement All data generated or analyzed during this study are included in this article. Further inquiries can be directed to the corresponding author. References 1. Newell-Price J, Bertagna X, Grossman AB, Nieman LK. Cushing’s syndrome. Lancet. 2006;367(9522):1605–17. 2. 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Cortical thickness abnormalities in long-term remitted Cushing’s disease. Transl Psychiatry. 2020;10(1):293. 55. Wang X, Zhou T, Wang P, Zhang L, Feng S, Meng X, et al. Dysregulation of resting-state functional connectivity in patients with Cushing’s disease. Neuroradiology. 2019;61(8):911–20. © 2023 The Author(s). Published by S. Karger AG, Basel Open Access License / Drug Dosage / Disclaimer This article is licensed under the Creative Commons Attribution-NonCommercial 4.0 International License (CC BY-NC). Usage and distribution for commercial purposes requires written permission. Drug Dosage: The authors and the publisher have exerted every effort to ensure that drug selection and dosage set forth in this text are in accord with current recommendations and practice at the time of publication. 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  15. The following is a summary of “Diurnal Range and Intra-patient Variability of ACTH Is Restored With Remission in Cushing’s Disease,” published in the November 2023 issue of Endocrinology by Alvarez, et al. Distinguishing Cushing’s disease (CD) remission from other conditions using single adrenocorticotropic hormone (ACTH) measurements poses challenges. For a study, researchers sought to analyze changes in ACTH levels before and after transsphenoidal surgery (TSS) to identify trends confirming remission and establish ACTH cutoffs for targeted clinical trials. A retrospective analysis involved 253 CD patients undergoing TSS at a referral center from 2005 to 2019. Remission outcomes were assessed based on postoperative ACTH levels. Among 253 patients, 223 achieved remission post-TSS. The remission group exhibited higher ACTH variability at morning (AM) (P = .02) and evening (PM) (P < .001) time points compared to the nonremission group. Nonremission cases had a significantly narrower diurnal ACTH range (P < .0001). A ≥50% decrease in plasma ACTH from mean preoperative levels, especially in PM values, predicted remission. Absolute plasma ACTH concentration and the ratio of preoperative to postoperative values were associated with nonremission (adj P < .001 and .001, respectively). ACTH variability suppression was observed in CD, with remission linked to restored variability. A ≥50% decrease in plasma ACTH may predict CD remission post-TSS. The insights can guide clinicians in developing rational outcome measures for interventions targeting CD adenomas. Source: academic.oup.com/jcem/article-abstract/108/11/2812/7187942?redirectedFrom=fulltext
  16. Dr. Theodore Friedman (the Wiz) will be giving a webinar on Optimal replacement for Hypopituitarism and Sheehan’s: Oxytocin, testosterone, growth hormone, stimulants and beyond Learn what most Endocrinologists don’t know about but will improve your quality of life Topics to be discussed include: • Oxytocin-the love hormone • Testosterone, not just for men • Stimulants to treat pituitary apathy • Growth hormone, not just for kids • Thyroid optimization • Cortisol, the right and wrong way to give • Learn about the common medicine you should never take if on growth hormone Wednesday • December 6th• 6 PM PST Via Zoom Click here to join the meeting or https://us02web.zoom.us/j/4209687343?pwd=amw4UzJLRDhBRXk1cS9ITU02V1pEQT09&omn=84521530646 OR +16699006833,,4209687343#,,,,*111116# Slides will be available before the webinar and recording after the meeting at slides or on Dr. Friedman’s YouTube channel OR Join on Facebook Live https://www.facebook.com/goodhormonehealth at 6 PM PST Meeting ID: 420 968 7343 Passcode: 111116 Your phone/computer will be muted on entry. There will be plenty of time for questions using the chat button. For more information, email us at mail@goodhormonehealth.com
  17. Background: Cushing’s disease (CD) poses significant challenges in its treatment due to the lack of reliable biomarkers for predicting tumor localization or postoperative clinical outcomes. Sphingosine-1-phosphate (S1P) has been shown to increase cortisol biosynthesis and is regulated by adrenocorticotropic hormone (ACTH). Methods: We employed bilateral inferior petrosal sinus sampling (BIPSS), which is considered the gold standard for diagnosing pituitary sources of CD, to obtain blood samples and explore the clinical predictive value of the S1P concentration ratio in determining tumor laterality and postoperative remission. We evaluated 50 samples from 25 patients who underwent BIPSS to measure S1P levels in the inferior petrosal sinuses bilaterally. Results: Serum S1P levels in patients with CD were significantly higher on the adenoma side of the inferior petrosal sinus than on the nonadenoma side (397.7 ± 15.4 vs. 261.9 ± 14.88; P < 0.05). The accuracy of diagnosing tumor laterality with the interpetrosal S1P and ACTH ratios and the combination of the two was 64%, 56% and 73%, respectively. The receiver operating characteristic curve analysis revealed that the combination of interpetrosal S1P and ACTH ratios, as a predictor of tumor laterality, exhibited a sensitivity of 81.82% and a specificity of 75%, with an area under the curve value of 84.09%. Moreover, we observed that a high interpetrosal S1P ratio was associated with nonremission after surgery. Correlation analyses demonstrated that the interpetrosal S1P ratio was associated with preoperative follicle-stimulating hormone (FSH), luteinizing hormone (LH), and postoperative ACTH 8 am levels (P < 0.05). Conclusion: Our study demonstrated a significant association between the interpetrosal S1P ratio and tumor laterality, as well as postoperative remission in CD, suggesting that the interpetrosal S1P ratio could serve as a valuable biomarker in clinical practice. 1 Introduction Cushing’s disease (CD), also known as adrenocorticotropic hormone (ACTH)-secreting pituitary adenoma, arises from the pituitary corticotroph cells and induces endogenous hypercortisolism by stimulating the adrenal glands to produce excessive amount of cortisol (1). Patients with CD typically exhibit symptoms of hypercortisolism, such as hypertension, diabetes, purplish skin striae, mental disturbances, hyposexuality, hirsutism, menstrual disorders, acne, fatigue, obesity, and osteoporosis (1). The overall mortality of patients with CD is twice that of the general population, and if left untreated, hypercortisolism resulting from CD increases this rate to approximately four times the expected value (2–4). Transsphenoidal surgery continues to be the primary treatment for CD (5). However, previous studies reported variable remission rates, ranging from 45% to 95% (6–8). Long-term follow-up data have revealed recurrence in 3–66% of patients who had initially achieved complete remission (9, 10). The rate of surgical remission in CD can be influenced by various factors, including the size and location of the tumor, expertise of the neurosurgeon, and criteria used for assessing remission (11). Preoperative clinical variables, such as age, gender, disease duration, and severity of clinical signs and symptoms, cannot reliably identify patients at a higher risk of nonremission (12, 13). Therefore, predicting postsurgical remission in CD remains a challenging goal. Accumulating evidence has shown that sphingosine-1-phosphate (S1P), an intracellular pleiotropic bioactive sphingolipid metabolite synthesized by sphingosine kinase 1 (SPHK1), plays a pivotal role in diverse endocrine disorders (14–16). Overexpression of SPHK1 promotes the progression of multiple neuroendocrine tumors (17, 18). ACTH can rapidly activate sphingolipid metabolism, causing an increase in S1P secretion in the adrenal cortex (19). Furthermore, the activation of S1P signaling in H295R cells, a human adrenocortical tumor cell line, has been suggested to induce increased transcription of hormone-sensitive lipase and steroidogenic acute regulatory protein, ultimately elevating cortisol production (20). Recently, surgical removal of ACTH-secreting adenoma has been reported to cause a decline in sphingomyelin levels (21). However, whether they have a similar role in the pituitary gland remains to be investigated. Bilateral inferior petrosal sinus sampling (BIPSS) is a highly effective procedure for diagnosing pituitary sources of ACTH in CD (22, 23). Contemporaneous differences in ACTH concentration during venous sampling between the two sides of the adenoma can predict the location of the adenoma within the pituitary (on the side of the gland with a microadenoma) and may guide surgical treatment in cases with inconclusive magnetic resonance imaging findings. Previous studies demonstrated that an ACTH gradient of ≥1.4 between the inferior petrosal sinuses can indicate microadenoma lateralization in patients with CD (24–26). However, the correct lateralization only occurs in 57–68% of all cases (27–29). Therefore, we analyzed the clinical behavior of a well-characterized cohort of patients with CD who underwent BIPSS before surgery. We measured the difference in the concentration of S1P in bilateral petrosal sinus blood samples and explored the clinical predictive value of the S1P concentration ratio in determining tumor laterality and postoperative remission. 2 Materials and methods 2.1 Patients and study design This study was conducted at a tertiary center, involving a cohort of 25 patients diagnosed with CD who had undergone BIPSS and surgery, with a minimum follow-up duration of 2 years. Comprehensive chart reviews were conducted to collect data on demographics, clinical characteristics, pituitary imaging findings, tumor pathology, and biochemical tests. The criteria used for diagnosing CD encompassed the presence of characteristic signs and symptoms of hypercortisolism, along with biochemical evaluation of two urinary free cortisol measurements exceeding the normal range for the respective assay, serum cortisol level >1.8 μg/dL (50 nmol/L) after an overnight 1-mg dexamethasone suppression test, and two late-night salivary cortisol measurements exceeding the normal range for the respective assay (30). A diagnosis of Cushing’s syndrome was established if the patient had positive test results for at least two of the three aforementioned tests. Adrenal insufficiency was diagnosed if patients exhibited symptoms or signs of adrenal insufficiency or if serum cortisol levels were ≤3 μg/dL, even in the absence of clinical signs or symptoms. Remission was defined as normalization of the levels of 24-h urinary free cortisol, late-night salivary cortisol, and overnight 1-mg dexamethasone suppression test in patients without concurrent central adrenal insufficiency after surgery (31). 2.2 Patients and tissue/serum samples Surgical specimens of CD-affected tissues were collected from Xiangya Hospital, Central South University. Three normal pituitary tissues were obtained from cadaveric organ donors without any history of endocrine disease (Central South University). A total of 25 CD tissue samples were obtained for immunohistochemistry analysis. This study was conducted in compliance with the Helsinki Declaration and was ethically approved by the Xiangya Hospital Ethics Committee, Xiangya Hospital (Changsha, China). Tumor samples and corresponding clinical materials were obtained with written consent from all patients. 2.3 BIPSS After obtaining informed consent, BIPSS was performed using standard techniques described in previous studies (32, 33). Briefly, the patient’s head was immobilized to ensure midline positioning and prevent any potential bias towards asymmetric pituitary drainage by the petrosal sinuses. After placing peripheral catheters and cannulating both inferior petrosal sinuses, blood samples were collected at baseline and at 3, 5, 10, and 15 min following intravenous administration of DDAVP, which stimulates pituitary production of ACTH. Additional samples for experimental purposes were collected immediately following the 15-min sample collection to avoid interference with the patient’s diagnostic study. 2.4 Measurement of baseline plasma S1P concentration Blood samples were obtained from both petrosal sinuses and were centrifuged to remove cellular components. Samples that exhibited hemolysis or coagulation were excluded from the study. Plasma samples were stored at −80°C. The S1P levels in plasma were analyzed using a S1P competitive ELISA kit (Echelon Biosciences, Salt Lake City, UT) according to the manufacturer’s instructions (34). 2.5 Immunofluorescence staining The pituitary tissues were post-fixed and dehydrated with alcohol as follows: 70% for 24 h, 80% for 3 h, 90% for 4 h, 95% for 3 h, and finally in absolute alcohol for 2 h. Tissue slices with a 5-μm thickness were cut using a microtome (Thermo Fisher Scientific), blocked with 3% BSA, and then treated with primary antibodies to SPHK1 (CST, #3297) and ACTH (Proteintech, CL488-66358). Subsequently, the tissue slides were incubated with Alexa Fluor 488-conjugated anti-rabbit (Invitrogen, A21206, 1:200) or Alexa Fluor 555-conjugated anti-rabbit (Invitrogen, A21428, 1:200) secondary antibodies. Specimens were visualized and imaged using a fluorescence microscope. 2.6 Statistical analysis The Mann–Whitney U test was used to assess the clinical–molecular associations in adenoma samples, whereas the chi-square test was used to compare categorical data. The Kruskal–Wallis analysis and ANOVA were conducted for multiple comparisons. Statistical analyses were conducted using SPSS v20 and GraphPad Prism version 7. All results were presented in graphs and tables as median ± interquartile range. The distribution of each parameter was presented as the minimum–maximum range. Parametric or nonparametric statistical tests were applied, as appropriate, after testing for normality. The receiver operating characteristic curve was used to determine the cut-off value for predicting tumor laterality. Pearson correlation analyses was used to examine the correlations between variables. Proportions were expressed as percentages, and significance was defined as P < 0.05. 3 Results 3.1 Clinical characteristics of remission and nonremission in patients with CD This study included 25 patients with CD who underwent BIPSS before surgery (Figure 1). Among them, 12 patients had microadenomas, whereas the remaining 13 had inconclusive magnetic resonance imaging findings; clinicopathological data are summarized in Supplementary Table 1. Table 1 displays the demographics of patients who achieved remission (n = 16) and those who did not (n = 9). No significant differences were observed in terms of sex, age at diagnosis, or radiological variables between patients who achieved and those who did not achieve remission (P > 0.05). Patients who achieved remission exhibited a higher prevalence of emotional lability (P < 0.05). However, no significant differences were observed in other parameters (P > 0.05). Figure 1 Figure 1 Flowchart of the screening process employed to select eligible participants for the study. Table 1 Table 1 Baseline clinical features of patients with pituitary tumors secreting adrenocorticotropin. Several recent studies have established morning cortisol level measured on postoperative day 1 (POD1) as a predictive biomarker for long-term remission of CD (35, 36). For biochemical features, patients who did not achieve remission exhibited higher serum cortisol (19.16 ± 5.55 vs. 5.95 ± 1.42; P = 0.014) and median serum (8 am) ACTH (10.26 ± 8.24 vs. 5.15 ± 3.68; P = 0.042) levels on POD1. No significant differences were observed in the preoperative baseline 4 pm serum cortisol levels, preoperative baseline 0 am serum cortisol levels, preoperative 8 pm ACTH levels, 4 pm ACTH levels, and 0 am ACTH levels (P > 0.05) (Table 2). In addition preoperative FT3, FT4, TSH, GH, FSH, LH, and PRL levels were comparable in patients with and without remission. Table 2 Table 2 Baseline clinical and biochemical features of patients with pituitary tumors secreting adrenocorticotropin. 3.2 Overexpression of SPHK1 and higher concentrations of serum S1P on the tumor side in patients with CD Prior studies have demonstrated that ACTH acutely activates SPHK1 to increase S1P concentrations (19). Upregulation of SPHK1 is associated with poor prognosis in endocrine-related cancer (17, 18, 21). To investigate the role of SPHK1 in CD, we performed a heatmap analysis of key genes involved in phospholipid metabolism and signaling pathways in CD adenomas and surrounding normal tissues using the GEO dataset (GEO208107). This analysis revealed the activation of crucial genes involved in phospholipid metabolism and signaling pathways in ACTH-secreting pituitary adenomas (Supplementary Figure 1). Subsequently, we compared the association between pituitary SPHK1 expression and proopiomelanocortin, corticotropin-releasing hormone, corticotropin releasing hormone receptor 1, and corticotropin releasing hormone receptor 2 in pituitary tumor tissues and identified a positive correlation between SPHK1 and ACTH tumor-related genes in the TNM plot database (Supplementary Figure 2). To investigate the potential role of SPHK1 in CD, we compared the expression values of SPHK1 in the normal pituitary tissues and those obtained from patients with CD in the remission/nonremission groups. Immunofluorescence staining (Figures 2A, B; Supplementary Figure 3) revealed an increased number of double-positive cells for SPHK1 and ACTH in CD-affected pituitary tissues than those in the normal pituitary tissues. Furthermore, the proportion of double-positive cells for SPHK1 and ACTH was significantly higher in the nonremission CD adenomas tissues than that in the remission CD adenomas. Furthermore, we investigated the concentration of S1P in bilateral petrosal sinus blood samples and observed that the concentration was significantly higher on the adenoma side than that on the nonadenoma side (397.7 ± 15.4 vs. 261.9 ± 14.88; P < 0.05, Figure 2C). Thus, these findings suggested a close association between S1P concentration and the development of ACTH-secreting tumor. Figure 2 Figure 2 (A) Representative images of immunofluorescence double staining for SPHK1 (green) and ACTH (pink) in normal pituitary glands and ACTH-secreting pituitary adenomas from the remission and nonremission groups (Normal: n = 3, ACTH pituitary adenoma: remission vs. nonremission: n = 16 vs. 9); scale bars: 100-μm upper and 50-μm lower. (B) Quantitative analysis; white arrows indicate double-positive cells for ACTH and SPHK1. (C) The concentration of S1P in the plasma obtained from the inferior petrosal sinus of the adenoma side and nonadenoma side. ***P < 0.001. Bar represents mean ± SD. 3.3 Combination of interpetrosal S1P and ACTH ratios improved the diagnostic performance for adenoma laterality The pathology of patients with CD was classified based on adenomatous tissue with ACTH-positive immunostaining into adenoma or nonadenoma sides. To evaluate the correlation between the interpetrosal S1P ratio lateralization and tumor location, we compared the accuracy of predicting tumor laterality using the interpetrosal S1P ratio (>1) and interpetrosal ACTH ratio (>1.4) (the interpetrosal ACTH ratio >1.4 is acknowledged for its positive role in predicting tumor laterality), as well as their combination. Our results indicated that using the interpetrosal S1P or ACTH ratios alone yielded accuracies of 64% and 56% respectively. Notably, the combination of both demonstrated a significantly improved accuracy of 73% (Figure 3A). Figure 3 Figure 3 (A) Bar graph illustrating the accuracy of predicting tumor laterality. (B) Receiver operating characteristic (ROC) curve analysis of interpetrosal ACTH ratio to predict tumor location. (C) ROC curve analysis of the interpetrosal S1P ratio to predict tumor location. (D) ROC curve analysis of the combination of the interpetrosal S1P and ACTH ratios to predict tumor location. Thereafter, the receiver operating characteristic analysis was performed to determine the role of predicting tumor laterality. In particular, the interpetrosal ACTH ratio with an AUC of 75.32% (95% CI: 60.06–97.46%, P < 0.05) and the interpetrosal S1P ratio demonstrated a clinically significant diagnostic accuracy for lateralization, with an AUC of 79.17% (95% CI: 44.40–85.84%, P < 0.05). Furthermore, combining the interpetrosal S1P and ACTH ratios generated an receiver operating characteristic curve with an AUC of 84.09% (95% CI: 52.3–96.77%, P < 0.05) for predicting lateralization with tumor location (cutoff value: interpetrosal S1P ratio ≥1.06, interpetrosal ACTH ratio ≥2.8, 81.82% sensitivity, and 75% specificity) (Figures 3B–D). 3.4 Interpetrosal S1P ratio serves as a predictive factor for early remission in CD To investigate whether the interpetrosal S1P ratio is associated with early postoperative remission in CD, we compared the baseline interpetrosal S1P ratio between patients with CD in the remission and nonremission groups. Interestingly, we observed that the nonremission group exhibited higher interpetrosal S1P ratios than those of the remission group (median, 1.28 ± 0.25 vs. 1.10 ± 0.09, P = 0.012) (Figure 4). Figure 4 Figure 4 Left picture: Scatter plot of bilateral S1P concentrations in the remission and nonremission groups; the slope represents the interpetrosal S1P ratio, blue dots represent the remission group, and red dots represent the nonremission group. Right picture: The interpetrosal S1P ratio in the remission and nonremission groups. *P < 0.05. Bar represents mean ± SD. To investigate potential factors affecting the interpetrosal S1P ratio, we compared the correlation between interpetrosal S1P ratio and various clinical indicators. This analysis revealed that the interpetrosal S1P ratio positively correlated with preoperative FSH and LH levels, as well as with postoperative 8 am ACTH levels. No significant difference was observed between the interpetrosal S1P ratio and other indicators (Supplementary Figure 4). 