Search the Community

Abstract Background The aim of this study was to investigate the clinical features and treatment options for pediatric adrenal incidentalomas(AIs) to guide the diagnosis and treatment of these tumors. Methods The clinical data of AI patients admitted to our hospital between December 2016 and December 2022 were collected and retrospectively analyzed. All patients were divided into neonatal and nonneonatal groups according to their age at the time of the initial consultation. Results In the neonatal group, 13 patients were observed and followed up, and the masses completely disappeared in 8 patients and were significantly reduced in size in 5 patients compared with the previous findings. Four patients ultimately underwent surgery, and the postoperative pathological diagnosis was neuroblastoma in three patients and teratoma in one patient. In the nonneonatal group, there were 18 cases of benign tumors, including 9 cases of ganglioneuroma, 2 cases of adrenocortical adenoma, 2 cases of adrenal cyst, 2 cases of teratoma, 1 case of pheochromocytoma, 1 case of nerve sheath tumor, and 1 case of adrenal hemorrhage; and 20 cases of malignant tumors, including 10 cases of neuroblastoma, 9 cases of ganglioneuroblastoma, and 1 case of adrenocortical carcinoma. Conclusions Neuroblastoma is the most common type of nonneonatal AI, and detailed laboratory investigations and imaging studies are recommended for aggressive evaluation and treatment in this population. The rate of spontaneous regression of AI is high in neonates, and close observation is feasible if the tumor is small, confined to the adrenal gland and has no distant metastasis. Peer Review reports Background The incidence of adrenal incidentaloma (AI) is increasing due to the increased frequency of imaging and improved imaging sensitivity [1]. AI is relatively common in adults, and several organizations, such as the American Association of Clinical Endocrinologists/American Association of Endocrine Surgeons and the European Society Endocrinology, have proposed specific protocols to guide the evaluation, treatment, and follow-up management of AI in adults [2]. Although AI, a nonfunctioning adrenocortical adenoma, is most common in adults, neuroblastoma is the most common incidental tumor of the adrenal gland in children. In addition, in the neonatal period, which is a more complex stage of childhood, the biology of adrenal masses found in this age group is also more specific, and the nature of these masses can range from spontaneous regression to rapid progression to aggressive disease with metastatic dissemination and even death. Given that AI is the most common malignant tumor, the management of AI in children cannot be simply based on the measurements used in adult AI. In this study, we retrospectively analyzed the clinical data of pediatric AI patients in a single center to investigate the clinical characteristics and management of AI in children. Methods A total of 66 children with adrenal tumors were diagnosed and treated at the Department of Urology of the Children’s Hospital of Nanjing Medical University from December 2016 to December 2022. A total of 55 cases were detected during physical examination, or the patients were diagnosed and received treatment for diseases other than adrenal disease after excluding adrenal tumors detected due to typical clinical manifestations or signs such as centripetal obesity and precocious puberty. Research protocols involving human materials were approved by the Medical Ethics Committee of the Children’s Hospital of Nanjing Medical University. All clinical information, radiological diagnosis, laboratory test results, intervention results, and follow-up data were collected from the department’s database. All the children underwent ultrasonography and CT scanning, and 11 children underwent MRI. In addition to routine tests such as blood routine and biochemical indexes, the examination and evaluation of adrenal endocrine hormones and tumor markers included (1) plasma cortisol and ACTH levels, (2) plasma catecholamine and metabolite determination, (3) plasma renin and plasma aldosterone, (4) urinary vanillylmandelic acid/homovanillic acid(VMA/HVA), and (5) AFP, CEA, NSE, and CA19-9. Five patients underwent a low-dose dexamethasone suppression test. Seventeen of the 55 patients were treated with watch-waiting therapy, 4 of the 17 ultimately underwent surgery, 4 of the 38 patients underwent tumor biopsy, and 34 underwent adrenalectomy. The data were analyzed using Graph Pad Prism 8. The measurement data are expressed as ‾x ± sd. The maximum diameter of the tumors, age of the patients with benign and malignant tumors, and maximum diameter of the tumors between the laparoscopic surgery group and the open surgery group were compared using paired t tests, and the percentages of the count data were compared using Fisher’s exact test. Results In this study, all patients were divided into two groups according to their age at the time of consultation: the neonate group and the nonneonate group. Neonate group: There were 7 male and 10 female patients, 7 of whom were diagnosed via prenatal examination and 10 of whom were diagnosed after birth. Five patients were diagnosed with lesions on the left side, 12 patients were diagnosed with lesions on the right side, and the maximal diameters of the masses ranged from 16 to 48 mm. The characteristics of the AIs in the neonate group are presented in Table 1. Table 1 Characteristics of AI in the neonates group Full size table Among the 17 patients, 8 had cystic masses with a maximum diameter of 16∼48 mm, 5 had cystic-solid masses with a maximum diameter of 33∼39 mm, and 4 had solid masses with a maximum diameter of 18∼45 mm. Two patients with solid adrenal gland masses suggested by CT scan had obvious elevations in serum NSE and maximum diameters of 44 and 45 mm, respectively. These patients underwent adrenal tumor resection, and the pathology diagnosed that they had neuroblastomas(NB). In one patient, the right adrenal gland was 26 × 24 × 27 mm in size with slightly elevated echogenicity at 38 weeks after delivery, and the mass increased to a size of 40 × 39 × 29 mm according to the 1-month postnatal review. MRI suggested that the adrenal gland tumor was associated with liver metastasis, and the pathology of the tumor suggested that it was NB associated with liver metastasis after surgical resection (stage 4 S, FH). One child was found to have 25 × 24 × 14 mm cystic echoes in the left adrenal region during an obstetric examination, and ultrasound revealed 18 × 11 mm cystic solid echoes 5 days after birth. Ultrasound revealed 24 × 15 mm cystic solid echoes at 2 months. Serum NSE and urinary VMA were normal, and the tumor was excised due to the request of the parents. Pathology suggested a teratoma in the postoperative period. A total of 13 children did not receive surgical treatment or regular review via ultrasound, serum NSE or urine VMA. The follow-up time ranged from 1 to 31 months, with a mean of 9.04 ± 7.61 months. Eight patients had complete swelling, and 5 patients were significantly younger than the previous patients. Nonneonate group: There were 24 male and 14 female patients in the nonneonate group; 24 patients had lesions on the left side, 14 patients had lesions on the right side, and the maximal diameters of the masses ranged from 17 to 131 mm. Most of these tumors were found during routine physical examinations or incidentally during examinations performed for various complaints, such as gastrointestinal symptoms, respiratory symptoms, or other related conditions. As shown in Table 2, abdominal pain was the most common risk factor (44.7%) for clinical onset, followed by routine physical examination and examination for respiratory symptoms. Table 2 Clinical presentations leading to discovery of AI in non-neonate group Full size table Among the 38 patients, 10 had NBs with maximum diameters ranging from 20 to 131 mm, 9 had ganglion cell neuroblastomas with maximum diameters ranging from 33.6 to 92 mm, 9 had ganglion cell neuromas with maximum diameters ranging from 33 to 62 mm, 2 had adrenal adenomas with maximum diameters ranging from 17 to 70 mm, 1 had a cortical carcinoma with a maximum diameter of 72 mm, 2 had adrenal cysts with maximum diameters ranging from 26 to 29 mm, 2 had mature teratomas with maximum diameters of 34 and 40 mm, 1 had a pheochromocytoma with a diameter of 29 mm, 1 had a nerve sheath tumor with a diameter of 29 mm, and 1 patient with postoperative pathological confirmation of partial hemorrhagic necrosis of the adrenal gland had focal calcification with a maximum diameter of 25 mm (Table 3). Table 3 Distribution of different pathologies among AI with various sizes in non-neonate group Full size table The mean age of children with malignant tumors was significantly lower than that of children with benign tumors (57.95 ± 37.20 months vs. 105.0 ± 23.85 months; t = 4.582, P < 0.0001). The maximum diameter of malignant tumors ranged from 20 to 131 mm, while that of benign tumors ranged from 17 to 72 mm, and the maximum diameter of malignant tumors was significantly greater than that of benign tumors (65.15 ± 27.61 mm v 37.59 ± 12.98 mm; t = 3.863, P = 0.0004). Four biopsies, 5 laparoscopic adrenal tumor resections and 11 open adrenal tumor resections were performed for malignant tumors, and 16 laparoscopic adrenal tumor resections and 2 open procedures were performed for benign tumors. The maximum diameter of the tumors ranged from 17 to 62 mm in 21 children who underwent laparoscopic surgery and from 34 to 99 mm in 13 children who underwent open resection; there was a statistically significant difference in the maximum diameter of the tumors between the laparoscopic surgery group and the open surgery group (35.63 ± 10.36 mm v 66.42 ± 20.60 mm; t = 5.798, P < 0.0001). Of the 42 children with definitive pathologic diagnoses at surgery, 23 had malignant tumors, and 19 had benign tumors. There were 15 malignant tumors with calcification on imaging and 5 benign tumors. The percentage of malignant tumors with calcifications in was significantly greater than that of benign tumors (65.22% v 26.32%; P = 0.0157). In the present study, all the children underwent CT, and 31 patients had postoperative pathological confirmation of NB. A total of 26 patients were considered to have neurogenic tumors according to preoperative CT, for a diagnostic compliance rate of 83.97%. Three children were considered to have cortical adenomas by preoperative CT, and 1 was ultimately diagnosed by postoperative pathology, for a diagnostic compliance rate of 33.33%. For 4 patients with teratomas and adrenal cysts, the CT findings were consistent with the postoperative pathology. According to our research, NB 9-110HU, GNB 15-39HU, GB 19-38HU, ACA 8HU, adrenal cyst 8HU, and cellular achwannoma 17HU. Discussion According to the clinical practice guidelines developed by the European Society of Endocrinology and European Network for the Study of Adrenal Tumors, AI is an adrenal mass incidentally detected on imaging not performed for a suspected adrenal disease [3]. The prevalence of AI is approximately 4%, and the incidence increases with age [4]. Most adult AIs are nonfunctioning benign adrenal adenomas (up to 75%), while others include functioning adrenal adenomas, pheochromocytomas, and adrenocortical carcinomas [5]. In contrast to the disease spectrum of adult AI cases, NB is the most common tumor type among children with AI, and benign cortical adenomas, which account for the vast majority of adult AI, accounting for less than 0.5% of cases in children [6]. According to several guidelines, urgent assessment of an AI is recommended in children because of a greater likelihood of malignancy [3, 7]. When an adult patient is initially diagnosed with AI, it should be clear whether the lesion is malignant and functional. In several studies, the use of noncontrast CT has been recommended as the initial imaging method for adrenal incidentaloma; a CT attenuation value ≤ 10 HU is used as the diagnostic criterion for benign adenomas; and these methods have a specificity of 71-79% and a sensitivity of 96-98% [8, 9]. A CT scan of tumors with diameters greater than 4 to 6 cm, irregular margins or heterogeneity, a CT attenuation value greater than 10 HU, or a relative contrast enhancement washout of less than 40% 10 or 15 min after administration of contrast media on enhanced CT is considered to indicate potential malignancy [7]. As the most common AI in children, NB often appears as a soft tissue mass with uneven density on CT, often accompanied by high-density calcified shadows, low-density cystic lesions or necrotic areas. CT scans can easily identify more typical NBs, and for those AIs that do not show typical calcified shadows on CT, it is sometimes difficult to differentiate neurogenic tumors from adenomas. In these patients, except for the 1 patient with adrenal cysts who had a CT value of 8 HU, very few of the remaining AI patients had a CT value less than 10 HU. Therefore, the CT value cannot be used simply as a criterion for determining the benign or malignant nature of AI, and additional imaging examinations, such as CT enhancement, MRI, and FDG-PET if necessary, should be performed immediately for AI in children. Initial hormonal testing is also needed for functional assessment, and aldosterone secretion should also be assessed when the patient is hypertensive or hypokalemic [7]. Patients with AI who are not suitable for surgery should be observed during the follow-up period, and if abnormal adrenal secretion is detected or suggestive of malignancy during this period, prompt adrenal tumor resection is needed. For adult patients with AI, laparoscopic adrenal tumor resection is one of the most effective treatments that has comparative advantages in terms of hospitalization time and postoperative recovery speed; however, there is still some controversy over whether to perform laparoscopic surgery for some malignant tumors with large diameters, especially adrenocortical carcinomas, and some studies have shown that patients who undergo laparoscopic surgery are more prone to peritoneal seeding of tumors [10]. The maximum diameter of an adult AI is a predictor of malignancy, and a study by the National Italian Study Group on Adrenal Tumors, which included 887 AIs, showed that adrenocortical carcinoma was significantly correlated with the size of the mass, and the sensitivity of detecting adrenocortical carcinoma with a threshold of 4 cm was 93% [11]. According to the National Institutes of Health, patients with tumors larger than 6 cm should undergo surgical treatment, while patients with tumors smaller than 4 cm should closely monitored; for patients with tumors between 4 and 6 cm, the choice of whether to be monitored or surgically treated can be based on other indicators, such as imaging [12]. A diameter of 4 cm is not the initial threshold for determining the benign or malignant nature of a mass in children. In a study of 26 children with AI, Masiakos et al. reported that 9 of 18 benign lesions had a maximal diameter less than 5 cm, 4 of 8 malignant lesions had a maximal diameters less than 5 cm, and 2 had a diameter less than 3 cm. The mean maximal diameter of benign lesions was 4.2 ± 1.7 cm, whereas the mean maximum diameter of malignant lesions was 5.1 ± 2.3 cm. There was no statistically significant difference between the two comparisons; therefore, this study concluded that children with AI diameters less than 5 cm cannot be treated expectantly [6]. Additionally, this study revealed that malignant lesions occurred significantly more frequently than benign lesions in younger children (mean age 1.7 ± 1.8 years v 7.8 ± 5.9 years; P = 0.02). In the nonneonatal group of this study, 20 patients with malignant tumors had maximum diameters ranging from 20 to 131 mm, 10 had malignant tumors larger than 60 mm, and 3 had tumors smaller than 40 cm; 18 patients with benign tumors had maximum diameters ranging from 17 to 70 mm, 5 had diameters ranging from 40 to 60 mm, and 5 had diameters larger than 60 mm. Therefore, it is not recommended to use the size of the largest diameter of the tumor to decide whether to wait and observe or intervene surgically for children with AI. Instead, it is necessary to consider the age of the child; laboratory test results, such as whether the tumor indices are elevated or not; whether the tumor has an endocrine function; etc.; and imaging test results to make comprehensive judgments and decisions. Preoperative aggressive evaluation and prompt surgical treatment are recommended for nonneonatal pediatric AI patients. Adrenal hematoma and NBs are the most common types of adrenal area masses in children, while pheochromocytoma, adrenal cyst, and teratoma are rarer masses [13]. In clinical practice, adrenal hematoma and NB are sometimes difficult to differentiate, especially when adrenal masses are found during the prenatal examination and neonatal period, and such children need to be managed with caution. The Children’s Oncology Group (COG ANBL00B1) implemented the watchful waiting treatment for children under 6 months of age with a solid adrenal mass < 3.1 cm in diameter (or a cystic mass < 5 cm) without evidence of distant metastasis, and if there is a > 50% increase in the adrenal mass volume, there is no return to the baseline VMA or HVA levels, or if there is a > 50% increase in the urinary VMA/HVA ratio or an inversion, surgical resection should be performed [14]. Eighty-three children in this study underwent expectant observation, 16 of whom ultimately underwent surgical resection (8 with INSS stage 1 NB, 1 with INSS stage 2B, 1 with INSS stage 4 S, 2 with low-grade adrenocortical neoplasm, 2 with adrenal hemorrhage, and 2 with extralobar pulmonary sequestration). Most of the children who were observed had a reduced adrenal mass volume. Of the 56 patients who completed the final 90 weeks of expectant observation, 27 (48%) had no residual mass, 13 (23%) had a residual mass volume of 0–1 ml, 8 (14%) had a mass volume of 1–2 ml, and 8 (14%) had a volume of > 2 ml; ultimately, 71% of the residual masses had a volume ≤ 1 ml and 86% had a residual volume ≤ 2 ml. In this study, a total of 16 patients were included in the watchful waiting treatment group; 3 patients underwent surgical treatment during the follow-up period, and 13 patients ultimately completed watchful waiting treatment. After 1–31 months of follow-up, 8 patients’ swelling completely disappeared, and 5 patients’ swelling significantly decreased. After strict screening for indications and thorough follow-up review, AIs in the neonatal period can be subjected to watchful waiting treatment, and satisfactory results can be achieved. For benign adrenal tumors, laparoscopic surgery is superior to open surgery in terms of successful resection, whereas the feasibility of minimally invasive surgery for AI with preoperative suspicion of malignancy is controversial. The European Cooperative Study Group for Pediatric Rare Tumors recommends that minimally invasive surgery be considered only for early childhood tumors and should be limited to small, localized tumors; additionally, imaging should suggest no invasion of surrounding tissue structures or lymph nodes; and this strategy requires surgeons with extensive experience in oncologic and adrenal surgery [15]. NB is the most common pediatric AI, and open tumor resection remains the mainstay of treatment. For small, early tumors without evidence of invasion on preoperative examination, laparoscopic resection may be considered if the principles of oncologic surgery can be adhered to. If the patient responds to chemotherapy, the decision to perform laparoscopic tumor resection can also be re-evaluated after chemotherapy. According to the current study, the recurrence and mortality rates of laparoscopic surgery are comparable to those of open surgery [16, 17]. The relative contraindications for laparoscopic NB resection include a tumor diameter greater than 6 cm, venous dilatation, and the involvement of adjacent organs or blood vessels [18]. Patients who undergo open adrenalectomy have higher overall survival and recurrence-free survival rates than patients who undergo laparoscopic adrenalectomy [19]. Open adrenalectomy remains the gold standard for surgical resection of adrenocortical carcinoma, whereas laparoscopic adrenalectomy should be reserved for highly selected patients and performed by surgeons with appropriate expertise [20]. Cortical tumors are particularly rare among children with AIs and are sometimes not clearly distinguishable from neurogenic tumors on preoperative imaging; in such patients, the presence of subclinical Cushing’s syndrome needs to be carefully evaluated preoperatively; otherwise, a perioperative adrenal crisis may occur [21]. In patients in whom the possibility of an adrenocortical tumor was considered preoperatively, the assessment for subclinical Cushing’s syndrome mainly involved assessing the serum dehydroepiandrosterone sulfate level and performing an overnight dexamethasone suppression test. A procedure for evaluating pediatric AI is shown in Fig. 1. Imaging is the first step in the evaluation of AI in children. CT can be used to clarify the nature of most tumors. MRI can be used to evaluate imaging risk factors (IDRFs) for NB. Bone marrow cytomorphology is recommended for all children with AI, along with microscopic residual neuroblastoma testing and further bone scanning if the bone marrow examination is positive. In addition, serum tumor marker levels and other relevant tests should be performed, and hormone levels should be evaluated. If adrenal adenomas cannot be completely excluded during the preoperative examination, a 1 mg overnight dexamethasone suppression test should be performed to exclude subclinical Cushing’s syndrome. In patients with hypertensive hypokalemia, the presence of aldosteronism should be evaluated by testing plasma aldosterone concentrations and plasma renin activity. Adrenal masses found in the neonatal period can be observed if the tumor is small, confined to the adrenal gland and shows no evidence of distant metastasis, while tumors that increase significantly in size during the follow-up period or that are associated with persistently elevated tumor markers require aggressive surgical treatment. Fig. 1 Algorithm for the evaluation and management of a pediatric adrenal incidentaloma. *DST overnight :20µg/kg dexamethasoneweight ˂40 kg,1 mg dexamethasone if ≥ 40 kg. CT = computed tomographic;MRI = magnetic resonance imaging;NSE = neuron-specific enolase;AFP = alpha-fetoprotein;CEA = carcinoembryonic antigen;CA19-9 = cancerantigen19-9;ACTH = adrenocorticotropic hormone;PAC = plasma aldosterone concentration; PRA = plasma renin activity;DST = dexamethasone suppression test Full size image Data availability The datasets analyzed during the current study are not public, but are available from the corresponding author on reasonable request. Abbreviations CT: computed tomographic MRI: magnetic resonance imaging ACTH: adrenocorticotropic hormone VMA: vanillylmandelic acid HVA: homovanillic Acid AFP: alpha-fetoprotein CEA: carcinoembryonic antigen NSE: neuron-specific enolase CA19-9: cancerantigen19-9 FH: favorable histology HU: Hounsfiled Unit COG: Children’s Oncology Group INSS: International Neuroblastoma Staging System References Barzon L, Sonino N, Fallo F, Palu G, Boscaro M. Prevalence and natural history of adrenal incidentalomas. Eur J Endocrinol. 2003;149(4):273–85. Article CAS PubMed Google Scholar Maas M, Nassiri N, Bhanvadia S, Carmichael JD, Duddalwar V, Daneshmand S. Discrepancies in the recommendedmanagement of adrenalincidentalomas by variousguidelines. J Urol. 2021;205(1):52–9. Article PubMed Google Scholar Fassnacht M, Tsagarakis S, Terzolo M, et al. European Society of Endocrinology clinical practice guidelines on the management of adrenal incidentalomas, in collaboration with the European network for the study of adrenal tumors. Eur J Endocrinol. 2023;189(1):G1–42. Article PubMed Google Scholar Young WFJr. Clinical practice. The incidentally discovered adrenal mass. N Engl J Med. 2007;356(6):601–10. Article Google Scholar Rowe NE, Kumar R, Schieda N, et al. Diagnosis, management, and follow-up of the incidentallydiscoveredadrenalmass: CUAguidelineendorsed by the AUA. J Urol. 2023;210(4):590–9. Article PubMed Google Scholar Masiakos PT, Gerstle JT, Cheang T, Viero S, Kim PC, Wales P. Is surgery necessary for incidentally discovered adrenal masses in children?J. Pediatr Surg. 2004;39(5):754–8. Article Google Scholar Lee JM, Kim MK, Ko SH et al. Clinical guidelines for the management of adrenal incidentaloma. Endocrinol Metab. 2017;32(2). Terzolo M, Stigliano A, Chiodini I, et al. AME position statement on adrenal incidentaloma. Eur J Endocrinol. 2011;164(6):851–70. Article CAS PubMed Google Scholar Boland GW, Blake MA, Hahn PF, Mayo-Smith WW. Incidental adrenal lesions: principles, techniques, and algorithms for imaging characterization. Radiology. 2008;249(3):756–75. Article PubMed Google Scholar Payabyab EC, Balasubramaniam S, Edgerly M, et al. Adrenocortical cancer: a molecularlycomplexdiseasewheresurgerymatters. Clin Cancer Res. 2016;22(20):4989–5000. Article CAS PubMed Google Scholar Angeli A, Osella G, Alì A, Terzolo M. Adrenal incidentaloma: an overview of clinical and epidemiological data from the National Italian Study Group. Horm Res. 1997;47(4–6):279–83. Article CAS PubMed Google Scholar Grumbach MM, Biller BM, Braunstein GD, et al. Management of the clinically inapparent adrenal mass (incidentaloma). Ann Intern Med. 2003;138(5):424–9. Article PubMed Google Scholar Zhang K, Zhang Y, Zhang Y, Chao M. A retrospective analysis of the clinical characteristics of 207 hospitalized children with adrenal masses. Front Pediatr. 2023;11:1215095. Article PubMed PubMed Central Google Scholar Nuchtern JG, London WB, Barnewolt CE, et al. A prospective study of expectant observation as primary therapy for neuroblastoma in young infants: a Children‘s oncology group study. Ann Surg. 2012;256(4):573–80. Article PubMed Google Scholar Virgone C, Roganovic J, Vorwerk P, et al. Adrenocortical tumours in children and adolescents: the EXPeRT/PARTNER diagnostic and therapeutic recommendations. Pediatr Blood Cancer. 2021;68(suppl 4):e29025. Article PubMed Google Scholar Chang S, Lin Y, Yang S, et al. Safety and feasibility of laparoscopic resection of abdominal neuroblastoma without image-defined risk factors: a single-center experience. World J Surg Oncol. 2023;21(1):113. Article PubMed PubMed Central Google Scholar Zenitani M, Yoshida M, Matsumoto S, et al. Feasibility and safety of laparoscopic tumor resection in children with abdominal neuroblastomas. Pediatr Surg Int. 2023;39(1):91. Article PubMed Google Scholar International Pediatric Endosurgery Group. IPEG guidelines for the surgical treatment of adrenal masses in children. J Laparoendosc Adv Surg Tech A. 2010;20(2):vii–ix. Google Scholar Nakanishi H, Miangul S, Wang R, et al. Open versuslaparoscopicsurgery in the management of adrenocorticalcarcinoma: a systematicreview and meta-analysis. Ann Surg Oncol. 2023;30(2):994–1005. Article PubMed Google Scholar Gaillard M, Razafinimanana M, Challine A, et al. Laparoscopic or openadrenalectomy for stage I-IIadrenocorticalcarcinoma: a retrospectivestudy. J Clin Med. 2023;12(11):3698. Article PubMed PubMed Central Google Scholar Utsumi T, Iijima S, Sugizaki Y, et al. Laparoscopic adrenalectomy for adrenal tumors with endocrine activity: perioperative management pathways for reduced complications and improved outcomes. Int J Urol. 2023;30(10):818–26. Article CAS PubMed Google Scholar Download references Acknowledgements We would like to express our deepest gratitude to all the patients and their parents who participated in this study. Their patience and cooperation were instrumental to the success of this research. We thank our colleagues in the Department of Radiology for their invaluable contributions in diagnosing and monitoring the progression of adrenal incidentalomas. We sincerely appreciate the hard work of the pathologists in diagnosing and classifying tumors, which laid the foundation for our study. Finally, we would like to thank our institution for providing the necessary resources and an enabling environment to conduct this research. Funding Not applicable. Author information Authors and Affiliations Department of Urology, Children’s Hospital of Nanjing Medical University, 72 Guangzhou Road, Nanjing, 210008, Jiangsu, China Xiaojiang Zhu, Saisai Liu, Yimin Yuan, Nannan Gu, Jintong Sha, Yunfei Guo & Yongji Deng Contributions X.J.Z. and Y.J.D designed the study; S.S.L., Y.M.Y., N.N.G., and J.T.S. carried out the study and collected important data; X.J.Z. analysed data and wrote the manuscript; Y.F.G. and Y.J.D.gave us a lot of very good advices and technical support; All authors read and approved the final manuscript. Corresponding author Correspondence to Yongji Deng. Ethics declarations Competing interests The authors declare no competing interests. Ethics approval and consent to participate Ethics approval for this study was granted by the Ethics Committee of Children’s Hospital of Nanjing Medical University. Informed written consent was obtained from all the guardians of the children and we co-signed the informed consent form with their parents before the study. We confirmed that all methods were performed in accordance with relevant guidelines and regulations. Conflict of interest There are no conflicts of interest. 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 From https://bmcpediatr.biomedcentral.com/articles/10.1186/s12887-024-04673-7

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

Highlights EAS should be considered in patients presenting with rapid progression of ACTH-dependent hypercortisolism causing severe clinical and metabolic abnormalities. Ectopic ACTH secretion by a pheochromocytoma should be suspected in cases of ACTH-dependent Cushing syndrome in the presence of an adrenal mass. If required, medical management with steroidogenesis inhibitors can be initiated at the time of EAS diagnosis to control clinical and metabolic derangements associated with severe hypercortisolemia In patients with ACTH-dependent Cushing syndrome from an ectopic source, inhibiting steroidogenesis should be reserved for cases where the initial diagnosis is unclear or patients who are not suitable candidates for surgery. Unilateral adrenalectomy is indicated in the management of ACTH/CRH-secreting pheochromocytomas and is typically curative. Catecholamine blockade should be started prior to surgical removal of catecholamines-secreting pheochromocytomas. A multidisciplinary approach is required to diagnose and manage this condition. Abstract Background/Objective Ectopic co-secretion of corticotropin-releasing hormone (CRH) and adrenocorticotropic hormone (ACTH) in silent (i.e., noncatecholamine-secreting) pheochromocytoma is a rare cause of Cushing Syndrome (CS). Case Report A 57-year-old woman rapidly developed hypercortisolism, clinically manifesting as fatigue, muscle weakness, weight gain, and worsening hypertension, and biochemically characterized by hypokalemia and marked elevation of serum cortisol and plasma ACTH. This acute presentation suggested a diagnosis of ectopic ACTH syndrome (EAS). Imaging studies revealed a right adrenal mass that enhanced after administration of the radioisotope 68Ga-DOTATATE. Plasma metanephrines were normal in two separate measurements. The possibility of a silent pheochromocytoma was considered. After controlling her hypercortisolism with metyrapone and surgical preparation with alpha blockade, the patient underwent elective right adrenalectomy. Pathology revealed a pheochromocytoma that stained focally for ACTH and CRH. Postoperatively, cortisol levels normalized, the hypothalamic–pituitary–adrenal (HPA) axis was not suppressed, and clinical symptoms from hypercortisolism abated. Discussion Patients who exhibit a rapid progression of ACTH-dependent hypercortisolism should be screened for ectopic ACTH syndrome (EAS). The use of functional imaging radioisotopes (such as gallium DOTA-peptides), improves the detection of ACTH-secreting tumors. Preoperative treatment with steroidogenesis inhibitors helps control clinical and metabolic derangements associated with severe hypercortisolemia, while alpha blockade prevents the onset of an adrenergic crisis. Conclusion We present a rare case of EAS due to a silent pheochromocytoma that co-secreted ACTH and CRH. Pheochromocytoma should be considered in patients with EAS who have an adrenal mass even in the absence of excessive catecholamine secretion. Key words ectopic ACTH syndrome Cushing Syndrome non-catecholamine-secreting pheochromocytoma Abbreviations EAS ectopic ACTH syndrome CS Cushing Syndrome CRH corticotropin-releasing hormone ACTH adrenocorticotropic hormone DHEA-S dehydroepiandrosterone sulfate UFC urine free cortisol PRA plasma renin activity Introduction Cushing Syndrome (CS) is rare, with an estimated incidence of 0.2-5.0 per million people per year, and prevalence of 39-79 per million (1). Ectopic ACTH Syndrome (EAS), a type of CS originating from extra-pituitary ACTH-secreting tumors, is uncommon. The prevalence of CS due to ACTH-secreting adrenal medullary lesions is not well established. However, EAS is observed in approximately 1.3% of all identified cases of pheochromocytoma (2). Recognizing EAS can be challenging due to its rarity, leading to delayed diagnosis. Neuroendocrine neoplasms can produce CRH, which can lead to the secretion of ACTH by the pituitary. In certain cases, co-secretion of ACTH and CRH by an adrenal neoplasm has been observed. Only two published cases have provided definitive biochemical and immunohistochemical evidence of exclusive CRH secretion (3). Case Report A 57-year-old woman with a history of well-controlled hypertension sought care due to a two-month history of 60 lb weight gain, facial rounding, easy bruising, muscle weakness, lower extremity edema and acne. Her blood pressure control had worsened, and laboratory tests showed a markedly low serum potassium level of 1.8 mmol/L while taking hydrochlorothiazide. To manage her blood pressure, she was prescribed a calcium channel blocker, an angiotensin receptor blocker, and potassium supplements. However, her symptoms worsened, and she was referred to our emergency department. Blood pressure at presentation to our hospital was 176/86 mmHg. She had characteristic features of CS, including face rounding, supraclavicular fullness, dorsocervical fat accumulation, pedal edema, oral candidiasis, multiple forearm ecchymoses, and acneiform skin eruptions. No visible abdominal striae were present. She had no family history of pheochromocytoma, or multiple endocrine neoplasia type 2. Serum cortisol level was 128 mcg/dL (normal range: 4.6-23.4) at 5 PM, with an ACTH level of 1055 pg/mL (normal range: 6-50); serum DHEA-S level was elevated at 445 mcg/dL (normal range: 8-188). Her 24-hour urine cortisol was at 12,566 mcg (normal range: 4.0-50.0). Plasma metanephrines were normal at <25 pg/mL (normal range: <57), and plasma normetanephrine was 44 (normal range: <148). A second plasma metanephrine measurement showed similar results. Serum aldosterone level and plasma renin activity were low at 2 ng/dL (normal range: 3-16) and 0.11 ng/mL/h (normal range: 0.25-5.82), respectively. Dopamine and methoxytyramine levels were not measured. An abdominal CT revealed a 4.8 x 4.5 x 5 cm right heterogeneously enhancing adrenal mass with a mean Hounsfield Unit of 68 in the non-contrast phase, and an absolute percentage washout of 30% (Fig 1A). The left adrenal gland appeared hyperplastic (Fig 1B). An Octreoscan, which was the in-hospital available nuclear medicine imaging modality, confirmed a 5.1 cm adrenal mass that was mild to moderately avid, with diffuse bilateral thickening of the adrenal glands and no other focal radiotracer avidity. A pituitary MRI did not show an adenoma, and EAS was suspected. Further evaluation with 68Ga-DOTATATE PET/CT (Fig 2) performed after her admission demonstrated an avid right adrenal mass consistent with a somatostatin receptor-positive lesion. No other suspicious tracer uptake was detected. These findings were consistent with a neuroendocrine tumor, such as pheochromocytoma. Download : Download high-res image (261KB) Download : Download full-size image Fig. 1. Preoperative abdominal computed tomography scan showing a 4.8 x 4.5 x 5 cm right heterogeneously enhancing adrenal mass with irregular borders (A) and a hyperplastic left adrenal gland (B). Download : Download high-res image (219KB) Download : Download full-size image Fig 2. 68Ga-DOTATATE PET/CT showing an avid right adrenal mass. To control her symptoms while undergoing workup, the patient received oral metyrapone 500 mg thrice daily and oral ketoconazole 200 mg twice daily. Ketoconazole was stopped due to an increase in transaminases. The dosage of metyrapone was increased to 500 mg four times daily and later decreased to alternating doses of 250 mg and 500 mg four times daily. Within 3 weeks of starting medical therapy, serum cortisol level normalized at 20 mcg/dL. The 24-hour UFC improved to 246.3 mcg/24h. She experienced gradual improvement in facial fullness, acne, and blood pressure control. The possibility of a silent pheochromocytoma was considered, and a-adrenergic blockade with doxazosin 1 mg daily was started 1 month prior surgery. She underwent surgery after two months of metyrapone therapy. With an unclear diagnosis and a large, heterogeneous adrenal mass, the surgical team elected to perform open adrenalectomy for en bloc resection due to concerns for an adrenal malignancy. The tumor was well-demarcated and did not invade surrounding structures (Figure 3A). H&E-stained sections showed classic morphologic features of a pheochromocytoma (Figure 3B), with immunohistochemistry demonstrating strong immunoreactivity for synaptophysin and chromogranin, and negative SF- I and inhibin stains excluding an adrenal cortical lesion. The sections analyzed by QuPath (4) revealed that approximately 4% of ce11s were ACTH cells, often found in isolation, and had a clear, high signal-to-noise staining (Figure 3C). CRH cells were less prevalent, comprising about 2.4% of the total analyzed cells, and tended to cluster together (Figure 3D). These cells had more background staining, resulting in a lower signal- to-noise ratio. Download : Download high-res image (663KB) Download : Download full-size image Figure 3. Gross and Histopathological analysis of the patient’s pheochromocytoma. (A) Image of the gross excised specimen. (B) H&E staining (200x final magnification) demonstrates prominent vascularity and cells with finely granular, eosinophilic cytoplasm and salt-and-pepper chromatin. (C) ACTH staining (200x final magnification) shows clear and isolated positive cells, representing about 4.0% of the section analyzed by QuPath. (D) CRH staining (200x final magnification) reveals tight clusters of positive cells, accounting for 2.4% of the total cells. Positive (human placenta and hypothalamus) and negative (thyroid gland) control tissues performed as expected (data not shown). The patient's postoperative recovery was uneventful, with a short course of hydrocortisone which was stopped 1 week after surgery after HPA axis evaluation showed normal results. After one month, hypercortisolism had resolved, as shown by a normal 24-hour UFC at 28 mcg. Administration of dexamethasone at 11 PM resulted in suppression of morning cortisol to 0.8 and 0.6 mcg/dL 1 and 7 months after surgery, respectively. Her liver function tests normalized, and blood pressure was well-controlled with amlodipine 10 mg daily and losartan 100 mg daily. Genetic testing for pheochromocytoma predisposition syndromes is currently planned. Discussion EAS accounts for 10-20% of cases of ACTH-dependent CS (5). This condition can be caused by several neuroendocrine neoplasms that produce bioactive ACTH (6) In the literature, we have found 99 documented cases of EAS caused by a pheochromocytoma. Of these, 93% showed ACTH expression. Only two cases have been reported with dual staining of ACTH and CRH (7). Exclusive CRH production has only been reported in two cases (8:9). However, the true prevalence of CRH-producing pheochromocytomas might be underestimated, as most cases testing for CRH expression was not performed. Although the clinical presentation of EAS may be highly variable, there is often a rapid onset of hypercortisolism accompanied by severe catabolic symptoms. The diagnostic process should focus on identifying the location of a potential neuroendocrine neoplasm responsible for the ACTH secretion. Sometimes the peripheral origin of ACTH must be confirmed by inferior petrosal sinus sampling (IPSS). In this case, given the clinical presentation consistent with EAS, negative pituitary MRI, and the presence of an adrenal mass that needed to be removed independently, IPSS was not performed. Neuroendocrine neoplasms express somatostatin receptors on their surface, which allow functional imaging using [11 lln]-pentetreotide (Octreoscan). However, Octreoscan has a low sensitivity in detecting occult EAS. In cases where the tumor is in the abdomen and pelvis, Octreoscan has limited utility in locating the source of ACTH (10). This increased risk of false negatives is caused by physiological tracer uptake by the liver, spleen, urinary tract, bowel, and gallbladder. The use of Gallium-68 labeled somatostatin receptor ligands (PET/CT 68Ga-DOTATATE) is more effective in detecting somatostatin receptors (SSTR2) than [11lln]-pentetreotide due to its higher spatial resolution and affinity (11)_ This test was performed after discharge form the hospital to rule out the presence of a second, smaller neuroendocrine tumor that the Octreoscan might have missed. A new molecular imaging technique targeting CRH receptors (68Ga CRH PET/CT) has shown potential in identifying tumors expressing CRH, but its availability remains limited (12). In our patient's case, both the Octreoscan and 68Ga- DOTATATE successfully identified the adrenal tumor as a potential ACTH/CRH secretion source. According to relevant guidelines, presurgical adrenergic blockade is recommended for patients with biochemical evidence of catecholamine excess (13, 14). Conversely, silent pheochromocytomas can generally be operated without alpha blockade (15). Despite this, we opted to administer pre-operative alpha blockade as a precautionary measure for this patient. Pathology examination confirmed the diagnosis of pheochromocytoma. ACTH and CRH staining demonstrated that clear and significant populations of two separate ACTH and CRH positive cells were present in the excised pheochromocytoma. ACTH/CRH cells were dispersed throughout various regions of the pheochromocytoma rather than being well-defined, separate histological entities. As a result, there is no indication that this resulted from collision tumors, but rather random mutation and expansion of tumor cells into ACTH or CRH secreting cells. These results have limitations, including variation in ACTH and CRH expressing regions due to tumor heterogeneity, nonspecific binding of polyclonal antibodies, and normal low-rate false negative/positive detection using QuPath. Post-surgical normal HPA activity was likely due to the de-suppression of the HPA axis by medical therapy, but it may also be explained by chronic stimulation of corticotroph cells induced by ectopic CRH secretion. The standard approach to managing EAS involves surgical intervention. However, surgery may not be a viable option in cases where the source of ACTH production is unknown. Medical therapy to reduce or block excess cortisol can be used in such circumstances. Conclusions In conclusion, a pheochromocytoma causing EAS should be considered even in the absence of elevated plasma metanephrines. These tumors may simultaneously express ACTH and CRH.CRH. References 1 C. Steffensen, A.M. Bak, K. Zøylner Rubeck, J.O.L. Jørgensen Epidemiology of Cushing’s syndrome Neuroendocrinology, 92 (2010), pp. 1-5 View PDF This article is free to access. CrossRefView in ScopusGoogle Scholar 2 H. Falhammar, J. Calissendorff, C. Höybye Frequency of Cushing’s syndrome due to ACTH-secreting adrenal medullary lesions: a retrospective study over 10 years from a single center Endocrine, 55 (2020), pp. 296-302 Google Scholar 3 K.B. Lois, A. Santhakumar, S. Vaikkakara, S. Mathew, A. Long, S.J. Johnson, et al. Phaeochromocytoma and ACTH-dependent Cushing’s syndrome: Tumour CRF secretion can mimic pituitary Cushing’s disease Clin Endocrinol (Oxf), 84 (2016), pp. 177-184 View article CrossRefView in ScopusGoogle Scholar 4 P. Bankhead, M. Loughrey, J. Fernandez, Y. Dombrowski, D. Mcart, P. Dunne, et al. QuPath: Open source software for digital pathology image analysis Sci. Rep, 7 (2017), pp. 1-7 Google Scholar 5 M. Savas, S. Mehta, N. Agrawal, E.F.C. van Rossum, R.A. Feelders Approach to the Patient: Diagnosis of Cushing Syndrome J Clin Endocrinol Metab, 107 (2022), pp. 3162-3174 View article CrossRefView in ScopusGoogle Scholar 6 A.M. Isidori, G.A. Kaltsas, C. Pozza, V. Frajese, J. Newell-Price, R.H. Reznek, et al. Extensive clinical experience - The ectopic adrenocorticotropin syndrome: Clinical features, diagnosis, management, and long-term follow-up J Clin Endocrinol Metab, 91 (2006), pp. 371-377 View article CrossRefView in ScopusGoogle Scholar 7 P.F. Elliott, T. Berhane, O. Ragnarsson, H. Falhammar Ectopic ACTH- and/or CRH-Producing Pheochromocytomas J. Clin. Endocr, 106 (2021), pp. 598-608 View article CrossRefView in ScopusGoogle Scholar 8 D.S. Jessop, D. Cunnah, J.G.B. Millar, E. Neville, P. Coates, I. Doniach, et al. A phaeochromocytoma presenting with Cushing’s syndrome associated with increased concentrations of circulating corticotrophin-releasing factor J. Endocrinol, 113 (1987), p. 133 View article CrossRefView in ScopusGoogle Scholar 9 T. O’Brien, W.F. Young, D.G. Davilla, B.W. Scheithauer, K. Kovacs, E. Horvath, et al. Cushing’s syndrome associated with ectopic production of corticotrophin-releasing hormone, corticotrophin and vasopressin by a phaeochromocytoma Clin Endocrinol (Oxf), 37 (1992), pp. 460-467 View article CrossRefView in ScopusGoogle Scholar 10 J. Young, M. Haissaguerre, O. Viera-Pinto, O. Chabre, E. Baudin, A. Tabarin Management of endocrine disease: Cushing’s syndrome due to ectopic ACTH secretion: an expert operational opinion Eur. J. Endocrinol, 182 (2020), pp. 29-58 View article CrossRefGoogle Scholar 11 A.M. Isidori, E. Sbardella, M.C. Zatelli, M. Boschetti, G. Vitale, A. Colao, et al. Conventional and Nuclear Medicine Imaging in Ectopic Cushing’s Syndrome: A Systematic Review J Clin Endocrinol Metab, 100 (2015), pp. 3231-3244 View article CrossRefView in ScopusGoogle Scholar 12 R. Walia, R. Gupta, A. Bhansali, R. Pivonello, R. Kumar, H. Singh, et al. Molecular Imaging Targeting Corticotropin-releasing Hormone Receptor for Corticotropinoma: A Changing Paradigm J. Clin. Endocr, 106 (2021), pp. 1816-1826 View article CrossRefGoogle Scholar 13 J.W.M. Lenders, M.N. Kerstens, L. Amar, et al. Genetics, diagnosis, management and future directions of research of phaeochromocytoma and paraganglioma: a position statement and consensus of the Working Group on Endocrine Hypertension of the European Society of Hypertension J Hypertens, 38 (2020), pp. 1443-1456 View article CrossRefView in ScopusGoogle Scholar 14 D. Taïeb, G.B. Wanna, M. Ahmad, C. Lussey-Lepoutre, N.D. Perrier, S. Nölting, et al. Clinical consensus guideline on the management of phaeochromocytoma and paraganglioma in patients harbouring germline SDHD pathogenic variants Lancet Diabetes Endocrinol, 11 (2023), pp. 345-361 View PDFView articleView in ScopusGoogle Scholar 15 K. Pacak Preoperative management of the pheochromocytoma patient J Clin Endocrinol Metab, 92 (2007), pp. 4069-4079 View article CrossRefView in ScopusGoogle Scholar Cited by (0) Sources of support: None Permission in the form of written consent from patient for use of actual test results was obtained. Cushing in silent pheochromocytoma Clinical Relevance This case highlights the importance of considering ectopic ACTH secretion by a pheochromocytoma in patients presenting with rapid progression and considerable clinical hypercortisolism concomitant with an adrenal mass and elevated plasma ACTH. This represents an unusual manifestation of a specific subtype of ACTH/CRH-secreting pheochromocytoma that did not exhibit catecholamine secretion 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 ∗ These 2 authors contributed equally to this work From https://www.sciencedirect.com/science/article/pii/S2376060524000075

In a recent study published in Hypertension Research, scientists examine the endocrine causes of hypertension (HTN) and investigate the efficacy of treatments to alleviate HTN. What is HTN? About 30% of the global population is affected by HTN. HTN is a modifiable cardiovascular (CV) risk factor that is associated with a significant number of deaths worldwide. There are two types of HTN known as primary and secondary HTN. As compared to primary HTN, secondary HTN causes greater morbidity and mortality. The most common endocrine causes of HTN include primary aldosteronism (PA), paragangliomas and pheochromocytomas (PGL), Cushing’s syndrome (CS), and acromegaly. Other causes include congenital adrenal hyperplasia, mineralocorticoid excess, cortisol resistance, Liddle syndrome, Gordon syndrome, and thyroid and parathyroid dysfunction. What is PA? PA is the most common endocrine cause of hypertension, which is associated with excessive aldosterone secretion by the adrenal gland and low renin secretion. It is difficult to estimate the true prevalence of PA due to the complexity of its diagnosis. Typically, the plasma aldosterone-to-renin ratio (ARR) is measured to diagnose PA. The diagnosis of PA can also be confirmed using other diagnostic tools like chemiluminescent enzyme immunoassays (CLEIAs) and radio immune assay (RIA). Continuous aldosterone secretion is associated with organ damage due to chronic activation of the mineralocorticoid (MR) receptor in many organs, including fibroblasts and cardiomyocytes. An elevated level of aldosterone causes diastolic dysfunction, endothelial dysfunction, left ventricular hypertrophy, and arterial stiffness. Increased aldosterone secretion also leads to obstructive sleep apnea and increases the risk of osteoporosis. This is why individuals with PA are at a higher risk of cardiovascular events (CVDs), including heart failure, myocardial infarction, coronary artery disease, and atrial fibrillation. PA is treated by focusing on normalizing potassium and optimizing HTN and aldosterone secretion. Unilateral adrenalectomy is a surgical procedure proposed to treat PA. Young patients who are willing to stop medication are recommended surgical treatment. The most common pharmaceutical treatment for PA includes mineralocorticoid receptor antagonists such as spironolactone and eplerenone. Pheochromocytomas and paragangliomas PGL are tumors that develop at the thoracic-abdominal-pelvic sympathetic ganglia, which are present along the spine, as well as in the parasympathetic ganglia located at the base of the skull. The incidence rate of PGL is about 0.6 for every 100,000 individuals each year. PGL tumors synthesize excessive catecholamines (CTN), which induce HTN. Some of the common symptoms linked to HTN associated with PGL are palpitations, sweating, and headache. PGL can be diagnosed by determining metanephrines (MN) levels, which are degraded products of CTN. Bio-imaging tools also play an important role in confirming the diagnosis of PGL. Excessive secretion of CTN increases the risk of CVDs, including Takotsubo adrenergic heart disease, ventricular or supraventricular rhythm disorders, hypertrophic obstructive or ischaemic cardiomyopathy, myocarditis, and hemorrhagic stroke. Excessive CTN secretion also causes left ventricular systolic and diastolic dysfunction. Typically, PGL treatment is associated with surgical procedures. Two weeks before the surgery, patients are treated with alpha-blockers. For these patients, beta-blockers are not used as the first line of treatment without prior use of alpha-adrenergic receptors. Patients with high CTN secretion are treated with metyrosine, as this can inhibit tyrosine hydroxylase. Hydroxylase converts tyrosine into dihydroxyphenylalanine, which is related to CTN synthesis. What is CS? CS, which arises due to persistent exposure to glucocorticoids, is a rare disease with an incidence rate of one in five million individuals each year. The most common symptoms of CS include weight gain, purple stretch marks, muscle weakness, acne, and hirsutism. A high cortisol level causes cardiovascular complications such as HTN, hypercholesterolemia, and diabetes. CS is diagnosed based on the presence of two or more biomarkers that can be identified through pathological tests, such as salivary nocturnal cortisol, 24-hour urinary-free cortisol, and dexamethasone suppression tests. CS is treated through surgical procedures based on the detected lesions. Patients with severe CS are treated with steroidogenic inhibitors, such as metyrapone, ketoconazole, osilodrostat, and mitotane. Pituitary radiotherapy and bilateral adrenalectomy are performed when other treatments are not effective. Acromegaly Acromegaly arises due to chronic exposure to growth hormone (GH), leading to excessive insulin-like growth factor 1 (IGF1) synthesis. This condition has a relatively higher incidence rate of 3.8 million person-years. Clinical symptoms of acromegaly include thickened lips, widened nose, a rectangular face, prominent cheekbones, soft tissue overgrowth, or skeletal deformities. Prolonged exposure to GH leads to increased water and sodium retention, insulin resistance, reduced glucose uptake, and increased systemic vascular resistance. These conditions increase the risk of HTN and diabetes in patients with acromegaly. Acromegalic patients are also at a higher risk of cancer, particularly those affecting the thyroid and colon. Acromegaly is diagnosed using the IGF1 assay, which determines IGF1 levels in serum. After confirming the presence of high IGF1 levels, a GH suppression test must be performed to confirm the diagnosis. Bioimaging is also conducted to locate adenoma. Acromegaly is commonly treated through surgical procedures. Patients who refuse this line of treatment are treated with somatostatin receptor ligands, growth hormone receptor antagonists, dopaminergic agonists, or radiotherapy. Journal reference: De Freminville, J., Amar, L., & Azizi, M. (2023) Endocrine causes of hypertension: Literature review and practical approach. Hypertension Research; 1-14. doi:10.1038/s41440-023-01461-1 From https://www.news-medical.net/news/20231015/Hormones-and-high-blood-pressure-Study-reveals-endocrine-culprits-and-targeted-treatments.aspx

Adrenal incidentalomas (AI) are associated with an increased risk of cardiometabolic complications due to adrenal hyperfunction. Obtaining accurate prevalence estimates of distinct types of functioning AIs is crucial for efficient resource allocation and effective management strategies. For a study, researchers sought to ascertain the prevalence of various forms of autonomous hormone secretion in individuals diagnosed with adrenal incidentaloma, including autonomous/possible autonomous cortisol secretion (ACS), primary aldosteronism (PA), pheochromocytoma (PHEO), and Cushing syndrome (CS). A comprehensive and systematic search was conducted across multiple databases (PubMed, Ovid MEDLINE, Web of Science) up to February 2022. Among the 1,661 publications initially screened at the title and abstract levels, 161 articles underwent full-text examination, and ultimately, 36 studies were included for analysis. Three independent reviewers meticulously extracted clinical data from these selected studies. The overarching prevalence of functioning adrenal incidentalomas was 27.5% (95% CI 23.0, 32.5). The highest prevalence was observed for ACS/possible ACS, with a rate of 11.7% (95% CI 8.6, 15.7), followed by PA at 4.4% (95% CI 3.1, 6.2). Subgroup analysis unveiled a greater prevalence of PA in patients from Asian regions than those from Europe/America. Conversely, the prevalence of ACS/possible ACS was comparatively lower in Asian countries. Meta-regression analysis elucidated that the proportion of female patients influenced the prevalence of ACS/possible ACS, while PA prevalence positively correlated with the proportion of patients with hypertension and the publication year. PHEO and CS demonstrated prevalences of 3.8% (95% CI 2.8, 5.0) and 3.1% (95% CI 2.3, 4.3), respectively. The comprehensive meta-analysis offered valuable insights into the prevalence rates of diverse types of functioning adrenal incidentalomas and identified influential factors contributing to heterogeneity in these estimates. The findings contributed significantly to understanding clinical implications and aided in devising effective management strategies for individuals diagnosed with these adrenal disorders. Source: academic.oup.com/jcem/article-abstract/108/7/1813/7015785?redirectedFrom=fulltext