4 Discussion The use of BIPSS involves collection of samples from each inferior petrosal sinus simultaneously, enabling a direct comparison of ACTH concentrations between the left and right petrosal sinuses. BIPSS is used for two purposes: 1) to assist in the differential diagnosis of Cushing’s syndrome; and 2) to determine which side of the pituitary gland contains an adenoma in patients with CD. The interpetrosal ACTH ratio is also useful in determining the location/lateralization of pituitary microadenomas (24, 30, 37), thereby providing guidance to the neurosurgeon during surgery. To our knowledge, this is the first study to demonstrate that serum S1P levels in patients with CD are significantly higher on the adenoma side of the inferior petrosal sinus than on the nonadenoma side. The interpetrosal S1P ratio exhibited a positive significance in predicting tumor laterality, and the predictive performance was improved when S1P was combined with the interpetrosal ACTH ratio. Notably, the interpetrosal S1P ratio exhibited a positive significance in predicting remission after surgery. Furthermore, the interpetrosal S1P ratio demonstrated a positive and significant correlation with preoperative FSH and LH levels, as well as 8 am ACTH levels on POD1. ACTH is recognized for its role in controlling the expression of genes involved in steroid production and cortisol synthesis in the human adrenal cortex through sphingolipid metabolism (19). Specifically, ACTH rapidly stimulates SPHK1 activity, leading to an increased in S1P levels, which in turn, increases the expression of multiple steroidogenic proteins (20). Our study demonstrated that higher S1P concentrations were present on the tumor side than on the nontumor side in patients with CD, indicating that the regulatory relationship between ACTH and S1P also exists in ACTH-secreting pituitary adenomas. Several pieces of evidence have supported the potential relationship between S1P and the occurrence of CD. Interestingly, SPHK1 and S1P are known to be integral to the regulation of epidermal growth factor receptor (EGFR) (38), which is highly expressed in human corticotropinomas, where it triggers proopiomelanocortin (the precursor of ACTH) transcription and ACTH synthesis (39). Blocking EGFR activity with an EGFR inhibitor can attenuate corticotroph tumor cell proliferation (40). Furthermore, SPHK1 and proopiomelanocortin share a common transcriptional coactivator, P300 (41, 42). Notably, S1P also directly binds to and inhibits histone deacetylase 2, thereby regulating histone acetylation and gene expression (43). Notably, histone deacetylase 2 expression is deficient in ACTH-pituitary adenomas in CD, contributing to glucocorticoid insensitivity (44), which is a hallmark of CD and a feature associated with nonremission. These studies further demonstrated an association between high S1P ratio and nonremission of CD. Our study, for the first time, established an association between SPHK1/S1P and ACTH adenoma. Nevertheless, further experimental verification is required to confirm the existence of common pathways linking SPHK1 and ACTH. Thus, these findings indicated that the S1P ratio can, to some extent, reflect the differences in ACTH levels and may serve as a surrogate marker for detecting ACTH-secreting pituitary adenomas. BIPSS is a highly effective procedure for diagnosing pituitary sources of ACTH in CD and remains the gold standard diagnostic method. However, some findings indicated certain limitations associated with the use of the inferior petrosal sinus sampling (IPSS) method in predicting tumor lateralization. The possible causes of error include asymmetrical or underdeveloped petrosal sinus anatomy and placement of the catheter (27). The present study revealed a notable increase in the interpetrosal ACTH ratio among patients with accurate predictions of tumor laterality than among those with inaccurate predictions, although the positive predictive value remained low. These findings suggested that other mechanisms may exist that contribute to false-positive results. The limitations on lateralization highlighted the need for further research to understand the underlying mechanisms contributing to the accuracy of IPSS in predicting tumor lateralization. Further investigation is required to understand these potential mechanisms and improve the accuracy of IPSS in predicting tumor lateralization. We observed that the interpetrosal S1P ratio was slightly more effective than the ACTH ratio in predicting tumor laterality. However, combining both methods significantly improved the diagnostic sensitivity and specificity. These results have important implications for clinical practice as accurate tumor lateralization is essential for the correct management and treatment of pituitary adenomas. Overall, these findings highlighted the importance of using multiple measures in predicting tumor lateralization and suggested that combining measures may be more effective than relying on any single measure alone. Future research should investigate additional measures to improve the accuracy of tumor lateralization and optimize the use of existing measures for making clinical decisions. The initial treatment recommendation for CD is surgery. However, long-term surveillance is necessary because of the high recurrence rate (12). Therefore, identifying patients who are at a greater recurrence risk would be helpful in establishing an effective surveillance strategy. Our study revealed that the expression of SPHK1 in pituitary tissue was higher in postoperative nonremission group than in postoperative remission group. Moreover, patients in the nonremission group exhibited significantly higher interpetrosal S1P ratios than those of patients in the remission group. SPHK1 catalyzes the direct phosphorylation synthesis of S1P, and the S1P ratio can thus reflect the expression level of SPHK1 in ACTH tumors. Since S1P can increase the expression of multiple steroidogenic proteins, including steroidogenic acute regulatory protein, 18-kDa translocator protein, low-density lipoprotein receptor, and scavenger receptor class B type I (20), the interpetrosal S1P ratios may be indicative of disease prognosis. This finding is consistent with previous findings indicating the overexpression of SPHK1 is associated with poor prognosis in various neuroendocrine tumors, as factors associated with tumor proliferation, S1P and SPHK1, may play a key role in the proliferation and survival of ACTH pituitary adenomas. The high proportions of SPHK1/ACTH double-positive cells are likely associated with greater phenotypic severity, and CD tumors with this phenotype may have a poor prognosis. These findings hold clinically significance for predicting early postoperative remission in patients with CD. As aforementioned, the interpetrosal S1P ratios have been suggested as a useful diagnostic tool for determining adenoma lateralization in CD, which can also serve as a prognostic indicator for postoperative remission. Pearson correlation analysis indicated that ACTH 8 am on POD1 and FSH/LH levels were significantly associated with the interpetrosal S1P ratio, suggesting that these pituitary dysfunctions may have a role in the early remission of CD. However, the sample size in this study was relatively small, and further studies with larger sample sizes are needed to confirm these findings. Additionally, other factors affecting surgical outcomes, such as the experience of the surgeon, extent of surgical resection, and use of adjuvant therapy, should be considered when predicting postoperative remission in patients with CD. This study has some limitations. First, the study was retrospective in design, which limited the control of confounding factors. Additionally, because of the limited sample size, we did not specifically investigate cases where the ACTH ratio failed to accurately identify the correct tumor location. Finally, we did not explore the functional evidence of a common pathway between SPHK1 and ACTH. Despite these limitations, the study contributes to our understanding of the potential utility of the interpetrosal S1P ratio as a biomarker for CD and provides a basis for future research in this area. In conclusion, our study demonstrated a significant association between the interpetrosal S1P ratio and tumor laterality, as well as in early remission in CD. These findings suggested that the interpetrosal S1P ratio could serve as a useful biomarker in clinical practice. Moreover, targeting genes and drugs related to SPHK1/S1P could provide novel therapeutic strategies for treating CD. Data availability statement The original contributions presented in the study are included in the article/Supplementary Material. Further inquiries can be directed to the corresponding author. Ethics statement The studies involving humans were approved by The Xiangya Hospital Ethics Committee, Xiangya Hospital (Changsha, China). The studies were conducted in accordance with the local legislation and institutional requirements. The participants provided their written informed consent to participate in this study. Author contributions HS: conceptualization, methodology, software, visualization, and investigation. CW and BH: software. YX: writing – review & editing. All authors contributed to the article and approved the submitted version. Funding The author(s) declare that no financial support was received for the research, authorship, and/or publication of this article. Acknowledgments The authors gratefully acknowledge contributions from the GEO databases and TNMplot database (https://www.tnmplot.com/). Conflict of interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Publisher’s note All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher. Supplementary material The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fendo.2023.1238573/full#supplementary-material References 1. Tritos NA, Miller K. Diagnosis and management of pituitary adenomas: A review. JAMA (2023) 329(16):1386–98. doi: 10.1001/jama.2023.5444 PubMed Abstract | CrossRef Full Text | Google Scholar 2. 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Genes Dev (2006) 20:2871–86. doi: 10.1101/gad.1444606 PubMed Abstract | CrossRef Full Text | Google Scholar Keywords: ipss, sphingosine-1-phosphate, Cushing’s disease, remission, tumor laterality Citation: Sun H, Wu C, Hu B and Xiao Y (2023) Interpetrosal sphingosine-1-phosphate ratio predicting Cushing’s disease tumor laterality and remission after surgery. Front. Endocrinol. 14:1238573. doi: 10.3389/fendo.2023.1238573 Received: 12 June 2023; Accepted: 17 October 2023; Published: 31 October 2023. Edited by: Anton Luger, Medical University of Vienna, Austria Reviewed by: Guangwei Wang, Hunan University of Medicine, China Marie Helene Schernthaner-Reiter, Medical University of Vienna, Austria Copyright © 2023 Sun, Wu, Hu and Xiao. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. *Correspondence: Yuan Xiao, xiaoyuan2021@csu.edu.cn Disclaimer: All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article or claim that may be made by its manufacturer is not guaranteed or endorsed by the publisher. From https://www.frontiersin.org/articles/10.3389/fendo.2023.1238573/full
  18. Abstract Background This single-center retrospective cohort study aimed to describe the findings and validity of Bilateral inferior petrosal sinus sampling (BIPSS) in the differential diagnosis of patients with ACTH-dependent Cushing’s syndrome (CS). Methods Eleven patients underwent BIPSS due to equivocal biochemical tests and imaging results. Blood samples were taken from the right inferior petrosal sinus (IPS), left IPS, and a peripheral vein before and after stimulation with desmopressin (DDAVP). ACTH and prolactin levels were measured. The diagnosis was based on the ACTH ratio between the IPS and the peripheral vein. Also, lateralization of pituitary adenoma in patients with Cushing’s disease (CD) was predicted. No significant complications were observed with BIPSS. Results Based on the pathology report, eight patients had CD, and three had ectopic ACTH syndrome (EAS). Unstimulated BIPSS resulted in a sensitivity of 87.5%, specificity of 100%, PPV of 100%, NPV of 75%, and accuracy of 91%. Stimulated BIPSS resulted in a sensitivity of 100%, specificity of 100%, PPV of 100%, NPV of 100%, and accuracy of 100%. However, pituitary magnetic resonance imaging (MRI) had a lower diagnostic accuracy (sensitivity:62.5%, specificity:33%, PPV:71%, NPV:25%, accuracy:54%). BIPSS accurately demonstrated pituitary adenoma lateralization in 75% of patients with CD. Conclusions This study suggests that BIPSS may be a reliable and low-complication technique in evaluating patients with ACTH-dependent CS who had inconclusive imaging and biochemical test results. The diagnostic accuracy is improved by DDAVP stimulation. Pituitary adenoma lateralization can be predicted with the aid of BIPSS. Peer Review reports Introduction All disorders with manifestations associated with glucocorticoid excess are called Cushing’s syndrome. Exogenous corticosteroids cause most CS cases, and endogenous CS cases are rare [1, 2]. The diagnosis of Cushing’s syndrome may be complicated, particularly in cases with ambiguous clinical findings, atypical presentations, and cyclic hypercortisolemia [3,4,5]. The initial laboratory tests for diagnosis of CS include 24-hour urinary free cortisol (UFC), late-night salivary cortisol, and low-dose dexamethasone suppression test (DST). These tests only represent hypercortisolemia [1, 2]. Once CS is diagnosed, further evaluations are needed to identify the etiology. The first step is to measure the plasma ACTH level. A low plasma ACTH level indicates ACTH-independent CS and a high level suggests ACTH-dependent CS. Normal ACTH can also occur in ACTH-dependent CS. Almost all cases of ACTH-dependent are due to pituitary adenoma (Cushing’s disease) or EAS [1, 2, 6]. Some ectopic sources include neuroendocrine tumors, bronchial carcinoma, and pancreatic carcinoma [7, 8]. Because of the high mortality in tumors associated with EAS, it is essential to differentiate CD from EAS. To distinguish CD from EAS, a high-dose dexamethasone suppression test (HDDST), corticotropin-releasing hormone (CRH), or DDAVP stimulation tests, or pituitary MRI is recommended [1, 2, 6, 9,10,11,12]. MRI can be equivocal in half of the patients, and only relatively large lesions (> 6 mm) detected on MRI reliably confirm the diagnosis of CD with biochemical confirmation and expected clinical symptoms [9]. Considering the relatively low sensitivity and specificity of non-invasive tests [13, 14] and the high complications of the surgery, it seems reasonable to use a test with high sensitivity and specificity and few complications before resection. BIPSS with CRH or DDAVP stimulation can be helpful for further evaluation [1, 2, 10, 15, 16]. The BIPSS procedure is the same in both stimulation methods. Due to its lower cost, availability, and comparable diagnostic accuracy, using DDAVP instead of CRH for BIPSS is an alternative [17, 18]. BIPSS has been reported to have high sensitivity and specificity and is a safe procedure when performed by experienced interventional radiologists [15, 16, 19, 20]. This case series describes the experience with BIPSS and examines the validity of BIPSS for differentiating CD from EAS in patients with ACTH-dependent CS who had ambiguous or equivocal results in non-invasive tests. Materials and methods Patients This retrospective cohort study included 11 patients with ACTH-dependent CS who underwent BIPSS between 2018 and 2020 in a tertiary care hospital. Data collection Well-trained nurses conducted anthropometric measurements, including height and weight. Standing height was measured with a portable stadiometer (rounded to the nearest 0.1 cm). Using a calibrated balance beam scale, this study measured weight in the upright position (rounded to the nearest 0.1 kg). Body mass index (BMI) was calculated by dividing weight (kg) by height squared (m2). Well-trained examiners measured blood pressure (systolic and diastolic) at the left arm in the sitting position after 5 min of rest using a calibrated mercury sphygmomanometer. The blood sample was taken, and fasting blood sugar (FBS), hemoglobin (Hb), potassium (K), and creatinine (Cr) were measured. All research was performed in accordance with the Declaration of Helsinki. Informed consent was obtained from all participants or their legal guardians. Biochemical tests and imaging Patients with signs and symptoms of CS underwent screening evaluations, and confirmatory tests were performed using serum cortisol and 24-hour UFC. After confirmation of CS, ACTH was measured using an immunoradiometric assay to categorize patients into ACTH-dependent or independent groups. ACTH test was performed with SIEMENS IMMULITE 2000 device with an analytical sensitivity of 5 pg/ml (1.1 pmol/l) and CV ∼7.5%. HDDST was conducted by administering 2 mg dexamethasone every 6 h for 48 h to all patients, and then serum cortisol and 24-hour UFC were rechecked. A pituitary MRI was performed with sagittal and coronal T1- and T2-weighted images before and after the gadolinium injection. BIPSS procedure After biochemical tests and imaging, an experienced interventional radiologist performed bilateral and simultaneous catheterization of the inferior petrosal sinuses. Venography was obtained to evaluate venous anatomy and catheter placement. The retrograde flow of contrast dye into the contralateral cavernous sinuses was used as a marker of adequate sampling. After the correct placement of catheters, blood samples were obtained from each of three ports (peripheral (P), left inferior petrosal sinus (IPS), and right IPS) at -15, -10, -5, and 0 min. The current study used DDAVP for stimulation. After peripheral injection of 10 micrograms of DDAVP, blood samples from these three sites were obtained at + 3, +5, + 10, and + 15 min. Three samples from these sites were also obtained to measure prolactin. Upon collection, BIPSS samples were placed in an ice-water bath. At the end of the procedure, samples were taken to the laboratory, where the plasma was separated and used for immediate measurement of ACTH. Specimens were refrigerated, centrifuged, frozen, and assayed within 24 h. After the samples were obtained, both femoral sheaths were removed, and manual compression was used to obtain hemostasis before transferring patients to the recovery room. The whole procedure took 1–2 h. Patients underwent strict bed rest for 4 h before discharge on the same day. All BIPSS were performed without significant complications, and only hematoma at the catheterization site was observed in some patients. BIPSS interpretation The ratio of IPS ACTH to peripheral ACTH level (IPS/P ACTH) for each side was calculated. Baseline sampling at minute 0 with IPS/P ≥ 2 or stimulated sampling at minute 3 with 1PS/P ≥ 3 is confirmatory for CD [1, 8]. Also, the IPS/P ratio was checked for prolactin level after DDAVP stimulation (stimulated IPS/P prolactin). A stimulated IPS/P prolactin ≥ 1.8 indicates successful catheterization, meaning the catheter is correctly placed in the IPS [21]. For further evaluation, the current study normalized the ACTH to the prolactin level by dividing stimulated IPS/P ACTH into stimulated IPS/P prolactin for each side. A normalized ACTH/prolactin IPS/P ratio ≥ 1.3 supports a pituitary ACTH source (Cushing’s disease), and a normalized ratio ≤ 0.7 an ectopic source (EAS) [22]. The values between 0.7 and 1.3 are equivocal. The inter-sinus ratio was defined as the ratio of the IPS/P ACTH level of one side with the higher level divided by the IPS/P ACTH level of the other side with the lower level, either before or after stimulation. An inter-sinus ratio ≥ 1.4 indicates lateralization to the side with a higher IPS/P ACTH level [23]. Statistical analysis This analysis used SPSS software version 18 (SPSS, Inc.) to perform analyses. Data were expressed as numbers and percentages. Continuous variables were presented as means (± SD). This study reported the median or range when the data did not follow a normal distribution. The Shapiro-Wilk test was used to test for normality. The nonparametric Mann-Whitney U Test was utilized to compare variables. The sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and accuracy of the tests were calculated based on standard statistical equations. Results Baseline characteristics and clinical manifestations This retrospective research studied 11 patients with ACTH-dependent CS, including eight females (72.7%) and three (27.3%) males. The median (Q1-Q3) age was 32.0 (22–45) years. The median (Q1-Q3) of BMI, systolic blood pressure (SBP), diastolic blood pressure (DBP), FBS, Hb, K, and Cr were 29.2 (24.8–33.3), 130.0 (125–140), 80.0 (80–95), 98.0 (88–103), 13.5 (12.4–13.9), 4.2 (3.9–4.5), and 1.0 (0.9–1.1), respectively. The demographic characteristics of patients are presented in Table 1. The Hb levels were not different in women and men (median 13.35 vs. 13.70, p-value = 0.776). In addition, no statistical difference between patients with a final diagnosis of CD and EAS was detected for Hb levels (Total: median 13.60 vs. 13.2, p-value > 0.05) (Women: median 13.5 vs. 13.2, p-value > 0.05) (Men: median 13.7 vs. 13.25, p-value > 0.05). Table 1 Demographic characteristics of the studied patients Full size table 90% of patients had at least one skin manifestation, such as striae, easy bruising, acne, hyperpigmentation, hirsutism, hair loss, edema, and hypertrichosis. Other symptoms were hypertension (HTN) (81%), reproductive dysfunction (81%), including infertility, oligomenorrhea, loss of libido, weight gain (72%), proximal muscle weakness (45%), and headache (27%) (Table 2). Table 2 Clinical manifestations of the studied patients Full size table Results of biochemical tests Biochemical tests results, including basal serum cortisol (median:26 mcg/dl, range:15-54.5 mcg/dl), basal 24-hour UFC (median:670 mcg/dl, range:422–1545 mcg/dl), ACTH (median:58.8 pg/ml, range:25–155 pg/ml), serum cortisol after HDDST (median:14.2 mcg/dl, range:2.63-36.0 mcg/dl), 24-hour UFC after HDDST (median:292 mcg/dl, range:29.5–581 mcg/dl) are presented in Table 3. According to the basal serum cortisol results, eight patients (Cases 1, 3, 5, 7, 8, 9, 10, and 11) had basal serum cortisol levels > 22 mcg/dl, which indicates hypercortisolemia. Other patients (Cases 2, 4, and 6) had basal serum cortisol in the normal range (5–25 mcg/dl) and were considered as false negative results of this test. Table 3 The results of biochemical tests in the studied patients Full size table All patients had elevated basal 24-hour UFC levels (422–1545 mcg/dl), indicative of hypercortisolemia (Table 3). There were six patients with elevated peripheral ACTH levels (> 58 pg/ml) (cases 5, 6, 8, 9, 10, and 11). Other patients had ACTH within the normal range (6–58 pg/ml) (cases 1, 2, 3, 4, 7) (Table 3). None of the patients showed suppression after 1 mg DST. After HDDST, cases 2, 3, 8, and 10 had more than 50% suppression of serum cortisol. In the other six patients, serum cortisol was not suppressed or suppressed by less than 50%. In one patient, serum cortisol levels were not measured (case 1) because the sample was not stored under standard test conditions. Also, eight patients had more than 50% 24-hour UFC suppression after HDDST (cases 1, 2, 3, 4, 6, 7, 9, and 10). In two patients, 24-hour UFC was suppressed less than 50% (cases 5 and 11), and in one patient (case 8), the 24-hour UFC sample was not tested due to the non-standard condition of the sample. BIPSS results BIPSS results before and after stimulation are shown in Table 4. The baseline value (sampling at minute 0) of IPS/P ACTH ≥ 2 confirms CD. According to this ratio, cases 1,3,4,5,6,7, and 8 were diagnosed as CD. The unilateral source for CD was confirmed in cases 1, 3, 7, and 8. BIPPS didn’t demonstrate lateralization in cases 4, 5, and 6. Table 4 Baseline and stimulated IPS/P ratio for ACTH and Prolactin in the studied patients Full size table The highest IPS/P ACTH ratio was 3 min after the DDAVP injection. A sampling at minute 3 with stimulated IPS/P ACTH ≥ 3 confirms CD. This ratio confirmed CD in cases 1–8 and showed a unilateral source for CD in cases 1, 2, 3, and 7. The ratio didn’t demonstrate lateralization in cases 4, 5, 6, and 8. The stimulated IPS/P prolactin was ≥ 1.8 in all cases. The variability in the IPS/P ACTH ratio in patients with CD is shown in Fig. 1. The peak of this ratio was 3 min after the DDAVP injection. In patients with EAS, there were no changes before or after the DDAVP stimulation. Fig. 1 Comparison of mean values of IPS/P ACTH in CD (Lt.) and EAS (Rt.). IPS; inferior petrosal sinus; P: peripheral; ACTH: adrenocorticotropic hormone; CD: Cushing’s disease; EAS: ectopic ACTH syndrome; Lt: left; Rt: right Full size image According to the Prolactin-normalized ACTH IPS/P ratios, eight patients (cases 1–8) were diagnosed as CD and three as EAS (cases 9–11). In cases 1, 2, 3, 7, and 8, unilateral sources of CD were confirmed, but in cases 4,5 and 6, bilateral sources were detected (Table 4). According to the inter-sinus ratio, BIPSS could lateralize the source of ACTH in all patients with CD. The inter-sinus ratio in patients with EAS could not lateralize any pituitary source for ACTH (Table 4). In five patients with CD and one with EAS, the highest peripheral ACTH level was observed 15 min after stimulation. Two patients with CD and one with EAS had the highest peripheral ACTH level 10 min after stimulation. Only one patient with CD and one with EAS had the highest peripheral ACTH level 5 min after stimulation. No patient had maximum peripheral ACTH levels in the first post-stimulation sample (minute 3). The larger numerator or smaller denominator produces a higher value in a ratio. In the samples obtained immediately after stimulation, the highest concentration of ACTH was in the IPS, and the lowest was in the peripheral blood. Therefore, as mentioned, the highest post-stimulation value of the IPS/P ACTH ratio was obtained at minute 3. MRI results MRI results showed pituitary adenoma in five patients, enhancement in one patient, pituitary mass and lesion in two patients, empty sella in two patients, and possible pituitary adenoma and adrenal mass in one patient (Table 5). Table 5 Final diagnosis, lateralization, MRI results, and management Full size table Immunohistochemistry (IHC) results According to the pathology report, eight patients were confirmed as CD (Table 5). The other two patients were EAS (one carcinoid tumor of the lung and one pheochromocytoma). One patient had no documented pathologic source of hypercortisolemia because the patient did not consent to surgery, and the diagnosis of EAS was made based on the results of biochemical tests. BIPSS vs. MRI results MRI results showed pituitary adenoma in five patients with CD. MRI and BIPSS showed the adenoma on a similar side in two of them. In the other three patients, MRI showed bilateral adenoma, but BIPSS lateralized the adenoma to one side. One of the other three patients had only left-sided enhancement but no overt adenoma on MRI, whereas BIPSS lateralized the adenoma to the right side. One patient had a low-signal pituitary mass on the right side on MRI, and BIPSS also lateralized to the right. Another patient with a history of transsphenoidal surgery (TSS), diagnosed as recurrent CD, had a partially empty sella. MRI was equivocal, but BIPSS lateralized to the left side. Among patients with EAS, one with an equivocal BIPSS result had an empty sella on MRI. Two other patients had pituitary lesions on MRI, but BIPSS results were equivocal. Comparison between BIPSS, MRI, and