Abstract Introduction and importance Pheochromocytoma and Cushing's syndrome are rare endocrine conditions caused by tumors in the adrenal gland. These conditions are classified under Multiple Endocrine Neoplasia (MEN) syndrome, characterized by the development of multiple tumors in the endocrine system. However, diagnosing these conditions can be challenging as they often lack clear symptoms, requiring careful evaluation, monitoring, and treatment to prevent complications. Case presentation A 23-year-old male recently presented with right-sided abdominal fullness and lipoma-like masses on the torso. Over a span of six months, the abdominal mass nearly doubled in size, accompanied by elevated levels of catecholamines, cortisol, parathyroid hormone (PTH), and calcitonin. Surprisingly, the patient remained asymptomatic despite these abnormal lab values. CT imaging revealed a substantial increase in the size of the mass in the right adrenal gland, from 6 × 7 cm to approximately 11.2 × 10.2 × 9 cm. Clinical discussion Pheochromocytoma secretes catecholamines and often leads to hypertension and related symptoms. Interestingly, most individuals with pheochromocytoma do not exhibit obvious symptoms, necessitating blood and urine tests, along with imaging studies, for accurate diagnosis. The size of the tumor does not necessarily indicate the severity of symptoms. MEN-2, a genetic syndrome, is characterized by pheochromocytoma, medullary thyroid carcinoma, and hyperparathyroidism. Additionally, methods for diagnosing Cushing's syndrome, caused by excess cortisol production, are discussed. Conclusion Early diagnosis and genetic counseling are crucial in preventing complications associated with these conditions. By identifying them, appropriate treatment can be ensured for positive outcomes of patients and their families. Previous article in issue Next article in issue Keywords Pheochromocytoma Multiple Endocrine Neoplasia (MEN) syndrome Cushing's syndrome Rare Case Report Abbreviations CT computed tomography MRI Magnetic resonance imaging USG Ultrasonography 131I-MIBG iodine 131 labeled meta-iodobenzylganidine RAAS Renin-angiotensin-aldosterone system 1. Introduction Pheochromocytoma are catecholamine secreting tumors of chromaffin cells of adrenal medulla. It can be found anywhere in the body, with the majority being intra-abdominal and those other than adrenal medulla are referred to as paragangliomas [1,2]. Pheochromocytoma typically secretes norepinephrine and epinephrine, with norepinephrine being the primary catecholamine. However, some tumors may only secrete one of the two, and rarely, some may secrete dopamine or dopa [3]. Vast majority >90 % of adrenal neoplasms are benign non-functional adenomas [4].About 10 % of pheochromocytomas are malignant and 10 % of cases are found on both sides. Additionally, approximately 40 % of pheochromocytomas are caused by genetic factors and can be associated with inherited syndromes [5]. Pheochromocytoma is found to be associated with MEN-2. MEN-2 is a hereditary genetic condition that is caused by a de novo mutation in the RET gene. It is inherited in an autosomal dominant fashion and is mainly characterized by medullary thyroid carcinoma, pheochromocytoma and parathyroid adenoma or hyperplasia [6]. MEN syndrome can be MEN-1, MEN-2A and MEN-2B. MEN-1 is characterized by pituitary tumors (prolactin or growth hormone), pancreatic endocrine tumors and parathyroid adenomas. Additionally, other tumors such as foregut carcinoids, adrenocortical adenomas, meningioma, lipomas, angiofibromas and collagenomas may also occur in MEN-1. MEN-2A is characterized by medullary thyroid carcinoma, pheochromocytoma, and parathyroid adenoma/hyperplasia; it can also be associated with cutaneous lichen amyloidosis and Hirschsprung disease. On the other hand, MEN-2B is characterized by familial medullary thyroid cancer, pheochromocytoma, mucosal neuromas, gastrointestinal tract issues, musculoskeletal and spinal problems. [7]. Cushing syndrome results from hypercortisolism and is characterized by hypertension, weight gain, easy bruising, and central obesity [4]. Cushing's disease refers to ACTH-dependent cortisol excess caused by a pituitary adenoma, while ACTH-independent cortisol excess due to non-pituitary causes such as excess use of glucocorticoids, adrenal adenoma, hyperplasia, or carcinoma is referred to as Cushing syndrome [8]. This case report has been written according to the SCARE checklist [9]. 2. Case presentation A 23-year-old male presented to our surgery department with the chief complaint of right sided abdominal fullness for six months. According to the patient a mass was incidentally reported six months back while he was under-evaluation for mild trauma due to road traffic accident. Six months back, the mass was approximately 6 × 7 cm, while at the time of presentation to our department the mass was approximately 11.2 × 10.2 × 9 cm (CT abdomen) which was globular in shape, had regular margin, and moved with respiration. He had no history of hypertension, headache, palpitation, sweating, pallor, recent weight loss, abdominal pain, psychological disturbance, dizziness, loss of consciousness, dark color urine, burning micturition, had normal bowel and bladder habit. Past history and family history were insignificant. He was not under any long-term medication and no known drug allergies. He occasionally smokes and consumes alcohol. On physical examination at the time of presentation, multiple soft, mobile, painless, subcutaneous nodules like lipoma were present over the torso. His height was 176.8 cm, weight 68 kg, BMI 21.8 kg/m2 (body mass index). He had blood pressure of 110/70 mm of Hg taken in left arm at sitting position, heart rate of 62 beats/min, respiratory rate of 24/min, temperature of 96.6 °F, SPO2 of 98 % at right hand. A mass was palpable on the right side of abdomen, otherwise abdomen was soft, non-tender, normal bowel sound was present. Chest, cardiac and neurologic examinations were all normal. Initial laboratory evaluation revealed 24 h. urine metanephrine of 5415 μg/24 h (normal: 25–312 μg/24 h.); 24 h. urine VMA of 32.2 mg/24 h. (normal: <13.60 mg/24 h.); serum cortisol of 535.16 nmol/l after overnight low dose dexamethasone(1 mg) suppression test (normal: <50 nmol/l);24 h. Urine free cortisol of 526.61 nmol/24 h. (normal: 30–145 nmol/24 h) PTH(intact) of 89.2 pg./ml (normal: 15–65 pg./ml); serum calcitonin of 15.2 pg./ml (normal: ≤8.4 pg./ml); serum CEA of 4.72 ng/ml (normal: 0.0–4.4 ng/ml); serum DHEA of 1.19 ng/ml (normal: 1.7–6.1 ng/ml). Baseline investigation: Hematology, urine routine/microscopic, electrolytes were within the normal range. Additional laboratory findings were as in the Table 1. Table 1. Lab evaluation Result Reference Unit Metanephrine, urine 24 h 5415 25–312 μg/24 h VMA, urine 24 h 32.2 <13.60 mg/24 h VMA, urine 12.88 – ng/l Cortisol, serum, overnight DST 535.16 <50 nmol/l Cortisol, urine 24 h 526.61 30–145 nmol/24 h ACTH, complete 28.3 7.2–63.3 pg/ml DHEA, serum 1.19 1.7–6.1 ng/ml CEA, serum 4.72 0.0–4.4 ng/ml Phosphorus, serum 3.0 2.5–4.5 mg/dl Albumin, serum 5.2 3.5–5.2 g/dl Calcitonin, serum 15.2 ≤8.4 pg/ml Calcium, serum 8.94 8.6–10.0 mg/dl PTH (intact) 89.2 15–65 pg/ml aldosterone 8.7 7.0–30 g/dl Plasma rennin activity 1.42 0.10–6.56 ng/ml/h Aldosterone-rennin ratio 6.13 ≤20 Creatinine, urine 36 – mg/dl DST - dexamethasone suppression test; VMA - vanilmandelic acid; ACTH - adrenocorticotropic hormone; DHEA - dehydroepiandrosterone; CEA - carcino-embryonic-antigen; PTH - parathyroid hormone. 2.1. USG abdomen USG abdomen (Fig. 1, Fig. 2) showed well defined mixed echoic area in Right adrenal region measuring 12.7 × 10.7 cm in size. There was presence of internal vascularity with multiple foci of cystic compound. The lesion displaced the right kidney inferiorly. Download : Download high-res image (102KB) Download : Download full-size image Fig. 1. USG abdomen. Download : Download high-res image (106KB) Download : Download full-size image Fig. 2. USG abdomen. 2.2. Plane and contrast CT scan of abdomen Plane and contrast CT scan of Abdomen (Fig. 3) showed approximately 11.2 × 10.2 × 9 cm sized, relatively well defined heterogeneous soft tissue density lesion with well-defined enhancing wall in right adrenal region. Non-enhancing areas were noted within the mass suggestive of necrosis. Few calcific foci were noted within the mass with no obvious hemorrhagic component. The lesion showed heterogeneous enhancement post contrast image. Download : Download high-res image (192KB) Download : Download full-size image Fig. 3. CT abdomen. After all the workup patient was given diagnosis of right sided Pheochromocytoma associated with MEN syndrome, with ACTH-independent Cushing's syndrome and right adrenalectomy was performed. 2.3. Pathology report 2.3.1. Gross descriptions The specimen was globular mass measuring 14.5 × 10 cm, with smooth outer surface. On sectioning, the mass was well circumscribed, soft and yellow-brown, predominantly solid with cyst formation. The size of cyst ranges from 0.3 to 3.5 cm in diameter. Areas of hemorrhages were noted. 2.3.2. Microscopic description Section showed tumor cells arranged in well-defined nests (Zellballen), alveolar and diffuse pattern with intervening fibrovascular stroma. The cells were intermediate to large sized, polygonal with finely granular amphophilic cytoplasm. The nuclei showed mild to moderate pleomorphism and were round to ovoid, with prominent nuclei noted. No capsular invasion, vascular invasion and necrosis. Areas of hemorrhage were seen. Mitosis 0–1/10 high power field was noted (Figs. 4 and 5). Download : Download high-res image (1MB) Download : Download full-size image Fig.a Diffuse Zellbalen pattern with intervening fibrous stroma. Fig.b Mild to moderate pleomorphic nuclei with abundant hemorrhage. Fig.c Low power field with intact capsule. Download : Download high-res image (352KB) Download : Download full-size image Figs. 4 and 5. Fig. 4 Intra-operative resection of tumor; Fig. 5 tumor after resection. 3. Discussion In Pheochromocytoma activation of the alpha-one adrenergic receptor by catecholamine in the vascular bed causes vasoconstriction and leads to a rise in blood pressure. Similarly, activation of the beta-one receptor in the heart enhances the chronotropic and inotropic effect of the myocardium, leading to an increase in heart rate and cardiac output. In addition, activation of the beta-one receptor in the juxtaglomerular cells of the kidney activates the RAAS system. These receptor activation result in cardiovascular and sympathetic changes, such as hypertension, palpitation, headache, sweating, trembling, and anxiety [10]. In Pheochromocytoma, the patient may have a 10-fold increase in plasma catecholamines, but the hemodynamic response can still fall within the normal range due to desensitization of the cardiovascular system. When catecholamine levels are elevated for a prolonged period, the alpha-one receptors in blood vessels may be down-regulated, making norepinephrine unresponsive in raising peripheral vascular resistance, which can lead to normal blood pressure. Similarly, a marked decrease in beta-one receptors in the heart could explain the normal heart rate, which was observed in our asymptomatic patient with Pheochromocytoma [11]. Sometimes in asymptomatic patients, the size of the tumor tends to be larger than in those with hyperfunctioning tumors [12]. However, medical interventions such as surgery, anesthesia induction, intravenous urography contrast, or manipulation of the tumor can trigger adrenergic and hypertensive crises, so biopsy is usually contraindicated in pheochromocytoma [13]. The diagnosis of pheochromocytoma is typically based on measuring plasma and urinary levels of catecholamines and their derivatives such as metanephrine and vanillylmandelic acid. The most reliable test is the measurement of urinary metanephrine as its excretion levels are relatively higher [13,14]. The combination of 131I-MIBG scintigraphy along with diagnostic urinary and blood tests can further enhance the sensitivity of the test. Specifically, the urinary normetanephrine test is considered the most sensitive single test for detecting Pheochromocytoma [15,16]. In addition to a 24-h urine test and blood test, if the lab results are positive for Pheochromocytoma or paragangliomas, further diagnostic tests may be recommended, such as a CT scan, MRI, m-iodobenzylganidine (MIBG) imaging, or positron emission tomography (PET) [16,17]. In our patient 24 h. urine metanephrine of 5415 μg/24 h (normal: 25–312 μg/24 h.); 24 h. urine VMA of 32.2 mg/24 h. (normal: <13.60 mg/24 h.) and imaging confirmation of right adrenal mass lead to the diagnosis of right sided pheochromocytoma. Our patient with pheochromocytoma was tested for parathyroid hormone and calcitonin due to the association of pheochromocytoma with MEN-2 [18]. MEN-2 can be diagnosed biochemically by measuring the baseline levels of calcitonin, parathyroid hormone and serum calcium along with blood tests for catecholamines and their metabolites to detect pheochromocytoma [19]. In our patient, multiple soft, mobile, painless, subcutaneous nodules like lipoma were present over the torso(MEN-1) and high levels of parathyroid hormone and calcitonin were detected(MEN-2). These findings can be correlated with MEN syndrome. USG of the neck revealed no abnormalities of thyroid and parathyroid gland in our patient so prophylactic thyroidectomy was not done, instead he was counseled for follow up if any symptoms or thyroid swelling appears. The diagnosis of Cushing's syndrome typically involves measuring the levels of 24-h urine free cortisol and assessing the suppression of cortisol in response to a 1 mg overnight dexamethasone test. If cortisol levels remain elevated despite the test, the next step is to measure serum ACTH levels. If ACTH levels are suppressed, it suggests an ACTH-independent cause of Cushing's syndrome, while elevated ACTH levels suggest an ACTH-dependent cause. Further evaluation may include a CT scan of the chest, abdomen, and pelvis to identify potential ectopic sources, as well as an MRI of the pituitary gland [8]. Our patient had a high level of 24 h. urine free cortisol of 526.61 nmol/24 h (reference range: 30–145 nmol/24 h) and serum cortisol of 535.16 nmol/L(reference range: <50 nmol/L) after overnight 1 mg dexamethasone suppression test, but normal level of ACTH of 28.3 pg./ml (reference range: 7.2–63.1 ng/ml), this suggests the diagnosis of ACTH independent Cushing's syndrome. 4. Conclusion Large Pheochromocytoma patients can be asymptomatic and can present in association with other endocrine disorders. So proper evaluation is necessary to find out associated conditions and manage accordingly to prevent the possible outcomes. Patient consent Written, informed consent was obtained from the patient for the publication of the report. Ethical approval It is exempted at my institution. We don't need to take approval from ethical committee for case report. Funding N/A. Author contribution Conceptualization: Sanjit Kumar Shah. Clinical diagnosis and patient management: Mahipendra Tiwari. Microscopic slide preparation: Sneh Acharya. Writing original draft: Sanjit Kumar Shah and Avish Shah. All authors were involved in reviewing, editing, supervision and in preparing the final manuscript. Guarantor Guarantor: Sanjit Kumar Shah Email: sanjitshah023@gmail.com Conflict of interest statement N/A. References [1] P.J. Klingler, T.P. Fox, D.M. Menke, J.M. Knudsen, J.T. Fulmer Pheochromocytoma in an incidentally discovered asymptomatic cystic adrenal mass Mayo Clin. Proc., 75 (5) (2000), pp. 517-520, 10.4065/75.5.517 View PDFView articleView in ScopusGoogle Scholar [2] K. Salmenkivi, J. Arola, R. Voutilainen, et al. Inhibin/activin betaB-subunit expression in pheochromocytomas favors benign diagnosis J. Clin. Endocrinol. Metab., 86 (5) (2001), pp. 2231-2235, 10.1210/jcem.86.5.7446 View article View in ScopusGoogle Scholar [3] W.M. Manger, R.W. Gifford Pheochromocytoma J. Clin. Hypertens (Greenwich)., 4 (1) (2002), pp. 62-72, 10.1111/j.1524-6175.2002.01452.x View PDF This article is free to access. Google Scholar [4] S.R. Pingle, F. Jalil, D. Millar, C.D. Malchoff, B.T. Ristau Isolated DHEAS production by an adrenal neoplasm: clinical, biochemical and pathologic characteristics Urol. Case Rep., 31 (2020), Article 101148 Published 2020 Feb 26 https://doi.org/10.1016/j.eucr.2020.101148 Published 2020 Feb 26 View PDFView articleView in ScopusGoogle Scholar [5] I. Jandou, A. Moataz, M. Dakir, A. Debbagh, R. Aboutaieb Malignant pheochromocytoma: a diagnostic and therapeutic dilemma Int. J. Surg. Case Rep., 83 (2021), Article 106009, 10.1016/j.ijscr.2021.106009 View PDFView articleView in ScopusGoogle Scholar [6] L. Canu, G. Parenti, G. De Filpo, M. Mannelli Pheochromocytomas and Paragangliomas as causes of endocrine hypertension Front. Endocrinol. (Lausanne)., 10 (2019), p. 333 Published 2019 Jun 4 https://doi.org/10.3389/fendo.2019.00333 Published 2019 Jun 4 View in ScopusGoogle Scholar [7] G.G. Callender, T.A. Rich, N.D. Perrier Multiple endocrine neoplasia syndromes Surg. Clin. North Am., 88 (4) (2008), p. 863 viii https://doi.org/10.1016/j.suc.2008.05.001 viii View PDFView articleView in ScopusGoogle Scholar [8] R. Pivonello, M. De Leo, A. Cozzolino, A. Colao The treatment of Cushing’s disease Endocr. Rev., 36 (4) (2015), pp. 385-486, 10.1210/er.2013-1048 View article View in ScopusGoogle Scholar [9] R.A. Agha, T. Franchi, C. Sohrab, G. Mathew, A. Kirwan, A. Thomas, et al. The SCARE 2020 guideline: updating consensus Surgical Case Report (SCARE) guidelines Int. J. Surg., 84 (1) (2020), pp. 226-230 View PDFView articleCrossRefView in ScopusGoogle Scholar [10] L. Canu, G. Parenti, G. De Filpo, M. Mannelli Pheochromocytomas and Paragangliomas as causes of endocrine hypertension Front. Endocrinol. (Lausanne)., 10 (2019), p. 333 Published 2019 Jun 4 https://doi.org/10.3389/fendo.2019.00333 Published 2019 Jun 4 View in ScopusGoogle Scholar [11] E. Bravo, F. Fouad-Tarazi, G. Rossi, et al. A reevaluation of the hemodynamics of pheochromocytoma Hypertension., 15 (2 Suppl) (1990), pp. I128-I131, 10.1161/01.hyp.15.2_suppl.i128 View article Google Scholar [12] M.A. Blake, S.K. Krishnamoorthy, G.W. Boland, et al. Low-density pheochromocytoma on CT: a mimicker of adrenal adenoma AJR Am. J. Roentgenol., 181 (6) (2003), pp. 1663-1668, 10.2214/ajr.181.6.1811663 View article View in ScopusGoogle Scholar [13] R. Yu, A. Pitts, M. Wei Small pheochromocytomas: significance, diagnosis, and outcome J. Clin. Hypertens (Greenwich)., 14 (5) (2012), pp. 307-315, 10.1111/j.1751-7176.2012.00604.x View PDF This article is free to access. View in ScopusGoogle Scholar [14] F. Zoghbi, C. Landault, N. Salmon, J.C. Legrand Les catécholamines et leurs dérivés dans l’exploration du phéochromocytome [Catecholamines and their derivatives in the study of pheochromocytoma] Ann. Med. Interne (Paris)., 134 (3) (1983), pp. 230-232 View in ScopusGoogle Scholar [15] U. Guller, J. Turek, S. Eubanks, E.R. Delong, D. Oertli, J.M. Feldman Detecting pheochromocytoma: defining the most sensitive test Ann. Surg., 243 (1) (2006), pp. 102-107, 10.1097/01.sla.0000193833.51108.24 View article View in ScopusGoogle Scholar [16] G. Eisenhofer, M. Peitzsch Laboratory evaluation of pheochromocytoma and paraganglioma Clin. Chem., 60 (12) (2014), pp. 1486-1499, 10.1373/clinchem.2014.224832 View PDF This article is free to access. View in ScopusGoogle Scholar [17] H.P. Neumann, D.P. Berger, G. Sigmund, et al. Pheochromocytomas, multiple endocrine neoplasia type 2, and von Hippel-Lindau disease [published correction appears in N Engl J Med 1994 Dec 1;331(22):1535] N. Engl. J. Med., 329 (21) (1993), pp. 1531-1538, 10.1056/NEJM199311183292103 View article View in ScopusGoogle Scholar [18] V. Amodru, D. Taieb, C. Guerin, et al. MEN2-related pheochromocytoma: current state of knowledge, specific characteristics in MEN2B, and perspectives [published correction appears in endocrine. 2020 Jun 2;:] [published correction appears in endocrine. 2020 Jul 14;:] Endocrine, 69 (3) (2020), pp. 496-503, 10.1007/s12020-020-02332-2 View article View in ScopusGoogle Scholar [19] C.J. Lips, J.W. Höppener, B.P. Van Nesselrooij, R.B. Van der Luijt Counselling in multiple endocrine neoplasia syndromes: from individual experience to general guidelines J. Intern. Med., 257 (1) (2005), pp. 69-77, 10.1111/j.1365-2796.2004.01429.x View PDF This article is free to access. View in ScopusGoogle Scholar From https://www.sciencedirect.com/science/article/pii/S2210261223005370

Abstract Summary Cushing’s syndrome due to ectopic adrenocorticotropic hormone (ACTH) secretion (EAS) by a pheochromocytoma is a challenging condition. A woman with hypertension and an anamnestic report of a ‘non-secreting’ left adrenal mass developed uncontrolled blood pressure (BP), hyperglycaemia and severe hypokalaemia. ACTH-dependent severe hypercortisolism was ascertained in the absence of Cushingoid features, and a psycho-organic syndrome developed. Brain imaging revealed a splenial lesion of the corpus callosum and a pituitary microadenoma. The adrenal mass displayed high uptake on both 18F-FDG PET/CT and 68Ga-DOTATOC PET/CT; urinary metanephrine levels were greatly increased. The combination of antihypertensive drugs, high-dose potassium infusion, insulin and steroidogenesis inhibitor normalized BP, metabolic parameters and cortisol levels; laparoscopic left adrenalectomy under intravenous hydrocortisone infusion was performed. On combined histology and immunohistochemistry, an ACTH-secreting pheochromocytoma was diagnosed. The patient's clinical condition improved and remission of both hypercortisolism and catecholamine hypersecretion ensued. Brain magnetic resonance imaging showed a reduction of the splenial lesion. Off-therapy BP and metabolic parameters remained normal. The patient was discharged on cortisone replacement therapy for post-surgical hypocortisolism. EAS due to pheochromocytoma displays multifaceted clinical features and requires prompt diagnosis and multidisciplinary management in order to overcome the related severe clinical derangements. Learning points A small but significant number of cases of adrenocorticotropic hormone (ACTH)-dependent Cushing’s syndrome are caused by ectopic ACTH secretion by neuroendocrine tumours, which is usually associated with severe hypercortisolism causing severe clinical and metabolic derangements. Ectopic ACTH secretion by a pheochromocytoma is exceedingly rare but can be life-threatening, owing to the simultaneous excess of both cortisol and catecholamines. 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. Immediate-acting steroidogenesis inhibitors are required for the treatment of hypercortisolism, and catecholamine excess should also be appropriately managed before surgical removal of the tumour. A multidisciplinary approach is required for the treatment of this challenging entity. Keywords: Adult; Female; White; Italy; Adrenal; Pituitary; Unique/unexpected symptoms or presentations of a disease; May; 2023 Background Cushing’s syndrome (CS) is a rare endocrine disease characterized by high levels of glucocorticoids; it increases morbidity and mortality due to cardiovascular and infectious diseases (1, 2, 3). To diagnose CS, adrenocorticotropic hormone (ACTH)-dependent disease must be distinguished from ACTH-independent disease, and pituitary ACTH production from ectopic production. About 20% of ACTH-dependent cases arise from ectopic ACTH secretion (EAS) (2, 3, 4). EAS is most often due to aberrant ACTH production by small-cell lung carcinoma or neuroendocrine tumours originating in the lungs or gastrointestinal tract; this, in turn, strongly increases cortisol production by the adrenal glands (3, 4, 5). Since the first-line treatment of EAS is the surgical removal of the ectopic ACTH-secreting tumour, its prompt and accurate localization is crucial. Rapid cortisol reduction by means of immediate-acting steroidogenesis inhibitors (4) is mandatory in order to treat the related endocrine, metabolic and electrolytic derangements. EAS by a pheochromocytoma is exceedingly rare and can be life-threatening. We describe the case of a woman with hypertension and a known ‘non-secreting’ left adrenal mass, who manifested uncontrolled blood pressure (BP), hyperglycaemia, hypokalaemia and psycho-organic syndrome associated with damage of the splenium of the corpus callosum. These findings were eventually seen to be related to an ACTH-secreting left pheochromocytoma, which was ascertained by hormonal evaluation and morphological and functional imaging assessment and confirmed by histopathology/immunostaining. Hormonal hypersecretion resolved after adrenalectomy and metabolic derangements normalized. Case presentation A 72-year-old woman with hypertension was taken to the emergency department because of increased BP (200/100 mm Hg). High BP (190/100 mmHg) was confirmed, whereas oxygen saturation (98%), heart rate (84 bpm) and lung and abdomen examination were normal. Electrocardiogram and chest x-ray were unremarkable. Captopril 50 mg orally, followed by intramuscular clonidine, normalized BP. The patient looked thin and reported significant weight loss (10 kg) over the previous 6 months; she was on antihypertensive therapy with bisoprolol 5 mg/day and irbesartan 150 mg/day, and ezetimibe 10 mg/day for dyslipidaemia. The patient’s records included a previous diagnosis in another hospital of normofunctioning multinodular goitre and a 2.5 cm-left solid inhomogeneous adrenal mass with well-defined margins, which was found on CT performed 6 years earlier during the work-up for hypertension. On the basis of hormonal data and absent uptake on 123I metaiodobenzylguanidine scintigraphy, the adrenal lesion had been deemed to be non-functioning and follow-up had been advised. Unfortunately, only initial cortisol (15.7 μg/dL) and 24-h urine-free cortisol (UFC) levels (32.5 μg/24 h) were retrievable; both proved normal. Investigations Blood chemistry showed neutrophilic leucocytosis, hyperglycaemia with increased glycated haemoglobin, severe hypokalaemia and metabolic alkalosis (Table 1). Potassium infusion (50 mEq in 500 mL saline/24 h) was rapidly started, together with a subcutaneous rapid-acting insulin analogue and prophylactic enoxaparin. The patient experienced mental confusion, hallucinations and restlessness; non-enhanced computed tomography (CT) of the brain revealed a hypodense area of the splenium of the corpus callosum, possibly due to metabolic damage (Fig. 1A). View Full Size Figure 1 Non-enhanced CT showing a hypodense area of the splenium of the corpus callosum (arrows), without mass effect (A, axial view). Contrast-enhanced MR image showing a hypointense pituitary lesion (arrow) which enhances more slowly than normal pituitary parenchyma, deemed suspicious for microadenoma (B, coronal view). FLAIR MR image showing hyperintense signal of the splenium of the corpus callosum (asterisk), which partially extended to the crux of the left fornix (arrow) (C, axial view). As the lesion showed no restricted diffusion on DWI (D, axial view), an ischaemic lesion was excluded. A progressive reduction in the extension of the hyperintense signal in the splenium of the corpus callosum (arrowheads) and in the crux of the left fornix (arrows) was observed on FLAIR MR images (2 months (E); 3 months (F); axial view). CT, computed tomography; DWI, diffusion-weighted imaging; FLAIR, fluid-attenuated inversion recovery; MR, magnetic resonance. Citation: Endocrinology, Diabetes & Metabolism Case Reports 2023, 2; 10.1530/EDM-22-0308 Download Figure Download figure as PowerPoint slide Table 1 Hormonal and biochemical evaluation of patient throughout hospitalization and follow-up. Normal range On hospital admission After surgery 10 days 2 months 3 months 6 months 9 months 12 months 16 months ACTH (pg/mL) 9–52 551 7 37 50 29.5 26 40.9 52 Morning cortisol† (µg/dL) 7–19.2 63.4 14 5.1 3.5 3.8 4.2 7.2 12.8 After 1 mg overnight dexamethasone  ACTH 583  Cortisol 60 DHEAS (µg/dL) 9.4–246 201 24-h urinalysis (µg/24 h)  Adrenaline 0–14.9 95.5  Noradrenaline 0–66 1133  Metanephrine 74–297 1927  Normetanephrine 105–354 1133 Chromogranin A 0–108 290 Renin (supine) (µU/mL) 2.4–29 3.9 14.6 Aldosterone (supine) (ng/dL) 3–15 3.4 12.5 LH (mIU/mL)* > 10 0.3 65.8 FSH (mIU/mL)* > 25 1.9 116 PRL (ng/mL) 3–24 13.7 FT4 (ng/dL) 0.9–1.7 1.1 1.2 FT3 (pg/mL) 1.8–4.6 1.1 2.7 TSH (µU/mL) 0.27–4.2 0.23 1.3 PTH (pg/mL) 15–65 166 Calcium (mg/dL) 8.2–10.2 8.2 Calcitonin (pg/mL) 0–10 1 Glycaemia (mg/dL) 60–110 212 69 73 83 Potassium (mEq/L) 3.5–5 2.4 3.3 3.9 4.2 3.7 5 4.4 3.9 Leucocytes (K/µL) 4.0–9.3 15.13 HbA1c (mmol/mol) 20–42 55 30 HCO3− (mEq/L) 22–26 41.8 *For menopausal age; †07:00–10:00 h. The patient was transferred to the internal medicine ward. Although potassium infusion was increased to 120 mEq/day, serum levels did not normalize; a mineralocorticoid receptor antagonist (potassium canreonate) was therefore introduced, but the effect was partial. In order to control BP, the irbersartan dose was increased (300 mg/day) and amlodipine (10 mg/day) was added. The combination of severe hypertension, newly occurring diabetes and resistant hypokalaemia prompted us to hypothesize a common endocrine aetiology. A thorough hormonal array showed very high ACTH and cortisol levels, whereas supine renin and aldosterone levels were in the low-normal range (Table 1). Since our patient proved repeatedly non-compliant with 24-h urine collection, UFC could not be measured. After an overnight 1 mg dexamethasone suppression test, cortisol levels remained unchanged, whereas ACTH levels slightly increased (Table 1). Notably, the patient showed no Cushingoid features. Gonadotropin levels were inappropriately low for the patient’s age; FT4 levels were normal, whereas FT3 and thyroid-stimulating hormone (TSH) levels were reduced and calcitonin levels were normal (Table 1). HbA1c levels were increased (Table 1). Finally, secondary hyperparathyroidism, associated with low-normal calcium levels and reduced vitamin D levels, was found (Table 1). Brain contrast-enhanced magnetic resonance (MR) imaging revealed a 5-mm median posterior pituitary microadenoma (Fig. 1B) and a hyperintense lesion of the splenium of the corpus callosum (Fig. 1C). Diffusion-weighted MR images of the lesion showed no restricted diffusion (Fig. 1D), thus excluding an ischaemic origin. Petrosal venous sampling for ACTH determination at baseline and after CRH stimulation was excluded, as it was deemed a high-risk procedure, given the patient's poor condition. Since the ACTH and cortisol levels were greatly increased and were associated with severe hypokalaemia, EAS was hypothesized; total-body contrast-enhanced CT revealed the left adrenal mass (3 cm), which showed regular margins and heterogeneous enhancement (Fig. 2A and B) and measured 25 Hounsfield units. There was no evidence of adrenal hyperplasia in the contralateral adrenal gland. The adrenal mass showed intense tracer uptake on both 18F-FDG PET/CT (Fig. 2C and D), suggestive of adrenal malignancy or functioning tumour, and 68Ga-DOTATOC PET/CT (Fig. 3), which is characteristic of a neuroendocrine lesion. No other sites of suspicious tracer uptake were detected. View Full Size Figure 2 Contrast-enhanced abdominal computed tomography showing a 3-cm left adrenal mass (arrow) with well-defined margins and inhomogeneus enhancement, deemed compatible with an adenoma (A, coronal view; B, axial view). The adrenal mass showed high uptake (SUV max: 7.3) on 18F-FDG PET/CT (C, coronal view; D, axial view). Citation: Endocrinology, Diabetes & Metabolism Case Reports 2023, 2; 10.1530/EDM-22-0308 Download Figure Download figure as PowerPoint slide View Full Size Figure 3 The left adrenal mass displaying very high uptake (SUV max: 40) on 68Ga-DOTATOC PET/CT (arrow, axial view). Citation: Endocrinology, Diabetes & Metabolism Case Reports 2023, 2; 10.1530/EDM-22-0308 Download Figure Download figure as PowerPoint slide Bisoprolol was withdrawn, and 24-h urinary catecholamine, metanephrine and normetanephrine levels proved significantly increased, as were chromogranin A levels (Table 1). In sum, an ACTH-secreting pheochromocytoma was suspected and the pituitary microadenoma was deemed a likely incidental finding. The patient’s mental state worsened, fluctuating from sopor to restlessness, which required parenteral neuroleptics and restraint. An electroencephalogram revealed a specific slowdown of cerebral electrical activity. Following rachicentesis, the cerebrospinal fluid showed pleocytosis (lympho-monocytosis), whereas a culture test and polymerase chain reaction for common neurotropic agents were negative. The neurologist hypothesized a psycho-organic syndrome secondary to severe metabolic derangement. Intravenous ampicillin, acyclovir and B vitamins were empirically started. The patient was transferred to the subintensive unit, where a nasogastric tube and central venous catheter were inserted, and enteral nutrition was started. Treatment Ketoconazole was started