surgery Among patients with CD, the final diagnosis based on surgery in three patients was consistent with MRI and BIPSS results and lateralized the adenoma on the same side. In one patient, the surgery result was similar to the MRI findings and showed bilateral adenoma, but BIPSS showed adenoma on the left side. In the patient with equivocal MRI findings and a history of TSS, IHC could not identify ACTH +, although BIPSS lateralized to the left side. In three other patients, surgery results were concordant with BIPSS and lateralized the adenoma on the same side, although MRI showed discordant results. Validity of BIPSS Baseline IPS/P ACTH resulted in a sensitivity of 87.5%, specificity of 100%, PPV of 100%, NPV of 75%, and accuracy of 91%. Stimulation with DDAVP improved validity. Both stimulated IPS/P ACTH and normalized ACTH/prolactin IPS/P ratio resulted in a sensitivity of 100%, specificity of 100%, PPV of 100%, NPV of 100%, and accuracy of 100%. BIPSS, either unstimulated or stimulated, had higher validity than MRI, with a sensitivity of 62.5%, specificity of 33%, PPV of 71%, NPV of 25%, and accuracy of 54%. BIPSS accurately predicted pituitary adenoma lateralization in 75% of patients with CD. Discussion In this study, BIPSS before stimulation showed a sensitivity of 87.5% and a specificity of 100%. However, BIPSS after stimulation showed a sensitivity of 100% and specificity of 100%. It has been demonstrated that the sensitivity of BIPSS can vary from 88 to 100%, and its specificity from 67 to 100% in the diagnosis of CD [24]. Previous studies have reported sensitivity and specificity of more than 80% and 90% for BIPSS, and the combination of BIPSS with stimulation by CRH or DDAVP improves the sensitivity and specificity to more than 95 and 100%, respectively [15, 19, 25]. Chen et al. suggested the optimal IPS:P cutoff value of 1.4 before and 2.8 after stimulation [20]. Considering these cutoffs, the only patient in this study who was negative for CD before stimulation becomes positive, and the sensitivity before stimulation increases from 87.5 to 100%. The diagnostic accuracy after stimulation remains unchanged. Results of the current study showed that BIPSS is highly valued in final diagnosis, even without stimulation. In this investigation, the utilization of Prolactin-normalized ACTH IPS/P ratios exhibited a sensitivity and specificity of 100% for the CD diagnosis. This finding aligns with research conducted by Detomas et al., which reported a sensitivity of 96% and specificity of 100% for the normalized ACTH: Prolactin IPS/P ratio [26]. It seems that concurrently assessing prolactin levels may potentially enhance the diagnostic accuracy of BIPSS. However, the current literature is inconsistent. Some studies do not support the use of prolactin to diagnose CD [27]. In all patients, the IPS/P ACTH ratio at minute 15 did not show a considerable difference from this ratio at minute 0. Previous studies have shown that sampling at minute 15 is not helpful for diagnosis [1, 15, 20, 28]. Unlike the IPS/P ACTH ratio, six patients had the highest peripheral ACTH level at minute 15 after stimulation, but no patient had it at minute 3 after stimulation. However, more studies are needed to obtain more precise results, and this study’s sample size was limited. BIPSS accurately lateralized the adenoma in six patients with CD, but MRI was able to lateralize the adenoma in two patients correctly. BIPSS had higher validity than MRI in differentiating CD from EAS, both with and without stimulation. The current literature is controversial. Colao et al. reported that adenoma could be accurately localized in 65% of patients using IPSS [23]. However, Lefournier et al. showed that the diagnostic accuracy of IPSS in identifying the side of the pituitary adenoma was 57% [28]. Wind et al. showed that the PPV for IPSS to identify the tumor side correctly was 69%. Additionally, MRI was more accurate than IPSS in tumor lateralization [29]. Earlier studies have shown that MRI may show a pituitary lesion, and BIPSS indicates a pituitary adenoma. However, the lesion observed on the MRI is not related to the pituitary adenoma [1, 15, 19, 25, 28]. Also, MRI may show pituitary lesions, while BIPSS indicates EAS. In the current study, the concordance of IHC results with BIPSS and MRI findings was inconclusive, possibly due to the limited number of patients. However, there is disagreement about the role of pathological study in diagnosis [19, 28]. Eight patients had elevated basal serum cortisol levels in this study (Sensitivity:73%). Instead, all patients had hypercortisolemia according to basal 24-hour UFC results, and no false-negative results were observed (Sensitivity:100%). This study’s findings were consistent with previous studies regarding low sensitivity for basal serum cortisol and high sensitivity for 24-hour UFC as screening tests for hypercortisolemia [6, 30, 31]. After HDDST, basal serum cortisol suppression was observed in three patients with CD (cases 2, 3, and 😎 but not in the others with CD. Also, serum cortisol levels were suppressed after HDDST in a patient with EAS who had a lung carcinoid tumor. Arnaldi et al. showed that some carcinoid tumors might be sensitive to HDDST, and suppression of serum cortisol may be observed after this test [1, 32]. After HDDST, six patients with CD had suppressed 24-hour UFC, but one did not show more than 50% suppression. Two patients with EAS had more than 50% 24-hour UFC suppression. According to the final pathology report, the sensitivity of serum and urine cortisol level tests after HDDST was 43% and 86%, and the specificity was 67% and 33%, respectively. PPV in both was 75%, NPV was 33% and 50%, and accuracy was 50% and 70%, respectively, which shows that these preliminary tests cannot be a good guide for the final diagnosis and subsequent treatment planning. Previous studies showed that more than one biochemical test could improve the accuracy for differentiating between CD and EAS [1, 5, 6, 9, 31]. The current study confirms the importance of using more than one biochemical test for diagnosing hypercortisolemia and diagnosing CD from EAS. Detomas et al. reported that Hb levels were high in females with CS while they were low in males with CS. Furthermore, there were lower levels of Hb in EAS than in CD in females [33]. In the current study, the Hb levels were not different in women and men. Furthermore, no statistical difference was observed for Hb levels between patients with a final diagnosis of CD and EAS. Hb levels did not contribute to diagnosing ACTH-dependent CS in this analysis. There were some limitations in this study. First, the sample size was relatively small. Second, it was a retrospective study. Further studies could investigate the BIPSS in a larger sample size and determine the validity of this method in patients with CS. Conclusions The current study suggests that BIPSS can be a reliable and low-complication method in evaluating patients with ACTH-dependent CS who had equivocal results in imaging and biochemical tests, even before stimulation. Stimulation with DDAVP increases diagnostic accuracy. BIPSS can be used to predict the lateralization of the pituitary adenoma. Data Availability All data generated or analyzed during this study are included in this published article. Abbreviations BIPSS: Bilateral inferior petrosal sinus sampling ACTH: Adrenocorticotropic hormone CS: Cushing’s syndrome IPS: Inferior petrosal sinus DDAVP: Desmopressin CD: Cushing’s disease EAS: Ectopic ACTH syndrome MRI: Magnetic resonance imaging UFC: Urinary free cortisol DST: Dexamethasone suppression test HDDST: High-dose dexamethasone suppression test CRH: Corticotropin-releasing hormone BMI: Body mass index FBS: Fasting blood glucose Hb: Hemoglobin Cr: Creatinine PPV: Positive predictive value NPV: Negative predictive value SBP: Systolic blood pressure DBP: Diastolic blood pressure K: Potassium HTN: Hypertension IHC: Immunohistochemistry TSS: Transsphenoidal surgery References Arnaldi G, Angeli A, Atkinson A, Bertagna X, Cavagnini F, Chrousos G, et al. Diagnosis and Complications of Cushing’s syndrome: a consensus statement. J Clin Endocrinol Metabolism. 2003;88(12):5593–602. Article CAS Google Scholar Sharma ST, Nieman LK, Feelders RA. 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Author information Authors and Affiliations Endocrinology and Metabolism Research Center (EMRC), Vali-Asr Hospital, Tehran University of Medical Sciences, Tehran, Iran Mohammadali Tavakoli Ardakani, Soghra Rabizadeh, Amirhossein Yadegar, Fatemeh Mohammadi, Sahar Karimpour Reyhan, Reihane Qahremani, Alireza Esteghamati & Manouchehr Nakhjavani Advanced Diagnostic and Interventional Radiology Research Center (ADIR), Tehran University of Medical Sciences, Tehran, Iran Hossein Ghanaati Contributions MN and MTA and SR: Conception and design of the study. AY and FM and HG: Acquisition of data. MTA and AY and SR: Analysis and interpretation of data. FM and RQ and SK: Drafting the article. MN and AE and AY: Critical revision of the article. All authors read and approved the final manuscript. Corresponding author Correspondence to Manouchehr Nakhjavani. Ethics declarations Ethics approval and consent to participate This study was performed in line with the principles of the Declaration of Helsinki. Informed consent was obtained from all participants or their legal guardians. Approval was granted by the Research Ethics Committee of Tehran University of Medical Sciences (Approval number: IR.TUMS.MEDICINE.REC.1398.707). Consent for publication In order to publish this study, written informed consent was obtained from each participant. A copy of the written consent form is available for review by the journal editor. Competing interests The authors declare no competing interests. Additional information Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Rights and permissions Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Reprints and Permissions Cite this article Ardakani, M.T., Rabizadeh, S., Yadegar, A. et al. Bilateral inferior petrosal sinus sampling: validity, diagnostic accuracy in lateralization of pituitary microadenoma, and treatment in eleven patients with Cushing’s syndrome – a single-center retrospective cohort study. BMC Endocr Disord 23, 232 (2023). https://doi.org/10.1186/s12902-023-01495-z Download citation Received05 July 2023 Accepted19 October 2023 Published23 October 2023 DOIhttps://doi.org/10.1186/s12902-023-01495-z Share this article Anyone you share the following link with will be able to read this content: Get shareable link Provided by the Springer Nature SharedIt content-sharing initiative Keywords BIPSS Bilateral inferior petrosal sinus sampling Cushing’s Disease Cushing’s syndrome EAS From https://bmcendocrdisord.biomedcentral.com/articles/10.1186/s12902-023-01495-z
  19. We have an opportunity for you to take part in a Cushing’s Disease, Type 2 Diabetes Study (M3_8994) for patients. Our project number for this study is M3_8994. Project Details: Web-assisted telephone interview (you must be by a computer with high-speed internet access while on the phone during the time of the interview) Interview is 60-minutes long 120 Dollar Reward Things to Note: We recommend using the web browsers Google Chrome or FireFox Study is open to patients Please do not share study links One participant per household only Want to share this opportunity? Let us know and we can provide a new link Please use a laptop/computer ONLY. No smartphones or tablets - Preliminary questions are mobile friendly! Save this email to reference if you have any questions about the study! If you have any problems, email tana.karamustafic@rarepatientvoice.com and reference the project number. If you are interested in this study, please click the link below to answer a few questions to see if you qualify. Study link: Start Here OR if the study hyperlink is not clickable above, please copy/paste this URL below. https://panel.rarepatientvoice.com/newdesign/site/rarepatientvoice/surveystart.php?surveyID=s4gl5f3v5tr8&panelMemberID=trfnbc7mvduh1gseff1h&invite=email Thanks as always for your participation! Please be aware that by entering this information you are not guaranteed that you will be selected to participate. As always, we do not share any of your contact information without your permission. We have recently updated our Privacy Policy. Please make sure you have read through it and agree to the terms and conditions before taking studies. Please contact us at research@rarepatientvoice.com if you have any questions.
  20. Objective: This study evaluated short- and long-term efficacy and safety of the second-generation somatostatin receptor ligand pasireotide alone or in combination with dopamine agonist cabergoline in patients with Cushing’s disease (CD). Study design: This is an open-label, multicenter, non-comparative, Phase II study comprising 35-week core phase and an optional extension phase. All patients started with pasireotide, and cabergoline was added if cortisol remained elevated. Eligible patients had active CD, with or without prior surgery, were pasireotide naïve at screening or had discontinued pasireotide for reasons other than safety. Primary endpoint was proportion of patients with a mean urinary free cortisol (mUFC) level not exceeding the upper limit of normal (ULN) at week 35 with missing data imputed using last available post-baseline assessments. Results: Of 68 patients enrolled, 26 (38.2%) received pasireotide monotherapy and 42 (61.8%) received pasireotide plus cabergoline during the core phase. Thirty-four patients (50.0%; 95% CI 37.6–62.4) achieved the primary endpoint, of whom 17 (50.0%) received pasireotide monotherapy and 17 (50.0%) received combination therapy. Proportion of patients with mUFC control remained stable during the extension phase up to week 99. Treatment with either mono or combination therapy provided sustained improvements in clinical symptoms of hypercortisolism up to week 99. Hyperglycemia and nausea (51.5% each), diarrhea (44.1%) and cholelithiasis (33.8%) were the most frequent adverse events. Conclusion: Addition of cabergoline in patients with persistently elevated mUFC on maximum tolerated doses of pasireotide is an effective and well-tolerated long-term strategy for enhancing control of hypercortisolism in some CD patients. Clinical trial registration: https://clinicaltrials.gov/ct2/show/NCT01915303, identifier NCT01915303. 1 Introduction Cushing’s disease (CD) is a rare condition arising from chronic overproduction of cortisol, secondary to an adrenocorticotropic hormone (ACTH)-secreting pituitary tumor (1). Untreated hypercortisolism results in substantial multisystem morbidity, impaired quality of life (QoL) and premature mortality (1–4). Pasireotide is a second-generation, multireceptor-targeted somatostatin receptor ligand (SRLs), with affinity for 4 of the 5 known somatostatin receptor subtypes (SSTRs) (5) and is approved for the treatment of patients with CD for whom surgery has failed or is not an option (6). Phase III trials of pasireotide monotherapy have shown sustained biochemical and clinical benefits up to 5 years (6–9). These benefits are also reflected in real-world evidence (10). Cabergoline, a potent dopamine agonist with high affinity for dopamine type 2 receptors (D2), is commonly used off-label for the treatment of CD (2). Small, retrospective, non-randomized studies have demonstrated long-term urinary free cortisol (UFC) control (24−;60 months) in 23−;40% of patients with CD, especially those with mild hypercortisolism (11–13). A meta-analysis of individual patient data from six observational studies (n=124) reported normalization of mean UFC (mUFC) levels in 34% of patients (14, 15). However, a short prospective study on cabergoline monotherapy showed a limited value in controlling UFC, possibly linked to short duration (16). As most corticotropinomas co­express SSTR5 and D2, combining pasireotide and cabergoline in a stepwise approach could potentially improve efficacy with achieving more rapid biochemical control (17), a premise supported by results from an 80-day pilot study of 17 patients with CD treated with cabergoline- pasireotide combination, and low-dose ketoconazole (in case of lack of complete control with the two-drug combination) (18). The current study aims to report the efficacy and safety of prolonged treatment with pasireotide alone or in combination with cabergoline from the largest prospective, multicentre study to date of a pituitary-targeting combination treatment regimen in patients with CD (NCT01915303). 2 Materials and methods 2.1 Patients Adults (≥18 years) with a confirmed diagnosis of CD or de novo CD, if they were not candidates for surgery or refused surgery were enrolled. Cushing’s disease was defined by a mean 24-hour (24h) UFC level greater than the upper limit of normal (ULN, 137.95 nmol/24h), calculated from three 24h samples collected within 2 weeks; a morning plasma ACTH level within or above the normal range; and a confirmed pituitary source of Cushing’s syndrome, determined by MRI confirmation of pituitary adenoma >6mm or inferior petrosal sinus sampling (IPSS) gradient >3 after CRH stimulation (or >2 if IPSS without CRH stimulation) for those patients with a tumor ≤6mm. For patients who had prior pituitary surgery, histopathology confirming an ACTH staining adenoma was considered confirmatory of CD. Key exclusion criteria included optic chiasm compression requiring surgery, poorly controlled diabetes (glycated hemoglobin [HbA1c] >8%) and having risk factors for torsades de pointes (for further details, see the Supplementary Appendix). 2.2 Study design This was a single-arm, open-label, multicenter, non-comparative, Phase II study. After 4 weeks of screening, patients were treated in a stepwise approach during the core phase. Patients received subcutaneous pasireotide 0.6 mg twice daily (bid) for 8 weeks. Patients with a mUFC level exceeding ULN after 8 weeks received pasireotide 0.9 mg bid for another 8 weeks. If mUFC level remained elevated with pasireotide 0.9 mg bid, oral cabergoline 0.5 mg once daily (qd) was added for 8 weeks and could be increased to 1.0 mg qd for another 8 weeks (Supplementary Figure S1). After 35 weeks of treatment in the core phase, patients could enter the extension phase of the trial. Addition or titration of cabergoline during the extension phase was at the discretion of investigators. Collection of extension data commenced from week 43, and patients continued their current study treatment up to study end (4 September 2019; date of last patient visit), week 257. Data beyond week 99 are not reported here because of small patient numbers. 2.3 End points and assessments The primary endpoint of the study was the proportion of patients with mUFC ≤ULN at week 35. Secondary endpoints (reported at 4-week intervals up to week 35 and 8-week intervals from week 43 to the date of the last patient visit) included changes from baseline in mUFC, plasma ACTH, serum cortisol, total cholesterol, and clinical signs (systolic/diastolic blood pressure, body mass index (BMI), weight, waist circumference, facial rubor, hirsutism, striae, supraclavicular and dorsal fat pads) and symptoms (CushingQoL). Treatment escape was defined as an increase in one UFC above the normal range during follow-up of complete responders (14). Cushing Quality of Life Questionnaire (CushingQoL) (19) scores were reported up to week 35 only. Details on the safety assessments are provided in the Supplementary Appendix. 2.4 Statistical analyses No formal hypothesis testing was performed because of the exploratory design of the study. Efficacy analyses were conducted on full analysis set, i.e., all patients to whom study treatment was assigned. Safety analyses were conducted on all patients who received ≥1 dose of pasireotide per day during the study. For patients with missing mUFC value at week 35, including those who discontinued, the last available assessment was carried forward. Details on the post hoc analyses and sample size estimation is provided in the Supplementary Appendix. Enrolled patients, who were observed for failed inclusion or exclusion criteria during the monitoring visits, were classified under protocol deviation. However, patients with no safety concerns were allowed to continue in the study and included in the full analysis set as intention to treat – assessing the study outcome, while some patients were excluded from the per protocol analysis. 3 Results 3.1 Study population A total of 68 patients were enrolled in the study. At baseline, 66 (97.1%) patients were pasireotide naïve, while 2 (2.9%) were treated with pasireotide previously with 4 weeks of washout period prior to screening (Table 1). Of 68 patients received treatment during the core phase, 26 (38.2%) received pasireotide monotherapy and 42 (61.8%) received combination therapy. Fifty-two (76.5%) patients completed the 35-week core phase while 16 (23.5%) discontinued (Figure 1). All 68 patients were included in the full analysis set based on the intention to treat (ITT) principle. One of the protocol deviations observed during the study, was inclusion of 3 patients with normal mUFC value at screening visit (baseline) and assigning a treatment. The deviation category for the 3 patients was ‘failed inclusion criteria’ with screening mUFC value ≤ULN (137.95 nmol/24h) or mUFC calculated using ❤️ UFC values or 2 out of 3 UFC values ≤ULN. One of these patients (baseline mUFC 37.37 nmol/24h ≤ULN) was discontinued from the study at Week 2 and due to lack of post-baseline mUFC assessment, was classified ‘non-responder’ at Week 35 assessment. The 2nd patient’s baseline mUFC value of 135.20 nmol/24h was close to ULN (137.95 nmol/24h) and was rescreened. Based on the rescreened mUFC value 306.5 nmol/24h, this patient was included in study, and the mUFC at Week 35 was 192.30 nmol/24h (non-responder at Week 35 assessment). For all study assessments, the scheduled screening visit’s first mUFC value (≤ULN) was used as baseline value. The 3rd patient (baseline mUFC value 131.77 nmol/24h) was discontinued from the study at Week 26 and was also observed for non-compliant schedule visit and medication dosages. The mUFC value recorded at Week 26 (88.95 nmol/24h) was ≤ULN and this last observation was carried forward to Week 35. Hence, the patient was classified ‘responder’, leaving one patient included in the study as responder as a protocol deviation. Table 1 Table 1 Patient demographics and baseline characteristics. Figure 1 Figure 1 Patient disposition. *If the study drugs were locally available at the end of the core phase, patients could switch over to the commercial supply and exit the extension phase. Only in countries where the drug was not locally available were patients given the option to enter the extension phase. Percentage for patients not entering the extension phase was calculated from the total number of patients enrolled in the study. Twenty-nine (42.6%) patients continued treatment in the extension phase; 10 (34.5%) received pasireotide monotherapy and 19 (65.5%) received combination therapy. Twelve (41.4%) patients completed the extension phase, while 17 (58.6%) discontinued treatment before study end, most commonly for unsatisfactory therapeutic effect (n=8). The most common reason for discontinuation was adverse events (AEs): 5 (17.2%) patients with pasireotide monotherapy and 2 (5.1%) patients with combination therapy. 3.2 Efficacy: biochemical response Overall, 34/68 (50.0%; 95% CI 37.6–62.4) patients achieved the primary endpoint, of whom 17 (50.0%) were receiving pasireotide monotherapy and 17 (50.0%) were receiving combination therapy. Patients with mild hypercortisolism (mUFC 1.0–<2.0 x ULN) at baseline were more likely to respond to both pasireotide monotherapy and combination therapy (n=15; 22.1%, Figure 2). Seven of 17 patients in the pasireotide monotherapy group met the primary endpoint based on their last available assessment prior to week 35. Even if the 3 patients who had mUFC ≤ULN at baseline were excluded from the primary analysis, 33/65 (50.7%; 95% CI 38.1–63.4) patients would have achieved the primary endpoint. The results are similar to the original analysis (34/68 (50.0%; 95% CI 37.6–62.4) based on the full analysis set. Figure 2 Figure 2 Patients achieving mUFC ≤ULN at week 35. †At baseline there were 23 patients with mild, 30 with moderate and 12 with severe hypercortisolism. mUFC, mean urine free cortisol; ULN, upper limit of normal. For the overall study population (n=68), mUFC rapidly decreased from 501.6 nmol/24h (3.6 x ULN; SD: 488.66 nmol/24h) to 242.1 nmol/24h (1.8 x ULN; SD: 203.47 nmol/24h) at week 4 and mUFC remained below baseline levels up to week 35 (184.8 nmol/24h; 1.3 x ULN; SD:140.13 nmol/24h). For patients who received pasireotide monotherapy (n=26), mUFC( ± SD) decreased from baseline (442.1± 557.13 nmol/24h [n=26]; 3.2 x ULN) to week 35 (136.6 ± 127.77 nmol/24h [n=14]; 1 x ULN) and at the end of the study (111.2 ± 40.39 nmol/24h [n=5]; 0.8 x ULN) using the last-observation-carried-forward (LOCF). For those who did not normalize on pasireotide monotherapy (n=42), mUFC ( ± SD) decreased from baseline, i.e., last observation before starting cabergoline (280.20 ± 129.03 nmol/24h [n=40]; 2.0 x ULN) to week 35 (206.6 ± 141.96 nmol/24h [n=31]; 1.5 x ULN) and at the end of the study (219.60 ± 83.78 nmol/24h [n=7]; 1.6 x ULN) using the LOCF. During the core phase, mean serum cortisol decreased from 738.6 nmol/L (1.3 x ULN) at baseline to 538.2 nmol/L (0.95 x ULN) and ACTH levels from 16.3 pmol/L (2.7 x ULN) to 11.0 pmol/L (1.8 x ULN) at week 35. During the extension phase, 25 patients had a mUFC assessment; of whom 12 (48%) had a mUFC ≤ULN at the end of the extension phase. During the extension phase, mUFC levels decreased slightly and fluctuated above and below the ULN up to the week 139 (Figure 3A), while mean serum cortisol remained below ULN (404 nmol/L; Figure 3B) and ACTH levels fluctuated from 8.2 pmol/L to 11.5 pmol/L) and remained above the ULN value (Figure 3C). Figure 3 Figure 3 Mean actual change over time in (A) mUFC (B) serum cortisol, and (C) ACTH. ACTH, adrenocorticotropic hormone; mUFC, mean urine free cortisol; ULN, upper limit of normal . Twenty-one of 38 (55%) patients achieved control with combination therapy at some point during the core or extension study, of whom 13 (62%) experienced escape (at least one UFC >ULN after previous control). The time to achieve control after starting cabergoline ranged from 14−;343 days. Notably, one patient received pasireotide 0.6 mg bid initially, dose increased to 0.9 mg bid at Week 17, followed by addition of cabergoline 0.5 mg od at Week 31. The patient achieved biochemical control (mUFC value of 120.15 nmol/24h) on the same day of the start of combination therapy. Clinically it is highly unlikely that biochemical control was achieved with single dose of cabergoline administration. Therefore, it could be considered that normalization was achieved while receiving pasireotide monotherapy. Also, the physician might have prescribed combination therapy before receiving the mUFC value of the (urinary) sample delivered on the morning of combination therapy initiation (while the patient was still on monotherapy). The patient continued combination therapy and maintained biochemical control up to Week 35 and beyond. Furthermore, at Week 59 the cabergoline dose was increased to 1.0 mg/day due to mUFC >ULN at previous visit (Week 51). The patient remained on pasireotide 0.9 mg bid/cabergoline 1.0 mg od combination therapy until the study end. The median time to escape after achieving control with the addition of cabergoline was 58 days (range 28−;344). 10/13patients regained biochemical control with combination therapy. No patients on pasireotide alone experienced escape, probably due to the short observation time. 3.3 Clinical signs and symptoms of CD Relative to baseline, pasireotide monotherapy was accompanied by reductions in median blood pressure, weight, BMI, waist circumference, and total cholesterol. Overall improvement in clinical measures persisted over time (Supplementary Table S1). Clinical improvements were also seen following the addition of cabergoline, particularly for hirsutism (Supplementary Figures S2, S3). Mean( ± SD) standardized CushingQoL score was 41.6(± 20.2) at baseline and increased to 47.6(± 20.8) at week 35 (Supplementary Table S2), indicating improvements in patients’ QoL (19). 3.4 Safety and tolerability Median duration of exposure to pasireotide was 35.0 weeks (range 0−;268), with a median dose of 1.53 mg/day (range 0.29−;1.80). Median duration of exposure to cabergoline was 16.9 weeks (range 1−;215), with a median dose of 0.50 mg/day (range 0.44−;0.97). All patients (N=68) reported at least one AE and 28/68 (41.2%) patients had a grade 3/4 AE (Table 2). The most common AEs (≥30%) were hyperglycemia and nausea (51.5% each), diarrhea (44.1%) and cholelithiasis (33.8%). Treatment-related AEs (TRAEs) were reported in 66/68 (97.1%) patients; the most frequent TRAEs (≥30%) were hyperglycemia and nausea (47.1% each), diarrhea (39.7%), and cholelithiasis (32.4%). Fourteen (20.6%) patients had ≥1 AE leading to discontinuation. Table 2 Table 2 Summary of adverse events (≥10%), overall and by treatment regimen. The most common AEs leading to discontinuation were increased gamma-glutamyl transferase (GGT) and hyperglycemia (two patients each, 2.9%). Twenty-three (33.8%) patients had ≥1 AE leading to dose adjustment or interruption. Details on special safety assessments such as hyperglycemia-related AEs, blood glucose, HbA1c, IGF-1 as well as hematological and biochemical abnormalities are presented in the Supplementary Appendix. Three (4.4%) patients died during the study, two (2.9%) during the core phase and one (1.5%) during the extension. All deaths were considered unrelated to study medication. The causes during the core phase were multi-organ dysfunction syndrome for one patient aged 79 years and unknown for the other aged 34 years. Uncontrolled hypertension was reported as the cause of death for the patient aged 47 during the extension phase. 4 Discussion The severe morbidity and increased mortality with uncontrolled CD highlight the importance of identifying an effective medical strategy. This study explored the potential of a synergistic benefit of the addition of cabergoline to pasireotide treatment in patients with CD. Complete normalization of cortisol production is required to reverse the risks of morbidity and mortality in patients with CD (1). Two small studies showed clinical improvement of normalized UFC when cabergoline and ketoconazole were combined (20, 21). Benefit has also been reported with triple therapy with pasireotide, cabergoline and ketoconazole (18) and triple therapy with ketoconazole, metyrapone and mitotane in severe CD (22). In the current study, 50% of patients achieved the primary endpoint of mUFC ≤ULN at week 35 and a similar proportion (48%) sustained biochemical control throughout the extension phase. Notably, combination treatment doubled the number of patients who attained mUFC ≤ULN from the core phase to the end of the extension phase. In particular, mUFC was rapidly reduced with treatment, i.e., in most patients within 2 months, while measures of patient-reported outcomes also improved including QoL. Twenty-three patients (33.8%) who completed the core phase did not enter the extension phase. This was because only patients from countries where a commercial supply was unavailable were given the option to enter the extension phase. This study confirms previous reports that patients with mild hypercortisolism at baseline were more likely to achieve mUFC control with pasireotide monotherapy than patients with moderate or severe hypercortisolism (6, 23). In addition, patients with moderate hypercortisolism at baseline were more likely to achieve mUFC control with the addition of cabergoline. This supports that a combination therapy can be effective for patients with a wider range of disease severity. Accordingly, in vitro data may indeed indicate synergism between SSTR and D2 that might increase therapeutic efficacy (24, 25). Improvements in clinical signs and symptoms with pasireotide monotherapy were consistent with published data (6, 10). In the core phase, an improvement of blood pressure and BMI was observed with pasireotide monotherapy and, to a lesser extent, with combination therapy which may related to the difference in duration of biochemical remission. The overall safety profile was consistent with that expected for pasireotide, with most AEs being mild/moderate (26, 27). There were no new safety signals identified with the addition of cabergoline. Common AEs including nausea, headache, dizziness, and fatigue are suggestive of steroid withdrawal symptoms associated with the decrease in UFC, although direct drug effects cannot fully be excluded. Adrenal insufficiency was not reported as side effect. Rates of hyperglycemia-related AEs (68%) were consistent with those in previous reports of pasireotide monotherapy (6, 10). FPG increased with pasireotide monotherapy during the first 8 weeks of treatment and stabilized for the remainder of the study, including following the addition of cabergoline. These data highlight the vital role of blood glucose monitoring in these patients. Both pasireotide and cabergoline are pituitary-targeted agents that act directly on the source of the disease via inhibition of ACTH release by the corticotroph tumor, which may be an advantage over steroid synthesis inhibitors. This study further confirms previous data reporting the benefits of pasireotide in combination with cabergoline in patients with CD (18). While not entirely elucidated, down-regulation of dopamine D2 receptors (D2R) expression, and post-receptor desensitization and/or tumor regrowth of corticotroph tumor cell were suggested as possible mechanisms for treatment escape (15). Moreover, different dopamine receptor patterns and/or D2R isoforms also influence the response and eventually the treatment escape. Treatment escape has been observed in some studies after long-term (7−;12 months) treatment with cabergoline (13), however it is possible that use of concomitant SRLs could potentially reduce the rate of escape. In this study, a total of 13 patients experienced treatment escape. However, 10 of these patients regained biochemical control. For 7 of these 10 patients, there was up titration of doses to a maximum of 1.8 mg/day of pasireotide and 1 mg/day of cabergolineAlthough pasireotide and cabergoline have shown long-term reduction in IGF-1 levels in patients with acromegaly (28, 29), there is little evidence for this effect in patients with CD (4, 30). One study (n=17) found significant decreases in IGF-1 after 28 days’ treatment with pasireotide that was independent of UFC reduction. One-third of patients had low IGF-1 (30). Our study showed that almost half of patients (47.6%) had IGF-1 levels either above ULN or below LLN prior to the addition of cabergoline, and IGF-1 levels decreased relative to the baseline, with majority of values within the normal range during the core and extension phases up to week 99. Baseline levels of IGF-1 may already be low because of the suppressive effect of excess cortisol on the somatotropic axis (31). Although clinicians have several therapeutic options at their disposal to treat hypercortisolemia associated with CD, the optimal treatment approach should be based on the individual clinical situation and the benefit–risk considerations for each patient. In this study, 13 patients had history of pituitary radiation, with a duration of at least 2.6 years (median 3.3 years) between the last radiation treatment and the observed response date. However, only 7/13 patients achieved the therapeutic target. Although there was a gap of > 2 years, we cannot exclude the role of radiation in normalizing UFC. Contrastingly, 6/13 patients treated with radiation did not achieve mUFC ≤ULN (responders) at Week 35. The impact of the adjuvant radiation therapy remains unclear. The strengths of this study are that this is the largest and longest prospective study with pituitary-directed pharmacotherapy, to date, evaluating the addition of cabergoline to pasireotide in patients with CD, and this stepwise approach reflects real-world clinical practice (18). The study is limited by the open-label design and the fact that it was not a head-to-head comparative study of pasireotide only versus pasireotide plus cabergoline. This may be of importance in interpreting patient-reported outcomes. Several patients continued treatment for almost 2 years; however, interpretation of long-term data should be made with caution because of the small patient numbers. Notably, the last available assessment was carried forward for patients with missing mUFC value at week 35 including those who discontinued and were considered for response analysis. It should also be noted that the definition of loss of response, also known as escape, used in this study (at least one UFC value >ULN after previously achieving UFC ≤ULN) may overestimate the rate of apparent escape as UFC values may have fluctuated about the ULN range or been marginally elevated. The definition of treatment escape differs across studies, and we have used a very stringent one in this study, requiring only a single high UFC to meet the classification as escape. Thus, it is likely that some loss of biochemical control interpreted as escape is actually fluctuation of cortisol around the upper limit of normal range. Other limitations include protocol deviations in including 3 patients with normal UFC at baseline (one patient was uncontrolled at rescreen, and one was discontinued at 2 weeks - both classified as non-responders), lack of data on impact of radiation therapy without study drug in patients who gained biochemical control with adjuvant radiation therapy, lack of pituitary magnetic resonance imaging to detect pituitary tumor changes, lack of data about effective cabergoline dose and absence of cardiac valve assessment for mild to moderate severity in the medium term. Both pasireotide and cabergoline can induce tumor shrinkage in CD (6, 9, 32–35) and it would be interesting to examine the combined effect on tumor size. This study used the subcutaneous formulation of pasireotide, whereas the most common usage currently is the long-acting formulation. Efficacy of long-acting pasireotide (36) seems higher compared to the subcutaneous formulation (7) and the effect of combination of long-acting pasireotide with cabergoline should be evaluated in future studies. No formal assessments were made for impulsive control disorders, which have been associated with dopamine agonists, including cabergoline (32, 33, 37, 38). The reason that several different terms were used for hyperglycemia-related AEs is that they were reported as per discretion of each investigator. No additional psychiatric AEs were reported, although they were not exhaustively searched. 5 Conclusions This is the first study demonstrating that pituitary-targeted combination treatment with pasireotide and cabergoline doubled the number of patients who attained mUFC ≤ULN. Both short- and long-term safety profile are consistent with known data for pasireotide and cabergoline. The low rate of discontinuation due to AEs suggests that pasireotide alone or as combination treatment is generally well-tolerated if appropriately monitored, even with prolonged treatment. The addition of cabergoline to pasireotide treatment in patients with persistently elevated mUFC could be an effective long-term strategy for enhancing the control of CD in a subset of patients, with close monitoring for possible escape. Data availability statement The original contributions presented in the study are included in the article/Supplementary Material. Further inquiries can be directed to the corresponding author. Ethics statement The studies involving humans were approved by Hospital Britanico, Buenos Aires, Argentina; Ethische commissie University Hospitals Leuven, Leuven, Belgium; Universitair Ziekenhuis Gent, Gent, Belgium; Comite de Etica em Pesquisa Hospital Moinhos de Vento, Porto Alegre-RS, Brazil; Comitê de Ética em Pesquisa do Hospital de Clı́nicas, Universidade Federal do Paraná, Curitiba-PR, Brazil; Comissão de Ética para Análise de Projetos de Pesquisa, São Paulo - SP, Brazil; Ethics Committee for clinical trials, Sofia, Bulgaria; Comité Corporativo de Ética en Investigación, Bogotá DC, Colombia; Comite De Protection Des Personnes, Groupe Hospitalier Pellegrin - Bat, Bordeaux Cedex, France; Friedrich-Alexander Universitat Erlangen-Nurnberg, Medizinische Fakultat, Erlangen, Germany;National Ethics Committee, Cholargos, Athens, Greece; Ethics Committee for Clinical Pharmacology (ECCP), Budapest, Hungary; Institute Ethics Committee, New Delhi, India; Institutional Review Board (IRB) Ethics Committee Silver, Christian Medical College, Vellore, Tamil Nadu, India; Institute Ethics Committee, PGIMER, Chandigarh, India; Comitato Etico Dell’irccs Istituto Auxologico Italiano Di Milano, Milano, Italy; Comitato Etico Universita’ Federico Ii Di Napoli, Napoli, Italy; Jawatankuasa Etika & Penyelidikan Perubatan (Medical Research and Ethics Committee), d/a Institut Pengurusan Keshatan Jalan Rumah Sakit, Kuala Lumpur, Malaysia; Institutd Nacional De Neurologia Y Neurocirugia, Mexico City, Mexico; Clinica Bajio (CLINBA), Guanajuato, Mexico; Medische Ethische Toetsings Commissie, Rotterdam; Netherlands; CEIm Provincial de Málaga, Málaga, Spain; Istanbul University Cerrahpasa Medical Faculty, Istanbul, Turkey; WIRB, Puyallup, WA, USA; Research Integrity Office, Oregon Health & Science University Portland, OR USA. The studies were conducted in accordance with local legislations and institutional requirements. The participants provided their written informed consent to participate in this study. Author contributions All authors directly participated in the planning, execution, or analysis, and have had full control of complete primary data, and hold responsibility for data integrity and accuracy. All authors contributed to the article and approved the submitted version. Acknowledgments We thank Julie Brown, Mudskipper Business Ltd, and Manojkumar Patel and Sashi Kiran Goteti, Novartis Healthcare Private Limited, for medical editorial assistance with this manuscript. We would also like to thank all investigators, sub-investigators, study nurses and coordinators, and patients who have made this study possible. Conflict of interest HP and RM were Novartis employees and owned Novartis stocks. AMP was employed by Novartis and Recordati. AC is a Novartis employee and owns Novartis stocks. RF received research grants from Strongbridge and Corcept, consulting fee from Recordati, honoraria and financial support for meetings and/or travel from HRA Pharma and Recordati, and attended advisory boards for Recordati. MF has received research support to Oregon Health & Science University as a principal investigator from Recordati and Xeris Strongbridge and has performed occasional scientific consultancy for Recordati, HRA Pharma, Sparrow, and Xeris Strongbridge. PK attended advisory boards for Recordati. MB’s institution received consulting fee and attended advisory boards from Recordati. DG-D received research grants from Recordati Rare Disease and Bayer, consulting fee from Abbott-Lafrancol, Biotoscana, PTC lab, Glaxo/Helou, Recordati Rare Disease, and Bayer, honoraria from Valentech Pharma, Sanofi, and Bayer, travel grants from Recordati Rare Disease, advocacy groups and other leadership roles from Asociación Colombiana de Endocrinologia and Asociación Colombiana de Osteoporosis y Metabolismo, and other financial and non-financial interests include Asociacion Colombiana de Endocrinologia y Metabolismo, Hospital Universitario Fundación Santa Fé de Bogota, and Asociación Colombiana de Osteoporosis y Metabolismo. CB received research grants from Novartis and Recordati, and consulting and speaker fee from Novartis. BB served as the principal investigator for grants to Massachusetts General Hospital from Cortendo/Strongbridge Xeris, Millendo, and Novartis and has occasionally consulted for Cortendo/Strongbridge Xeris, HRA Pharma, Novartis Recordati, and Sparrow. RP and his institution received research grants and honoraria from Pfizer, Ipsen, Novartis, Merck Serono, IBSA Farmaceutici, Corcept, Shire, HRA Pharma, ICON, Covance, Neuroendocrine CAH, Camurus, Recordati, Janssen Cilag, and CMED Clinical Services, received consulting fee from Recordati Rare Disease, Organon Italia, Siunergos Pharma, Corcept, S&R Farmaceutici S.p.A., DAMOR Farmaceutici, and Pfizer, attended advisory boards from Crinetics Pharmaceuticals, Recordati Rare Disease, Pfizer, and HRA Pharma. The remaining author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The authors declare that this study received funding from Novartis Pharma AG. Novartis was involved in the study design, analysis, interpretation of data, and providing financial support for medical editorial assistance of this article. 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(2018) 41(1):19–75. doi: 10.1007/s40264-017-0590-6 PubMed Abstract | CrossRef Full Text | Google Scholar Keywords: somatostatin, pasireotide, cabergoline, Cushing’s disease, hypercortisolism Citation: Feelders RA, Fleseriu M, Kadioglu P, Bex M, González-Devia D, Boguszewski CL, Yavuz DG, Patino H, Pedroncelli AM, Maamari R, Chattopadhyay A, Biller BMK and Pivonello R (2023) Long-term efficacy and safety of subcutaneous pasireotide alone or in combination with cabergoline in Cushing’s disease. Front. Endocrinol. 14:1165681. doi: 10.3389/fendo.2023.1165681 Received: 14 February 2023; Accepted: 11 August 2023; Published: 09 October 2023. Edited by: Renato Cozzi, Endocrinology Unit Ospedale Niguarda, Italy Reviewed by: Przemyslaw Witek, Warsaw Medical University, Poland Athanasios Fountas, General Hospital of Athens G. Genimatas, Greece Copyright © 2023 Feelders, Fleseriu, Kadioglu, Bex, González-Devia, Boguszewski, Yavuz, Patino, Pedroncelli, Maamari, Chattopadhyay, Biller and Pivonello. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. *Correspondence: Richard A. Feelders, r.feelders@erasmusmc.nl †Present addresses: Alberto M. Pedroncelli, Chief Medical Office, Camurus AB, Lund, SwedenRicardo Maamari, Global Medical Affairs, Mayne Pharma, Raleigh, NC, United States ‡These authors have contributed equally to this work Disclaimer: All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. 