at a dosage of 200 mg twice daily; both cortisol and ACTH levels significantly decreased over a few days (Fig. 4), with a progressive decrease in glucose levels and normalization of potassium levels and BP on therapy. Subsequently, ketoconazole was titrated to 600 mg/day owing to a new increase in cortisol levels, which eventually normalized (Fig. 4). Of note, ACTH levels partially decreased on ketoconazole treatment (Fig. 4). View Full Size Figure 4 ACTH and cortisol levels throughout the patient’s hospitalization and follow-up. Citation: Endocrinology, Diabetes & Metabolism Case Reports 2023, 2; 10.1530/EDM-22-0308 Download Figure Download figure as PowerPoint slide Doxazosin 2 mg/day was added and the patient's systolic BP blood settled at around 100 mm Hg; after a few days, bisoprolol was restarted. Contrast-enhanced MR showed a partial reduction of the hyperintense splenial lesion (Fig. 1E). Despite the severe clinical condition and the high risks of adrenal surgery, the patient’s relatives strongly requested the procedure and laparoscopic left adrenalectomy was planned. Alpha-blocker and fluid infusion were continued, ketoconazole was withdrawn the day before surgery, and a 100 mg IV bolus of hydrocortisone was administered just before the operation, followed by 200 mg/day, at first in continuous infusion, then as a 100 mg bolus every 8 h. After the removal of the left adrenal mass, noradrenaline infusion was required, owing to the occurrence of severe hypotension. Outcome and follow-up Pathology revealed a 2.5 cm reddish-brown encapsulated tumour, which was compatible with pheochromocytoma (Fig. 5A and B); ACTH immunostaining was positive in about 30% of tumour cells (Fig. 5C). This confirmed the diagnostic hypothesis of an ACTH-secreting pheochromocytoma. The tumour was stained for Chromogranin A (Fig. 5D). There were no signs of adrenal cortex hyperplasia in the resected gland. Thorough germinal genetic testing, comprising the commonest pheochromocytoma/paraganglioma genes: CDKN1B, KIF1B, MEN1, RET, SDHA, SDHB, SDHC, SDHD, SDHAF2 and TMEM127, was negative. View Full Size Figure 5 Histological images of adrenal pheochromocytoma: the tumour is composed of well-defined nests of cells (‘zellballen’) (A; haematoxylin-eosin stain (HE), ×20) with pleomorphic nuclei with prominent nucleoli, basophilic or granular amphophilic cytoplasm (B; HE, ×40). The mitotic index was low: 1 mitosis per 30 high-power fields, and Ki-67 was 1%. On immunohistochemistry, cytoplasmatic ACTH staining was found in about 30% of tumour cells (C; ×20), whereas most tumour cells were stained for chromogranin A (D; ×20). Citation: Endocrinology, Diabetes & Metabolism Case Reports 2023, 2; 10.1530/EDM-22-0308 Download Figure Download figure as PowerPoint slide One week after surgery ACTH levels had dropped to a low-normal value: 7 pg/mL, and cortisol levels (before morning hydrocortisone bolus administration) were normal: 14 µg/dL (Fig. 4). The patient’s clinical status slowly improved and the nasogastric tube was removed; intravenous hydrocortisone was carefully tapered until withdrawal and high-dose oral cortisone acetate (62.5 mg/day) was started. This dose was initially required since BP remained low (systolic: 90 mm Hg); thereafter, cortisone was reduced to 37.5 mg/day. Plasma cortisol levels before morning cortisone administration were reduced (Fig. 4). A new MR of the brain showed a further partial reduction of the splenial lesion (Fig. 1F). The patient was discharged with normal off-therapy BP and metabolic parameters. During follow-up, she fully recovered, and BP and metabolic parameters remained normal. Gonadotropin levels became adequate for the patient’s age, and TSH and renin/aldosterone levels normalized (Table 1). Hypoadrenalism, however, persisted for more than 1 year; as the last hormonal evaluation, 16 months after surgery, showed normal baseline cortisol levels, the cortisone dose was tapered (12.5 mg/day) and further hormonal examination was scheduled (Table 1). ACTH and cortisol levels throughout the patient’s hospitalization and follow-up are shown in Fig. 4. Discussion The diagnosis of EAS is challenging and requires two steps: confirmation of increased ACTH and cortisol levels and anatomic distinction from pituitary sources of ACTH overproduction. Besides metabolic derangements (hyperglycaemia, hypertension), EAS-related severe hypercortisolism may cause profound hypokalaemia (3, 4, 5). In our patient, the combination of worsening hypertension, newly occurring diabetes and resistant hypokalaemia raised the suspicion of a common endocrine cause. ACTH-dependent severe hypercortisolism was ascertained, and subsequent brain MR revealed a pituitary microadenoma. The diagnosis of CS requires the combination of two abnormal test results: 24-h UFC, midnight salivary cortisol and/or abnormal 1 mg dexamethasone suppression testing (2, 6). ACTH evaluation (low/normal-high) is fundamental to tailoring the imaging technique. The very high cortisol levels found in our patient were unchanged after overnight dexamethasone testing, whereas UFC could not be assessed owing to the lack of compliance with urine collection. The accuracy of the UFC assays, however, may be impaired by cortisol precursors and metabolites. Salivary cortisol assessment was not performed since the specific assay is not available in our hospital. The combination of ACTH-dependent severe hypercortisolism and hypokalaemia prompted us to suspect EAS. The differential diagnosis between pituitary and ectopic ACTH-dependent CS involves high-dose (8 mg) dexamethasone suppression testing, which has relatively low diagnostic accuracy (6). Owing to the patient's very high cortisol levels and severe hypokalaemia, this testing was not performed, on account of the risks of administering corticosteroids in a patient already exposed to excessive levels (6). Furthermore, owing to the increase in ACTH levels observed after overnight dexamethasone testing, we postulated the possible occurrence of glucocorticoid-driven positive feedback on ACTH secretion, which has been described in EAS, including cases of pheochromocytoma (7). Finally, in the case of EAS suspected of being caused by pheochromocytoma, we do not recommend performing high-dose dexamethasone suppression testing, owing to the risk of triggering a catecholaminergic crisis (8). The dynamic tests commonly used to distinguish patients with EAS from those with Cushing's disease are the CRH stimulation test and the desmopressin stimulation test, either alone or in combination with CRH testing (6). Owing to the rapid worsening of our patient’s condition, dynamic testing was not done; however, the clinical picture and hormonal/biochemical data were suggestive of EAS. EAS is mainly (45–50%) due to neuroendocrine tumours, mostly of the lung (small-cell lung cancer and bronchial tumours), thymus or gastrointestinal tract; however, up to 20% of ACTH-secreting tumours remain occult (3, 4, 5). ACTH-secreting pheochromocytomas are responsible for about 5% of cases of EAS (3, 4, 9, 10). Indeed, this rate ranges widely, from 2.5% (11) to 15% (12), according to the different case series. Patients with EAS due to pheochromocytoma present with severe CS, overt diabetes mellitus, hypertension and hypokalaemia (3); symptoms of catecholamine excess may be unapparent (3), making the diagnosis more challenging. A recent review of 99 patients with ACTH- and/or CRH-secreting pheochromocytomas found that the vast majority displayed a Cushingoid phenotype (10); by contrast, another review of 24 patients reported that typical Cushingoid features were observed in only 30% of patients, whereas weight loss was a prevalent clinical finding (13). We hypothesized that the significant weight loss reported by our patient was largely due to the hypermetabolic state induced by catecholamines, which directly reduce visceral and subcutaneous fat, as recently reported (14). Our patient showed no classic stigmata of CS, owing to the rapid onset of severe hypercortisolism (10, 13), whereas she had worsening hypertension and newly occurring diabetes mellitus, which were related to both cortisol and catecholamine hypersecretion; hypokalaemia was deemed to be secondary to severe hypercortisolism. Indeed, greatly increased cortisol levels act on the mineralocorticoid receptors of the distal tubule after saturating 11β-hydroxysteroid dehydrogenase type 2, leading to hypokalaemia (4). Consequently, hypokalaemia is much more common (74–95% of patients) in EAS than in classic Cushing’s disease (10%) (3, 4, 10). This apparent mineralocorticoid excess suppresses renin and aldosterone secretion, as was ascertained in our patient. In this setting, the most effective way to manage hypokalaemia is to treat the hypercortisolism itself by administering immediate-acting steroidogenesis inhibitors, combined with potassium infusion and a mineralocorticoid receptor-antagonist (e.g. spironolactone) at an appropriate dosage (100–300 mg/day) (4). In ACTH-secreting pheochromocytoma, cortisol hypersecretion potentiates catecholamine-induced hypertension by stimulating the phenol-etholamine-N-methyl–transferase enzyme, which transforms noradrenaline to adrenaline (4). Indeed, in our patient, the significant ketoconazole-induced reduction in cortisol secretion led to satisfactory BP control on antihypertensive drugs. After the biochemical diagnosis of pheochromocytoma, a selective alpha-blocker was added, and after a few days, a beta-blocker was restarted in order to control reflex tachycardia (15). Our patient had greatly increased ACTH levels (>500 pg/mL) associated with very high cortisol levels (>60 µg/dL), which, together with the finding of hypokalaemia, prompted us to hypothesize EAS. With regard to these findings, ACTH levels are usually higher (>200 pg/mL) in patients with EAS than in those with CS due to a pituitary adenoma; however, considerable overlapping occurs (3, 11, 16). Most patients with ACTH-secreting pheochromocytomas in those series had ACTH levels >300 pg/mL, and a few had normal ACTH levels (9), thus complicating the diagnosis. In addition, patients with EAS usually have higher cortisol levels than those with ACTH-secreting adenomas (3, 11). In our patient, the left adrenal mass was deemed the culprit of EAS, and owing to very high urinary metanephrine levels, a pheochromocytoma was suspected. It can be assumed that the adrenal tumour, which was anamnestically reported as ‘non-secreting’, but on which only part of the initial hormonal data were available, was actually a pheochromocytoma at the time of the first diagnosis but displayed a silent clinical and hormonal behaviour. The mass subsequently showed significant uptake on both 18F-FDG PET/CT and 68Ga-DOTATOC PET/CT (4, 5). It is claimed that 68Ga-DOTATOC PET/CT provides a high grade (90%) of sensitivity and specificity in the diagnosis of tumours that cause EAS (4, 5); nevertheless, a recent systematic review reported much lower sensitivity (64%), which increased to 76% in histologically confirmed cases (17). In patients with EAS, immediate-acting steroidogenesis inhibitors are required in order to achieve prompt control of severe hypercortisolism (4). Ketoconazole is one of the drugs of choice since it inhibits adrenal steroidogenesis at several steps. In our patient, ketoconazole rapidly reduced cortisol levels to normal values, without causing hepatic toxicity (4). Moreover, ketoconazole proved effective at a moderate dosage (600 mg/day), which falls within the mean literature range (18, 19). However, dosages up to 1200–1600 mg/day are sometimes required in severe cases (usually EAS) (18, 19). Speculatively, our results might reflect an enhanced inhibitory action of ketoconazole at the adrenal level, which was able to override the strong ectopic ACTH stimulation. In addition, the finding that, following cortisol reduction, ACTH levels paradoxically decreased suggests an additive and direct effect of the drug. This effect has been observed in a few patients with EAS (20) and is supported by in vitro studies showing a direct anti-proliferative and pro-apoptotic effect of ketoconazole on ectopic ACTH secretion by tumours (21). Finally, the reduction in ACTH levels during treatment with steroidogenesis inhibitors prompts us to postulate the presence of glucocorticoid-driven positive feedback on ACTH secretion, as already described in neuroendocrine tumours (7, 20, 21). The coexistence of EAS and ACTH-producing pituitary adenoma is very rare but must be taken into account. In our case, we deemed the pituitary mass found on MR to be a non-secreting microadenoma. This hypothesis was strengthened by the finding that, following exeresis of the ACTH-secreting pheochromocytoma, ACTH normalized, hypercortisolism vanished and pituitary function recovered. These findings suggest that: (i) altered pituitary function at the baseline was secondary to the inhibitory effect of hypercortisolism; (ii) the excessive production of cortisol was driven by ACTH overproduction outside the pituitary gland, specifically within the adrenal gland tumour. In our patient, a few days after surgery, morning cortisol levels before hydrocortisone bolus administration were ‘normal’. Owing to both the half-life of hydrocortisone (8–12 h) and the supraphysiological dosage used, it is likely that a residual part of the drug, which cross-reacts in the cortisol assay, was still circulating at the time of blood collection, thus resulting in ‘normal’ cortisol values. Following the switch to oral cortisone, cortisol levels before therapy were low, thus confirming post-surgical hypocortisolism. Hypocortisolism remained throughout the first year after surgery, and glucocorticoid therapy was continued. Sixteen months after surgery, baseline cortisol levels returned to the normal range; cortisone therapy was therefore tapered and a further hormonal check was scheduled. Assessment of the cortisol response to ACTH stimulation testing would be helpful in order to check the resumption of the residual adrenal function. A peculiar aspect of our case was the occurrence of a psycho-organic syndrome together with the finding of a splenial lesion on brain imaging, which was deemed secondary to metabolic injury. Indeed, the increased cortisol levels present in patients with Cushing’s disease are detrimental to the white matter of the brain, including the corpus collosum, causing subsequent clinical derangements (22). Besides the direct effects of hypercortisolism, the splenial damage was also probably due to long-standing hypertension, worsened by newly occurring catecholamine hypersecretion and diabetes. Together with the normalization of cortisol and glycaemic levels, and of BP, a partial reduction in the splenial damage was observed on two subsequent MR examinations, and the patient's neurological condition slowly improved until she fully recovered. In our patient, thorough germinal genetic testing for the commonest pheochromocytoma/paraganglioma (PPGL) genes proved negative. Since approximately 40% of these tumours have germline mutations, genetic testing is recommended regardless of the patient’s age and family history. In the absence of syndromic, familial or metastatic presentation, the selection of genes for testing may be guided by the tumour location and biochemical phenotype. Alterations of the PPGL genes can be divided into two groups: 10 genes (RET, VHL, NF1, SDHD, SDHAF2, SDHC, SDHB, SDHA, TMEM127 and MAX) that have well-defined genotype–phenotype correlations, thus allowing to tailor imaging procedures and medical management, and a group of other emerging genes, which lack established genotype–phenotype associations; for patients in whom mutations of genes belonging to this second group are detected, and hence hereditary predisposition is established, only general medical surveillance and family screening can be planned (23, 24). In conclusion, our case highlights the importance of investigating patients with hypertension and metabolic derangements such as diabetes and hypokalaemia, since these findings may be a sign of newly occurring EAS, which, in rare cases, may be due to an ACTH-secreting pheochromocytoma. Since the additive effect of cortisol and catecholamine can cause dramatic clinical consequences, the possibility of an ACTH-secreting pheochromocytoma should be taken into account in the presence of an adrenal mass. EAS must be considered an endocrine emergency requiring urgent multi-specialist treatment. Surgery, whenever possible, is usually curative, and anatomic brain damage, as ascertained in our patient, may be at least partially reversible. Declaration of interest The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported. Funding This study did not receive any specific grant from any funding agency in the public, commercial or not-for-profit sector. The study was approved by the Local Ethics Committee (no: 732/2022). Patient consent The patient provided written informed consent. Author contribution statement All authors contributed equally to the conception, writing and editing of the manuscript. L Foppiani took care of the patient during hospitalization and in the outpatient department, performed the metabolic and endocrine work-up, conceived the study, analysed the data and wrote the manuscript. MG Poeta evaluated the patient during hospitalization with regard to neurological problems and planned the related work-up (brain imaging procedures and rachicentesis). M Rutigliani analysed the histological specimens and performed immunohistochemical studies. S Parodi performed CT and MR scans and analysed the related images. U Catrambone performed the left adrenalectomy. L Cavalleri performed general anaesthesia and assisted the patient during the surgical and post-surgical periods. G Antonucci revised the manuscript. P Del Monte helped in the endocrine work-up, in the evaluation of hormonal data and in the revision of the manuscript. A Piccardo performed 18F-FDG PET/CT and analysed the related images. Acknowledgement The work of Prof Silvia Morbelli in performing and analysing 68Ga-DOTATOC PET/CT is gratefully acknowledged. References 1↑ Pivonello R, Isidori AM, De Martino MC, Newell-Price J, Biller BMK, Colao A. Complications of Cushing's syndrome: state of the art. The Lancet Diabetes & Endocrinology 2016 4 611–629. (https://doi.org/10.1016/S2213-8587(1600086-3) Search Google Scholar Export Citation 2↑ Fleseriu M, Auchus R, Bancos I, Ben-Shlomo A, Bertherat J, Biermasz NR, Boguszewski CL, Bronstein MD, Buchfelder M, Carmichael JD, et al.Consensus on diagnosis and management of Cushing's disease: a guideline update. Lancet. Diabetes and Endocrinology 2021 9 847–875. (https://doi.org/10.1016/S2213-8587(2100235-7) PubMed Search Google Scholar Export Citation 3↑ Gabi JN, Milhem MM, Tovar YE, Karem ES, Gabi AY, & Khthir RA. Severe Cushing syndrome due to an ACTH-producing pheochromocytoma: a case presentation and review of the literature. Journal of the Endocrine Society 2018 2 621–630. (https://doi.org/10.1210/js.2018-00086) Search Google Scholar Export Citation 4↑ Young J, Haissaguerre M, Viera-Pinto O, Chabre O, Baudin E, & Tabarin A. Management of endocrine disease: Cushing's syndrome due to ectopic ACTH secretion: an expert operational opinion. European Journal of Endocrinology 2020 182 R29–R58. (https://doi.org/10.1530/EJE-19-0877) PubMed Search Google Scholar Export Citation 5↑ Hayes AR, & Grossman AB. The ectopic adrenocorticotropic hormone syndrome: rarely easy, always challenging. Endocrinology and Metabolism Clinics of North America 2018 47 409–425. (https://doi.org/10.1016/j.ecl.2018.01.005) PubMed Search Google Scholar Export Citation 6↑ Hayes AR, & Grossman AB. Distinguishing Cushing's disease from the ectopic ACTH syndrome: needles in a haystack or hiding in plain sight? Journal of Neuroendocrinology 2022 34 e13137. (https://doi.org/10.1111/jne.13137) Search Google Scholar Export Citation 7↑ Sakuma I, Higuchi S, Fujimoto M, Takiguchi T, Nakayama A, Tamura A, Kohno T, Komai E, Akina Shiga A, Nagano H, et al.Cushing syndrome due to ACTH-secreting pheochromocytoma, aggravated by glucocorticoid-driven positive-feedback loop. Journal of Clinical Endocrinology and Metabolism 2016 101 841–846. (https://doi.org/10.1210/jc.2015-2855) PubMed Search Google Scholar Export Citation 8↑ Barrett C, van Uum SH, & Lender JW. Risk of catecholaminergic crises following glucocorticoid administration in patients with an adrenal mass: a literature review. Clinical Endocrinology 2015 83 622–628. (https://doi.org/10.1111/cen.12813) Search Google Scholar Export Citation 9↑ Ballav C, Naziat A, Mihai R, Karavitaki N, Ansorge O, & Grossman AB. Mini-review: pheochromocytomas causing the ectopic ACTH syndrome. Endocrine 2012 42 69–73. (https://doi.org/10.1007/s12020-012-9646-7) PubMed Search Google Scholar Export Citation 10↑ Elliott PF, Berhane T, Ragnarsson O, & Falhammar H. Ectopic ACTH- and/or CRH-Producing pheochromocytomas. Journal of Clinical Endocrinology and Metabolism 2021 106 598–608. (https://doi.org/10.1210/clinem/dgaa488) Search Google Scholar Export Citation 11↑ Isidori AM, Kaltsas GA, Pozza C, Frajese V, Newell-Price J, Reznek RH, Jenkins PJ, Monson JP, Grossman AB, & Besser GM. The ectopic adrenocorticotropin syndrome: clinical features, diagnosis, management, and long-term follow-up. Journal of Clinical Endocrinology and Metabolism 2006 91 371–377.. (https://doi.org/10.1210/jc.2005-1542) PubMed Search Google Scholar Export Citation 12↑ Salgado LR, Fragoso MCB, Knoepfelmacher M, Machado MC, Domenice S, Pereira MA, & de Mendonça BB. Ectopic ACTH syndrome: our experience with 25 cases. European Journal of Endocrinology 2006 155 725–733. (https://doi.org/10.1530/eje.1.02278) PubMed Search Google Scholar Export Citation 13↑ Paleń-Tytko JE, Przybylik-Mazurek EM, Rzepka EJ, Pach DM, Sowa-Staszczak AS, Gilis-Januszewska A, & Hubalewska-Dydejczyk AB. Ectopic ACTH syndrome of different origin: diagnostic approach and clinical outcome. Experience of one clinical centre. PLoS One 2020 15 e0242679. (https://doi.org/10.1371/journal.pone.0242679) PubMed Search Google Scholar Export Citation 14↑ Krumeich LN, Cucchiara AJ, Nathanson KL, Kelz RR, Fishbein L, Fraker DL, Roses RE, Cohen DL, & Wachtel H. Correlation between plasma catecholamines, weight, and diabetes in pheochromocytoma and paraganglioma. Journal of Clinical Endocrinology and Metabolism 2021 106 e4028–e4038. (https://doi.org/10.1210/clinem/dgab401) PubMed Search Google Scholar Export Citation 15↑ Bihain F, Nomine-Criqui C, Guerci P, Gasman S, Klein M, & Brunaud L. Management of patients with treatment of pheochromocytoma: a critical appraisal. Cancers (Basel) 2022 14 3845. (https://doi.org/10.3390/cancers14163845) PubMed Search Google Scholar Export Citation 16↑ Ilias I, Torpy DJ, Pacak K, Mullen N, Wesley RA, & Nieman LK. Cushing’s syndrome due to ectopic corticotropin secretion: 20 years’ experience at the National Institute of Health. Journal of Clinical Endocrinology and Metabolism 2005 90 4955–4962. (https://doi.org/10.1210/jc.2004-2527) Search Google Scholar Export Citation 17↑ Varlamov E, Hinojosa-Amaya JM, Stack M, & Fleseriu M. Diagnostic utility of gallium-68-somatostatin receptor PET/CT in ectopic ACTH-secreting tumours: a systematic literature review and single-center clinical experience. Pituitary 2019 22 445–455. (https://doi.org/10.1007/s11102-019-00972-w) PubMed Search Google Scholar Export Citation 18↑ Varlamov EV, Han AJ, & Fleseriu M. Updates in adrenal steroidogenesis inhibitors for Cushing's syndrome. A practical guide. Best Practice and Research. Clinical Endocrinology and Metabolism 2021 35 101490. (https://doi.org/10.1016/j.beem.2021.101490) PubMed Search Google Scholar Export Citation 19↑ Marques JVO, & Boguszewski CL. Medical therapy in severe hypercortisolism. Best Practice and Research. Clinical Endocrinology and Metabolism 2021 35 101487. (https://doi.org/10.1016/j.beem.2021.101487) Search Google Scholar Export Citation 20↑ Sharma ST, & Nieman LK. Prolonged remission after long-term treatment with steroidogenesis inhibitors in Cushing's syndrome caused by ectopic ACTH secretion. European Journal of Endocrinology 2012 166 531–536. (https://doi.org/10.1530/EJE-11-0949) PubMed Search Google Scholar Export Citation 21↑ Martinez AD, Feelders RA, de Herder WW, Castaño JP, Moreno MAG, Dogan F, van Dungen R, van Koetsveld P, & Hofland LJ. Effects of ketoconazole on ACTH-producing and non-ACTH-producing neuroendocrine tumor cells. Hormones and Cancer 2019 107–119. (https://doi.org/10.1007/s12672-019-00361-6) Search Google Scholar Export Citation 22↑ Cui M, Zhou T, Feng S, Liu X, Wang F, Zhang Y, & Yu X. Altered microstructural pattern of white matter in Cushing’s disease identified by automated fiber quantification. NeuroImage. Clinical 2021 31 102770. (https://doi.org/10.1016/j.nicl.2021.102770) PubMed Search Google Scholar Export Citation 23↑ Lenders JWM, Duh QY, Eisenhofer G, Gimenez-Roqueplo AP, Grebe SKG, Hassan Murad MH, Naruse M, Pacak K, Young WF Jr & Endocrine Society. Pheochromocytoma and paraganglioma: an endocrine society clinical practice guideline. Journal of Clinical Endocrinology and Metabolism 2014 99 1915–1942. (https://doi.org/10.1210/jc.2014-1498) PubMed Search Google Scholar Export Citation 24↑ Sarkadi B, Saskoi E, Butz H, & Patocs A. Genetics of pheochromocytomas and paragangliomas determine the therapeutical approach. International Journal of Molecular Sciences 2022 23 1450. (https://doi.org/10.3390/ijms23031450) PubMed Search Google Scholar Export Citation From https://edm.bioscientifica.com/view/journals/edm/2023/2/EDM22-0308.xml

The Carling Adrenal Center, a worldwide destination for the surgical treatment of adrenal tumors, becomes the first center to offer the use of amniotic membrane during adrenal surgery which saves functional adrenal tissue in patients undergoing adrenal surgery. This novel technique enables more patients to have a partial adrenalectomy thereby preserving some normal adrenal physiology, potentially eliminating life-long adrenal hormone replacement. Preliminary clinical data from the Carling Adrenal Center suggest that the use of a human amniotic membrane allograph on the adrenal gland remnant following partial adrenal surgery leads to faster recovery of normal adrenal gland function. Rather than removing the entire adrenal gland—which has been standard of care for decades—a portion of the adrenal gland is able to be salvaged with amniotic membrane placed upon the remnant as a biologic covering. The preliminary data from an ongoing clinical trial shows this technique translates into fewer patients needing steroid hormone replacement following adrenal surgery, and if they do, it is for a significantly shorter period of time. "Sometimes it is possible, and preferable, to remove the adrenal tumor without removing the entire adrenal gland. This is called partial adrenal surgery and our study shows this technique is more successful when amniotic membrane is used," said Dr. Carling. He further stresses that "removing only part of the adrenal gland is a more advanced operation and is typically only performed by expert adrenal surgeons. The goal is to leave some normal adrenal tissue so that the patient can avoid adrenal insufficiency which requires a daily dose of several adrenal hormones and steroids. Partial adrenal surgery is especially beneficial for patients with pheochromocytoma, as well as Conn's and Cushing's syndrome. Avoiding daily steroids is life-changing for these patients so this is a major breakthrough." So how does it work? The increased viability of the adrenal gland remnant is presumed to be related to the release of growth factors known to be present in amniotic tissue which is in direct contact with the adrenal gland remnant as a covering. The results are improved rates of viable adrenal cortical tissues with faster regeneration and recovery to normal endocrine physiology by the adrenal cortical cells. These findings come during Adrenal Disease Awareness Month. Adrenal gland diseases cause many debilitating symptoms like chronic headaches, anxiety, depression, fatigue, brain fog, memory loss, dangerously high blood pressure, heart arrythmia, weight gain, tremors, and more, yet they are often misdiagnosed or improperly treated. Since many doctors are inexperienced in the workup of adrenal hormone problems and only see a handful of adrenal tumors during their careers, it is important for patients to know about the symptoms of adrenal tumor disease and request their doctor measure adrenal hormones. Adrenal.com is the leading resource for adrenal gland function, tumors and cancers, and an award-winning resource for adrenal gland surgery. The diagnosis and surgical treatment of all types of adrenal tumor types are discussed. Adrenal.com is edited by Dr. Tobias Carling who has performed more adrenal surgery than any other surgeon and has published some of the most important scientific studies of adrenal disease and adrenal surgery including the understanding of the pathogenesis of pheochromocytoma and adrenal tumors causing Conn's and Cushing's syndrome. Established by Dr. Tobias Carling in 2020, the Carling Adrenal Center located at the Hospital for Endocrine Surgery in Tampa FL, is the highest volume adrenal surgical center in the world. The Center now averages nearly 20 adrenal tumor patients every week. Dr Carling was the Director of Endocrine Surgery at Yale University prior to opening the Center in Tampa. At the new Hospital for Endocrine Surgery, Dr Carling joins the Norman Parathyroid Center, the Clayman Thyroid Center and the Scarless Thyroid Surgery Center as the highest volume endocrine surgery center in the world. About the Carling Adrenal Center: Founded by Dr. Tobias Carling, one of the world's leading experts in adrenal gland surgery, the Carling Adrenal Center is a worldwide destination for the surgical treatment of adrenal tumors. Dr. Carling spent nearly 20 years at Yale University, including 7 as the Chief of Endocrine Surgery before leaving in 2020 to open to Carling Adrenal Center, which performs more adrenal operations than any other hospital in the world. (813) 972-0000. More about partial adrenalectomy for adrenal tumors can be found at the Center's website www.adrenal.com. From https://www.streetinsider.com/PRNewswire/Novel+application+of+amniotic+membrane+saves+adrenal+tissue+in+patients+undergoing+adrenal+surgery/19915274.html