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  21. Abstract Mifepristone and misoprostol are globally used medications that have become disparaged through the stigmatization of reproductive healthcare. Patients are hindered from receiving prompt treatment in clinical scenarios where misoprostol and mifepristone are the drugs of choice. It is no exaggeration to emphasize that in cases where reproductive healthcare is concerned. The aim of this paper is to discuss the different indications of mifepristone and to delineate where the discrepancy in accessibility arises. For this systematic review, we included publications citing clinical trials involving the use and efficacy of mifepristone published in English within the date range of 2000 to 2023. Five databases were searched to identify relevant sources. These databases are Google Scholar, MEDLINE with full text through EBSCO, and three National Center for Biotechnology Information (NCBI) databases (NCBI Bookshelf, PubMed, and PubMed Central). Twenty-three records were ultimately included in this review. Mifepristone has been shown to have therapeutic effects in the treatment of psychiatric disorders, such as major depressive disorder and psychotic depression. There was a significant decrease in depression and psychiatric rating symptoms for patients taking mifepristone versus placebo with no adverse events. Mifepristone has also been shown to improve treatment course in patients with Cushing’s disease (CD) who failed or are unable to undergo surgical treatment. In addition, mifepristone has been shown to be a successful treatment option for adenomyosis and leiomyomas. Patients had a statistically significant decrease in uterine volumes following mifepristone treatment, which aided in the alleviation of other symptoms, such as blood loss and pelvic discomfort. Mifepristone is a synthetic steroid that has immense potential to provide symptomatic relief in patients suffering from a wide array of complicated diseases. Historically, mifepristone has been proven to have an incredible safety profile. While further research is certainly needed, the politicization of its medical use for only one of its many indications has unfortunately led to the willful ignorance of its potential despite its evidence-based safety profile and efficacy. Introduction & Background Mifepristone is a synthetic steroid derived from norethindrone and therefore has antagonistic activity against progesterone and glucocorticoid receptors. Misoprostol is a synthetic prostaglandin E1 analog that works through the direct stimulation of prostaglandin E1 receptors. Recently, these medications have become disparaged due to their associations with the controversial medical procedure known as abortion. Abortions, however, have been so common that one out of four women will have had an abortion by the time they reach the age of 45 [1]. It is estimated that 3.7 million women have used mifepristone and misoprostol for medication abortions since they were first approved by the Food and Drug Administration (FDA) in 2000 [1]. Mifepristone followed by misoprostol is up to 14 times safer than carrying the patient’s pregnancy to term [1]. Aside from abortion, mifepristone is used for both gynecologic and obstetric conditions. Obstetric conditions include induction of labor, postpartum hemorrhage, intrauterine fetal demise, ectopic pregnancies, and miscarriages [2]. Gynecological conditions that can be treated with mifepristone include abnormal uterine bleeding, post-coital contraception, and treatment of gynecological cancers [3]. Due to the stigmatized nature of abortion, however, patients are hindered from receiving prompt treatment in clinical scenarios where mifepristone is the drug of choice. It is no exaggeration to emphasize that in cases where reproductive healthcare is concerned, every second counts [3]. Legislation that varies across states further impacts patients who risk their lives and health as they attempt to navigate their care plan across borders. Travel costs, time-off, childcare, transportation, and living accommodations are just a few more of the factors patients must take into consideration when they are forced to seek life-saving care outside of their homes [3]. Mifepristone is a medication that has multiple therapeutic applications, such as treating leiomyomas, psychotic depression, and post-traumatic stress disorder (PTSD). However, its use is restricted in many countries because of its abortifacient effect. This is a logical fallacy that deprives patients of a beneficial and safe treatment option. This systematic review aims to explore the evidence-based uses of mifepristone and how it can improve patients' health outcomes. The clinical indications that will be discussed are adenomyosis, leiomyomas, psychotic depression, PTSD, and Cushing's disease (CD). Review Methods Eligibility Criteria For this systematic review, we included publications of clinical trials and systematic reviews citing clinical trials relating to the clinical use of mifepristone and published in English within the date range of 2000 to 2023. Info Sources Five databases were searched to identify relevant sources. These databases include Google Scholar, MEDLINE with full text through EBSCO, and three National Center for Biotechnology Information (NCBI) databases (NCBI Bookshelf, PubMed, and PubMed Central). Search Strategy For each database, we inputted “clinical use of mifepristone” as our search term. The populated results were then narrowed down to those published in the English language and within the date range of 2000 to 2023 using automated search tools. Selection Process The titles and abstracts of the remaining records were then screened, and those deemed relevant to clinical uses of mifepristone and its efficacy were included for comprehensive review. This initial record search in three of the four databases (Google Scholar, MEDLINE, and PubMed) was completed by three separate reviewers. The initial record search in the remaining two databases (NCBI Bookshelf and PubMed Central) was completed by another individual reviewer. Data Collection Process After the initial record search, 60 records were deemed relevant to the study topic and compiled for a more comprehensive review. Two records were found to be duplicates and removed. Each of the four reviewers read the remaining 58 records and voted on the eligibility of the publication for inclusion in our review. Older publications that were expanded upon in more recent study trials were excluded to reduce redundancy. In addition, for records with similar study protocols, only the more recently published record was included. Ten records were excluded from the review due to ineligible study design. For those records that were not unanimously accepted (at least one reviewer voted for exclusion), the record was excluded. To ensure that the data utilized in this review were backed by sufficient evidence, the reviewers organized the remaining records into groups based on the disease mifepristone was being studied to treat. After further discussion, it was decided to exclude the records in the groups that lacked at least three separate clinical trials on the use of mifepristone in the treatment of the disease. Thirty articles were excluded. Seven of the 18 remaining records were systematic reviews, and citation searching of the records found four additional records that met the eligibility criteria. The remaining 23 records were included for further review. Data Items Of the remaining 23 records deemed acceptable for inclusion, only studies with statistically significant findings regarding the clinical use of mifepristone were included for detailed analysis. One record was excluded due to early termination of the trial. Our records include two open-label studies, four retrospective studies, seven reviews (systematic, meta-analysis), one wet lab (human specimen was used), five long-term safety extension articles, and seven randomized control experimental trials. Study Risk-of-Bias Assessment We assessed the risk of bias (RoB) in the studies included in the review using the revised Cochrane RoB tool for randomized trials (RoB 2). The five domains assessed were (1) RoB arising from the randomization process, (2) RoB due to deviations from the intended interventions (effect of assignment to intervention and effect of adhering to intervention), (3) missing outcome data, (4) RoB in the measurement of the outcome, and (5) RoB in the selection of the reported result. Each randomized control trial included in this review was assessed for RoB by two authors working independently using the RoB 2. For those studies in which the assessing authors came to different conclusions, the remaining two authors completed independent RoB 2 assessments of the study in question, and the majority of findings was accepted. Utilizing the methodology for assigning the overall RoB for each study as outlined by the RoB 2 tool, each study was designated as having “low risk of bias” or “high risk of bias.” After an initial assessment, both authors deemed the nine randomized control studies had a low RoB. Effect Measures Analysis of the studies included a focus on statistically significant findings that varied between control and intervention groups as defined by a p-value less than 0.5. As each study had its own parameters and primary and secondary endpoints, we focused our analysis on the safety and clinical efficacy of mifepristone as measured and reported by the authors of the studies included. Synthesis Methods As previously mentioned, as the studies included in this review vary widely in their study population and intervention design, our analysis focused on qualitative synthesis of study outcomes. These outcomes were categorized as the clinical efficacy and safety of mifepristone for CD, psychiatric disorders, and select gynecological diseases (adenomyosis and leiomyomas). Certainty Assessment To assess the certainty of the body of evidence regarding the studies included in our review, two reviewers applied the five Grading of Recommendations, Assessment, Development, and Evaluations (GRADE) considerations (study limitations, inconsistency of results, indirectness of evidence, imprecision, and publication bias) to each study. Accordingly, the included studies were categorized as having high, moderate, low, or very low certainty of evidence based on the GRADE criteria. After the assessment, both reviewers deemed that all records had high certainty of evidence. Figure 1: PRISMA 2020 flow diagram for new systematic reviews that included searches of databases, registers, and other sources *Consider, if feasible to do so, reporting the number of records identified from each database or register searched (rather than the total number across all databases/registers). **If automation tools were used, indicate how many records were excluded by a human and how many were excluded by automation tools. PRISMA: Preferred Reporting Items for Systematic Reviews and Meta-Analyses Results Psychiatric Implications Based on the analyses, numerous trials demonstrated the profound therapeutic effect that mifepristone can have on psychiatric disorders. In a double-blind study following 19 patients with bipolar disorder, researchers studied neurocognitive function and mood in patients treated with mifepristone vs. the placebo [4]. Significant improvements in verbal fluency and spatial working memory were seen in the group treated with mifepristone. The Hamilton Depression Rating Scale (HDRS) and Montgomery-Asberg Depression Rating Scale (MADRS) scores also improved from baseline (i.e., lower scores) measurements in these patients. It is worth noting that these improvements were seen in as little as two weeks, which is quicker than what is normally seen with typical therapeutic agents for bipolar disorder (lithium/valproic acid) [4]. The most extensive research demonstrated the benefits of using mifepristone with major or psychotic depression [5]. It is important to note that approximately 20% of patients living with major depression experience psychotic symptoms [6]. A randomized, double-blind study looked at 30 participants with psychotic major depression (PMD) and treated them with mifepristone 600 mg or a placebo for eight days. Using the HDRS and Brief Psychiatric Rating Scale (BPRS) to quantify baseline levels of symptoms, results from eight days later showed that mifepristone was significantly more effective in reducing psychotic symptoms compared to the placebo group [6]. By day 8, nearly half of the participants attained a 50% reduction in the BPRS compared to the placebo group (p<0.046) in addition to lower HDRS scores (although this was not found to be significant). Moreover, when researchers looked further into the use of mifepristone in psychotic depression disorders, they discovered a correlation between higher plasma levels of mifepristone and a reduction in psychotic symptoms [7]. More specifically, the strongest reduction in psychosis symptoms was found to be associated with doses of 1200 mg/day of mifepristone, which resulted in a statistically significant reduction in psychotic symptoms (p<0.0004) [7]. The drug was also well tolerated and demonstrated a large safety margin in contrast to the numerous common adverse effects that patients experience when placed on standard treatment options (i.e., antipsychotics). In another double-blind, placebo-controlled study that took place over four days, five participants diagnosed with psychotic major depression were administered 600 mg of mifepristone [5]. The HDRS and BPRS scores were used, and the results showed that all five participants' depression ratings decreased - a nearly statistically significant finding (p<.07) [5]. Likewise, four out of the five BPRS scores declined, approximating to a 32.5% decline, which is comparable to the 40% decline seen with traditional antipsychotic treatments that span six to eight weeks. Once again, no adverse effects were reported. The use of mifepristone has been explored in many cognitive disorders, including Alzheimer's disease. One study found that patients with mild to moderate Alzheimer’s disease displayed improvement on the Alzheimer’s disease assessment cognitive subtest - by 2.67 as opposed to the 1.67 decline in patients treated with a placebo [5]. Although not statistically significant, this finding encourages further studies to continue exploring the psychiatric and neurologic use of mifepristone. Cushing’s Disease Multiple trials have been conducted regarding the use and efficacy of mifepristone in the treatment of CD. Although surgical intervention to remove the source of excess cortisol production is the current mainstay of treatment, clinical trials have focused on the treatment with mifepristone for medical therapy, especially in patients who have failed surgical intervention or for those who are not good candidates for surgery. Accordingly, a retrospective study of 20 patients with hypercortisolism (12 with adrenocortical carcinoma, three with ectopic adrenocorticotropic hormone (ACTH) secretion, four with CD, and one with bilateral adrenal hyperplasia) found clinically significant improvement in excess cortisol-induced symptoms in 15 out of 20 patients [4]. Patient responses to mifepristone treatment were monitored by clinical signs of hypercortisolism (signs of hypercortisolism, blood pressure measurements, and signs of adrenal insufficiency) and serum potassium and glucose. The study found that 15 out of 20 patients showed significant clinical improvement in excess cortisol-induced symptoms. Psychiatric symptoms and blood glucose levels also improved in the patients [4]. Of note, 11 out of 20 trial participants exhibited moderate to severe hypokalemia as a side effect, although only one patient had to leave the study early due to severe adverse effects [4]. In another well-known study, 50 patients were assessed at baseline and during intervention (total of six times) for 24 weeks, referred to as the SEISMIC study [8]. Changes in oral glucose tolerance tests over time were used to assess the mifepristone effect in type 2 diabetes millets (T2DM)/impaired glucose tolerance patients. Changes in diastolic blood pressure (BP) over time were used to measure the effect of mifepristone in hypertensive cardiogenic shock (CS) patients [8]. Results found a statistically significant improvement in symptoms in both groups: diabetic patients had improvement in response to oral glucose test, decreased A1C, and decreased fasting glucose, and hypertensive patients had decreased diastolic BP or reduction in antihypertensive medications [8]. In addition, the waist circumference and hemoglobin A1C (HbA1C) also improved, and study findings concluded that mifepristone use has an acceptable risk-benefit ratio for six months of treatment [8]. Several extension studies were later performed utilizing the data found during the SEISMIC study [9]. One such study assessing weight loss in patients who participated in the SEISMIC study also found statistically significant improvement in patients with CD. After one-week mifepristone period (patients who chose to participate in this follow-up study had to be assessed to ensure it was safe for them to enroll in this study), 30 patients were enrolled and started on once daily mifepristone at the dose they were taking when the SEISMIC study concluded [9]. The patient's weight was assessed at baseline and week 24 of the SEISMIC study, and for this study, the follow-up weight was taken at months 6, 12, 18, and 24 and a final visit. Data were assessed for 29 of the participants and statistically significant decreases in weight were found for all participants from baseline to end of the SEISMIC study, and the maintenance of weight loss was statistically significant in all participants at their final visit to this study as well [9]. Another SEISMIC extension study focused on monitoring the effects of mifepristone treatment in CD on ACTH levels and pituitary MRI findings [10]. Serum ACTH, urinary, and salivary cortisol levels were monitored during the SEISMIC study (baseline, day 14, and weeks 6, 10, 16, and 24) and once after a six-week mifepristone-free "washout" period. ACTH levels were then monitored one month later and then routinely every three months during the intervention period, which varied per participant [10]. Serum cortisol measures were assessed during the SEISMIC study at the intervals mentioned previously and then every six months during the extension study. Pituitary MRI studies were taken prior to mifepristone administration during the SEISMIC study and at weeks 10 and 24 [10]. Repeat imaging was then taken every six months during the extension study. On average, ACTH levels increased greater than twofold (2.76 ± 1.65-fold over baseline; p<0.0001 vs. baseline) in patients during the SEISMIC and extension study periods and decreased to near baseline levels after six weeks of mifepristone discontinuation [10]. Serum cortisol levels in both the initial intervention and extension period increased as well, although a higher mean cortisol level was seen during the extension study intervention (SEISMIC: 1.97 ± 1.02-fold increase; p<0.0001 vs. baseline; extension study: 2.85 ± 1.05-fold increase; p<0.0001 vs. baseline) [10]. In comparing the baseline and post-intervention MRI images, 30 out of 36 patients showed no progression in pituitary tumor size with mifepristone intervention, two patients showed regression of tumor size, and three patients showed evidence of tumor progression. One patient was found to have a tumor post-intervention despite a negative initial MRI at baseline [10]. A retrospective analysis of data collected during the SEISMIC study utilized oral glucose tolerance test data to assess the mifepristone treatment effect on the total body insulin sensitivity, beta cell function, weight, waist circumference, and additional parameters [11]. The analysis found improved total body insulin sensitivity in all participants, with the greatest improvement occurring from baseline to week 6. The weight and waist circumference both decreased by week 24 [11]. An additional important six-month study was done on 46 patients with refractory CS and either DM2, impaired glucose tolerance, or diagnosis of HTN in which mifepristone treatment was administered daily [12]. Patients were examined by three separate reviewers using global clinical response assessments (-1 = worsening, 0 = no change, 1 = improving) measured by eight clinical categories: glucose control, lipids, blood pressure, body composition, clinical appearance, strength, psychiatric/cognitive symptoms, and quality of life at weeks 6, 10, 16, and 24. A positive correlation with increasing GCR scores was found by week 24, with 88% of participants showing statistically significant improvement (p<0.001) [12]. Adenomyosis/Leiomyoma Adenomyosis and leiomyomas are common gynecological conditions that affect large portions of the female population. Multiple trials have proven mifepristone’s success in treating endometriosis and various forms of cancer. Current data shows that mifepristone is well tolerated and has mild side effects in certain long-term clinical settings. In one trial following mifepristone and its effects on adenomyosis, 20 patients were treated with 5 mg oral mifepristone/day for three months [13]. After the three-month trial, patients demonstrated a statistically significant (p<0.001) reduction in uterine volume as was measured through transvaginal ultrasound. These patients were also found to have significantly decreased CA-125 markers (a marker of adenomyosis and an increase in uterine size) and significantly increased hemoglobin concentration The patient’s endometrial tissue was then obtained from each patient during their hysterectomy [13]. The endometrial tissue samples were treated with varying concentrations of mifepristone for 48 hours. They found that mifepristone significantly decreased the viability of endometrial epithelial and stromal cells in adenomyosis and can induce their apoptosis as well [13]. This concentration-dependent inhibitory effect was most significantly seen with concentrations of mifepristone above 50 μmol/L at 48 hours. The same study showed that mifepristone demonstrated another dose-dependent relationship in the inhibition of the migration of ectopic endometrial and stromal cells. This finding is significant as the migratory nature of the patient’s endometrial and stromal cells is the pathogenesis behind adenomyosis [13]. Another study looked at the effect of mifepristone in combination with high-intensity focused ultrasound (HIFU) and levonorgestrel-releasing intrauterine system (LNG-IUS) in the treatment of adenomyosis [13]. Out of 123 patients, 34 patients were treated with HIFU alone, 29 patients were treated with HIFU combined with mifepristone, 10 patients with HIFU combined with LNG-IUS, and 50 patients with HIFU combined with mifepristone and LNG-IUS [13]. In the group treated with HIFU combined with mifepristone and LNG-IUS, the uterine volume was significantly reduced after treatment at 3, 6, 12, and 24 months compared to the previous treatment (p<0.05). Dysmenorrhea was measured using a visual analog score (VAS). In the combination group of mifepristone, HIFU, and LNG-IUS, VAS scores decreased from 80.82 ± 12.49 to 29.58 ± 9.29 at 24 months [13]. This was significantly lower than the three other treatment groups (p<0.05). The combination group of mifepristone, HIFU, and LNG-IUS also demonstrated statistically significant decreases in the menstrual volume and CA-125 serum markers [13]. Hemoglobin levels were not statistically different among the four treatment groups, but it is postulated that this could have been due to the fact that the patients who were anemic had been treated with different medications to improve their Hb aside from the trial medications [13]. Uterine leiomyomas are another gynecological condition that has been found to improve with the use of mifepristone as well. Insulin-like growth factor 1 (IGF-1) has been found to be overexpressed in leiomyomas [14]. This study showed that mifepristone inhibited the gene expression of IGF-1, and the reduction in symptoms correlated with a decrease in IGF-1 expression although the mechanism is not fully understood [14]. A meta-analysis studied the effects of mifepristone on uterine and leiomyoma volumes of 780 women from 11 randomized controlled trials. Mifepristone at doses from 2.5, 5, and 10 mg was found to effectively reduce uterine and leiomyoma volumes and alleviate leiomyoma symptoms at six months [6]. Pelvic pain, pelvic pressure, and dysmenorrhea were found to be alleviated after three months of treatment. Mifepristone also decreased the mean loss of blood during menstruation and a statistically significant increase in hemoglobin. No significant difference was found among varying dosages of 2.5, 5, and 10 mg other than increased frequency of hot flashes in patients of the 10 mg group. Another review investigated six clinical trials involving 166 women and the effects of 5-50 mg mifepristone for three to six months on leiomyomas [3]. The review demonstrated that daily treatment with all doses of mifepristone resulted in reductions in pelvic pain, pelvic pressure, dysmenorrhea, and uterine and leiomyoma volume size by 26-74%. Even doses of 2.5 mg of mifepristone resulted in significant improvement in the quality of life scores although there was little reduction in leiomyoma size at this dose [3]. This review also reported the rapid correction of uterine bleeding, amenorrhea, and increases in hemoglobin levels following treatment with 50 mg of mifepristone on alternating days. Even vaginal mifepristone has demonstrated efficacious results in the improvement of leiomyomas. In one such trial, the effects of daily 10 mg vaginal mifepristone were studied in 33 women from the ages of 30-53 [15]. Vaginal mifepristone significantly reduced leiomyoma volume and reduced the effects of symptoms on the patient’s quality of life as measured by the Uterine-Fibroid Symptoms Quality of Life questionnaire (UFS-QoL). It is important to note that the only significant side effect found in this review of trials was hot flashes at doses of mifepristone at 10 mg or more. Mifepristone was otherwise generally well tolerated with minimal if any adverse effects [15]. Discussion Adenomyosis is a gynecologic condition that is characterized by the growth of endometrial cells into the myometrium, resulting in a globally enlarged uterus and an associated increase in CA-125 [16]. This marker is classically known to be an ovarian tumor marker; however, in this class, it reflects the increase in uterine glandular size. Although it is often labeled as a “benign” disease, it affects around 20% of reproductive-aged women. This condition can lead to dysmenorrhea, infertility, and menorrhagia in addition to detrimental effects on a patient’s mental health [16]. Despite 20% of affected patients being under the age of 40, the gold standard of treatment is a hysterectomy. Hysterectomies may often not be wanted by patients as it is an invasive surgery that comes with several potential complications of its own. It is important to note that due to the large percentage of patients with adenomyosis who are of reproductive age, hysterectomies may not be an appropriate standard method of treatment. To rob patients of their fertility without attempting medication therapy with mifepristone first is an act of injustice. Surgery alone comes with many complications and the possibility of recurrence. The ability of physicians to manage their patient’s pain and symptoms should be guided medically before surgical sterilization is considered. Many of these patients are forced to seek alternative non-invasive treatments instead of medication therapies to preserve their fertility. HIFU and LNG-IUS are noninvasive therapies for adenomyosis that can be used in patients who refuse hysterectomies or for those who are not good candidates [16]. The pitfalls of these procedures include the fact that 20% of patients on HIFU alone end up relapsing, and LNG-IUS cannot be used in patients with a uterine size that is >12 weeks gestation or a uterine cavity depth that is >9 cm. Because adenomyosis is an estrogen-dependent disease, gonadotropin-releasing hormone agonists (GnRH-a) are also often used in combination with HIFU and LNG-US. Through the inhibition of the secretion of estrogen, GnRH-as facilitate reduced pelvic pain, reduced bleeding, and reduced uterine cavity size [16]. Reduction in cavity size is significant as this alone can lead to improved pain and reduced bleeding and allows patients to qualify for LNG-US where their previous uterine cavity size would have prevented their candidacy. Its current limitations include price (>$200/month), induction of premenopausal syndrome, and high rates of relapse following drug cessation [16]. Mifepristone offers a cheaper alternative (<$4/month) with significantly improved outcomes in reduced uterine cavity size, decreased dysmenorrhea pain scale score, and lower menstruation volume scores [16]. Mifepristone is also able to provide such results without the bone loss that is commonly associated with GnRH-analogs [3]. This is because mifepristone allows for serum estradiol to remain within the patient’s physiologic follicular phase range [3]. In addition, mifepristone is able to significantly reduce serum levels of CA-125 and improve hemoglobin levels in patients with menorrhagia. These reductions in CA-125 demonstrate marked reductions in the size of glands of the uterus of these patients. Through the reduction of cavity size, mifepristone can not only offer therapeutic relief but also allow patients to qualify for noninvasive LNG-US procedures, which can offer further therapeutic benefits. Patients should have the option to explore all potential medical therapies before opting for surgical correction. Leiomyomas, or uterine fibroids, are another commonly encountered gynecologic condition and represent the most common benign tumors found in the female population. These benign smooth muscle tumors are estrogen-sensitive and can rarely develop into malignant leiomyosarcomas. Nearly 20-50% of patients with these fibroids experience symptoms, such as abnormal uterine bleeding (AUB), infertility, pelvic pain, and miscarriages [17]. Currently, the only treatment for this common condition is surgery. Two medications that are commonly used for preoperative reductions in leiomyoma size are mifepristone and enantone. Enantone is a gonadotropin-releasing hormone analog that has shown significant improvement in leiomyoma shrinkage, correction of anemia, and correction of AUB [17]. Through its MOA, however, enantone can lead to harmful adverse effects, such as menopausal symptoms and bone mineral loss. Using hormone supplementation to negate these side effects leads to reduced effectiveness of enantone in fibroid size reduction. Several studies have shown that progesterone plays a large role in the proliferation of leiomyoma growth [17]. Mifepristone, therefore, offers an effective alternate solution by producing the same results without enantone’s adverse effects. When comparing enantone to mifepristone, the two medications both resulted in statistically significant reductions in fibroid size, reduction in dysmenorrhea, reduction in non-menstrual abdominal pain, and increased Hgb/Hct/and RBC count despite differences in dosage [17]. However, mifepristone was able to maintain the patients’ premenopausal levels of estrogen, whereas patients on enantone were found to have estrogen levels of menopausal patients. Furthermore, patients who were treated with enantone also reported more adverse events compared to those in the mifepristone group [17]. Vaginal use of mifepristone has also been shown to significantly reduce leiomyoma size and improve symptoms of anemia while lowering systemic bioavailability of mifepristone [15]. Through its concentrated distribution to uterine tissue, vaginal mifepristone can lead to increased improvement in its clinical outcomes. Vaginal mifepristone showed statistically significant improvements in leiomyoma volume change, USF-QoL, and decreased bleeding intensity at the end of the three-month trial and three months after treatment [15]. For these reasons, mifepristone can be used effectively for conservative therapy in patients suffering from leiomyomas and should be considered a viable option for patients not wishing to undergo surgery. CD refers to hypercortisolism that is caused by pituitary adenomas, adrenal neoplasias, or paraneoplastic ACTH secretion. Hypercortisolism in these patients leads to the development of skin changes, HTN, obesity, insulin resistance, dyslipidemia, anovulation, skeletal disorders, and neuropsychiatric disorders [18]. Patients suffering from these conditions endure a severely decreased quality of life and increased morbidity and mortality. The syndromic nature of this disease prompts delayed diagnosis and further increases the mortality and morbidity of this population [18]. CS therefore necessitates effective and rapid treatment options to diminish harm and clinical burden. The current first-line treatment for CD is pituitary surgery despite its nearly ⅓ relapse rate within 10 years postoperatively [18]. In these patients and patients with recurrent CD, further treatment options are necessitated. These options include adrenal surgery, pituitary radiotherapy, or medication therapy. Radiotherapy further delays symptomatic relief as it usually takes years before excess cortisol levels are managed. It also carries the risk of the patient developing hypopituitarism due to subsequent pituitary damage [18]. While surgery of the adrenal glands can quickly achieve control of excess cortisol, it also carries a risk of permanent adrenal insufficiency. Medication therapy can be used preoperatively, postoperatively, and as adjunctive therapy to radiotherapy. These drug classes include somatostatin analogs, dopamine agonists, and adrenal steroidogenesis inhibitors [18]. The most commonly used medication is the adrenal steroidogenesis inhibitor ketoconazole. While it has been proven to be effective and rapid in its success, doses may need to be frequently increased due to the cortisol blockade that occurs in CD patients [8]. In fact, due to the hormonal imbalances in CD patients, many medications often have to be dose adjusted to achieve therapeutic effect. It is also important to note that many of the medications that are used are not easily tolerated when doses are increased or adjusted frequently. The use of mifepristone has demonstrated statistically significant results in weight reduction, insulin resistance, depression, HTN, and quality of life in CD patients [10]. Furthermore, mifepristone can also be used effectively in patients experiencing cortisol-induced psychosis during acute exacerbations of hypercortisolism. While not included in the classes of more commonly used drugs for CD, mifepristone has been approved by the FDA for the treatment of CD when associated with disorders of glucose metabolism. This is undoubtedly due to the stigmatization of mifepristone and the subsequent reluctance of clinicians to incorporate it into their treatment plans. Neuropsychiatric disorders have been investigated for their associations with dysregulations of the hypothalamic-pituitary-adrenal axis (HPA) and increases in cortisol levels. Studies have shown that patients suffering from depression, schizophrenia, and psychotic depression have elevated levels of cortisol and increased activity of their HPA [19]. The role of cortisol in psychiatric disorders is evidenced by the adverse psychiatric effects that patients can develop in response to exogenous glucocorticoid use through subsequent increases in cortisol. These include delirium, depression, mania, or psychosis. When functioning normally, HPA activity and cortisol secretion are maintained through sensitive negative feedback systems involving glucocorticoid receptors (GCRs) and mineralocorticoid receptors (MCR) [19]. At low doses, cortisol preferentially binds to MCR. As cortisol levels rise, it begins to bind to GCR and thereby initiates the negative feedback loop. Antipsychotics that are typically used work by reducing cortisol levels. Mifepristone, when dosed at >200 mg/day, selectively binds only to GCR and has no effect on MCR [19]. Through its sole inhibition of GCR, it ensures that normal cortisol homeostasis is maintained while ensuring that excess high levels of cortisol are blocked. This was evidenced by the statistically significant correlation between rising plasma concentrations of mifepristone and improvement of psychotic symptoms [20]. The hippocampus is a region of the temporal lobe that is most notably recognized for its role in learning and memory. Further studies have shown correlations between hippocampal atrophy and patients with severe depression, PTSD, and schizophrenia. It is postulated that this hippocampal atrophy leads to persistently high levels of cortisol, worsening these patient’s psychiatric symptoms. Administration of mifepristone to patients with combat-related PTSD demonstrated significant benefits in quality of life and psychiatric improvement. Psychotic major depression is another psychiatric condition that affects around 20% of patients with major depression [7]. When mifepristone was used to treat psychotic depression, patients were able to achieve rapid antipsychotic effects that lasted for weeks after the medication therapy ended. It should be noted that patients suffering from PMD generally have increased cortisol levels even with standard antidepressant therapy alone [7]. Some patients are even unresponsive to electroconvulsive therapy. The ability of patients suffering from psychotic depression to achieve rapid relief is imperative as these patients are more susceptible to suicidal ideation, especially during an episode of psychosis [7]. Bipolar disorder is another mood disorder that has been found to be associated with high levels of cortisol, dysfunction of the HPA axis, and GR dysfunction. Several neuroendocrine studies demonstrated that around 43% of bipolar patients with depression were also dexamethasone-suppression-test (DST) nonsuppressors [7]. Further studies found that bipolar patients suffering through relapse and recovery had abnormal dexamethasone/corticotropin-releasing hormone (dex/CRH) test results [21]. These abnormal (dex/CRH) findings were also seen in healthy patients who had certain genetic predispositions for mood disorders [21]. Regarding these HPA dysfunctions, GR has been implicated in being an important modulator of neurocognitive function and mood. This can be evidenced through research findings that report increased GR number and GR binding in brain tissue following the administration of antidepressants in depressed patients [21]. Mifepristone’s unique advantage is that its selective role as a GR antagonist was also found to increase both MR and GR binding in the frontal cortex. In fact, data from Young et al. [21] reveals significant improvement in frontal cortex functioning following clinical mifepristone trials. These results were seen through improvements in spatial working memory function and reductions in the HDRS17 and MADRS. They also demonstrated significant improvement in verbal fluency from baseline. These improvements in neurocognitive functioning were measured when the subjects’ mood was similar to their baseline or did not vary when compared to the placebo group [21]. This key finding suggests that improvements in neurocognitive functioning were not solely related to improvements in mood or depression. Mifepristone achieves these improvements in neurocognitive function through its selective activity towards GR within the frontal cortex. Furthermore, patients are also able to achieve symptomatic improvement two weeks after the initiation of treatment [21]. The rapid nature of mifepristone adds further clinical benefit as classic bipolar treatments take longer to achieve therapy and the fact that treatment plans for patients with bipolar disorder are tricky to individualize. Other commonly known psychiatric disorders are treated with antipsychotics. While these medications often come with a large array of adverse effects, weight gain, metabolic derangements, and glucose intolerance have been a few of the more frequently reported negative effects. While the exact cause of the weight gain is unknown, mifepristone was shown to significantly reduce weight gain in patients when taken alongside risperidone or olanzapine [21]. As discussed previously, mifepristone also has the ability to significantly improve insulin resistance, thereby further improving the AE patients may experience on antipsychotics. Therefore, through mifepristone’s selective activity as a GCR antagonist, it has immense potential as a psychiatric therapeutic agent. Conclusions Mifepristone is a synthetic steroid that has immense potential to provide symptomatic relief in patients suffering from a wide array of complicated diseases. Prednisone, dexamethasone, and anabolic steroids are also synthetic steroids that are commonly used. Despite being a part of the same class as mifepristone, none of these medications fall under as much legal, political, and social duress as mifepristone. This is in spite of the fact that mifepristone has been proven to have an incredible safety profile since its introduction to the public in the 1980s. In fact, its mortality rate is significantly lower than that of Tylenol, NSAIDs, penicillin, and phosphodiesterase inhibitors. While further research is certainly needed, its involvement in politics has unfortunately led to the willful ignorance of its medical potential despite its evidence-based safety profile and efficacy. References Beaman J, Prifti C, Schwarz EB, Sobota M: Medication to manage abortion and miscarriage. J Gen Intern Med. 2020, 35:2398-405. 10.1007/s11606-020-05836-9 Hagey JM, Givens M, Bryant AG: Clinical update on uses for Mifepristone in obstetrics and gynecology. Obstet Gynecol Surv. 2022, 77:611-23. 10.1097/OGX.0000000000001063 Spitz IM: Mifepristone: where do we come from and where are we going? Clinical development over a quarter of a century. Contraception. 2010, 82:442-52. 10.1016/j.contraception.2009.12.012 Castinetti F, Fassnacht M, Johanssen S, et al.: Merits and pitfalls of mifepristone in Cushing's syndrome. Eur J Endocrinol. 2009, 160:1003-10. 10.1530/EJE-09-0098 Belanoff JK, Flores BH, Kalezhan M, et al.: Rapid reversal of psychotic depression using mifepristone. J Clin Psychopharmacol. 2001, 21:516-21. Eisinger SH, Meldrum S, Fiscella K, et al.: Low-dose mifepristone for uterine leiomyomata. Obstet Gynecol. 2003, 101:243-50. 10.1016/S0029-7844(02)02511-5 Flores BH, Kenna H, Keller J, Solvason HB, Schatzberg AF: Clinical and biological effects of mifepristone treatment for psychotic depression. Neuropsychopharmacology. 2006, 31:628-36. 10.1038/sj.npp.1300884 Fleseriu M, Biller BM, Findling JW, Molitch ME, Schteingart DE, Gross 😄 Mifepristone, a glucocorticoid receptor antagonist, produces clinical and metabolic benefits in patients with Cushing's syndrome. J Clin Endocrinol Metab. 2012, 97:2039-49. 10.1210/jc.2011-3350 Fein HG, Vaughan TB 3rd, Kushner H, Cram D, Nguyen 😧 Sustained weight loss in patients treated with mifepristone for Cushing's syndrome: a follow-up analysis of the SEISMIC study and long-term extension. BMC Endocr Disord. 2015, 15:63. 10.1186/s12902-015-0059-5 Fleseriu M, Findling JW, Koch CA, Schlaffer SM, Buchfelder M, Gross 😄 Changes in plasma ACTH levels and corticotroph tumor size in patients with Cushing's disease during long-term treatment with the glucocorticoid receptor antagonist mifepristone. J Clin Endocrinol Metab. 2014, 99:3718-27. 10.1210/jc.2014-1843 Wallia A, Colleran K, Purnell JQ, Gross C, Molitch ME: Improvement in insulin sensitivity during mifepristone treatment of Cushing syndrome: early and late effects. Diabetes Care. 2013, 36:e147-8. 10.2337/dc13-0246 Katznelson L, Loriaux DL, Feldman D, Braunstein GD, Schteingart DE, Gross 😄 Global clinical response in Cushing's syndrome patients treated with mifepristone. Clin Endocrinol (Oxf). 2014, 80:562-9. 10.1111/cen.12332 Che X, Wang J, He J, et al.: A new trick for an old dog: the application of mifepristone in the treatment of adenomyosis. J Cell Mol Med. 2020, 24:1724-37. 10.1111/jcmm.14866 Shen Q, Zou S, Sheng B, et al.: Mifepristone inhibits IGF-1 signaling pathway in the treatment of uterine leiomyomas. Drug Des Devel Ther. 2019, 14:3161-70. Yerushalmi GM, Gilboa Y, Jakobson-Setton A, Tadir Y, Goldchmit C, Katz D, Seidman DS: Vaginal mifepristone for the treatment of symptomatic uterine leiomyomata: an open-label study. Fertil Steril. 2014, 101:496-500. 10.1016/j.fertnstert.2013.10.015 Zhu H, Ma Q, Dong G, Yang L, Li Y, Song S, Mu Y: Clinical evaluation of high-intensity focused ultrasound ablation combined with mifepristone and levonorgestrel-releasing intrauterine system to treat symptomatic adenomyosis. Int J Hyperthermia. 2023, 40:10.1080/02656736.2022.2161641 Liu C, Lu Q, Qu H, et al.: Different dosages of mifepristone versus enantone to treat uterine fibroids: a multicenter randomized controlled trial. Medicine (Baltimore). 2017, 96:e6124. 10.1097/MD.0000000000006124 Pivonello R, De Leo M, Cozzolino A, Colao A: The treatment of Cushing's disease. Endocr Rev. 2015, 36:385-486. 10.1210/er.2013-1048 Hartmann J, Bajaj T, Klengel C, et al.: Mineralocorticoid receptors dampen glucocorticoid receptor sensitivity to stress via regulation of FKBP5. Cell Rep. 2021, 35:109185. 10.1016/j.celrep.2021.109185 Block TS, Kushner H, Kalin N, Nelson C, Belanoff J, Schatzberg A: Combined analysis of mifepristone for psychotic depression: plasma levels associated with clinical response. Biol Psychiatry. 2018, 84:46-54. 10.1016/j.biopsych.2018.01.008 Young AH, Gallagher P, Watson S, Del-Estal D, Owen BM, Ferrier IN: Improvements in neurocognitive function and mood following adjunctive treatment with mifepristone (RU-486) in bipolar disorder. Neuropsychopharmacology. 2004, 29:1538-45. 10.1038/sj.npp.1300471 From https://www.cureus.com/articles/191397-multiple-clinical-indications-of-mifepristone-a-systematic-review#!/
  22. Abstract Objective Since Cushing's disease (CD) is less common in the paediatric age group than in adults, data on this subject are relatively limited in children. Herein, we aim to share the clinical, diagnostic and therapeutic features of paediatric CD cases. Design National, multicenter and retrospective study. Patients All centres were asked to complete a form including questions regarding initial complaints, physical examination findings, diagnostic tests, treatment modalities and follow-up data of the children with CD between December 2015 and March 2017. Measurements Diagnostic tests of CD and tumour size. Results Thirty-four patients (M:F = 16:18) from 15 tertiary centres were enrolled. The most frequent complaint and physical examination finding were rapid weight gain, and round face with plethora, respectively. Late-night serum cortisol level was the most sensitive test for the diagnosis of hypercortisolism and morning adrenocorticotropic hormone (ACTH) level to demonstrate the pituitary origin (100% and 96.8%, respectively). Adenoma was detected on magnetic resonance imaging (MRI) in 70.5% of the patients. Transsphenoidal adenomectomy (TSA) was the most preferred treatment (78.1%). At follow-up, 6 (24%) of the patients who underwent TSA were reoperated due to recurrence or surgical failure. Conclusions Herein, national data of the clinical experience on paediatric CD have been presented. Our findings highlight that presenting complaints may be subtle in children, the sensitivities of the diagnostic tests are very variable and require a careful interpretation, and MRI fails to detect adenoma in approximately one-third of cases. Finally, clinicians should be aware of the recurrence of the disease during the follow-up after surgery. From https://onlinelibrary.wiley.com/doi/10.1111/cen.14980
  23. Abstract Cushing’s syndrome is a condition leading to overproducing of cortisol by the adrenal glands. If the pituitary gland overproduces cortisol, it is called Cushing’s disease. Cushing’s syndrome and even Cushing’s disease during and after pregnancy are rare events. There is not enough literature and guidance for managing and treating these patients. The diagnosis of Cushing’s syndrome in pregnancy is often delayed because the symptoms overlap. We presented a thin 31-year-old woman, admitted 2 months after a normal-term delivery, with an atypical presentation of Cushing’s disease, unusual clinical features, and a challenging clinical course. She had no clinical discriminatory features of Cushing’s syndrome. Given that the patient only presented with psychosis and proximal myopathy and had an uncomplicated pregnancy, our case was considered unusual. The patients also had hyperpigmentation and severe muscle weakness which are among the less common presentations of Cushing’s syndrome. Our findings suggest that an early diagnosis of Cushing’s disease is important in pregnancy period for its prevalent fetal and maternal complications, and it should be treated early to optimize fetal and maternal outcomes as there is an increasing trend toward live births in treated participants. Introduction Cushing’s syndrome is a condition that originates from excessive production of glucocorticoids. The condition is most common in women of childbearing age and is characterized by altered distribution of the adipose tissue to the central and upper regions of the trunk (central obesity and buffalo hump), face (moon face), capillary wall integrity (easy bruising), hyperglycemia, hypertension, mental status changes and psychiatric symptoms, muscle weakness, signs associated with hyperandrogenism (acne and hirsutism), and violaceous striae among other signs. Hypercortisolism and hyperandrogenism suppress the production of the pituitary gonadotropins, which in turn leads to menstrual irregularities and infertility.1-3 Moreover, the main common cause of developing Cushing’s syndrome is the use of exogenic steroid.3 Cushing’s disease is a form of Cushing’s syndrome with overproduction of adrenocorticotropic hormone (ACTH) due to pituitary adenoma. The diagnosis is made using clinical features and paraclinical tests including urinary free cortisol (UFC), serum ACTH, dexamethasone suppression tests (DSTs), pituitary magnetic resonance imaging (MRI), and sometimes by inferior petrosal sinus sampling (IPSS).4 Although women with Cushing’s disease are less likely to become pregnant, timely diagnosis and appropriate management are especially important during possible pregnancy, preventing neonatal and maternal complications and death. The diagnosis is challenging due to the overlap of the disease symptoms with the changes associated with a normal pregnancy. Moreover, the hormonal milieu during pregnancy has recently been proposed as a potential trigger for Cushing’s disease in some cases; hence, the term “pregnancy-associated Cushing’s disease” has been used for the disease in the recent literature. In this study, we presented a thin 31-year-old woman who was referred to our clinic 2 months after a normal delivery, with an atypical presentation of Cushing’s disease, unusual clinical features, and a challenging clinical course. Case Presentation Our patient was a 31-year-old woman who presented 2 months after the delivery of her second child. She had a history of type 2 diabetes mellitus and hypertension in the past 2 years prior to her presentation. She had been admitted to another center following an episode of falling and muscle weakness. Two weeks later, she was admitted to our center with an impression of pulmonary thromboembolism due to tachypnea, tachycardia, and dyspnea. During follow-up, she was found to have leukocytosis, hyperglycemia (random blood sugar: 415 mg/d; normal level: up to 180 mg/dL) and hypokalemic metabolic alkalosis (PH: 7.5, HCO3 [bicarbonate]: 44.7 mEq/L, paO2 [partial pressure of oxygen]: 73 mm Hg, pCO2: 51.7 mm Hg, potassium: 2.7 mEq/L [normal range: 3.5-5.1 mEq/L]), which was refractory to the treatment; therefore, an endocrinology consultation was first requested. On physical examination, the patient was agitated, confused, and psychotic. Her vital signs were: blood pressure 155/100 mm Hg, heart rate: 130 bpm, and respiratory rate: 22 bpm, temperature: 39°C. As it has shown in Figure 1A, her face is not typical for moon face of Cushing’s syndrome, but facial hirsutism (Figure 1A) and generalized hyperpigmentation is obvious (Figure 1A-C). She was a thin lady and had a normal weight and distribution of adiposity (Body Mass Index [BMI] = 16.4 kg/m2; weight: 40 kg, and height: 156 cm). Aside from thinness of skin, she did not have the cutaneous features of Cushing’s syndrome (e.g. purpura, acne, and violaceous striae) and did not have supraclavicular