Single-cell transcriptome analysis identifies a unique tumor cell type producing multiple hormones in ectopic ACTH and CRH secreting pheochromocytoma Abstract Ectopic Cushing’s syndrome due to ectopic ACTH&CRH-secreting by pheochromocytoma is extremely rare and can be fatal if not properly diagnosed. It remains unclear whether a unique cell type is responsible for multiple hormones secreting. In this work, we performed single-cell RNA sequencing to three different anatomic tumor tissues and one peritumoral tissue based on a rare case with ectopic ACTH&CRH-secreting pheochromocytoma. And in addition to that, three adrenal tumor specimens from common pheochromocytoma and adrenocortical adenomas were also involved in the comparison of tumor cellular heterogeneity. A total of 16 cell types in the tumor microenvironment were identified by unbiased cell clustering of single-cell transcriptomic profiles from all specimens. Notably, we identified a novel multi-functionally chromaffin-like cell type with high expression of both POMC (the precursor of ACTH) and CRH, called ACTH+&CRH + pheochromocyte. We hypothesized that the molecular mechanism of the rare case harbor Cushing’s syndrome is due to the identified novel tumor cell type, that is, the secretion of ACTH had a direct effect on the adrenal gland to produce cortisol, while the secretion of CRH can indirectly stimulate the secretion of ACTH from the anterior pituitary. Besides, a new potential marker (GAL) co-expressed with ACTH and CRH might be involved in the regulation of ACTH secretion. The immunohistochemistry results confirmed its multi-functionally chromaffin-like properties with positive staining for CRH, POMC, ACTH, GAL, TH, and CgA. Our findings also proved to some extent the heterogeneity of endothelial and immune microenvironment in different adrenal tumor subtypes. Editor's evaluation The study described an extremely rare type of adrenal pheochromocytoma that secretes both ACTH and CRH, in addition to catecholamines. Single-cell RNA sequencing of the tumor and other tumors revealed a group of cells that are responsible for the hormone secretion. We believe that this work will provide an interesting example of functional endocrine tumors and how they are formed. https://doi.org/10.7554/eLife.68436.sa0 Introduction Cushing’s syndrome (CS) is a rare disorder caused by long-term exposure to excessive glucocorticoids, with an annual incidence of about 0.2–5.0 per million (Lacroix et al., 2015; Newell-Price et al., 2006; Lindholm et al., 2001; Steffensen et al., 2010; Bolland et al., 2011; Valassi et al., 2011). About 80% of CS cases are due to ACTH secretion by a pituitary adenoma, about 20% are due to ACTH secretion by nonpituitary tumors (ectopic ACTH syndrome [EAS]), and 1% are caused by corticotropin-releasing hormone (CRH)-secreting tumors (Alexandraki and Grossman, 2010; Ejaz et al., 2011; Ballav et al., 2012). Most EAS tumors (~60%) are more common intrathoracic tumors, only 2.5–5% of all EAS are caused by a pheochromocytoma (Alexandraki and Grossman, 2010; Isidori et al., 2006; Ilias et al., 2005; Aniszewski et al., 2001). Pheochromocytoma, a catecholamine-producing tumor, becomes even rarer when it is capable of both secreting ACTH and CRH (Lenders et al., 2005; Zelinka et al., 2007). By 2020, only two cases with pheochromocytoma secreted both ACTH and CRH were reported (Elliott et al., 2021; O’Brien et al., 1992; Jessop et al., 1987). As one of the largest adrenal tumor treatment centers in China, our hospital, Peking Union Medical College Hospital (PUMCH) receives more than 500 adrenal surgery performed per year, with almost 100 cases undergoing pheochromocytoma surgery. But so far, we have encountered only one case of pheochromocytoma secreting both ACTH and CRH, which was first reported in this study. Since the combination of dual ACTH/CRH secreting pheochromocytoma with CS is extremely rare, there is limited knowledge about the diagnosis and management of this disease. Ectopic secretion hormones ACTH and CRH may complicate the presentation of pheochromocytoma, and this tumor usually leads to CS, which can be fatal if not properly diagnosed and managed (Ballav et al., 2012; Ilias et al., 2005; Lenders et al., 2014; Lase et al., 2020). Surgical resection of the pheochromocytoma is the primary treatment option. Although previous studies have reported ectopic ACTH and CRH secreting pheochromocytomas, it was unclear whether a unique cell type that produces multiple hormones influences CS. The concept of ‘one cell, one hormone, and one neuron one transmitter,’ which is known as Dale’s Principle (Dale in 1934; for detailed discussion, see Burnstock, 1976), has dominated the understanding of neurotransmission for many years (Burnstock, 1976). Currently, single-cell RNA-sequencing (scRNA-seq) can examine the expression profiles of a single cell and is recognized as the gold standard for defining cell states and phenotypes (Tang et al., 2009; Tammela and Sage, 2020; Kolodziejczyk et al., 2015; Patel et al., 2014; Tirosh et al., 2016b; Tirosh et al., 2016a; Puram et al., 2017; Venteicher et al., 2017; Young et al., 2018; Bernard et al., 2019; Segerstolpe et al., 2016; Reichert and Rustgi, 2011). It can reveal the presence of rare and novel unique cell types, such as CFTR-expressing pulmonary ionocytes on lung airway epithelia (Montoro et al., 2018; Plasschaert et al., 2018). It also provides an unbiased method to better understand the diversity of immune cells in the complex tumor microenvironment (Papalexi and Satija, 2018; Stubbington et al., 2017). In this study, we reported a rare case of CRH/ACTH-secreting pheochromocytoma infiltrating the kidney and psoas muscle tissue. scRNA-seq identified a unique chromaffin-like cell type, called ACTH+&CRH + pheochromocyte, with both high expression of POMC (precursor for ACTH) and CRH pheochromocyte as well as TH (tyrosine hydroxylase, a key enzyme for catecholamine synthesization). Immunocytochemical and immunofluorescence staining showed all for these markers, which confirmed the tumor capable of multiple hormones secreting characteristics. We determined that the expression of POMC directly causes the secretion of ACTH, and the expression of CRH indirectly promotes the secretion of ACTH hormone, which ultimately leads to CS. After the tumor resection, clinical manifestations also showed complete remission of CS. For comparison, other adrenal tumor subtypes were also collected and studied, namely, a common pheochromocytoma (without ectopic ACTH or CRH secretion function) and two adrenocortical adenomas. We used a scRNA-seq approach to obtain transcriptomic profiles for all collected samples and identified a list of differentially expressed genes (DEGs) through cell clustering and markers finding. Notably, GAL, co-expressed with ACTH and CRH, could be a new candidate marker to detect the rare ectopic ACTH+&CRH + secreting pheochromocytes by comparing ACTH+&CRH + pheochromocyte with common pheochromocyte and cortical cell clusters. It suggested that GAL, which encodes small neuroendocrine peptides, may be locally involved in the regulation of the hypothalamic-pituitary-adrenal (HPA) axis. Results Single-cell profiling and unbiased clustering of collecting specimens We applied scRNA-seq methods to perform large-scale transcriptome profiling of seven prospectively collected samples from tumors and peritumoral tissue of three adrenal tumor patients (Figure 1A). Case 1 suffered from a rare pheochromocytoma with typical Cushingoid features. The laboratory results showed high levels of cortisol, ACTH, and catecholamines. The abdominal contrast-enhanced computer tomography scanning revealed bilateral adrenocortical hyperplasia and irregular tumor within the left adrenal. After the resection, we collected three dissected tumor specimens (esPHEO_T1, esPHEO_T2, and esPHEO_T3) from different anatomic sites of the tumor and an adrenal tissue adjacent to the tumor (esPHEO_Adj). For comparison, we also collected other adrenal tumors, namely, a common pheochromocytoma (PHEO_T) from Case 2 and two adrenocortical adenomas (ACA_T1 and ACA_T2) from Case 3. Case 2 showed elevated catecholamines and normal levels of cortisol and ACTH. Case 3 showed a high level of cortisol, a low level of ACTH, and an intermediate level of catecholamines. The detailed clinical information for the three cases was summarized in Appendix 1—table 1. To investigate the difference of the secretory function, we performed the immunohistochemistry (IHC) staining of selected markers, CgA (chromogranin A) and ACTH in esPHEO_T1, PHEO_T, and esPHEO_Adj samples (Figure 1B). We observed that CgA positive cells were present in both pheochromocytomas (esPHEO_T1 and PHEO_T), but ACTH positive cells were only observed in the rare pheochromocytoma (esPHEO_T1) with the ACTH-secreting cellular characteristics. As expected, there were no CgA and ACTH positive cells in the adjacent sample (esPHEO_Adj). Thus, at the clinical stage, our histopathology results confirmed that Case 1 was a rare ectopic ACTH secreting pheochromocytoma which stained positively for both ACTH and CgA. Figure 1 Download asset Open asset Clinical sample collection of adrenal tumor and adjacent specimen for scRNA-seq analysis. (A) scRNA-seq workflow for three tumor specimens (esPHEO_T1, esPHEO_T2, and esPHEO_T3) and one adjacent specimen (esPHEO_Adj) from the rare pheochromocytoma with ectopic ACTH and CRH secretion (Case … see more Then, we applied scRNA-seq approaches to selected seven specimen samples (six tumors and one sample adjacent to the tumor). The tissues after resection were rapidly digested into a single-cell suspension, and the 3′-scRNA-seq protocol (Chromium Single Cell 3′ v2 Libraries) was performed for each sample unbiasedly. After quality control filtering to remove cells with low gene detection, high mitochondrial gene coverage, and doublets filtration, we compiled a unified cells-by-genes expression matrix of a total of 44,511 individual cells (Supplementary file 1, Appendix 1—figure 2). Then the SCT-transformed normalization, principal component analysis (PCA), was employed to perform unsupervised dimensionality reduction. Then, the cells were clustered based on the graph-based clustering analysis, and visualized in the distinguished diagram using the Uniform Manifold Approximation and Projection (UMAP) method. The marker genes were calculated to identify each cell cluster by performing differential gene expression analysis (Supplementary file 2). As shown in Figure 2A, the distinct cell clusters were identified and the conventional cell lineage gene markers were employed to annotate the clusters, such as CHGA and CHGB for adrenal chromaffin cell, cytochrome P450 superfamily for adrenocortical cell, S100B for sustentacular cell, GNLY for NK cell, MS4A1 for B cell, CD8A for CD8+ T cell, and IL7R for CD4+ T cell. Based on the expression of gene markers, we recognized a total of 16 main cell groups: ACTH+&CRH + pheochromocyte, pheochromocyte, adrenocortical, sustentacular, erythroblast/granulosa, endothelial, fibroblast, neutrophil, monocyte, macrophage, plasma, B, NK, CD8+ T&NKT, CD8+ T, and CD4+ T, among which the endothelial cell group was composed of four endothelial cell subgroups. The heatmap showed the expression levels of specific cluster markers for each cell phenotype that we identified (Figure 2B). For this analysis, we specifically focused on the four types of adrenal cells and showed their markers in a heatmap (Appendix 1—figure 3). Additionally, we detected the transcription factors alongside their candidate target genes, which are jointly called regulons. The analysis scored the activity of regulon for each cell (Appendix 1—figure 4A) and yielded specific regulons for each cellular cluster (Appendix 1—figure 4B). We also specifically focused on the adrenal cells and found XBP1 as the top regulons for ACTH+&CRH + pheochromocyte and adrenocortical cell type (Appendix 1—figure 4C). Figure 2 Download asset Open asset Different cell types and their highly expressed genes through single-cell transcriptomic analysis. (A) The t-distributed stochastic neighbor embedding (t-SNE) plot shows 16 main cell types from all specimens. (B) Heatmap shows the scaled expression patterns of the top 10 marker genes in each cell … see more Identification of a previously unrecognized cell type The presence of heterogeneous cell populations in different adrenal tumor specimens and the peritumoral sample (Figure 3A) prompted us to investigate their cellular compositions and characteristics. As shown in Figure 3B, different sources of specimens represented distinct cell type compositions. Notably, although the size of the cell clusters of the adrenal gland was relatively small, four distinct subtypes of adrenal cells were observed, including ACTH+&CRH + pheochromocyte, pheochromocyte, adrenocortical cells, and sustentacular cells. The ACTH+&CRH + pheochromocytoma cell subtype was specific to three tumor samples, esPHEO_T1, esPHEO_T2, and esPHEO_T3 from Case 1, but was not observed in the peritumoral sample (esPHEO_Adj) and other adrenal tumor samples from Case 2 (PHEO_T) and Case 3 (ACA_T1 and ACA_T2). This result was consistent with the clinical symptoms in our earlier reports that ACTH was only over-secreted in pheochromocytoma of Case 1. The cell cluster of ACTH+&CRH + pheochromocyte was supported by the specific expression of the markers POMC (proopiomelanocortin) and CRH (corticotropin-releasing hormone) (Figure 3C). POMC is a precursor of ACTH, and CRH is the most important regulator of ACTH secretion. We also detected another specific expression signal, GAL, for the cell cluster of ACTH+&CRH + pheochromocyte (Figure 3C). GAL encodes small neuroendocrine peptides and can regulate diverse physiologic functions, including growth hormone, insulin release, and adrenal secretion (Ottlecz et al., 1988; McKnight et al., 1992; Murakami et al., 1989; Hooi et al., 1990). A study found that GAL and ACTH were co-expressed in human pituitary and pituitary adenomas, and suggested that GAL may be locally involved in the regulation of the HPA axis (Hsu et al., 1991). We demonstrated that GAL was expressed in the ACTH+&CRH + pheochromocyte and might participate in the regulation ATCH secretion (Figure 3C). Then we examined the known adrenal chromaffin cell markers (CHGA and CHGB) and the markers for catecholamine-synthesizing enzymes (TH and PNMT) (Figure 3C). These known markers and another new candidate marker CARTPT were observed in both ACTH+&CRH + pheochromocyte and pheochromocyte cell subtypes. The CYP17A1 and CYP21A2, the typical markers of the adrenal cortical cell subtype, were also investigated (Figure 3C). They are members of the cytochrome P450 superfamily, encoding key enzymes, and maybe the precursors of cortisol in the adrenal glucocorticoids biosynthesis pathway (Auchus et al., 1998; Petrunak et al., 2014). Finally, a subtype of cells with positive expression of S100B was identified, called sustentacular cells. Sustentacular cells were found near chromaffin cells and nerve terminations. Several studies have shown that sustentacular cells exhibit stem-like characteristics (Pardal et al., 2007; Fitzgerald et al., 2009; Poli et al., 2019; Scriba et al., 2020). Figure 3 Download asset Open asset A unique tumor cell type was revealed by the composition analysis of cell types in each sample. The results validated an ectopic ACTH and CRH secreting pheochromocytoma. (A) Cell clusters shown in UMAP map can be subdivided by different specimens. (B) Frequency distribution of cell types among … see more Our scRNA-seq analysis validated that the mRNA expression of POMC (precursor for ACTH) and CRH in pheochromocyte triggered the pathophysiology of ectopic ACTH and CRH syndromes, thereby stimulating the adrenal glands to release cortisol. The overexpression of TH and PNMT was responsible for the excessive secretion of catecholamines in the ACTH+&CRH + pheochromocyte and pheochromocyte cell subtypes. Tumor samples (esPHEO_T1, esPHEO_T2, and esPHEO_T3) from Case 1 and PHEO_T from Case 2 were demonstrated to have the function of producing catecholamine. These genes related to catecholamine secretion were all negative for adrenocortical cell subtypes because the catecholamine-producing pheochromocytomas originated from chromaffin cells in the adrenal medulla rather than the adrenal cortex. Our laboratory tests were consistent with these results, that is, both Case 1 and Case 2 had a high level of catecholamines in plasma and 24 hr urine while Case 3 had a normal level. We also found CARTPT was similar to PNMT and can be used as a marker for ACTH+&CRH + pheochromocyte and pheochromocyte. Chromaffin cell markers CHGA and CHGB were mainly characterized in PHEO_T and three tumor samples from Case 1. Adrenocortical cell clusters mainly existed in ACA_T1 and ACA_T2, but a few existed in esPHEO_Adj. S100B was specifically identified in PHEO_T. An absence of S100-positive sustentacular cells has been previously confirmed in most malignant adrenal pheochromocytomas, and the locally aggressive or recurrent group usually contains a large number of these cells (Unger et al., 1991). It suggests that PHEO_T from Case 2 might be a locally aggressive case, while Case 1 is the opposite. To validate this finding, we performed additional IHC staining experiments on paraffin-embedded serial slices with similar tissue regions from the tumor specimen esPHEO_T3 using antibodies against CgA, ACTH, POMC, CRH, TH, and GAL. We did find that these markers were all positive in the tumor tissue, which further indicated that the special rare pheochromocytoma exhibited multiple hormone-secreting characteristics, including ACTH, CRH, and catecholamines (Figure 3D, Appendix 1—figure 8). We also prepared two serial slices for immunofluorescence co-staining for POMC&CRH and POMC&TH. The legible co-localization signals were observed, where the green signal was for POMC, and the red signal was for CRH and TH (Figure 3E, Appendix 1—figure 9). This result confirmed the ACTH and CRH secreting pheochromocytoma from Case 1 contained a unique multi-functional chromaffin-like cell type, which was consistent with the analysis result by scRNA-seq. Differential expression genes show adrenal tumor cell-type specificity Next, we analyzed the DEGs between ACTH+&CRH + pheochromocyte and the other two subtypes of adrenal tumor cells (pheochromocyte and adrenocortical cells). It is worth noting that many genes were dramatically upregulated specifically in ACTH+&CRH + pheochromocyte when compared with the other tumor cell types, such as GAL, POMC, PNMT, and CARTPT (Figure 4A). Using these upregulated or downregulated genes, we performed functional enrichment analysis based on gene ontology (GO) annotation to further characterize the molecular characteristics of different tumor cell types. In comparison with adrenocortical cell types, the highly upregulated genes of ACTH+&CRH + pheochromocyte were mainly enriched in the neuropeptide signaling pathway, hormone secretion, and transport, while the downregulated genes were mostly enriched in the pathway of adrenocortical hormones (Figure 4B). Comparing the two types of pheochromocyte, GO functional enrichment analysis for the biology process (BP) revealed that the upregulated genes for ACTH+&CRH + pheochromocyte were also enriched in the neuropeptide signaling pathway, while the enrichment of the downregulated genes from the GO functional result hardly reach statistical significance. Interestingly, compared with adrenocortical cells, a total of 248 upregulated and 198 downregulated genes were detected in ACTH+&CRH + pheochromocyte, while only 95 upregulated and 111 downregulated genes were detected in ACTH+&CRH + pheochromocyte when compared with pheochromocyte (Figure 4C), which suggested that the difference between ACTH+&CRH + pheochromocyte and pheochromocyte was relatively small. The known adrenal chromaffin cell markers (CHGA and CHGB) were differential expressed significantly between ACTH+&CRH + pheochromocyte and adrenocortical cells, but not observed significant difference between two subtypes of pheochromocytes. Besides, the co-upregulated genes, such as CARTPT, PNMT, POMC, GAL, and CRH, were responsible for the production of a variety of hormones and involved in neuropeptide signaling pathways. Of which, the product of PNMT catalyzes the last step of the catecholamine biosynthesis pathway, methylating norepinephrine to form epinephrine. The overexpression of PNMT was responsible for the significantly elevated epinephrine (Appendix 1—table 1) of the rare Case 1 with ectopic ACTH and CRH secretory pheochromocytoma. The elevated plasma ACTH (Appendix 1—table 1) of the rare Case 1 could be explained by specific high expression signals of GAL, POMC, and CRH. In details, POMC is the precursor of ACTH; CRH is the most important regulator of ACTH secretion; and GAL was co-expressed in the ACTH+&CRH + pheochromocyte, which might be locally involved in the regulation of the HPA axis. Therefore, we concluded that the tumor cell type of ACTH+&CRH + pheochromocyte from Case 1 had multiple hormone secretion functions, namely, CRH secretion function, ACTH secretion function, and catecholamine secretion function. Furthermore, we believed that the rare Case 1 harbor the ACTH-dependent CS is due to the presence of the identified novel tumor cell type of ACTH+&CRH + pheochromocyte, which secretes both ACTH and CRH. The secretion of ACTH had a direct effect on the adrenal gland to produce cortisol, while the secretion of CRH can indirectly stimulate the secretion of ACTH from the anterior pituitary (Figure 4D). Figure 4 Download asset Open asset Altered functions in POMC+&CRH + pheochromocyte revealed by differential gene expression analysis. (A) Volcano plot of changes in gene expression between POMC+&CRH + pheochromocytes and other adrenal cell types (pheochromocytes and adrenocortical cells). The x-axis specifies the natural logarithm … see more RNA velocity analysis To investigate dynamic information in individual cells, we performed RNA velocity analysis using velocyto.py for spliced or unspliced transcripts annotation followed by scVelo pipeline for RNA dynamics modeling. RNA velocity is the time derivative of the measured mRNA abundance (spliced/unspliced transcripts) and allows to estimate the future developmental directionality of each cell (La Manno et al., 2018). We observed the ratios of spliced and unspliced mRNA, and sustentacular cell type was ranking first with 36% unspliced proportions among non-immune cell types (Figure 5A and B). The balance of unspliced and spliced mRNA abundance is an indicator of the future state of mature mRNA abundance, and thus the future state of the cell (Bergen et al., 2020). Previously study had observed unspliced transcripts were enriched in genes involved in DNA binding and RNA processing in hematopoietic stem cells (Bowman et al., 2006). For the high proportions of unspliced/spliced transcripts, stem-like characteristics of sustentacular cells were supported. There were more spliced transcripts proportions in POMC+&CRH + pheochromocytes than in pheochromocytes (Figure 5B). Then, we estimated pseudotime grounded on transcriptional dynamics and generated velocity streamlines that account for speed and direction of motion. As observed in the pseudotime of four adrenal cell subtypes, medullary cells are earlier than cortical cells (Figure 5C). From velocity streamlines, we found the four adrenal cell subtypes, that is, POMC+&CRH + pheochromocytes, pheochromocytes adrenocortical cells, and sustentacular cells, were independent respectively and not directed toward other cell types (Figure 5D). Newly transcribed, unspliced pre-mRNAs were distinguished from mature, spliced mRNAs by detecting the presence of introns. Genes, like POMC and CRH, only contain one coding sequence (CDS) region, were all detected as spliced (Appendix 1—figure 5). It indicated that the actual values of RNA velocity for POMC+&CRH + pheochromocytes might be larger than the predicted ones. Furthermore, the spliced versus unspliced phase for CHGA, CHGB, and TH demonstrated a clear more dynamics expression in POMC+&CRH + pheochromocytes than in pheochromocytes (Appendix 1—figure 5). Figure 5 Download asset Open asset RNA velocity analysis supported sustentacular cells as root and indicated four adrenal cell subtypes were independent respectively and not directed toward other cell types. RNA velocity is the time derivative of the measured mRNA abundance (spliced/unspliced transcripts) and allows to estimate the future developmental directionality of each cell. (A) The total ratios … see more Lineage tracing analysis confirms the plasticity of adrenal tumor cell subsets We performed the pseudotime analysis for the adrenal tumor cell subsets to determine the pattern of the dynamic cell transitional states. We used the recommended strategy of Monocle to order cells based on genes that differ between clusters. The sustentacular cells were in an early state in pseudotime analysis (Figure 6A, B and C), which was in accordance with their exhibited stem-like properties and the highest unspliced proportion among non-immune cell types in the RNA velocity analysis. The results also showed a transition from sustentacular cells to pheochromocytes and then to ACTH+&CRH + pheochromocyte, and adrenocortical cells were on another branch (Figure 6A, B and C). To determine whether specific gene modules might be responsible for this cell plasticity, we calculated the expression levels of all the genes in the single-cell transcriptome identified the DEGs on the different paths through the entire trajectory (Figure 6D), which showed the dynamic changes of each gene over pseudotime. Figure 6 Download asset Open asset Pseudotime analysis of adrenal cells inferred by Monocle. We ran reduce dimension with t-SNE for four types of adrenal cells and sorted cells along pseudotime using Monocle. The single-cell pseudotime trajectories by ordering cells were constructed based … see more scRNA-seq reveals distinct immune and endothelial cell type in the tumor microenvironment scRNA-seq allowed us to use an unbiased approach to discover the composition of immune cell populations of the adrenal tumor specimens. Analysis of our transcriptional profiles revealed that from the frequency distribution of cell clusters, immune cells accounted for more than ~50% of total cells (Figure 3B). We identified and annotated the immune cell types based on the expression of conventional markers, such as B cells with MS4A1, NK cells with GNLY, and Neutrophil with S100A8 and S100A9 (Figure 7A). The various frequency distribution of immune cell sub-clusters was observed among different samples (Figure 7B). Due to the identical tumor microenvironment, all three tumor specimens one peritumoral specimen from the rare case had similar immune cell composition. Interestingly, the CD4 T cells, B cells, and macrophages are mainly presented in two adrenal cortical adenomas (ACA_T1 and ACA_T2), while the CD8 T cells mostly resided in the microenvironment of other pheochromocytoma tumor and the peritumoral specimen. We found the heterogeneity of T cells in different adrenal tumor subtypes, that is, compared with CD4 T cells in adrenocortical adenomas, the pheochromocytoma types were mostly manifested by activated CD8+, especially in the anatomic specimens from the ectopic ACTH&CRH secreting pheochromocytoma. Figure 7 Download asset Open asset Diverse immune microenvironments in different adrenal tumor subtypes and tumor-adjacent tissue. (A) The UMAP diagram shows the expression levels of well-known marker genes of immune cell types. (B) Frequency distribution of immune cell sub-clusters in different adrenal tumors and … see more Endothelial cells consisted of four distinct sub-clusters: vascular endothelial cells, lymphatic endothelial cells, cortical endothelial cells, and other endothelial cells, as shown in the cell cluster distribution map highlighted by endothelial cells (Figure 8A, Supplementary file 3). Various adrenal tumor subtypes had different endothelial compositions (Figure 8B). Vascular endothelial cells were mainly identified in pheochromocytoma samples (esPHEO_T1, esPHEO_T2, esPHEO_T3, and PHEO_T), because pheochromocytoma is a tumor arising in the adrenal medulla, and vascular endothelial cells might be detected from the medullary capillary. Cortical endothelial cells were mainly detected in adrenocortical adenomas (ACA_T1 and ACA_T2). Lymphatic endothelial cells were found in the adjacent adrenal specimen of the rare ACTH+&CRH + pheochromocytoma (esPHEO_Adj). Then, by comparing vascular endothelial cells with two other subclusters (lymphatic endothelial cells and cortical endothelial cells), we found the markers across the subclusters of endothelial cells and annotated GO function of differentially expressed genes (Figure 8C and D). Vascular endothelial cells are the barrier between the blood and vascular wall and have the functions of organizing the extracellular matrix and regulating the metabolism of vasoactive substances. Lymphatic endothelial cells are responsible for chemokine-mediated pathways. Cortical endothelial cells express TFF3 and FABP4, which are involved in repairing and maintaining stable functions. Figure 8 Download asset Open asset Differential gene expression analysis shows changes in endothelial cell functions. (A) The UMAP diagram shows four different endothelial cell sub-clusters. (B) Frequency distribution of endothelial cell sub-clusters among different adrenal tumors and tumor-adjacent specimen. (C) … see more Discussion Both CS and pheochromocytoma are serious clinical conditions. In this study, we reported an extremely rare patient (Case 1) with ATCH-dependent CS due to an ectopic ACTH&CRH secreting pheochromocytoma. Surgery is the most common treatment