and dorsocervical fat pad (buffalo hump), or plethora. In other words, she had no clinical discriminatory features of Cushing’s syndrome despite the high levels of cortisol, as confirmed by severely elevated UFC (5000 μg/24 h and 8000 μg/24 h; normal level: 4-40 μg/24 h). In addition, as will be mentioned later, the patient had axonal neuropathy which is a very rare finding in Cushing’s syndrome. Figure 1. Clinical finding of our case with Cushing’s disease. (A) Hirsutism, (B) muscle atrophy seen in proximal portion of lower limbs, and (C) hyperpigmentation specially on the skin of the abdominal region. OPEN IN VIEWER She had a markedly diminished proximal muscle force of 1 out of 5 across all extremities; the rest of the physical examinations revealed no significant abnormalities (Figure 1B). On the contrary, based on her muscle weakness, hirsutism, psychosis and hyperpigmentation and refractory hypokalemic alkalosis, hyperglycemia, and hypertension, Cushing’s syndrome was suspected; therefore, 24-hour UFC level was checked that the results showed a severely elevated urinary cortisol (5000 μg/24 h and 8000 μg/24 h; normal level: 4-40 μg/24 h). Serum ACTH level was also inappropriately elevated (45 pg/mL; normal range: 10-60 pg/mL). High-dose dexamethasone failed to suppress plasma cortisol level and 24-hour urine cortisol level. A subsequent pituitary MRI showed an 8-mm pituitary mass, making a diagnosis of Cushing’s disease more probable. Meanwhile, the patient was suffering from severe muscle weakness that did not improve after the correction of hypokalemia. Then, a neurology consultation was requested. The neurology team evaluated laboratory data as well as EMG (Electromyography) and NCV (Nerve Conduction Velocity) of the patient, and based on their findings, “axonal neuropathy” was diagnosed for her weakness; so they ruled out the other neuromuscular diseases. A 5-day course of intravenous immunoglobulin (IVIG) was started for her neuropathy; however, the treatment did not improve her symptoms and the patient developed fungal sepsis and septic shock. Therefore, she was processed with broad-spectrum antibiotics and antifungal agents and recovered from the infection. Mitotane was started for the patient before definitive surgical treatment to suppress hormonal production due to her poor general condition. Despite the 8-mm size of the pituitary mass which is likely to be a source of ACTH, our patient was underweight and showed the atypical clinical presentation of Cushing’s disease, making us suspect an ectopic source for the ACTH. Therefore, a Gallium dotatate scan was performed to find any probable ectopic sources; however, the results were unremarkable. The patient underwent Trans-Sphenoidal Surgery (TSS) to resect the pituitary adenoma because it was not possible to perform IPSS in our center. Finally, the patient’s condition including electrolyte imbalance, muscle weakness, blood pressure, and hyperglycemia started to improve significantly. The pathologist confirmed the diagnosis of a corticotropic adenoma. Nevertheless, the patient suddenly died while having her meal a week after her surgery; most likely due to a thromboembolic event causing a cardiac accident. Discussion Our patient was significantly different from other patients with Cushing’s disease because of her atypical phenotype. She was unexpectedly thin and had psychosis, hyperpigmentation, proximal myopathy, axonal neuropathy and no clinical discriminatory features of Cushing’s syndrome such as central adiposity, dorsocervical or supraclavicular fat pad, plethora or striae. She had also a history of type 2 diabetes and hypertension 2 years before her admission. The patient was diagnosed with Cushing’s later. From what was presented, the patient did not know she had Cushing’s until after her delivery and despite the highly elevated UFC, and she completed a normal-term delivery. Given that she only presented with psychosis and proximal myopathy, her pregnancy was considered unusual. Her clinical features such as hyperpigmentation and severe muscle weakness are among less common presentations.5 11β-hydroxysteroid dehydrogenase type 1 (11-βHSD1) is an enzyme responsible for converting cortisone (inactive glucocorticoid) into cortisol (active). It is speculated that this enzyme has a role in obesity (Figure 2).6,7 Figure 2. The enzymatic actions of 11β-hydroxysteroid dehydrogenase on its substrate interconverting inactive and active glucocorticoid. OPEN IN VIEWER In a case reported by Tomlinson, a 20-year-old female was diagnosed with Cushing’s disease despite not having the classical features of the disease. It has been suggested that the mechanism is a partial defect in 11β-HSD1 activity and concomitant increase in cortisol clearance rate. Thus, the patient did not have a classic phenotype; the defect in the conversion of cortisone to cortisol rises cortisol clearance and protects the patient from the effects of cortisol excess. This observation may help explain individual susceptibility to the side effects of glucocorticoids.6 Further studies of Tomlinson et al showed that a deficit in the function of (and not a mutation related to) 11β-HSD2 might have been responsible for the absence of typical Cushing’s symptoms. 11-HSD2 keeps safe the mineralocorticoid receptor from excess cortisol. Mutation in the HSD11B2 gene explains an inherited form of hypertension, apparent mineralocorticoid excess syndrome, in which Cushing’s disease results in cortisol-mediated mineralocorticoid excess affecting the kidney and leads to both hypokalemia and hypertension.8 It is frequent in Cushing’s syndrome that the patients usually have no mineralocorticoid hypertension; however, it is still proposed that a defect in 11β-HSD1 can be responsible for the presence of mineralocorticoid hypertension in a subgroup of patients. In fact, 11β-HSD1 is expressed in several tissues like the liver, kidneys, placenta, fatty tissues and gonads,9 meaning that this enzyme may potentially affect the results of cortisol excess in Cushing’s syndrome/disease. Abnormality in the function of this enzyme could explain the absence of the symptoms like central obesity, easy bruising, and typical striae during Cushing’s disease. Several factors affect the action of glucocorticoids. In this regard, the impact of the different types and levels of impairment in glucocorticoid receptors have been highlighted in some studies, as it can lead to different levels of response to glucocorticoids10 as well as a variety in the symptoms observed in Cushing’s disease. The predominant reaction of the NADP(H)-dependent enzyme 11-Tukey’s honestly significant difference (HSD)1 happens through the catalysis of the conversion of inactive cortisol into receptor-active cortisol. The reverse reaction is mediated through the unidirectional NAD-dependent 11-HSD type 2 (Figure 2).11 In another case reported by Ved V. Gossein, a 41-year-old female was evaluated for hirsutism and irregular menstrual cycles. Her BMI was 22.6 kg/m2. The patient had no signs or symptoms of overnight recurrent Cushing’s syndrome, the 48-hour DST failed to suppress cortisol levels, and 24-hour urinary cortisol levels were persistently elevated on multiple occasions. Adrenocorticotropic hormone levels were unreasonably normal, suggesting ACTH-dependent hypercortisolism. Despite these disorders, she had 2 children. Magnetic resonance imaging (MRI) of the pituitary did not show any abnormalities. Moreover, abdominal MRI did not show adrenal mass or enlargement. Genetic testing to determine glucocorticoid resistance syndrome showed no mutation.12 Primary generalized glucocorticoid resistance is a rare genetic disorder characterized by generalized or partial insensitivity of target tissues to glucocorticoids.13-17 There is a compensatory increase in hypothalamic-pituitary activity due to decreased sensitivity of peripheral tissues to glucocorticoids systems.13-17 Excessive ACTH secretion leads to high secretion of cortisol and mineralocorticoids and/or androgens. However, the clinical features of Cushing’s syndrome do not develop after resistance to the effects of cortisol. Generalized glucocorticoid resistance is a rare condition characterized by high cortisol levels but no scarring of Cushing’s syndrome.18 An important aspect of our case was her pregnancy. Our patient had a history of hypertension and diabetes type 2, 2 years before her presentation to our center that could be because of an undiagnosed Cushing’s disease. The patient’s pregnancy terminated 2 months prior the admission and she had a normal vaginal delivery. So, we suspect that she become pregnant while involved with the disease. Aside from focusing on how this can happen in a patient with such high levels of glucocorticoids, more attention should be paid to occurring pregnancy in the background of Cushing’s disease. In fact, up to 250 patients were reported, of which less than 100 were actively treated.19-22 Cushing’s disease is associated with serious complications in up to 70% of the cases coinciding with pregnancy.21 The most frequent maternal complications reported in the literature are hypertension and impaired glucose tolerance, followed by preeclampsia, osteoporosis, severe psychiatric complications, and maternal death (in about 2% of the cases). Prematurity and intrauterine growth retardation account for the most prevalent fetal complications. Stillbirth, intrauterine deaths, intrauterine hemorrhage, and hypoadrenalism have also been reported.23 Early diagnosis is especially challenging during pregnancy because of many clinical and biochemical shared features of the 2 conditions.23,24 These features include an increase in ACTH production, corticosteroid-binding globulin (CBG) 1 level, level of cortisol (urinary, plasma and free), hyperglycemia, weight gain, and an increased chance for occurrence of bruising, hypertension (mistaken with preeclampsia), gestational diabetes mellitus, weight gain, and mood swings.3 There are some suggestions proposed in the studies that help in screening and differentiation of Cushing’s from the normal and abnormal effects of pregnancy and Cushing’s disease from Cushing’s syndrome in suspected pregnant patients. Contrary to Cushing’s syndrome, the nocturnal minimum level of cortisol is preserved in pregnancy.23,25 There is not yet a diagnostic cut-off determined on mentioned level; however, a few studies elucidate the evaluation of hypercortisolemia in a pregnant patient.26-28 Urinary free cortisol, a measure that reflects the amount of free cortisol in circulation, normally increases during pregnancy, and it can increase up to 8 times the normal level with Cushing’s disease during the second and the third trimesters,23,29 which is a useful tool to evaluate cortisol levels in a suspected pregnant woman. Because the suppression of both UFC and plasma cortisol is decreased in pregnancy,23,30 a low-dose DST is not very helpful for screening Cushing’s disease in pregnant patients. However, a high-dose DST with a <80% cortisol suppression might only indicate Cushing’s disease.3,31 Thus, it helps differentiating between ectopic ACTH syndrome and Cushing’s disease.32 The use of high-dose DST can distinguish between adrenal and pituitary sources of CS in pregnancy. Owing to the limited evidence available and the lack of data on normal pregnancies, the use of corticotropin-releasing hormone (CRH), desmopressin, and high-dose DST in pregnancy is not recommended yet.33 More timely diagnosis as well as timely intervention may have saved the life of our patient. To differentiate between ectopic ACTH syndrome and Cushing’s disease, adrenal imaging should be considered. For higher plasma levels, combined employment of CRH stimulation test and an 8-mg DST can be helpful.3 Bilateral inferior petrosal sinus sampling (B-IPSS) might be needed when the findings are not in accordance with other results, but it is recommended to perform B-IPSS only if the noninvasive studies are inconclusive and only if there is enough expertise, experience, and technique for its performance.3 Although axonal neuropathy has been reported as a rare syndrome associated with paraneoplastic ectopic Cushing’s syndrome and exogenous Cushing’s syndrome, its association with Cushing’s disease has not been reported.5,32 Our patient had severe muscle weakness that we initially attributed it to myopathy and hypokalemia associated with Cushing’s syndrome. In our study, the diagnosis of axonal neuropathy was made based on electrophysiological studies by a neurology consultant and then IVIG was administered; however, the patient’s weakness did not improve after this treatment. The co-occurrence of Guillain-Barré syndrome which may also be classified as axonal neuropathy has also been reported in a pregnant woman with ectopic Cushing’s syndrome.34,35 Whether this finding is coincidental or the result of complex immune reactions driven by Cushing’s disease, or the direct effect of steroids, these results cannot be deduced from current data.36 Some data suggest that the fluctuations and inferior petrosal sinus sampling may trigger the flare of autoimmune processes, specifically when the cortisol levels start to decline during the course of Cushing’s syndrome.35,8 Also, due to COVID-19 pandemic affecting vital organs like kidney, paying attention to COVID-19 is suggested.37-40 Conclusions We presented a thin young female with psychosis, proximal myopathy, and axonal neuropathy with Cushing’s disease who had a recent pregnancy that was terminated without any fetal or maternal complications despite the repeated elevated serum cortisol and 24-hour UFC; therefore, we suggest that she might have glucocorticoid resistance. Glucocorticoid resistance is a rare disease in which the majority, but not all, of patients have a genetic mutation in the hGR-NR3C1 gene. As we did not perform genetic testing for our patient, the data are lacking. Another clue to the absence of the classic Cushing’s disease phenotype in our case is the role of isoenzymes of 11-HSD1 and 11-HSD2. Other mechanisms, such as the defect somewhere in the glucocorticoid pathway of action such as a decreased number of receptors, a reduction in ligand affinity, or a postreceptor defect, play an important role in nonclassical clinical manifestations of Cushing’s syndrome. Acknowledgments The authors thank the patient for allowing us to publish this case report. The authors show their gratitude to the of the staff of the Rasool Akram Medical Complex Clinical Research Development Center (RCRDC) specially Mrs. Farahnaz Nikkhah for its technical and editorial assists. Ethics Approval Our institution does not require ethical approval for reporting individual cases or case series. Informed Consent Written informed consent was obtained from the patient and for her anonymized information to be published in this article. Declaration of Conflicting Interests The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article. Funding The author(s) received no financial support for the research, authorship, and/or publication of this article. References 1. Guilhaume B, Sanson ML, Billaud L, Bertagna X, Laudat MH, Luton JP. Cushing’s syndrome and pregnancy: aetiologies and prognosis in twenty-two patients. Eur J Med. 1992; 1(2):83-89. GO TO REFERENCE PubMed Google Scholar 2. Lin W, Huang HB, Wen JP, et al. Approach to Cushing’s syndrome in pregnancy: two cases of Cushing’s syndrome in pregnancy and a review of the literature. Ann Transl Med. 2019; 7(18):490. 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  24. Abstract Cushing’s disease is a rare neuroendocrine disorder with excessive endogenous cortisol, impaired cognition, and psychiatric symptoms. Evidence from resting-state fMRI revealed the abnormalities of static brain connectivity in patients with Cushing’s disease (CD patients). However, it is unknown whether the CD patients’ dynamic functional connectivity would be abnormal and whether the dynamic features are associated with deficits in cognition and psychopathological symptoms. Here, we evaluated 50 patients with Cushing’s disease and 57 healthy participants by using resting-state fMRI and dynamic functional connectivity (dFNC) approach. We focused on the dynamic features of default mode network (DMN), salience network (SN), and central executive network (CEN) because these are binding sites for the cognitive-affective process, as well as vital in understanding the pathophysiology of psychiatric disorders. The dFNC was further clustered into four states by k-mean clustering. CD patients showed more dwell time in State 1 but less time in State 4. Intriguingly, group differences in dwell time in these two states can explain the cognitive deficits of CD patients. Moreover, the inter-network connections between DMN and SN and the engagement time in State 4 negatively correlated with anxiety and depression but positively correlated with cognitive performance. Finally, the classifier trained by the dynamic features of these networks successfully classified CD patients from healthy participants. Together, our study revealed the dynamic features of CD patients’ brains and found their associations with impaired cognition and emotional symptoms, which may open new avenues for understanding the cognitive and affective deficits induced by Cushing’s disease. Introduction Cushing’s disease is characterized by excess endogenous cortisol secretion [1] and served as a unique and natural model for investigating the effects of elevated endogenous cortisol levels on brain functions and structure [2]. It is also a good model for unraveling the linkage between stress-related brain dysfunctions and psychiatric symptoms [3]. Long-term exposure to hypercortisolism negatively affects patients’ physical and mental health, such as depression, anxiety, and psychosis [1, 4], as well as shows deleterious effects on cognitive function including impaired executive function, working memory, and attention [5,6,7]. Research progress on Cushing’s disease, which depends on static resting-state fMRI, revealed that patients with Cushing’s disease showed increased functional connectivity between the default mode network (DMN) and left lateral occipital cortex [2], and hippocampus [8]. Cortisol increase would induce connectivity changes within the DMN and salience network (SN) [9], and the DMN’s activity correlated with the morning cortisol level of patients with Cushing’s disease [10]. Despite these advances leading to an improved understanding of Cushing’s disease, it remains enigmatic how the abnormal brain connectivity within large-scale networks and how the different brain networks interact would contribute to the deficits in impaired cognitive function, as well as psychopathological symptoms. Furthermore, recent years have witnessed an increasing number of studies providing solid evidence that the brain is a dynamic system rather than a static one on a micro-time scale [11, 12]. Dynamic functional connectivity (dFNC), which is implemented by the sliding window method [13], is an ideal approach to characterize the dynamic nature of brain [11], as well as detect and predict diseases [14, 15]. However, to our knowledge, no studies have ever investigated dynamic brain functional connectivity for patients with CD. We focus here on dynamic functional connectivity and emphasize the role of default mode network (DMN), salience network (SN), and central executive network (CEN). These large-scale neurocognitive networks are critical for cognitive and affective processing [16] and are highly related to stress and cortisol level. Deficits or abnormal connectivity within these three networks are associated with a wide range of stress-related psychiatric disorders [17], as well as the high level of cortisol production [18, 19]. For example, the network-connectivity changes between SN and DMN [20, 21], SN and CEN [22] corresponded to increased cortisol levels. Furthermore, our previous studies also identified that CD patients would show dysregulations of resting-state functional connectivity patterns with DMN [10, 23]. Since CD patients also suffer from cognitive impairment and neuropsychological symptoms, including depression and anxiety, which DMN, SN, and CEN mainly modulate, we hypothesized that these three networks are critical to understanding Cushing’s disease and its comorbidity. Here we aimed to investigate two research questions. First, whether there are group differences (CD patients vs. healthy controls) in the dynamic functional connectivity within DMN, SN, and CEN; second, whether the differences can explain the psychiatric symptoms and cognitive impairments in CD patients. We configure our design with a sliding-window approach [11, 13] to portray the features of dynamic functional connectivity (dFNC) within DMN, SN, and CEN among patients with Cushing’s disease (N = 50) and healthy controls (N = 57). We first compared the temporal properties between healthy and CD patients. Then we conducted correlation and mediation analysis to see whether and how the differences in dFNC would contribute to patients’ psychiatric and physiological symptoms and cognitive deficits. We finally implemented a classification machine learning algorithm based on dynamic FNC features within these three networks to see whether these dynamic features would identity CD patients successfully. Materials and methods Ethic approval The experimental protocol was in accordance with principles of the Declaration of Helsinki and approved by a local research ethics Committee of The First Medical Center of Chinese PLA General Hospital (Beijing, China). All participants provided written informed consent after the experimental procedure had been fully explained and were reminded of their right to withdraw at any time during the study. Participants The current study recruited 50 patients with Cushing’s disease (CD patients) and 57 healthy controls (HC) who were matched in age, gender, and education (Table 1). The CD patients were recruited from the Department of Neurosurgery, The First Medical Center of Chinese PLA General Hospital, between May 2017 and November 2019. The following criteria confirmed Cushing’s disease and its etiology: clinical features (e.g., moon face, supraclavicular fat pad, truncal obesity), elevated 24-h urinary free cortisol (24-h UFC, reference range 98.0–500.1 nmol/24 h), absence of normal cortisol circadian rhythm, elevated ACTH levels (reference range at 0800 h: <10.12 pmol/L), elevated cortisol secretion rates (reference range of cortisol level at 0800 h, 198.7–797.5 nmol/L), absence of normal suppression in midnight (1 mg) dexamethasone suppression test and low dose (2 mg) dexamethasone suppression test (but >50% suppression with a high dose (8 mg) of dexamethasone), and a central to peripheral ACTH ratio >2 for petrosal sinus sampling and pathology after surgery. Healthy controls (HC) were recruited from the local community through poster advertisements and were interviewed by experienced psychiatrists to ensure the absence of current or history of any mental disorder. Demographic information and clinical characteristics of all CD patients and healthy controls were shown in Table 1. Table 1 Demographic and clinical data from healthy controls and CD patients. Full size table Clinical data acquisition, neuropsychological and neuropsychiatric assessment Biometric measurements of the CD patients, including 24-h urinary free cortisol (UFC) levels, plasma Cortisol level (at 0000 h, 0800 h, 1600 h) and adrenocorticotropin (ACTH) level (at 0000 h, 0800 h, 1600 h) from a peripheral vein. Clinical severity of CD patients was obtained using the Cushing Quality of Life Scale (Cushing QOL) [24]. We also included the neuropsychological and neuropsychiatric assessments such as Self-Rating Depression Scale (SDS) [25], Self-Rating Anxiety Scale (SAS) [26], Montreal Cognitive Assessment-Beijing Version (MoCA-BJ) [27], and Chinese version of neuropsychiatric inventory (CNPI) [28]. Image acquisition Functional brain images were acquired using a 3-Tesla GE750 scanner at the First Medical Center of Chinese PLA General Hospital (Beijing, China). Blood oxygen level-dependent (BOLD) gradient echo planar images (EPIs) were obtained using an 8-channel head coil [64 × 64 × 36 matrix with 3.5 × 3.5 × 3.5 mm spatial resolution, repetition time (TR) = 2000 ms, echo time (TE) = 30 ms, flip angle = 90°, field of view (FOV) = 256 × 256 mm2]. A high-resolution T1-weighted structural image (256 × 256 × 144 matrix with a spatial resolution of 1 × 1 × 1 mm, repetition time (TR) = 6700 ms, echo time (TE) = 29 ms, flip angle = 7°) was subsequently acquired. During scanning, all participants were fitted with soft earplugs, and were requested to keep their eyes closed, to stay awake and not to think of anything. Data preprocessing The fMRI data was preprocessed using SPM12 (Wellcome Trust Centre for Neuroimaging, London). The first 10 volume