strategy for this type of tumor. After the operation, our clinical manifestations of Case 1 showed the complete remission of CS. The IHC of the dissected tumor confirmed the diagnosis with positive staining for CRH and ACTH. In this study, scRNA-seq was used for the first time to identify the rare ACTH+&CRH + pheochromocyte cell subset. Compared with other subtypes of adrenal tumors, the common pheochromocytoma (from Case 2) and adrenal cortical cells (from Case 3), the DEGs in Case 1 were further characterized. Case 2 was examined to have normal levels of cortisol and ACTH, but Case 3 showed a Cushingoid appearance. The molecular mechanism of CS in Case 3 was different, which was attributed to two cortical adenomas on the left adrenal, showing ACTH-independent hypercortisolemia. In addition, to investigate the genetic driver for Case 1, we supplemented whole-exome sequencing experiments for all rest specimens, that is, tumors (esPHEO_T2 and esPHEO_T3) and controls (esPHEO_Adj and esPHEO_Blood) from the rare case with ectopic ACTH&CRH-secreting pheochromocytoma. Filtered germline and somatic mutations were listed in Supplementary file 4 including detailed annotations. Genetic mutations of phaeochromocytoma and paraganglioma are mainly classified into two major clusters, that is, pseudo hypoxic pathway and kinase signaling pathways (Pillai et al., 2016; Nölting and Grossman, 2012). We did not find any gene mutations that were related to these two major clusters. We only identified one shared somatic variant of ACAN (c.5951T > A:p.L1984Q) comparing variants in tumor samples to controls but Sanger sequencing only confirmed the presence in esPHEO_T3 which was not observed in esPHEO_T2 (Appendix 1—figure 7). ACAN, encoding a major component of the extracellular matrix, is a member of the aggrecan/versican proteoglycan family. Mutations of ACAN were reported related to steroid levels (Yousri et al., 2018). It is well-established that circulating steroid levels are linked to inflammation diseases such as arthritis, because arthritis as well as most autoimmune disorders results from a combination of several predisposing factors including the stress response system such as hypothalamic-pituitary-adrenocortical axis (Cutolo et al., 2003). But no direct evidence related to ACAN to phaeochromocytoma. Therefore, no obvious genetic driver was found to explain the rare case of ACTH/CRH-secreting phaeochromocytoma. Further investigations would be needed to uncover the relation between ACAN and phaeochromocytoma. For many years, the understanding of neurotransmission has been dominated by the concept of ‘one cell, one hormone, and one neuron one transmitter,’ which is known as Dale’s Principle (Dale in 1934; for detailed discussion, see Burnstock, 1976; Burnstock, 1976). Sakuma et al., 2016 reported an ectopic ACTH pheochromocytoma case and proved that ACTH and catecholamine were produced by two functionally distinct chromaffin-like tumor cell types through immunohistochemical analysis Sakuma et al., 2016. However, more and more evidence has emerged that Dale’s principle is incorrect because existing studies have shown that these cells are multi-messenger systems (Hakanson and Sundler, 1983; Apergis-Schoute et al., 2019; Svensson et al., 2018). Based on scRNA-seq results, we concluded that the tumor cells from Case 1 had multiple hormone secretion functions, namely, CRH secretion function, ACTH secretion function, and catecholamine secretion function. CRH is the most important regulator of ACTH secretion. Therefore, we believed that the secretion of both CRH and ACTH of this tumor led to ACTH-dependent CS. Besides, the secretion of ACTH had a direct impact on the adrenal gland to produce cortisol, and the secretion of CRH indirectly stimulated the secretion of ACTH by the anterior pituitary. Jessop et al., 1987 also draw the same conclusion in their report in 1987. However, in the reported case, the histological immunostained result was shown only for the corticotropin-releasing factor (CRF-41), but not for ACTH (Jessop et al., 1987). Adrenal glands are composed of two main tissue types, namely, the cortex and the medulla, which are responsible for producing steroid and catecholamine hormones, respectively. The inner medulla is derived from neuroectodermal cells of neural crest origin, while the outer cortex is derived from the intermediate mesoderm. In the adrenal pheochromocytomas, a third cell type with the positive expression of S100B was identified, called ‘sustentacular’ cells (Suzuki and Kachi, 1995; Lloyd et al., 1985). By evaluating 17 malignant and recurrent or locally aggressive adrenal pheochromocytomas, Unger et al., 1991 found that sustentacular cells were absent in most malignant cases (Unger et al., 1991). Because there are no sustentacular cells in ACTH&CRH secreting pheochromocytoma, ACTH&CRH secreting pheochromocytoma is more serious than the common pheochromocytoma. Furthermore, several studies have demonstrated that sustentacular cells exhibit stem-like characteristics (Pardal et al., 2007; Fitzgerald et al., 2009; Poli et al., 2019; Scriba et al., 2020). A unique case of a tumor originating from S100-positive sustentacular cells was previously reported (Lau et al., 2006). The RNA velocity estimation and pseudo-time analysis of different adrenal cell subtypes supported the sustentacular cells exhibiting stem-like properties. Although pheochromocyte was prior to ACTH&CRH secreting pheochromocyte in pseudotime order, the RNA velocity prediction of POMC+&CRH+ pheochromocytes might be under-estimated because the transcripts of POMC and CRH were all predicted as spliced ones. Based on the spliced versus unspliced phase for CHGA, CHGB, and TH, it showed a clear more dynamics expression in POMC+&CRH+ pheochromocytes than in pheochromocytes. We assumed that ACTH&CRH secreting pheochromocyte have more hormone-producing functions, retain stem- and endocrine-differentiation ability. But further experiments are needed to validate our hypothesis. There are bidirectional communications between the immune system and the neuroendocrine system (Blalock, 1989). Hormones produced in the endocrine system, especially glucocorticoids, affect the immune system to modulate its function (Imura and Fukata, 1994). Other hormones, such as growth hormone (GH) and prolactin (PRL), also modulate the immune system (Blalock, 1989). It has been proved that the exogenous production of cytokines can stimulate and mediate the release of multiple hormones including ACTH, CRH (Rivier et al., 1989; Bernton et al., 1987), and induce the activation of the HPA axis (Gisslinger et al., 1993; Fukata et al., 1994; Kakucska et al., 1993; Murakami N Fukata et al., 1992). Human T cells coordinate the adaptive immunity of different anatomic compartments by producing cytokines and effector molecules (Szabo et al., 2019). The activation of naive T cells through the antigen-specific T cell receptor (TCR) can initiate transcriptional programs that can drive the differentiation of lineage-specific effector functions. CD4+ T cells secrete cytokines to recruit and activate other immune cells, while CD8+ T cells have cytotoxic functions and can directly kill infected or tumor cells. Recent studies have shown that the composition of the T cell subset is related to the specific tissue locations (Carpenter et al., 2018; Thome et al., 2014). scRNA-seq can be used to deconvolve the immune system heterogeneity with high resolution. Compared with adrenocortical adenomas which were in CD4+ (with the expression of cytokine receptors, such as the IL-7R) state, T cells in pheochromocytoma, especially T cells in the ectopic ACTH&CRH secreting pheochromocytoma were inactivated CD8+ state, suggesting different tumor microenvironments between adrenocortical adenomas and pheochromocytoma. Previous studies have shown that signaling through IL-7R is essential in the developmental process and regulation of lymphoid cells (Kondrack et al., 2003; Tan et al., 2001; Tan et al., 2002; Lenz et al., 2004; Li et al., 2003; Seddon et al., 2003), and disruption of the IL-7R signaling pathway may lead to skewed T cell distribution and cause immunodeficiency (Maraskovsky et al., 1996; Kaech et al., 2003; Carini et al., 1994). Our results indicated the heterogeneity of the immune system between different samples, and CD4+ T cells with the high expression level of IL-7R might be related to adrenal tumor progression, apoptosis, or factors influencing progression such as immune activation. Although we have shown the heterogeneity of immune cell types in different adrenal tumor subtypes, it is unclear how T cells influence different markers, including effector states and interferon-response states. In addition to composition differences, a deeper understanding of the complex interactions between adrenal tumor tissues and immune systems is a key issue in neuroendocrine tumor research. Overall, we reported a rare case in which ectopic ACTH&CRH-secreting pheochromocytoma on the left adrenal that infiltrated around the kidney and psoas major tissues. We applied scRNA-seq to identify this rare and special adrenal tumor cell. Thus, the majority of our analysis focused on the validation of novel tumor cell type and their multiple hormones-secreting functions, namely, CRH secretion function, ACTH secretion function, and catecholamine secretion function. Also, GAL could be a candidate marker to detect the rare ectopic ACTH+&CRH + secreting pheochromocytes. For future studies, on one hand, we are very concerned about similar suspicious cases in the clinic. On the other hand, we are going for following research for further downstream experiments to validate the molecular mechanism for secreting multiple hormones. Materials and methods Clinical specimens collection Request a detailed protocol Our study included three adrenal tumor patients, that is, pheochromocytoma with ectopic ACTH and CRH secretion, common pheochromocytoma, and adrenocortical adenoma. All three patients had signed the consent forms at the General Surgery Department of Peking Union Medical College Hospital (PUMCH). The enhanced CT scanning images and laboratory test (ACTH, 24 hr urine-free cortisol, Catecholamines) of relevant patients are listed in Appendix 1. Fresh tumor specimens were collected during surgical resection. For the case of ACTH and CRH secreting pheochromocytoma, we performed the surgical resection of the tumor at left adrenal (esPHEO_T1) and its infiltrating tissues located in the kidney (esPHEO_T3) and masses (esPHEO_T2), and obtained three tumor specimens. The peritumor sample (esPHEO_Adj) was collected from the left adrenal tissue under the supervision of a qualified pathologist. The other two patients underwent left adrenalectomy and provided the other three tumor specimens. In details, one tumor specimen was obtained from the patient with common pheochromocytoma and two tumor specimens were obtained from the patient with adrenocortical adenoma. A total of seven specimens were carefully dissected under the microscope and confirmed by a qualified pathologist. Single-cell transcriptome library preparation and sequencing Request a detailed protocol After the resection, tissue specimens were rapidly processed for single-cell RNA sequencing. Single-cell suspensions were prepared according to the protocol of Chromium Single Cell 3′ Solution (V2 chemistry). All specimens were washed two times with cold 1× phosphate-buffered saline (PBS). Haemocytometer (Thermo Fisher Scientific) was used to evaluate cell viability rates. Then, we used Countess (Thermo Fisher Scientific) to count the concentration of single-cell suspension, and adjust the concentration to 1000 cells/μl. Samples that were lower than the required cell concentration defined in the user guide (i.e., <400 cells/µl) were pelleted and re-suspended in a reduced volume; and then the concentration of the new solution was counted again. Finally, the cells of the sample were loaded, and the libraries were constructed using a Chromium Single-Cell Kit (version 2). Single-cell libraries were submitted to 150 bp paired-end sequencing on the Illumina NavoSeq platform. Single-cell RNA-seq data pre-processing and quality control Request a detailed protocol After obtaining the paired-end raw reads, we used CellRanger (10× Genomics, v3.1.0) to pre-process the single-cell RNA-seq data. Cell barcodes and unique molecular identifiers (UMIs) of the library were extracted from read1. Then, the reads were split according to their cell (barcode) IDs, and the UMI sequences from read2 were simultaneously recorded for each cell. Quality control on these raw readings was subsequently performed to eliminate adapter contamination, duplicates, and low-quality bases. After filtering barcodes and low-quality readings that were not related to cells, we used STAR (version 2.5.1b) to map the cleaned readings to the human genome (hg19) and retained the uniquely mapped readings for UMIs counts. Next, we estimated the accurate molecular counts and generated a UMI count matrix for each cell by counting UMIs for each sample. Finally, we generated a gene-barcode matrix that showed the barcoded cells and gene expression counts. Based on the number of total reads, the number of detected gene features, and the percentage of mitochondrial genes, we performed quality control filtering through Seurat (v3.1.5) (Butler et al., 2018; Stuart et al., 2019) to discard low-quality cells. Briefly, mitochondrial genes inside one cell were calculated lower than 20%, and total reads in one cell were below 40,000. Also, the cells were further filtered according to the following criteria: PHEO, ACA, and esPHEO samples with no more than 5000, 3000, and 2500 genes were detected, respectively, and at least 200 genes were detected per cell in any sample. Low-quality cells and outliers were discarded, and the single cells that passed the QC criteria were used for downstream analyses. Doublets were predicted by DoubletFinder (v2.0) (McGinnis et al., 2019) and DoubletDecon (v1.1.6) (DePasquale et al., 2019; Appendix 1—figure 2). Clustering analysis and cell phenotype recognition Request a detailed protocol Seurat (Butler et al., 2018; Stuart et al., 2019) software package was used to perform cell clustering analysis to identify major cell types. All Seurat objects constructed from the filtered UMI-based gene expression matrixes of given samples were merged. We first applied ‘SCTransform’ function to implement normalization, variance stabilization, and feature selection through a regularized negative binomial model. Then, we reduced dimensionality through PCA. According to standard steps implemented in Seurat, highly variable numbers of principal components (PCs) 1–20 were selected and used for clustering using the t-distributed stochastic neighbor embedding method (t-SNE). We identified cell types of these groups based on the expression of canonic cell type markers or inferred by CellMarker database (Zhang et al., 2019). Finally, the four groups of endothelial cells were combined to a larger endothelial cell cluster for downstream analysis. Cellular cluster statistics were added in Supplementary file 2, which presented cell counts for each cellular cluster in different samples and top 10 gene markers. Endothelial cell cluster statistics were added in Supplementary file 3, which presented cell counts for each endothelial cell cluster in different samples and top 10 gene markers. DEG analysis Request a detailed protocol The cell-type-specific genes were identified by running Seurat (Butler et al., 2018; Stuart et al., 2019) containing the function of ‘FindAllMarkers’ on a log-transformed expression matrix with the following parameter settings: min.pct=0.25, logfc.threshold=0.25 (i.e., there is at least 0.25 log-scale fold change between the cells inside and outside a cluster), and only.pos=TRUE (i.e., only positive markers are returned). For heatmap and violin plots, the SCT-transformed data from Seurat pipeline were used. Using the Seurat ‘FindMarkers’ function, we found the DEGs between two cell types. We also used R package of clusterProfiler with default parameters to identify gene sets that exhibited significant and consistent differences between two given biological states. RNA velocity estimation Request a detailed protocol We used the velocyto python package (v0.17.17) (La Manno et al., 2018) for distinguishing transcripts as spliced or unspliced mRNAs based on the presence or absence of intronic regions in the transcript. We took aligned reads of BAM file for each sample as input. After per sample abundance estimation, it generated a LOOM file with the loompy package. Then, we used the scVelo (v0.2.3; Bergen et al., 2020) to combine each sample abundance data as well as cell cluster information from the Seurat object. We showed the proportions of abundances for each sample using scvelo.pl.proportions function. The RNA velocity was estimated for each cell for an individual gene at a given time point based on the ratio of its spliced and unspliced transcript. RNA velocity graph was visualized on a UMAP plot, with vector fields representing the averaged velocity of nearby cells. We also visualized some marker genes dynamics portraits with scv.pl.velocity to examine their spliced versus unspliced phase in different cell types. Pseudotime analysis Request a detailed protocol The Monocle2 packages (v2.14.0) (Trapnell et al., 2014) for R were used to determine the pseudotimes of the differentiation of four different cell subtypes, that is, POMC+/CRH + pheochromocytoma, pheochromocytoma, adrenocortical, and sustentacular cells. We converted a Seurat3 integrated object into a Monocle cds object and distributed the composed cell clusters to the Monocle cds partitions. Then, we used Monocle2 to perform trajectory graph learning and pseudotemporal sorting analysis by specifying the sustentacular cells as the root nodes. To identify genes that are significantly regulated as the cells differentiate along the cell-to-cell distance trajectory, we used the differentialGeneTest() function implemented in Monocle2 (Trapnell et al., 2014). Finally, we selected the genes that were differentially expressed on different paths through the trajectory and plotted the pseudotime_heatmap. Gene regulatory network (regulon) analysis Request a detailed protocol We used R package SCENIC (v1.1.2) (Aibar et al., 2017) for gene regulatory network inference. Normalized log counts were used as input to identify co-expression modules by the GRNBoost2 algorithm. Following which, regulons were derived by identifying the direct-binding TF target genes while pruning others based on motif enrichment around transcription start site (TSS) with cisTarget databases. Using aucell, the regulon activity score was measured as the area under the recovery curve (AUC). Additionally, regulon specificity score (RSS) was used for the detection of the cell-type-specific regulons. Cell-cell communication analysis Request a detailed protocol Given the diverse immune and endothelial cell types in the tumor microenvironment, we performed cell-cell communication analysis using CellPhoneDB Python package (2.1.7) (Efremova et al., 2020). We visualized the potential cell-cell interactions among various immune cells, endothelial cells, and other cell types in the different tumor microenvironment (esPHEO, esPHEO_Adj, PHEO, and ACA) (Appendix 1—figure 6). Whole-exome sequencing Request a detailed protocol Genomic DNA extracted from whole blood (esPHEO_Blood), esPHEO_T2, esPHEO_T3, and esPHEO_Adj of the rare Case 1 were sent for whole-exome sequencing. The exomes were captured using the Agilent SureSelect Human All Exon V6 Kit and the enriched exome libraries were constructed and sequenced on the Illumina NovaSeq 6000 platform to generate WES data (150 bp paired-end reads, >100×) according to standard manufacturer protocols. The cleaned reads were aligned to the human reference genome sequence NCBI Build 38 (hg38) using Burrows-Wheeler Aligner (BWA) (v0.7.17) (Li and Durbin, 2009). All aligned BAM were then performed through the same bioinformatics pipeline according to GATK Best Practices (v4.2) (McKenna et al., 2010). We obtained germline variants shared by all tumors and control samples based on variant calling from GATK-HaplotypeCaller. We then used GATK-MuTect2 to call somatic variants in tumors and obtained a high-confidence mutation set after rigorous filtering by GATK-FilterMutectCalls. All variants were annotated using ANNOVAR (v2018Apr16) (Wang et al., 2010). The criteria for filtering variants were as follows: (1) only retained variants located on exon or splice site, and excluded synonymous variants; (2) retained rare variants with minor allele frequencies <5% in any ancestry population groups from public databases (1000 Genomes, ESP6500, ExAC, or the GnomAD); (3) For germline variants, excluded common variants in dbSNP (Build 138) and predicted benign missense variants by SIFT, Polyphen2, and Mutation Taster. Immunocytochemistry and Immunofluorescence Request a detailed protocol Immunocytochemical and immunofluorescent staining experiments were conducted according to standard protocols using antibodies against malinfixed paraffin-embedded (FFPE) tissue specimens. The antibodies and reagents used in the experiments are listed as follows: ACTH (Abcam, ab199007), POMC (ProteinTech, 66358-1-Ig), TH (Abcam, ab112), CRH (ProteinTech, 10944-1-AP), CgA (ProteinTech, 60135-1-Ig), and Human Galanin Antibody (R&D, MAB5854). Appendix 1 Clinical samples description Case 1: A 39-year-old lady underwent laparoscopic left adrenal tumor resection in July 2012 at a local hospital. She had a 2-year history of headache, generalized swelling, and palpitations. She was noted to have hypertensive (BP 240/120 mmHg) and typical Cushingoid characteristics, including asthenia, supraclavicular fat deposits, bruises, purple striae, proximal myopathy, and hyperpigmentation. Histopathology confirmed an adrenomedullary chromaffin tumor. During tumor immunostaining, the tumor stained positively for ACTH. After the adrenal surgery, her Cushingoid characteristics, hypokalemia, and hypertension were all relieved. However, the patient experienced recurrence of symptoms and signs in January 2019 and was admitted to our hospital. It was found that urine and plasma metanephrine were significantly elevated, and plasma ACTH was also high. Enhanced CT scanning of the abdomen revealed bilateral adrenocortical hyperplasia and multiple masses in the left adrenal and around the left kidney. The largest mass lesion was 2.3×1.6 cm2, which invaded upper pole of left kidney. But the I123-MIBG scintigraphy was negative. We performed a surgery to remove left adrenal, kidney, and masses. After the surgery, the patient’s clinical features and symptoms were improved, and the excessive hypercortisolemia and catecholamine eventually returned to normal. IHC revealed positive staining for chromogranin A, ACTH, and CRH, confirming the diagnosis of pheochromocytoma secreting both ACTH and CRH. Case 2: A 42-year-old male with a 3-year history of headache and palpitations, and a 6-month history of hypertension was admitted to our hospital. Laboratory tests showed that the plasma and urine catecholamines and their metabolites were elevated, and cortisol and ACTH were at the normal level. Enhanced CT showed a 67×70 mm2 left adrenal tumor, and I123-MIBG scintigraphy exhibited positive. We performed a surgery to remove the left adrenal gland. After the surgery, the patient’s clinical features and symptoms were relieved. IHC confirmed the diagnosis of pheochromocytoma. Case 3: A 50-year-old female came to our hospital with hypertension, hyperkalemia, and Cushingoid symptoms (moon face and central obesity). Enhanced CT scanning revealed a 19×36 mm2 irregular mass in left adrenal gland. The laboratory tests showed ACTH-independent hypercortisolemia. The left adrenal gland was removed, and Cushing’s syndrome was relieved. Resected specimen revealed two tumors in the left adrenal gland, and IHC confirmed the diagnosis of adrenal adenoma. Appendix 1—table 1 Summary of laboratory test for three cases. Laboratory test Case 1 Case 2 Case 3 Reference range ACTH 519.0 24.0 <5 0–46.0 pg/ml 24 hr urine-free cortisol 2024.4 332.4 12.3–103.5 μg/24 hr Catecholamines Plasma metanephrines Normetanephrine 3.28 10.81 0.4 <0.9 nmol/L Metanephrine 3.44 11.55 0.2 <0.5 nmol/L 24 hr urine Epinephrine 397.63 56.23 1.92 1.74–6.42 μg/24 hr Norepinephrine 475.43 82.29 26.17 16.69–40.65 μg/24 hr Dopamine 432.21 301.71 240.5 120.93–330.5 μg/24 hr Appendix 1—figure 1 Download asset Open asset Enhanced CT scanning image for three cases. (A) Enhanced CT scanning for Case 1 with pheochromocytoma secreting both ACTH and CRH. The abdomen revealed bilateral adrenocortical hyperplasia and multiple masses in the left adrenal and around … see more Appendix 1—figure 2 Download asset Open asset Quality control plots and doublet detection for this scRNA-seq study. Violin plots showing number of total RNAs (A), number of genes (B), and percentage of mitochondrial (mito) genes (C) for cells in seven samples. Doublets were predicted by DoubletFinder (D) and … see more Appendix 1—figure 3 Download asset Open asset Four adrenal cell types and their highly expressed genes through single-cell transcriptomic analysis. Heatmap shows the scaled expression patterns of top 10 marker genes in each cell type. The color keys from white to red indicate relative expression levels from low to high. Appendix 1—figure 4 Download asset Open asset Transcription factors detection using SCENIC pipeline. (A) Binarized heatmap showing the AUC score (area under the recovery curve, scoring the activity of regulons) of the identified regulons plotted for each cell. (B) For each cellular cluster, dot … see more Appendix 1—figure 5 Download asset Open asset The spliced versus unspliced phase for marker genes in four types of adrenal cells. Transcripts were marked as either spliced or unspliced based on the presence or absence of intronic regions in the transcript. For each gene, the scatter plot shows spliced and unspliced ratios in a … see more Appendix 1—figure 6 Download asset Open asset Ligand-receptor interaction analysis for CD4+ T cells, CD8+ T cells, and endothelial cells in different tumor microenvironments. Overview of ligand-receptor interactions between the CD4+ T cells (A), CD8+ T cells (B), endothelial (C), and the other cell types in the different tumor microenvironments. p-values are represented … see more Appendix 1—figure 7 Download asset Open asset Whole-exome sequencing identified one shared somatic variant of ACAN comparing variants in tumor samples to controls and Sanger sequencing only confirmed the presence in esPHEO_T3 but not observed in esPHEO_T2. (A) Distribution of somatic mutations for the rare case with ectopic ACTH&CRH-secreting pheochromocytoma. OncoPrint plots were generated using the R package Maftools for somatic mutations from five … see more Appendix 1—figure 8 Download asset Open asset Immunohistochemistry of CgA, ACTH, POMC, CRH, TH, or GAL on serial biopsies from tumor specimen infiltrating tissues located in the kidney (esPHEO_T3). We observed positive staining signal at tumor left in each slice, while the adjacent kidney was un-stained could be negative controls. The magnification is 0.5×, 2.5×, 10×, and 40× from left to … see more Appendix 1—figure 9 Download asset Open asset Immunofluorescence co-staining for POMC&CRH and POMC&TH on two serial biopsies from tumor specimen esPHEO_T3. The magnification is 10× (top) and 40× (bottom). Red rectangular indicates the magnified area of the location, as shown in Figure 3E. Data availability The raw data of scRNA-seq sequencing reads generated in this study were deposited in The National Genomics Data Center (NGDC, https://bigd.big.ac.cn/) under the accession number: PRJCA003766. References Aibar S González-Blas CB Moerman T Huynh-Thu VA Imrichova H Hulselmans G Rambow F Marine J-C Geurts P Aerts J van den Oord J Atak ZK Wouters J Aerts S (2017) SCENIC: single-cell regulatory network inference and clustering Nature Methods 14:1083–1086. https://doi.org/10.1038/nmeth.4463 PubMed Google Scholar Alexandraki KI Grossman AB (2010) The ectopic ACTH syndrome Reviews in Endocrine & Metabolic Disorders 11:117–126. https://doi.org/10.1007/s11154-010-9139-z PubMed Google Scholar Aniszewski JP Young WF Jr Thompson GB Grant CS van Heerden JA (2001) Cushing syndrome due to ectopic adrenocorticotropic hormone secretion World Journal of Surgery 25:934–940. https://doi.org/10.1007/s00268-001-0032-5 PubMed Google Scholar Apergis-Schoute J Burnstock G Nusbaum MP Parker D Morales MA Trudeau LE Svensson E (2019) Editorial: Neuronal Co-transmission Frontiers in Neural Circuits 13:19. https://doi.org/10.3389/fncir.2019.00019 PubMed Google Scholar Auchus RJ Lee TC Miller WL (1998) Cytochrome b5 augments the 17,20-lyase activity of human P450c17 without direct electron transfer The Journal of Biological Chemistry 273:3158–3165. https://doi.org/10.1074/jbc.273.6.3158 PubMed Google Scholar Ballav C Naziat A Mihai R Karavitaki N Ansorge O Grossman AB (2012) Mini-review: pheochromocytomas causing the ectopic ACTH syndrome Endocrine 42:69–73. https://doi.org/10.1007/s12020-012-9646-7 PubMed Google Scholar Bergen V Lange M Peidli S Wolf FA Theis FJ (2020) Generalizing RNA velocity to transient cell states through dynamical modeling Nature Biotechnology 38:1408–1414. https://doi.org/10.1038/s41587-020-0591-3 PubMed Google Scholar Bernard V Semaan A Huang J San Lucas FA Mulu FC Stephens BM Guerrero PA Huang Y Zhao J Kamyabi N Sen S Scheet PA Taniguchi CM Kim MP Tzeng C-W Katz MH Singhi AD Maitra A Alvarez HA (2019) Single-Cell Transcriptomics of Pancreatic Cancer Precursors Demonstrates Epithelial and Microenvironmental Heterogeneity as an Early Event in Neoplastic Progression Clinical Cancer Research 25:2194–2205. https://doi.org/10.1158/1078-0432.CCR-18-1955 PubMed Google Scholar Bernton EW Beach JE Holaday JW Smallridge RC Fein HG (1987) Release of multiple hormones by a direct action of interleukin-1 on pituitary cells Science 