of the functional images were discarded to avoid initial steady-state problems. Then functional images were spatially realigned to the first image for motion correction and corrected for slice acquisition temporal delay. Subsequently, functional images were co-registered to each participant’s segmented gray matter T1 image, then spatially normalized to the Montreal Neurological Institute (MNI) coordinate system, resampled to 3 × 3 × 3 mm voxels. Finally, all functional images were spatially smoothed with an isotropic 4 mm FWHM Gaussian kernel. Group ICA and post-processing Preprocessed data were decomposed into functional components that exhibited a unique time course profile using the group-level spatial independent component analysis, which was implemented in the GIFT toolbox (http://mialab.mrn.org/software/gift/) [29]. First, a subject-specific data reduction principal component analysis (PCA) was performed in which 120 principal components remained. Then at group level, we adopted a high model order ICA to reduce the resting state data into 100 group independent components [30] using the expectation-maximization (EM) algorithm [31] in GIFT. Further, the Infomax ICA algorithm in ICASSO [32] was repeated 20 times [33] to ensure the reliability and stability. Subject-specific spatial maps and time-courses were estimated using the back-reconstruction approach (GICA) [34]. We characterized 50 components as intrinsic connectivity networks (ICNs) by applying the following criteria:[13, 35] whether the peak activation coordinates of the functional components were primarily located in gray matter, and with minimal spatial overlap with white matter structures, vascular, ventricular, edge regions corresponding to artefacts, and susceptibility artifacts. We sorted these 50 meaningful independent components into the interested functional networks including: default mode network (DMN), central executive network (CEN) and salience network (SN) (Fig. 1) according to the spatial correlation values between independent components and the given template [36]. Additional post-processing was conducted to remove remaining noise. Time-courses of the seven components were detrended, despiked and low-pass filtered with a high-frequency cutoff of 0.15 Hz [13]. Moreover, we regressed out the six parameters of head movement. Fig. 1: Composite map of the three networks. And the pipeline of dynamic functional connectivity and clustering analyses. A The three brain networks, default mode network (DMN, including 7 components), central executive network (CEN, including 9 components) and salience network (SN, including 7 components) are derived from group spatial independent components analyses among all participants. B First, for each participant, the dynamic functional connectivity (FNC) matrices are estimated on each sliding window (200 windows) of a set of components within the three networks. Then we applied k-means clustering algorithm on the dynamic FNC matrices across all subjects to assess the reoccurring FNC’s states. Optimal number of states was determined by elbow method. We showed the averaged FNC pattern and the corresponding total number of windows in each state, percentage of each occurrence was presented in parentheses. The color bar represents the z value of FNC. Full size image Dynamic functional connectivity Sliding window approach is the most common way to investigate the nonstationary nature of functional connectivity (FC) of fMRI data. We conducted dynamic FC analysis using the DFC network toolbox in GIFT. In line with previous studies [13, 36], a window of 60 s width (30 TR), sliding in steps of one repetition time was applied to divide the time-courses of each independent components into 200 windows. As covariance estimation using time series of shorter length can be noisy, the regularized inverse covariance matrix (ICOV) was adopted [37]. Following graphic LASSO framework [38], we imposed an additional L1 norm of the precision matrix to enforce sparsity. Clustering analysis Based on previous studies, we applied a k-means clustering algorithm on windowed functional connectivity matrices [39] to assess the frequency and structure of reoccurring functional connectivity patterns (states) across all subjects. We used Manhattan distance function to estimate the similarity between different time windows of FC matrices, which had been demonstrated as an effective measure for high-dimensional data [40]. To obtain the optimal number of states, a cluster validity analysis (silhouette) was conducted on the exemplars of all the subjects. To avoid cost function convergence to the local optimal solution, all clustering analyses were iterated 5 times in GIFT, and the best result was used. Finally, we determined the optimal number of clusters as equal to four (k = 4). According to the clustering results, three temporal properties of dynamic FC states derived from each subject’s state vector were calculated: (i) mean dwell time, measured as the average number of consecutive windows belonging to one state; (ii) fraction of time, measured as the proportions of total windows in one state; (iii) number of transitions, defined as the number of state transitions during the entire scan. Mediation analyses Bootstrapping method was used to estimate the mediation effect. Bootstrapping is a nonparametric approach to effect-size estimation and hypothesis testing that is increasingly recommended for many types of analyses, including mediation [41, 42]. Bootstrapping generates an empirical approximation of the sampling distribution of a statistic by repeated random resampling from the available data and uses this distribution to calculate p-values and construct confidence intervals (5000 resamples were taken for these analyses). Moreover, this procedure supplies superior confidence intervals (CIs) that are bias-corrected and accelerated [43, 44]. Classification analyses using dynamic functional connectivity We conducted classification analyses based on dynamic FNC features [35] to classify each kind of patients. Specifically, we firstly formed a regression matrix, Rgroups × cluster centroids, then regressed out the windowed FNC matrices at each time window using the regression matrix for each participant. These analyses end up with eight β coefficients for each time window for each participant. Next, we computed the mean β coefficients for all time windows. Thus, we got eight mean β coefficients for each participant. These mean β coefficients served as the dynamic FNC features for the classification analysis. The classification analysis using supervised machine learning method, linear support vector machine algorithm (http://www.csie.ntu.edu.tw/~cjlin/libsvm/) with a standard 10-fold cross-validation. We randomly divided the data into 10 subgroups, used the trained classifier from the nine subgroups to predict the performance on the left one subgroup, and repeated the procedure for 100 times. We reported the averaged classification accuracy for each group across these 100 times. Results Neuropsychological and neuropsychiatric difference between healthy controls and CD patients Patients with Cushing’s disease reported higher depression, anxiety, and higher frequency and severity mental illness than healthy controls. Additionally, CD patients also behaved impaired cognitive ability than healthy controls (see Table 1) Functional connectivity within DMN, CEN and SN networks in the four states Spatial map of default mode network, central executive network and salience network identified using the group independent component analysis was shown in Fig. 1A. Independent components were grouped based on their anatomical and presumed functional properties: default mode network (ICs, 9, 12, 27, 28, 32, 44, 74), central executive network (ICs, 15, 21, 26, 48, 50, 63, 85, 89, 97), and salience network (ICs, 20, 43, 57, 59, 76, 82, 92). We adopted a k-means clustering algorithm on the dynamic functional connectivity (dFNC) from all subjects into four connectivity states. Figure 1B shows the cluster centroid and the percentage of occurrences of each state (arranged in the order of emergence). Different temporal properties between HC and CD patients We firstly compared the mean dwell time between healthy controls and CD patients in each state (Fig. 2A–D). Using independent T test, we found that the CD patients had higher mean dwell time than HC in State 1 (CD patients: 89.040 ± 59.216 vs. HC: 57.491 ± 40.671; t(105) = 3.244, p = 0.002), but less mean dwell time than HC in State 4 (CD patients: 31.300 ± 39.413 vs. HC: 66.438 ± 45.734; t(105) = −4.227, p < 0.001). We did not observe significant difference in State 2 (CD patients vs. HC: t(105) = 1.700, p = 0.092), nor in State3 (CD patients vs. HC: t(105) = −1.517, p = 0.132). For the switch time (i.e., the number of transitions), CD patients revealed less transition number than healthy controls did (CD patients: 6.600 ± 3.187 vs. HC: 7.824 ± 3.059; t(105) = −2.205, p = 0.045; Fig. 2E). Multiple comparisons were corrected by false-discovery rate (FDR), p < 0.05. All contrasts remained the same after FDR correction excepted the results of switch time became marginally significant, FDR corrected p = 0.075. Group difference on fraction of time in each state was similar with the mean dwell time (see Supplementary Table S1). Levene’s test is used to check that variances are equal for all samples. Fig. 2: Mean dwell time of dynamic FNC states and number of transitions between CD patients and healthy controls. A In State 1, CD patients engaged higher mean dwell time than healthy control did. B, C In State 2 and State 3, no difference was found between CD patients and healthy controls. D In State 4, CD patients showed significant less mean dwell time than healthy controls. E There was marginally significant difference (after FDR correction) on number of transitions between CD patients and healthy controls. Multiple comparisons were corrected by FDR, p < 0.05 (Error bars represent standard error. p < 0.01**, p < 0.001***, p < 0.08+, N.S not significant). HC Healthy controls, CD patients with Cushing’s disease. Full size image Correlation between dynamic FNC properties and clinical characteristics To examined whether the dynamic FNC properties were associated with clinical characteristics, we did Pearson correlation analyses. Since the group differences were found in State 1 and State 4, we only restricted our analyses on these two states. Notably, we found that the dwell time in State 1 positively correlated with the self-reported anxiety (SAS), and cortisol level at 8:00, 16:00, 00:00, ACTH at 8:00, 16:00, as well as elevated 24-h urinary free cortisol. That is, the longer time spent on State 1 which with more sparsely connected pattern, the worse the mental health and higher cortisol level. We also detected a robust negative correlation between dwell time of State 1 and global cognitive scales (MoCA), which indicated that more time spent in State 1, the worse cognitive ability would be. In the contrary, dwell time in State 4 showed significant negative correlation with the self-reported depression, anxiety, and cortisol level at 8:00, 16:00, 00:00. More dwell time in State 4 predicted better cognitive performance measured by MoCA (all results see Table 2). Multiple comparisons were conducted by FDR, p < 0.05. Table 2 Correlations between dynamic functional connectivity temporal properties in cognitive control network and clinical data. Full size table Dwell time in State 1 and State 4 within cognitive control network mediate group difference in cognitive performance Interestingly, we found the dwell time in State 1 and State 4 significantly mediated the difference between individuals with excessive high cortisol level (CD patients) and healthy controls on cognitive performance. That is, lower cognitive performance in CD patients was linked with more dwell time in State 1 (Fig. 3A), and less dwell time in State 4 (Fig. 3B) within the three networks. Fig. 3: Mediation effect of dwell time in State 1 and State 4 on group difference on cognitive performance. A Dwell time in State 1 and B dwell time in State 4 significant partially mediated the difference between CD patients and healthy controls on cognitive performance measured by MoCA. HC Healthy controls, CD patients with Cushing’s disease. Full size image Distinct network-based functional connectivity between CD patients and healthy controls and its associations with psychiatric symptoms and cognitive performance We have already known that the difference on dwell time in State 1 and State 4 can explain the group difference (i.e., CD patients vs. healthy controls) on cognitive performance. We further characterized the State 1 and State 4 by analyzing functional connectivity between the three networks, as well as the functional connectivity within each network. Results showed that in State 1, the CD patients had weaker connectivity within DMN (t(104)1 = −2.584, p = 0.011), and the connections between CEN and DMN (t(104) = −5.141, p < 0.001), CEN and SN (t(104) = −4.732, p < 0.001) were also weaker than healthy controls. And in State 4, CD patients showed weaker functional connections between DMN and SN (t (84)2 = −4.203, p < 0.001), as well as DMN and CEN (t(84) = −3.547, p = 0.001). Moreover, in State 4, functional connection between DMN and SN was negatively correlated with anxiety level measured by SAS (r(68) = −0.336, p = 0.005), and depression level measured by SDS (r(68) = −0.320, p = 0.008), but positively correlated with cognitive performance measured by MoCA (r(65) = 0.421, p < 0.001). Since CD patients showed decreased connection between DMN and SN, these results may suggest that the connection between DMN and SN was critical for understanding the psychiatric symptoms and cognitive deficits in CD patients. All significant results reported here were survived after FDR (p < 0.05) correction. We did not find significant associations between functional connectivity of neither inter-network and intra-network and psychiatric symptoms and cognitive deficits in State 1. No significant correlation results were found between the inter-network and intra-network connectivity and physiological indices (i.e., cortisol, ACTH, and UFC) in these two states, which may suggest that the dwell time in specific state would be more sensitive to physiological change. Classification results based on dynamic FNC features The support vector machine (SVM) based on dynamic FNC approach (Fig. 4A, details see Method) showed classification accuracy of 84.76% for CD patients, 88.98% for healthy controls (Fig. 4B). The classification scores were evaluated using a receiver operating characteristic (ROC) curve aiming to visualize the performance of the classifier. The classification results may further indicate that the dynamic functional connectivity pattern within these three networks would be the potential biomarker of individuals with excessive higher cortisol level. Fig. 4: The results of classification. A An overview of classification approach. We first extracted the averaged FNC pattern for each state for each group. Then we performed Pearson correlation between the FNC in each window and the FNC pattern in all states among all groups. These procedures ended up with 8 averaged features for each participant. B Receiver Operating Characteristic (ROC) curves for classification. SVM support vector machine, AUC area under the curve. Full size image Discussion In the current study, we adopted independent component analysis (ICA) and dynamic functional connectivity (FNC) approaches to reveal the difference in dynamic FNC within DMN, SN, and CEN networks between CD patients and healthy controls. Using clustering algorithm, we defined four reoccurring FNC states during resting-state scanning. Wherein State 1 and State 4 exhibited significant differences between healthy control and CD patients. Patients generally showed more dwell time in State 1 but less in State 4 than healthy controls. Specifically, in State 1, the CD patients showed weaker connections within DMN, as well as weaker intra-network connectivity between DMN and CEN, SN and CEN than healthy controls. In State 4, connections between DMN and SN, DMN and CEN showed weaker connection in CD patients than in healthy participants. Further correlation and mediation analyses showed that the dwell time in State 1 significantly negatively correlated with cognitive performance. While dwell time in State 4, as well as the connections between DMN and SN in State 4, were found to positively correlate with cognitive performance, and negatively associated with depression and anxiety symptoms. Both states were associated with physiological indices including cortisol, ACTH and 24-hour UFC. Importantly, results from mediation analysis indicated the difference between CD patients and healthy controls on dwell time in State 1 and State 4 can be used to explain their cognitive performance difference. Intriguingly, adopting support vector machine algorithm based on dynamic FNC within DMN, SN and CEN network generally showed ideal classification accuracy for CD patients and healthy controls. These findings begin to delineate the dynamic properties of the three brain networks, which are critical for cognitive and neuropsychiatric, and open new avenues for understanding and explaining the impaired cognitive performance and psychiatric symptoms induced by Cushing’s disease. We found two distinct functional connectivity states across two groups. State 1 can be characterized as having weak connections among the three networks, while State 4 showed relatively strong inter-network and intra-network connections. We observed that in patients with Cushing’s disease, State 1 occurred more often, while State 4 occurred less than in healthy controls. These results help to confirm CD patients’ weaker connections within DMN, SN, and CEN. Previous studies identified that white matter integrity was generally decreased throughout the whole brain rather than just on individual fasciculus [45,46,47]. One possible explanation is that the extensive decline in white matter structural integrity leads to the decreased connectivity of the three networks, which are critical for the cognitive-affective process. We found that in State 1, CD patients showed decreased local synchronization (i.e., within network connectivity) of DMN, and weak inter-network connections between CEN and DMN, CEN and SN. The DMN’s integrity appears crucial for cognitive performance. For example, patients with Alzheimer’s disease showed decreased connectivity within DMN [48]. Since dwell time of State 1 was negatively correlated with MoCA and mediated the group differences on MoCA. We may infer that cognitive deficit may be due to that CD patients engaged more time in State 1 with weak connections of DMN. Interestingly, the more dwell time in State 4, the less anxiety and depression symptoms individuals would have. Moreover, our further analyses found that connections between DMN and SN during State 4 would also negatively affect anxiety and depression. And the CD patients had weaker DMN-SN connections than healthy controls in this state. In line with previous studies, effective connectivity from DMN to SN was lower in major depression disorders compared to healthy controls when processing negative information [49]. And the inter-network connections between the SN and DMN were inversely associated with trait anxiety levels [50]. Therefore, the time engaged in State 4 and the weak inter-network connectivity between SN and DMN may contribute to psychopathological symptoms in CD patients. Dynamic functional connectivity provides time-varying rather than static features over time [11], and it is more effective to capture various aspects of brain connectivity. The dFNC approach has obvious advantages for classification purposes [35]. For example, previous research showed high classification accuracy for psychiatric diseases such as schizophrenia [51], and bipolar [35]. In our study, the SVM based on dynamic functional connectivity features within DMN, SN and CEN showed high classification accuracy for CD patients and healthy controls, which may indicate that the dynamic properties in these three networks would be potential biomarkers for individuals with excessive higher cortisol level. The long-term remitted CD (LTRCD)-patients still suffered from cognitive impairments and emotional symptoms such as anxiety and depression, even though their cortisol levels back to normal after the removal of the adenoma [2, 52, 53]. We revealed that the dynamic features in DMN, SN, and CEN correlate with depression and anxiety symptoms in CD patients and are strongly associated with cognitive performance. Our findings may contribute to developing further neuro-modulation targets to help CD patients improve cognitive ability and mental health. Several limitations of the present study should be mentioned. First, Cushing’s disease is rare, and it is more common in women [1, 3]. We only showed results based on a female sample (healthy controls were all female). Therefore, our conclusion may not be adaptive for the male population. Second, some research suggested that the dynamic functional connectivity analyses should be performed in resting state acquisitions of at least ten minutes [54]. The length of current resting-state scan was eight minutes, although many previous studies studied dynamic FNC based on resting-state data in eight minutes or even less [13, 20, 51], further studies should consider longer scanning to capture more dynamic spontaneous features. Thirdly, our results revealed that cortisol concentrations were significantly associated with dwell time in State 1 and 4 but were not correlated with inter-network or intra-network connections. Human cortisol secretion has apparent circadian rhythmicity [55], but our resting state acquisitions were not collected multiple times. Our conclusions may not be informative to understand the relationships between dynamic functional connections and dynamic cortisol levels. In conclusion, our study delineates the differences in dynamic properties between CD patients and healthy participants. It unravels its associations with cognitive deficits, impaired affective processes, and physiological indices in CD patients. We believe the temporal dynamics of functional connectivity within the three crucial cognitive and affective brain networks could be a promising imaging biomarker to monitor cognitive changes and psychiatric symptoms in Cushing’s disease. Data availability All datasets are available on figshare. https://figshare.com/projects/Dynamic_functional_connectivity_changes_associated_with_psychiatric_traits_and_cognitive_deficits_in_Cushing_s_disease/170343. Code availability All code used for all analyses and plots are publicly available on GitHub at https://github.com/psywalkeryanxy/paper_CD_ICA. References Lacroix A, Feelders RA, Stratakis CA, Nieman LK. Cushing’s syndrome. Lancet. 2015;386:913–27. Article CAS PubMed Google Scholar van der Werff SJA, Pannekoek JN, Andela CD, Meijer OC, van Buchem MA, Rombouts SARB, et al. Resting-state functional connectivity in patients with long-term remission of Cushing’s disease. Neuropsychopharmacology. 2015;40:1888–98. Article PubMed PubMed Central Google Scholar Swearingen B, Biller BMK, editors. Cushing’s disease. vol. 31, US: Springer; 2011. Piasecka M, Papakokkinou E, Valassi E, Santos A, Webb SM, Vries F, et al. 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Article CAS PubMed Google Scholar Download references Acknowledgements This work was supported by the National Natural Science Foundation of China (No. 82001798 and No. 81871087) and China Brain Project (2021ZD0200407). Author information Authors and Affiliations Department of Neurosurgery, Chinese PLA General Hospital, Haidian District, Beijing, PR China Zhebin Feng, Tao Zhou, Xinguang Yu & Yanyang Zhang Department of Respiratory Medicine, Anhui Provincial Children’s Hospital, Hefei, Anhui, PR China Haitao Zhang Neurosurgery Institute, Chinese PLA General Hospital, Beijing, PR China Xinguang Yu & Yanyang Zhang Department of Psychiatry, University of Minnesota Medical School, Minneapolis, MN, USA Xinyuan Yan Contributions YZ and XGY, TZ conceived the project and designed research, HZ performed research, XY and ZF, YZ analyzed data and interpreted results, ZF and XY wrote the paper. All authors approved the final version of the manuscript for submission. Corresponding authors Correspondence to Yanyang Zhang or Xinyuan Yan. Ethics declarations Competing interests The authors declare no competing interests. Additional information Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Supplementary information Supporting information From https://www.nature.com/articles/s41398-023-02615-y
  25. 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
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