238:519–521. https://doi.org/10.1126/science.2821620 PubMed Google Scholar Blalock JE (1989) A molecular basis for bidirectional communication between the immune and neuroendocrine systems Physiological Reviews 69:1–32. https://doi.org/10.1152/physrev.1989.69.1.1 PubMed Google Scholar Bolland MJ Holdaway IM Berkeley JE Lim S Dransfield WJ Conaglen JV Croxson MS Gamble GD Hunt PJ Toomath RJ (2011) Mortality and morbidity in Cushing’s syndrome in New Zealand Clinical Endocrinology 75:436–442. https://doi.org/10.1111/j.1365-2265.2011.04124.x Google Scholar Bowman TV McCooey AJ Merchant AA Ramos CA Fonseca P Poindexter A Bradfute SB Oliveira DM Green R Zheng Y Jackson KA Chambers SM McKinney-Freeman SL Norwood KG Darlington G Gunaratne PH Steffen D Goodell MA (2006) Differential mRNA processing in hematopoietic stem cells Stem Cells 24:662–670. https://doi.org/10.1634/stemcells.2005-0552 PubMed Google Scholar Burnstock G (1976) Do some nerve cells release more than one transmitter? Neuroscience 1:239–248. https://doi.org/10.1016/0306-4522(76)90054-3 PubMed Google Scholar Butler A Hoffman P Smibert P Papalexi E Satija R (2018) Integrating single-cell transcriptomic data across different conditions, technologies, and species Nature Biotechnology 36:411–420. https://doi.org/10.1038/nbt.4096 PubMed Google Scholar Carini C McLane MF Mayer KH Essex M (1994) Dysregulation of interleukin-7 receptor may generate loss of cytotoxic T cell response in human immunodeficiency virus type 1 infection European Journal of Immunology 24:2927–2934. https://doi.org/10.1002/eji.1830241202 PubMed Google Scholar Carpenter DJ Granot T Matsuoka N Senda T Kumar BV Thome JJC Gordon CL Miron M Weiner J Connors T Lerner H Friedman A Kato T Griesemer AD Farber DL (2018) Human immunology studies using organ donors: Impact of clinical variations on immune parameters in tissues and circulation American Journal of Transplantation 18:74–88. https://doi.org/10.1111/ajt.14434 PubMed Google Scholar Cutolo M Sulli A Pizzorni C Craviotto C Straub RH (2003) Hypothalamic-pituitary-adrenocortical and gonadal functions in rheumatoid arthritis Annals of the New York Academy of Sciences 992:107–117. https://doi.org/10.1111/j.1749-6632.2003.tb03142.x PubMed Google Scholar DePasquale EAK Schnell DJ Van Camp PJ Valiente-Alandí Í Blaxall BC Grimes HL Singh H Salomonis N (2019) DoubletDecon: Deconvoluting Doublets from Single-Cell RNA-Sequencing Data Cell Reports 29:1718–1727. https://doi.org/10.1016/j.celrep.2019.09.082 PubMed Google Scholar Efremova M Vento-Tormo M Teichmann SA Vento-Tormo R (2020) CellPhoneDB: inferring cell-cell communication from combined expression of multi-subunit ligand-receptor complexes Nature Protocols 15:1484–1506. https://doi.org/10.1038/s41596-020-0292-x PubMed Google Scholar Ejaz S Vassilopoulou-Sellin R Busaidy NL Hu MI Waguespack SG Jimenez C Ying AK Cabanillas M Abbara M Habra MA (2011) Cushing syndrome secondary to ectopic adrenocorticotropic hormone secretion: the University of Texas MD Anderson Cancer Center Experience Cancer 117:4381–4389. https://doi.org/10.1002/cncr.26029 PubMed Google Scholar Elliott PF Berhane T Ragnarsson O Falhammar H (2021) Ectopic ACTH- and/or CRH-Producing Pheochromocytomas The Journal of Clinical Endocrinology and Metabolism 106:598–608. https://doi.org/10.1210/clinem/dgaa488 PubMed Google Scholar Fitzgerald RS Eyzaguirre C Zapata P (2009) Fifty years of progress in carotid body physiology--invited article Advances in Experimental Medicine and Biology 648:19–28. https://doi.org/10.1007/978-90-481-2259-2_2 PubMed Google Scholar Fukata J Imura H Nakao K (1994) Cytokines as mediators in the regulation of the hypothalamic-pituitary-adrenocortical function Journal of Endocrinological Investigation 17:141–155. https://doi.org/10.1007/BF03347705 PubMed Google Scholar Gisslinger H Svoboda T Clodi M Gilly B Ludwig H Havelec L Luger A (1993) Interferon-alpha stimulates the hypothalamic-pituitary-adrenal axis in vivo and in vitro Neuroendocrinology 57:489–495. https://doi.org/10.1159/000126396 PubMed Google Scholar Hakanson R Sundler F (1983) The design of the neuroendocrine system: a unifying concept and its consequences Trends in Pharmacological Sciences 4:41–44. https://doi.org/10.1016/0165-6147(83)90275-4 Google Scholar Hooi SC Maiter DM Martin JB Koenig JI (1990) Galaninergic mechanisms are involved in the regulation of corticotropin and thyrotropin secretion in the rat Endocrinology 127:2281–2289. https://doi.org/10.1210/endo-127-5-2281 PubMed Google Scholar Hsu DW Hooi SC Hedley-Whyte ET Strauss RM Kaplan LM (1991) Coexpression of galanin and adrenocorticotropic hormone in human pituitary and pituitary adenomas The American Journal of Pathology 138:897–909. PubMed Google Scholar Ilias I Torpy DJ Pacak K Mullen N Wesley RA Nieman LK (2005) Cushing’s syndrome due to ectopic corticotropin secretion: twenty years’ experience at the National Institutes of Health The Journal of Clinical Endocrinology and Metabolism 90:4955–4962. https://doi.org/10.1210/jc.2004-2527 PubMed Google Scholar Imura H Fukata J (1994) Endocrine-paracrine interaction in communication between the immune and endocrine systems: Activation of the hypothalamic-pituitary-adrenal axis in inflammation European Journal of Endocrinology 130:32–37. https://doi.org/10.1530/eje.0.1300032 PubMed Google Scholar Isidori AM Kaltsas GA Pozza C Frajese V Newell-Price J Reznek RH Jenkins PJ Monson JP Grossman AB Besser GM (2006) The ectopic adrenocorticotropin syndrome: clinical features, diagnosis, management, and long-term follow-up The Journal of Clinical Endocrinology and Metabolism 91:371–377. https://doi.org/10.1210/jc.2005-1542 PubMed Google Scholar Jessop DS Cunnah D Millar JG Neville E Coates P Doniach I Besser GM Rees LH (1987) A phaeochromocytoma presenting with Cushing’s syndrome associated with increased concentrations of circulating corticotrophin-releasing factor The Journal of Endocrinology 113:133–138. https://doi.org/10.1677/joe.0.1130133 PubMed Google Scholar Kaech SM Tan JT Wherry EJ Konieczny BT Surh CD Ahmed R (2003) Selective expression of the interleukin 7 receptor identifies effector CD8 T cells that give rise to long-lived memory cells Nature Immunology 4:1191–1198. https://doi.org/10.1038/ni1009 PubMed Google Scholar Kakucska I Qi Y Clark BD Lechan RM (1993) Endotoxin-induced corticotropin-releasing hormone gene expression in the hypothalamic paraventricular nucleus is mediated centrally by interleukin-1 Endocrinology 133:815–821. https://doi.org/10.1210/endo.133.2.8344218 PubMed Google Scholar Kolodziejczyk AA Kim JK Svensson V Marioni JC Teichmann SA (2015) The technology and biology of single-cell RNA sequencing Molecular Cell 58:610–620. https://doi.org/10.1016/j.molcel.2015.04.005 PubMed Google Scholar Kondrack RM Harbertson J Tan JT McBreen ME Surh CD Bradley LM (2003) Interleukin 7 regulates the survival and generation of memory CD4 cells The Journal of Experimental Medicine 198:1797–1806. https://doi.org/10.1084/jem.20030735 PubMed Google Scholar La Manno G Soldatov R Zeisel A Braun E Hochgerner H Petukhov V Lidschreiber K Kastriti ME Lönnerberg P Furlan A Fan J Borm LE Liu Z van Bruggen D Guo J He X Barker R Sundström E Castelo-Branco G Cramer P Adameyko I Linnarsson S Kharchenko PV (2018) RNA velocity of single cells Nature 560:494–498. https://doi.org/10.1038/s41586-018-0414-6 PubMed Google Scholar Lacroix A Feelders RA Stratakis CA Nieman LK (2015) Cushing’s syndrome Lancet 386:913–927. https://doi.org/10.1016/S0140-6736(14)61375-1 PubMed Google Scholar Lase I Strele I Grönberg M Kozlovacki G Welin S Janson ET (2020) Multiple hormone secretion may indicate worse prognosis in patients with ectopic Cushing’s syndrome Hormones 19:351–360. https://doi.org/10.1007/s42000-019-00163-z PubMed Google Scholar Lau SK Romansky SG Weiss LM (2006) Sustentaculoma: report of a case of a distinctive neoplasm of the adrenal medulla The American Journal of Surgical Pathology 30:268–273. https://doi.org/10.1097/01.pas.0000178095.07513.38 PubMed Google Scholar Lenders JWM Eisenhofer G Mannelli M Pacak K (2005) Phaeochromocytoma Lancet 366:665–675. https://doi.org/10.1016/S0140-6736(05)67139-5 PubMed Google Scholar Lenders JWM Duh QY Eisenhofer G Gimenez-Roqueplo AP Grebe SKG Murad MH Naruse M Pacak K Young WF Society E (2014) Pheochromocytoma and paraganglioma: an endocrine society clinical practice guideline The Journal of Clinical Endocrinology and Metabolism 99:1915–1942. https://doi.org/10.1210/jc.2014-1498 PubMed Google Scholar Lenz DC Kurz SK Lemmens E Schoenberger SP Sprent J Oldstone MBA Homann D (2004) IL-7 regulates basal homeostatic proliferation of antiviral CD4+T cell memory PNAS 101:9357–9362. https://doi.org/10.1073/pnas.0400640101 PubMed Google Scholar Li J Huston G Swain SL (2003) IL-7 promotes the transition of CD4 effectors to persistent memory cells The Journal of Experimental Medicine 198:1807–1815. https://doi.org/10.1084/jem.20030725 PubMed Google Scholar Li H Durbin R (2009) Fast and accurate short read alignment with Burrows-Wheeler transform Bioinformatics 25:1754–1760. https://doi.org/10.1093/bioinformatics/btp324 PubMed Google Scholar Lindholm J Juul S Jørgensen JO Astrup J Bjerre P Feldt-Rasmussen U Hagen C Jørgensen J Kosteljanetz M Kristensen L Laurberg P Schmidt K Weeke J (2001) Incidence and late prognosis of cushing’s syndrome: a population-based study The Journal of Clinical Endocrinology and Metabolism 86:117–123. https://doi.org/10.1210/jcem.86.1.7093 PubMed Google Scholar Lloyd RV Blaivas M Wilson BS (1985) Distribution of chromogranin and S100 protein in normal and abnormal adrenal medullary tissues Archives of Pathology & Laboratory Medicine 109:633–635. PubMed Google Scholar Maraskovsky E Teepe M Morrissey PJ Braddy S Miller RE Lynch DH Peschon JJ (1996) Impaired survival and proliferation in IL-7 receptor-deficient peripheral T cells Journal of Immunology 157:5315–5323. PubMed Google Scholar McGinnis CS Murrow LM Gartner ZJ (2019) DoubletFinder: Doublet Detection in Single-Cell RNA Sequencing Data Using Artificial Nearest Neighbors Cell Systems 8:329–337. https://doi.org/10.1016/j.cels.2019.03.003 PubMed Google Scholar McKenna A Hanna M Banks E Sivachenko A Cibulskis K Kernytsky A Garimella K Altshuler D Gabriel S Daly M DePristo MA (2010) The Genome Analysis Toolkit: a MapReduce framework for analyzing next-generation DNA sequencing data Genome Research 20:1297–1303. https://doi.org/10.1101/gr.107524.110 PubMed Google Scholar McKnight GL Karlsen AE Kowalyk S Mathewes SL Sheppard PO O’Hara PJ Taborsky GJ (1992) Sequence of human galanin and its inhibition of glucose-stimulated insulin secretion from RIN cells Diabetes 41:82–87. https://doi.org/10.2337/diab.41.1.82 PubMed Google Scholar Montoro DT Haber AL Biton M Vinarsky V Lin B Birket SE Yuan F Chen S Leung HM Villoria J Rogel N Burgin G Tsankov AM Waghray A Slyper M Waldman J Nguyen L Dionne D Rozenblatt-Rosen O Tata PR Mou H Shivaraju M Bihler H Mense M Tearney GJ Rowe SM Engelhardt JF Regev A Rajagopal J (2018) A revised airway epithelial hierarchy includes CFTR-expressing ionocytes Nature 560:319–324. https://doi.org/10.1038/s41586-018-0393-7 PubMed Google Scholar Murakami Y Kato Y Shimatsu A Koshiyama H Hattori N Yanaihara N Imura H (1989) Possible mechanisms involved in growth hormone secretion induced by galanin in the rat Endocrinology 124:1224–1229. https://doi.org/10.1210/endo-124-3-1224 PubMed Google Scholar Conference Murakami N Fukata J Tsukada T Nakai Y. Imura H (1992) Abstr of 9th International Congress of Endocrinology Endotoxin-induced changes in hormone and interleukin synthesis in the hypothalamic-pituitary-adrenal axis. e1. Google Scholar Newell-Price J Bertagna X Grossman AB Nieman LK (2006) Cushing’s syndrome Lancet 367:1605–1617. https://doi.org/10.1016/S0140-6736(06)68699-6 PubMed Google Scholar Nölting S Grossman AB (2012) Signaling pathways in pheochromocytomas and paragangliomas: prospects for future therapies Endocrine Pathology 23:21–33. https://doi.org/10.1007/s12022-012-9199-6 PubMed Google Scholar Ottlecz A Snyder GD McCann SM (1988) Regulatory role of galanin in control of hypothalamic-anterior pituitary function PNAS 85:9861–9865. https://doi.org/10.1073/pnas.85.24.9861 PubMed Google Scholar O’Brien T Young WF Jr Davila DG Scheithauer BW Kovacs K Horvath E Vale W van Heerden JA (1992) Cushing’s syndrome associated with ectopic production of corticotrophin-releasing hormone, corticotrophin and vasopressin by a phaeochromocytoma Clinical Endocrinology 37:460–467. https://doi.org/10.1111/j.1365-2265.1992.tb02359.x PubMed Google Scholar Papalexi E Satija R (2018) Single-cell RNA sequencing to explore immune cell heterogeneity Nature Reviews. Immunology 18:35–45. https://doi.org/10.1038/nri.2017.76 PubMed Google Scholar Pardal R Ortega-Sáenz P Durán R López-Barneo J (2007) Glia-like stem cells sustain physiologic neurogenesis in the adult mammalian carotid body Cell 131:364–377. https://doi.org/10.1016/j.cell.2007.07.043 PubMed Google Scholar Patel AP Tirosh I Trombetta JJ Shalek AK Gillespie SM Wakimoto H Cahill DP Nahed BV Curry WT Martuza RL Louis DN Rozenblatt-Rosen O Suvà ML Regev A Bernstein BE (2014) Single-cell RNA-seq highlights intratumoral heterogeneity in primary glioblastoma Science 344:1396–1401. https://doi.org/10.1126/science.1254257 PubMed Google Scholar Petrunak EM DeVore NM Porubsky PR Scott EE (2014) Structures of human steroidogenic cytochrome P450 17A1 with substrates The Journal of Biological Chemistry 289:32952–32964. https://doi.org/10.1074/jbc.M114.610998 PubMed Google Scholar Pillai S Gopalan V Smith RA Lam AK-Y (2016) Updates on the genetics and the clinical impacts on phaeochromocytoma and paraganglioma in the new era Critical Reviews in Oncology/Hematology 100:190–208. https://doi.org/10.1016/j.critrevonc.2016.01.022 PubMed Google Scholar Plasschaert LW Žilionis R Choo-Wing R Savova V Knehr J Roma G Klein AM Jaffe AB (2018) A single-cell atlas of the airway epithelium reveals the CFTR-rich pulmonary ionocyte Nature 560:377–381. https://doi.org/10.1038/s41586-018-0394-6 PubMed Google Scholar Poli G Sarchielli E Guasti D Benvenuti S Ballerini L Mazzanti B Armignacco R Cantini G Lulli M Chortis V Arlt W Romagnoli P Vannelli GB Mannelli M Luconi M (2019) Human fetal adrenal cells retain age-related stem- and endocrine-differentiation potential in culture FASEB Journal 33:2263–2277. https://doi.org/10.1096/fj.201801028RR PubMed Google Scholar Puram SV Tirosh I Parikh AS Patel AP Yizhak K Gillespie S Rodman C Luo CL Mroz EA Emerick KS Deschler DG Varvares MA Mylvaganam R Rozenblatt-Rosen O Rocco JW Faquin WC Lin DT Regev A Bernstein BE (2017) Single-Cell Transcriptomic Analysis of Primary and Metastatic Tumor Ecosystems in Head and Neck Cancer Cell 171:1611–1624. https://doi.org/10.1016/j.cell.2017.10.044 PubMed Google Scholar Reichert M Rustgi AK (2011) Pancreatic ductal cells in development, regeneration, and neoplasia The Journal of Clinical Investigation 121:4572–4578. https://doi.org/10.1172/JCI57131 PubMed Google Scholar Rivier C Chizzonite R Vale W (1989) In the mouse, the activation of the hypothalamic-pituitary-adrenal axis by a lipopolysaccharide (endotoxin) is mediated through interleukin-1 Endocrinology 125:2800–2805. https://doi.org/10.1210/endo-125-6-2800 PubMed Google Scholar Sakuma I Higuchi S Fujimoto M Takiguchi T Nakayama A Tamura A Kohno T Komai E Shiga A Nagano H Hashimoto N Suzuki S Mayama T Koide H Ono K Sasano H Tatsuno I Yokote K Tanaka T (2016) Cushing Syndrome Due to ACTH-Secreting Pheochromocytoma, Aggravated by Glucocorticoid-Driven Positive-Feedback Loop The Journal of Clinical Endocrinology and Metabolism 101:841–846. https://doi.org/10.1210/jc.2015-2855 PubMed Google Scholar Scriba LD Bornstein SR Santambrogio A Mueller G Huebner A Hauer J Schedl A Wielockx B Eisenhofer G Andoniadou CL Steenblock C (2020) Cancer Stem Cells in Pheochromocytoma and Paraganglioma Frontiers in Endocrinology 11:79. https://doi.org/10.3389/fendo.2020.00079 PubMed Google Scholar Seddon B Tomlinson P Zamoyska R (2003) Interleukin 7 and T cell receptor signals regulate homeostasis of CD4 memory cells Nature Immunology 4:680–686. https://doi.org/10.1038/ni946 PubMed Google Scholar Segerstolpe Å Palasantza A Eliasson P Andersson E-M Andréasson A-C Sun X Picelli S Sabirsh A Clausen M Bjursell MK Smith DM Kasper M Ämmälä C Sandberg R (2016) Single-Cell Transcriptome Profiling of Human Pancreatic Islets in Health and Type 2 Diabetes Cell Metabolism 24:593–607. https://doi.org/10.1016/j.cmet.2016.08.020 PubMed Google Scholar Steffensen C Bak AM Rubeck KZ Jørgensen JOL (2010) Epidemiology of Cushing’s syndrome Neuroendocrinology 92 Suppl 1:1–5. https://doi.org/10.1159/000314297 PubMed Google Scholar Stuart T Butler A Hoffman P Hafemeister C Papalexi E Mauck WM Hao Y Stoeckius M Smibert P Satija R (2019) Comprehensive Integration of Single-Cell Data Cell 177:1888–1902. https://doi.org/10.1016/j.cell.2019.05.031 PubMed Google Scholar Stubbington MJT Rozenblatt-Rosen O Regev A Teichmann SA (2017) Single-cell transcriptomics to explore the immune system in health and disease Science 358:58–63. https://doi.org/10.1126/science.aan6828 PubMed Google Scholar Suzuki T Kachi T (1995) Immunohistochemical studies on supporting cells in the adrenal medulla and pineal gland of adult rat, especially on S-100 protein, glial fibrillary acidic protein and vimentin Kaibogaku Zasshi. Journal of Anatomy 70:130–139. https://doi.org/10.5603/FM.a2016.0066 PubMed Google Scholar Svensson E Apergis-Schoute J Burnstock G Nusbaum MP Parker D Schiöth HB (2018) General Principles of Neuronal Co-transmission: Insights From Multiple Model Systems Frontiers in Neural Circuits 12:117. https://doi.org/10.3389/fncir.2018.00117 PubMed Google Scholar Szabo PA Levitin HM Miron M Snyder ME Senda T Yuan J Cheng YL Bush EC Dogra P Thapa P Farber DL Sims PA (2019) Single-cell transcriptomics of human T cells reveals tissue and activation signatures in health and disease Nature Communications 10:4706. https://doi.org/10.1038/s41467-019-12464-3 PubMed Google Scholar Tammela T Sage J (2020) Investigating Tumor Heterogeneity in Mouse Models Annual Review of Cancer Biology 4:99–119. https://doi.org/10.1146/annurev-cancerbio-030419-033413 PubMed Google Scholar Tan JT Dudl E LeRoy E Murray R Sprent J Weinberg KI Surh CD (2001) IL-7 is critical for homeostatic proliferation and survival of naive T cells PNAS 98:8732–8737. https://doi.org/10.1073/pnas.161126098 PubMed Google Scholar Tan JT Ernst B Kieper WC LeRoy E Sprent J Surh CD (2002) Interleukin (IL)-15 and IL-7 jointly regulate homeostatic proliferation of memory phenotype CD8+ cells but are not required for memory phenotype CD4+ cells The Journal of Experimental Medicine 195:1523–1532. https://doi.org/10.1084/jem.20020066 PubMed Google Scholar Tang F Barbacioru C Wang Y Nordman E Lee C Xu N Wang X Bodeau J Tuch BB Siddiqui A Lao K Surani MA (2009) mRNA-Seq whole-transcriptome analysis of a single cell Nature Methods 6:377–382. https://doi.org/10.1038/nmeth.1315 PubMed Google Scholar Thome JJC Yudanin N Ohmura Y Kubota M Grinshpun B Sathaliyawala T Kato T Lerner H Shen Y Farber DL (2014) Spatial map of human T cell compartmentalization and maintenance over decades of life Cell 159:814–828. https://doi.org/10.1016/j.cell.2014.10.026 PubMed Google Scholar Tirosh I Izar B Prakadan SM Wadsworth MH Treacy D Trombetta JJ Rotem A Rodman C Lian C Murphy G Fallahi-Sichani M Dutton-Regester K Lin JR Cohen O Shah P Lu D Genshaft AS Hughes TK Ziegler CGK Kazer SW Gaillard A Kolb KE Villani AC Johannessen CM Andreev AY Van Allen EM Bertagnolli M Sorger PK Sullivan RJ Flaherty KT Frederick DT Jané-Valbuena J Yoon CH Rozenblatt-Rosen O Shalek AK Regev A Garraway LA (2016a) Dissecting the multicellular ecosystem of metastatic melanoma by single-cell RNA-seq Science 352:189–196. https://doi.org/10.1126/science.aad0501 PubMed Google Scholar Tirosh I Venteicher AS Hebert C Escalante LE Patel AP Yizhak K Fisher JM Rodman C Mount C Filbin MG Neftel C Desai N Nyman J Izar B Luo CC Francis JM Patel AA Onozato ML Riggi N Livak KJ Gennert D Satija R Nahed BV Curry WT Martuza RL Mylvaganam R Iafrate AJ Frosch MP Golub TR Rivera MN Getz G Rozenblatt-Rosen O Cahill DP Monje M Bernstein BE Louis DN Regev A Suvà ML (2016b) Single-cell RNA-seq supports a developmental hierarchy in human oligodendroglioma Nature 539:309–313. https://doi.org/10.1038/nature20123 PubMed Google Scholar Trapnell C Cacchiarelli D Grimsby J Pokharel P Li S Morse M Lennon NJ Livak KJ Mikkelsen TS Rinn JL (2014) The dynamics and regulators of cell fate decisions are revealed by pseudotemporal ordering of single cells Nature Biotechnology 32:381–386. https://doi.org/10.1038/nbt.2859 PubMed Google Scholar Unger P Hoffman K Pertsemlidis D Thung S Wolfe D Kaneko M (1991) S100 protein-positive sustentacular cells in malignant and locally aggressive adrenal pheochromocytomas Archives of Pathology & Laboratory Medicine 115:484–487. PubMed Google Scholar Valassi E Santos A Yaneva M (2011) The European Registry on Cushing’s syndrome: 2-year experience Baseline Demographic and Clinical Characteristics. Eur J Endocrinol 165:383–392. https://doi.org/10.1530/EJE-11-0272 Google Scholar Venteicher AS Tirosh I Hebert C Yizhak K Neftel C Filbin MG Hovestadt V Escalante LE Shaw ML Rodman C Gillespie SM Dionne D Luo CC Ravichandran H Mylvaganam R Mount C Onozato ML Nahed BV Wakimoto H Curry WT Iafrate AJ Rivera MN Frosch MP Golub TR Brastianos PK Getz G Patel AP Monje M Cahill DP Rozenblatt-Rosen O Louis DN Bernstein BE Regev A Suvà ML (2017) Decoupling genetics, lineages, and microenvironment in IDH-mutant gliomas by single-cell RNA-seq Science 355:eaai8478. https://doi.org/10.1126/science.aai8478 PubMed Google Scholar Wang K Li M Hakonarson H (2010) ANNOVAR: functional annotation of genetic variants from high-throughput sequencing data Nucleic Acids Research 38:e164. https://doi.org/10.1093/nar/gkq603 PubMed Google Scholar Young MD Mitchell TJ Vieira Braga FA Tran MGB Stewart BJ Ferdinand JR Collord G Botting RA Popescu D-M Loudon KW Vento-Tormo R Stephenson E Cagan A Farndon SJ Del Castillo Velasco-Herrera M Guzzo C Richoz N Mamanova L Aho T Armitage JN Riddick ACP Mushtaq I Farrell S Rampling D Nicholson J Filby A Burge J Lisgo S Maxwell PH Lindsay S Warren AY Stewart GD Sebire N Coleman N Haniffa M Teichmann SA Clatworthy M Behjati S (2018) Single-cell transcriptomes from human kidneys reveal the cellular identity of renal tumors Science 361:594–599. https://doi.org/10.1126/science.aat1699 PubMed Google Scholar Yousri NA Fakhro KA Robay A Rodriguez-Flores JL Mohney RP Zeriri H Odeh T Kader SA Aldous EK Thareja G Kumar M Al-Shakaki A Chidiac OM Mohamoud YA Mezey JG Malek JA Crystal RG Suhre K (2018) Whole-exome sequencing identifies common and rare variant metabolic QTLs in a Middle Eastern population Nature Communications 9:333. https://doi.org/10.1038/s41467-017-01972-9 PubMed Google Scholar Zelinka T Eisenhofer G Pacak K (2007) Pheochromocytoma as a catecholamine producing tumor: implications for clinical practice Stress 10:195–203. https://doi.org/10.1080/10253890701395896 PubMed Google Scholar Zhang X Lan Y Xu J Quan F Zhao E Deng C Luo T Xu L Liao G Yan M Ping Y Li F Shi A Bai J Zhao T Li X Xiao Y (2019) CellMarker: a manually curated resource of cell markers in human and mouse Nucleic Acids Research 47:D721–D728. https://doi.org/10.1093/nar/gky900 PubMed Google Scholar Decision letter Murim Choi Reviewing Editor; Seoul National University, Republic of Korea Mone Zaidi Senior Editor; Icahn School of Medicine at Mount Sinai, United States Murim Choi Reviewer; Seoul National University, Republic of Korea In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses. Decision letter after peer review: Thank you for submitting your work entitled "Single-cell transcriptome analysis identifies a unique tumor cell type producing multiple hormones in ectopic ACTH and CRH secreting pheochromocytoma" for further consideration by eLife. Your article has been reviewed by 3 peer reviewers, one of whom is a member of our Board of Reviewing Editors, and the evaluation has been overseen by Mone Zaidi as the Senior Editor. Reviewer #1: The authors identified an extremely rare case of ATCH-dependent Cushing syndrome due to ACTH&CRH secreting pheochromocytoma. They retrieved sugically resected samples from the tumor and subjected them to scRNA-seq, which led them to identify a group of cells that are double-positive for ACTH&CRH. They then performed a series of expriments to confirm that the cells are indeed present in the tissue, and attempted to identify genes that may lie upstream of the process. Perhaps the most important point of the study is the identification of the double-positive (DP) cells from the patient. However, evidence supporting this observation is relatively scarce other than showing a cell cluster that express POMC, CRH etc (as displayed in Figure 3A, C). Gene expression pattern shown in Figure 3C supports that the DP cells share molecular characteristics with those of pheochromocytes. But in the t-SNE plot, these cells are located far from pheochromocytes in PHEO_T. Rather, the DP cell cluster seems to be branched out from immune cells. If I didn't read the t-SNP plot wrong, I wonder why the identity of DP cells is closer to the immune cells. Also, it needs to be clarified if the DP cells could be doublets? The authors did not show basic statistics and QA/QC data of the scRNA-seq experiment (as supplementary data for example). They should show that the DP cells are not technical doublet cells. Another critical question would be what is the genetic driver that induces expression of both hormones in the DP cells? They propose GAL, but the evidence supporting its direct role is not strong and remains speculative. Comments for the authors: Overall, this study requires more carefully designed expriments and interpretation. Otherwise, it remains as a descriptive study with vague conclusions, leaving the uniqueness of the sample being the only strength of the study. 1. Colors in Figure 3A are confusing. 2. Figure 5 does not add much to the molecular mechanism. Rather it merely describes physiological consequences by the presence of DP cells. Please consider strengthen or remove it. 3. Isn't Figure 7B a duplication of Figure 3B? 4. IHC data in Figure 3E, F lack negative controls. And the readers need additional markers to be guided of its anatomical location. 5. Figure 4 compared DEGs between DP cells and other tumor cells. Since the cell groups that were being compared are too different, observing such dramatic differences is not unexpected and hard to coin physiological relevance. Wouldn't it be more meaningful to compare them to pheochromocytes? 6. The pseudotime analysis in Figure 6 does not answer the question of how the DP cells originated. It should be performed in a such way to suggest genes that marks critical points during the pseudotime branching or proceeding. Reviewer #2: In this manuscript Zhang et al. generated single cell RNA sequencing data for the adrenal gland tumors including extremely rare type of tumor, ACTH & CRH-secreting pheochromocytoma. Unbiased clustering analysis discovered a unique tumor cell type that expresses multiple hormones unlike normal adrenal gland cells and other tumor cell types that produce a single hormone. By comparing with other type of tumor cells, they identified specific marker genes of the novel tumor cell type. They also revealed the distinct immune and endothelial cell populations in the microenvironment of different tumor samples. Although the gene expression profiles of novel cell type can be utilized to reveal the molecular mechanism of this rare tumor associated with Cushing's syndrome, the data was generated from only a single patient and have not validated in other samples. In addition, the results only provide the list of genes that were specifically expressed in the novel tumor cell type and their potentially related biological pathways, but not detail molecular and cellular characters of the cells. The single cell gene expression profiling data are definitely useful for the researches. Comments for the authors: I have several concerns and suggestions, which if addressed would improve the manuscript. 1. The major finding of this manuscript is the presence of multi-functional tumor cell type which produce multiple hormones such as POMC, the precursor of ACTH and CRH. But, this finding was only derived from a single sample and experimentally validated using the same tissue. I understand the sample is very rare, but could the authors validate the result in different tumor samples at least using IHC or IF? If sample is not available, the limitation of the study should be mentioned. 2. Please consider providing full list of marker genes that were used for cell type annotation. 3. Figure 3C does not seem to support the statement "We demonstrated that GAL was expressed in the ACTH+&CRH+ pheochromocyte and 'regulated the secretion of ACTH'". 4. The authors identified a unique and important multi-functional cell type but current analyses (differentially expressed genes identification and gene ontology analysis) seem insufficient to characterize molecular feature of ACTH+&CRH+ pheochromocyte. The authors could perform additional comprehensive analysis such as SCENIC analysis in order to identify the master transcription regulator of the cell type. 5. The pseudo-time analysis indicated that sustentacular cells transform to ACTH+&CRH+ pehochromocytes and then to pheochromocyte. The authors utilized Monocle3 in which user has to define the starting points. The authors can validate the result using RNA velocity analysis which also predicts cell transition without the need of prior knowledge about starting point cell type. 6. Given the diverse immune and endothelial cell type in the tumor microenvironment, it would be interesting to perform the cell-cell interaction analysis using the programs such as CellPhoneDB to see if they have distinct regulatory role in different tumor microenvironment. 7. How did the authors define the four subclusters of endothelial cells? Please consider providing list of marker genes. 8. In the method part, how did the authors determine different criteria for the maximum number of genes (no more than 5000, 3000, and 2500 genes for PHEO, ACA, and esPHEO samples, respectively)? Reviewer #3: Zhang et al. perform single cell RNA sequencing (scRNA-Seq) of one rare ACTH+CRH-secreting phenochromocytoma (3 anatomically distinct sites from the tumor and one peritumoral site), one typical pheochromocytoma, and two typical adrenocortical adenomas. Their main findings are as follows: (1) They identify a unique cell type, which they term ACTH+CRH+ pheochromocyte, which appears to be the tumor cell present in the rare ACTH+CRH+ tumor (2) Marker gene analysis reveals that while known adrenal chromaffin markers (CHGA, PNMT) are present in both pheochromocytes and ACTH+CRH+ pheochromocyte, the latter has some unique markers such as GAL and POMC. They validate the marker genes with IHC. (3) Profiling of the non-tumor populations reveals distinct immune microenvironment profile and endothelial cell profile to the rare tumor compared with classical pheochromocytoma and adrenalocortical adenoma. The main strength of this manuscript is that it involves single-cell profiling of an exceptionally rare tumor type and a distinction from the more common adrenal tumors (pheochromocytoma and adrenocortical adenoma). The broader implication of the authors' findings is with respect to Dale's principle, which states that a given neuron releases only one type of neurotransmitter. However, in the case of this tumor, single cell analysis clearly shows that ACTH, CRH, and chatacholemines are being released from the same cell. This is quite interesting and significant. The data will also potentially be valuable to others in the field for analysis in future studies. There remain some unanswered questions – namely: (1) What is the cell in normal physiology that gives rise to this ACTH+CRH+ pheochromocytoma? (2) Do conventional phenochromocytomas differ from the ACTH+CRH+ pheochromocytoma in terms of the cell of origin that is transformed, or in the spectrum of genetic alterations that result in transformation? Comments for the authors: Overall, I think this study is of broad interest given the rarity of this tumor type. My comments to the authors to improve the manuscript are as follows: 1. Given how rare the ACTH+CRH+ pheochromocytoma is, I think the study would be substantially strengthened if the authors could perform DNA sequencing (WGS or WES) and describe how, if at all, the genomic landscape differs from conventional pheochromocytoma. 2. Can the authors comment on whether the hypothesis is whether the ACTH+CRH+ pheochromocytoma originates from a rare progenitor cell that is distinct from the chromaffin cell giving rise to pheochromocytoma? If so, can the authors stain a panel of normal adrenal glands with some of their marker genes to try and identify this cell in normal tissues? 3. While the tumor type is interesting for its rarity, the analysis performed is quite standard and comes across as a bit superficial in parts. Although it is understandable that the authors have only one ACTH+CRH+ sample I think they can do more with the data and this would significantly strengthen the manuscript. For example, it would be interesting if the authors can point to specific master regulatory factors that drive the distinct programs in pheochromocytes vs. ACTH+CRH+ pheochromocytes. The immune microenvironment analysis, while inherently descriptive, is also somewhat superficial. [Editors' note: further revisions were suggested prior to acceptance, as described below.] Thank you for submitting your revised article "Single-cell transcriptome analysis identifies a unique tumor cell type producing multiple hormones in ectopic ACTH and CRH secreting pheochromocytoma" for consideration by eLife. Your article has been reviewed by 3 peer reviewers, including Murim Choi as the Reviewing Editor and Reviewer #1, and the evaluation has been overseen by Mone Zaidi as the Senior Editor. The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission. Essential revisions: Although the reviewers thought that many issues were addressed, they still concerned on the superficial analysis results. Nonetheless, they agreed that the manuscript contains a common interest for publication in eLife as the tumor is an extremely rare case. Please address reviewers' concerns below. Reviewer #1: Although the authors could not address all the questions, especially regarding the origin of DP cells and genetic driver for DP cells, it appears reasonable that they are hard to address as the tumor sample was extremely rare. Reviewer #2: Although the authors have satisfactorily addressed most of my points, there are remaining concerns about RNA velocity data. Please cite any reference for the statement "For the high proportions of unspliced/spliced transcripts, stem-like characteristics of sustentacular cells were supported." Can global ratio of unspliced/spliced transcripts support stem-like characteristics? Please elaborate Figure 5 C-F. Currently, they don't seem to add any information. Reviewer #3: In the revised manuscript Zhang et al. have included additional data and analyses including more exhaustive QC, RNA velocity analysis, regulome analysis, and have performed WES of the ACTH/CRH-secreting pheochromocytoma. They have generally addressed my technical concerns from the prior review. I maintain that the analysis remains somewhat superficial and descriptive in parts and this may be somewhat of a missed opportunity to more deeply explore the underlying biology of this unique case, understanding the caveats of its rarity. Nonetheless, I think a description of this tumor at single-cell resolution and availability of the dataset is of value to the scientific community. However, I would like to see a more careful analysis of the WES data prior to publication. I do not see any basic metrics (mutation rate etc.), description of pathogenicity filtering/annotation, or copy number analysis. The mutations shown are primarily missense and I do not really see any obvious driver genes – how many of these are putative driver vs. passenger mutations? ACAN is mentioned, but what is its significance, if any? The somatic landscape should be discussed in comparison to typical phenochromocytomas and adrenocortical carcinomas, which have been more extensively sequenced. If there is no obvious genetic driver of this ACTH/CRH-secreting phenochromocytoma, that should be stated. If the claim is that ACAN alterations are somehow related to this tumor type, that needs to be substantiated. Or if the implication is that ACAN is a passenger alteration, that needs to be stated explicitly also. https://doi.org/10.7554/eLife.68436.sa1 Author response Reviewer #1: The authors identified an extremely rare case of ATCH-dependent Cushing syndrome due to ACTH&CRH secreting pheochromocytoma. They retrieved surgically resected samples from the tumor and subjected them to scRNA-seq, which led them to identify a group of cells that are double-positive for ACTH&CRH. They then performed a series of experiments to confirm that the cells are indeed present in the tissue, and attempted to identify genes that may lie upstream of the process. We thank the reviewer for carefully reviewing the manuscript. We updated graphs, added supplementary files of raw data QC and cell cluster statistics, and performed RNA velocity analysis, scenic analysis for the single cell RNA sequencing experiments to response the reviewer’s critiques and strengthen the manuscript. In addition, to investigate the genetic driver for Case 1, we supplemented whole-exome sequencing experiments for all rest specimens, that is, tumors (esPHEO_T2, esPHEO_T3) and controls (esPHEO_Adj, esPHEO_Blood) from the rare case with ectopic ACTH&CRH-secreting pheochromocytoma. Perhaps the most important point of the study is the identification of the double-positive (DP) cells from the patient. However, evidence supporting this observation is relatively scarce other than showing a cell cluster that express POMC, CRH etc (as displayed in Figure 3A, C). Gene expression pattern shown in Figure 3C supports that the DP cells share molecular characteristics with those of pheochromocytes. But in the t-SNE plot, these cells are located far from pheochromocytes in PHEO_T. Rather, the DP cell cluster seems to be branched out from immune cells. If I didn't read the t-SNP plot wrong, I wonder why the identity of DP cells is closer to the immune cells. Also, it needs to be clarified if the DP cells could be doublets? The authors did not show basic statistics and QA/QC data of the scRNA-seq experiment (as supplementary data for example). They should show that the DP cells are not technical doublet cells. We thank the reviewer for raising the concerns and providing these helpful suggestions. First, we updated the colors mapped to 16 cellular clusters in Figure 2A and Figure 3A to enhance the color difference between doublet-positive (DP) cells and immune cells. Then, the new analysis based on RNA velocity was performed in the revision, and the results showed that DP cluster was isolated and not branched out from other cell types (including immune cells) from velocity streamlines (Figure 5F). In addition, we added the raw data QC and doublet prediction results of the scRNA-seq experiment as shown in Appendix 1—figure 2 and Supplementary File 1. From the doublets predicted by DoubletFinder and DoubletDecon, it is clarified that almost noDP cells were defined as doublets. Cellular cluster statistics were shown in Supplementary File 2, which presented cell counts for each cellular cluster in different samples and top10 gene markers. Another critical question would be what is the genetic driver that induces expression of both hormones in the DP cells? They propose GAL, but the evidence supporting its direct role is not strong and remains speculative. We thank the reviewer for raising these important concerns, and we agree with the reviewer that the presentation about the genetic driver in the previous version of the manuscript is not sufficient enough. We changed the conclusion statement "We demonstrated that GAL was expressed in the ACTH+&CRH+ pheochromocyte and regulated the secretion of ACTH" to "We demonstrated that GAL was expressed in the ACTH+&CRH+ pheochromocyte and might participate in the regulation of ACTH secretion". (Page 7 line 175-182) We provided more description and additional analysis about putative genetic driver in the DP cells, as follows: First, we found GAL co-expressed with POMC and CRH, could be a candidate marker to detect the rare ectopic ACTH+&CRH+ secreting pheochromocytes. It might be involved in the regulation of the hypothalamic-pituitary-adrenal axis. (Page 7 line 175-182, Figure 3, Figure 4). Second, we also found an additional weak signal of transcription regulons for the DP cells (Page 6 line 153-157, Appendix 1—figure 4). It showed XPBP1 as the specific regulons for ACTH+&CRH+ pheochromocyte and adrenocortical cell type. Third, to investigate the genetic driver, we supplemented whole-exome sequencing experiments for tumors (esPHEO_T2, esPHEO_T3) and controls (esPHEO_Adj, esPHEO_Blood) from the rare case with ectopic ACTH&CRH-secreting pheochromocytoma. We identified 1 shared somatic variant of ACAN (c.5951T>A:p.L1984Q) comparing variants in tumor samples to controls but Sanger sequencing only confirmed the presence in esPHEO_T3 which was not observed in esPHEO_T2 (Page 13 line 352-358, Appendix 1—figure 7). Comments for the authors: Overall, this study requires more carefully designed experiments and interpretation. Otherwise, it remains as a descriptive study with vague conclusions, leaving the uniqueness of the sample being the only strength of the study. We thank the reviewer for carefully reviewing and helpful suggestions. We updated graphs and tables, implemented supplementary analysis for the single-cell RNA sequencing data. Because this case is particularly rare, fresh tissue samples are lacking, currently, frozen tissue samples cannot be assayed by flow cytometry. For all rest of the samples, we can only supplement the whole-exome sequencing experiments for tumors (esPHEO_T2, esPHEO_T3) and controls (esPHEO_Adj, esPHEO_Blood) from the rare case with ectopic ACTH&CRH-secreting pheochromocytoma to make our results more comprehensive. Lastly, on one hand, we are very concerned about similar suspicious cases in the clinic. On the other hand, we are going for the following research for further downstream experiments to validate the molecular mechanism for secreting multiple hormones. 1. Colors in Figure 3A are confusing. We have updated the colors mapped to 16 cellular clusters in Figure 2 and Figure 3 to enhance the color difference between doublet-positive (DP) cells and immune cells. 2. Figure 5 does not add much to the molecular mechanism. Rather it merely describes physiological consequences by the presence of DP cells. Please consider strengthen or remove it. Due to the previous Figure 5 mainly describe the physiological consequences by the presence of DP cells as the reviewer commented. We have moved it to Figure 4D, because the differential expressed genes between DP cells and other adrenal cell types were shown in Figure 4A and Figure 4C. Combining these figures into a group could complement each other and clarify the secreting functions of the DP cells. 3. Isn't Figure 7B a duplication of Figure 3B? Figure 3B presents the frequency distribution of all cell types among different samples, while in Figure 7B we specifically focused on the immune microenvironments and showed statistics of immune cell types. To some extent, they are repetitive since both describe the percentage of immune cells. But the denominators are different for percentage calculation, that is, one is the total number of cells in Figure 3B, the other is the total number of immune cells in Figure 7B. 4. IHC data in Figure 3E, F lack negative controls. And the readers need additional markers to be guided of its anatomical location. We supplemented IHC figures of CgA, ACTH, POMC, CRH, TH or GAL with magnification (0.5x, 2.5x, 10x, 40x) from tumor specimen infiltrating tissues located in the kidney (esPHEO_T3) in Appendix 1—figure 8. We observed positive staining signal at tumor left in each slice, while the adjacent kidney was un-stained could be negative controls. Red rectangular indicates the magnified area of the location as shown in Figure 3D. The. We supplemented the immunofluorescence (IF) co-staining figures with magnification (10x, 40x) for POMC&CRH and POMC&TH from tumor specimen esPHEO_T3 in Appendix 1—figure 9, where red rectangular indicates the magnified area of the location in Figure 3E. 5. Figure 4 compared DEGs between DP cells and other tumor cells. Since the cell groups that were being compared are too different, observing such dramatic differences is not unexpected and hard to coin physiological relevance. Wouldn't it be more meaningful to compare them to pheochromocytes? We analyzed the differentially expressed genes (DEGs) between ACTH+&CRH+ pheochromocyte and the other two subtypes of adrenal tumor cells (pheochromocyte and adrenocortical cells) (Page 9 line 241-245). Such dramatic differences were observed because we set the statistically significant differences as a cut-off p-value < 0.05 and a fold change ≥ 1.5 ( which means a log2 fold change |logFC| ≥ 0.585 ) (Figure 4A). It could more strict such as a cut-off p-value <0.01 and a fold change ≥ 2 ( which means a log2 fold change |logFC| ≥ 1 ). But the top significantly differentially expressed genes were POMC, CRH, GAL etc, as marked in Figure 4A. There is a relatively larger difference in gene expression between DP cells and adrenocortical cells than that between DP cells and pheochromocytes (Figure 4C). Since we didn’t identify any pheochromocytes in esPHEO_adj, we could not compare the DP cells to their adjacent pheochromocytes (Supplementary File 2). Reviewer #2: In this manuscript Zhang et al. generated single cell RNA sequencing data for the adrenal gland tumors including extremely rare type of tumor, ACTH & CRH-secreting pheochromocytoma. Unbiased clustering analysis discovered a unique tumor cell type that expresses multiple hormones unlike normal adrenal gland cells and other tumor cell types that produce a single hormone. By comparing with other type of tumor cells, they identified specific marker genes of the novel tumor cell type. They also revealed the distinct immune and endothelial cell populations in the microenvironment of different tumor samples. Although the gene expression profiles of novel cell type can be utilized to reveal the molecular mechanism of this rare tumor associated with Cushing's syndrome, the data was generated from only a single patient and have not validated in other samples. In addition, the results only provide the list of genes that were specifically expressed in the novel tumor cell type and their potentially related biological pathways, but not detail molecular and cellular characters of the cells. The single cell gene expression profiling data are definitely useful for the researches. We thank the reviewer for carefully reviewing and raising insightful critiques. In this study, we reported a rare case in which ectopic ACTH&CRH-secreting pheochromocytoma in the left adrenal. To identify the hormones-secreting cells, we sent specimens for single-cell transcriptome sequencing immediately after the resection. Thus, the majority of our analysis focused on the validation of novel tumor cell type and their multiple hormones-secreting functions. For future studies, on one hand, we are very concerned about similar suspicious cases in the clinic. On the other hand, we are going for following research for further downstream experiments to validate the molecular mechanism for secreting multiple hormones. Comments for the authors:I have several concerns and suggestions, which if addressed would improve the manuscript. 1. The major finding of this manuscript is the presence of multi-functional tumor cell type which produce multiple hormones such as POMC, the precursor of ACTH and CRH. But, this finding was only derived from a single sample and experimentally validated using the same tissue. I understand the sample is very rare, but could the authors validate the result in different tumor samples at least using IHC or IF? If sample is not available, the limitation of the study should be mentioned. For the case of ACTH and CRH secreting pheochromocytoma, we performed the surgical resection of the tumor at left adrenal (esPHEO_T1) and its infiltrating tissues located in the kidney (esPHEO_T3) and masses (esPHEO_T2), and obtained 3 tumor specimens. The peritumor sample (esPHEO_Adj) was collected from the left adrenal tissue under the supervision of a qualified pathologist. At first, we performed immunohistochemistry (IHC) staining with chromogranin A (CgA) and ACTH markers for esPHEO_T1 and adjacent specimen (esPHEO_Adj) (Figure 1B). To validate our discovery from scRNA-seq data we implemented IHC of CgA, ACTH, POMC, CRH or TH (Figure 3D) on serial biopsies from another tumor specimen (esPHEO_T3) and added immunofluorescence co-staining for POMC&CRH and POMC&TH on two serial biopsies from esPHEO_T3 (Figure 3E). The frozen tissue of esPHEO_T1 is unavailable and a few remaining for esPHEO_T2. For all rest of tissue samples, we supplemented with the whole-exome sequencing experiments for tumors (esPHEO_T2, esPHEO_T3) and controls (esPHEO_Adj) from the rare case with ectopic ACTH&CRH-secreting pheochromocytoma. 2. Please consider providing full list of marker genes that were used for cell type annotation. We add row annotations for top10 marker genes at the heatmap showing different cellular clusters and their highly expressed genes (Figure 2B). Cellular cluster statistics were supplemented in Supplementary File 2, which presented cell counts for each cellular cluster in different samples and top10 gene markers. 3. Figure 3C does not seem to support the statement "We demonstrated that GAL was expressed in the ACTH+&CRH+ pheochromocyte and 'regulated the secretion of ACTH'". We changed the conclusion sentence to "We demonstrated that GAL was expressed in the ACTH+&CRH+ pheochromocyte and might participate in the regulation of ACTH secretion". We’re trying to express that: [We found GAL co-expressed with POMC and CRH, could be a candidate marker to detect the rare ectopic ACTH+&CRH+ secreting pheochromocytes. As previous research reported, it might be involved in the regulation of the hypothalamic-pituitary-adrenal axis.] 4. The authors identified a unique and important multi-functional cell type but current analyses (differentially expressed genes identification and gene ontology analysis) seem insufficient to characterize molecular feature of ACTH+&CRH+ pheochromocyte. The authors could perform additional comprehensive analysis such as SCENIC analysis in order to identify the master transcription regulator of the cell type. We have performed additional analysis (Page 18 line 519-570), including RNA velocity analysis, SCENIC analysis etc. In addition, whole-exome sequencing experiments for tumors (esPHEO_T2, esPHEO_T3) and controls (esPHEO_Adj, esPHEO_Blood) from the rare case with ectopic ACTH&CRH-secreting pheochromocytoma were performed to make our results more comprehensive. First, based on differentially expressed genes identification, we mainly found GAL co-expressed with POMC and CRH, could be a candidate marker to detect the rare ectopic ACTH+&CRH+ secreting pheochromocytes. It might be involved in the regulation of the hypothalamic-pituitary-adrenal axis. (Page 7 line 175-182, Figure 3, Figure 4). Second, applied the SCENIC pipeline, we found an additional weak signal of transcription regulons for the DP cells (Page 6 line 153-157, Appendix 1—figure 4). It showed XPBP1 as the specific regulons for ACTH+&CRH+ pheochromocyte and adrenocortical cell type. Third, the spliced vs. unspliced phase for CHGA, CHGB, and TH from RNA velocity analysis demonstrated a clear more dynamics expression in POMC+&CRH+ pheochromocytes than in pheochromocytes (Appendix 1—figure 5). Lastly, to investigate the genetic driver, the whole exome sequencing identified 1 shared somatic variant of ACAN (c.5951T>A:p.L1984Q) comparing variants in tumor samples to controls but Sanger sequencing only confirmed the presence in esPHEO_T3 which not observed in esPHEO_T2 (Page 13 line 352-358, Appendix 1—figure 7). 5. The pseudo-time analysis indicated that sustentacular cells transform to ACTH+&CRH+ pehochromocytes and then to pheochromocyte. The authors utilized Monocle3 in which user has to define the starting points. The authors can validate the result using RNA velocity analysis which also predicts cell transition without the need of prior knowledge about starting point cell type. At first, we have added RNA velocity analysis (Figure 5B, Page 10 line 268-286). For the high proportions of unspliced/spliced transcripts in Figure 5B, stem-like characteristics of sustentacular cells were supported. We performed the pseudo-time analysis for the adrenal tumor cell subsets to determine the pattern of the dynamic cell transitional states. Then, we re-run the pseudo-time analysis and used the recommended strategy of Monocel to order cells based on genes that differ between clusters. The sustentacular cells were also in an early stage (Figure 6). 6. Given the diverse immune and endothelial cell type in the tumor microenvironment, it would be interesting to perform the cell-cell interaction analysis using the programs such as CellPhoneDB to see if they have distinct regulatory role in different tumor microenvironment. To investigate the potential cell-cell interactions among various immune cells, endothelial cells, and other cell types in the different tumor microenvironment (esPHEO, esPHEO_Adj, PHEO, and ACA), we performed additional analysis using the CellPhoneDB Python package in the revised version of our manuscript. As shown in the new Appendix 1—figure 6, we observed very distinct patterns of ligand-receptor pairs for cell-cell interactions in the different tumor microenvironments. Notably, the diverse cell clusters within PHEO tumors exhibited a relatively high abundance of cell-cell connections between different cell types, while the cell-cell interactions within esPHEO_Adj samples were totally different. For example, MIF, one of the most enigmatic regulators of innate and adaptive immune responses, was shown as a specific regulator in esPHEO and PHEO, in contrast to ACA. 7. How did the authors define the four subclusters of endothelial cells? Please consider providing list of marker genes. The four groups of endothelial cells were combined to a larger endothelial cell cluster for downstream analysis. Endothelial cell cluster statistics were added in Supplementary File 3, which presented cell counts for each endothelial cell cluster in different samples and top10 gene markers. 8. In the method part, how did the authors determine different criteria for the maximum number of genes (no more than 5000, 3000, and 2500 genes for PHEO, ACA, and esPHEO samples, respectively)? We set the different criteria for the maximum number of genes (no more than 5000, 3000, and 2500 genes for PHEO, ACA and esPHEO samples respectively) based on QC violin plot showing the number of detected genes (Appendix 1—figure 2B). Reviewer #3: Zhang et al. perform single cell RNA sequencing (scRNA-Seq) of one rare ACTH+CRH-secreting phenochromocytoma (3 anatomically distinct sites from the tumor and one peritumoral site), one typical pheochromocytoma, and two typical adrenocortical adenomas. Their main findings are as follows: (1) They identify a unique cell type, which they term ACTH+CRH+ pheochromocyte, which appears to be the tumor cell present in the rare ACTH+CRH+ tumor (2) Marker gene analysis reveals that while known adrenal chromaffin markers (CHGA, PNMT) are present in both pheochromocytes and ACTH+CRH+ pheochromocyte, the latter has some unique markers such as GAL and POMC. They validate the marker genes with IHC. (3) Profiling of the non-tumor populations reveals distinct immune microenvironment profile and endothelial cell profile to the rare tumor compared with classical pheochromocytoma and adrenalocortical adenoma. The main strength of this manuscript is that it involves single-cell profiling of an exceptionally rare tumor type and a distinction from the more common adrenal tumors (pheochromocytoma and adrenocortical adenoma). The broader implication of the authors' findings is with respect to Dale's principle, which states that a given neuron releases only one type of neurotransmitter. However, in the case of this tumor, single cell analysis clearly shows that ACTH, CRH, and chatacholemines are being released from the same cell. This is quite interesting and significant. The data will also potentially be valuable to others in the field for analysis in future studies. There remain some unanswered questions – namely: (1) What is the cell in normal physiology that gives rise to this ACTH+CRH+ pheochromocytoma? (2) Do conventional phenochromocytomas differ from the ACTH+CRH+ pheochromocytoma in terms of the cell of origin that is transformed, or in the spectrum of genetic alterations that result in transformation? We thank the reviewer for carefully reviewing the manuscript and raising insightful questions. To response the reviewer’s questions and strengthen the manuscript, we supplemented analysis and experiments as much as possible. First, we performed RNA velocity analysis (Figure 5, Page 10 line 268-286) to investigate dynamic information in individual cells. For the high proportions of unspliced/spliced transcripts in Figure 5B, stem-like characteristics of sustentacular cells were supported. Also, the spliced vs. unspliced phase for CHGA, CHGB, and TH from RNA velocity analysis demonstrated a clear more dynamics expression in POMC+&CRH+ pheochromocytes than in pheochromocytes (Appendix 1—figure 5). Second, we re-run the pseudo-time analysis (Page 10 line 288-300) and used the recommended strategy of Monocel to order cells based on genes that differ between clusters. The sustentacular cells were also in an early state (Figure 6), which was in accordance with their exhibited stem-like properties and the highest unspliced proportion among non-immune cell types in the RNA velocity analysis (Figure 5B). The results also showed a transition from sustentacular cells to pheochromocytes and then to ACTH+&CRH+ pheochromocyte, and adrenocortical cells were on another branch (Figure 6). As we discussed in manuscript (Page 14 line 391-398), although pheochromocyte was prior to ACTH&CRH secreting pheochromocyte in pseudotime order, we assumed that ACTH&CRH secreting pheochromocyte have more hormone-producing functions, retain stem- and endocrine-differentiation ability. But further experiments are needed to validate our hypothesis. Third, we applied SCENIC analysis pipeline (Page 6 line 153-157, Appendix 1—figure 4) to detect the transcription factors (which are jointly called regulons) alongside their candidate target genes, and yield specific regulons for each cellular cluster. We observed an additional weak signal of transcription regulons (XPBP1) for the ACTH+CRH+ pheochromocytoma and adrenocortical cell type. Furthermore, to investigate the genetic driver, we supplemented with the whole-exome sequencing (WES) experiments for all rest of tissue samples (esPHEO_T2, esPHEO_T3 and esPHEO_Adj) from the rare case with ectopic ACTH&CRH-secreting pheochromocytoma and the blood sample (esPHEO_Blood). Based on WES data, we identified 1 shared somatic variant of ACAN (c.5951T>A:p.L1984Q) comparing variants in tumor samples to controls but Sanger sequencing only confirmed the presence in esPHEO_T3 which not observed in esPHEO_T2 (Page 13 line 352-358, Appendix 1—figure 7). Overall, additional analyses and experiments have presented more comprehensive results which appropriately address the questions raised by the reviewer. But they also provide new hypothesis remaining unanswered questions. For future studies, on one hand, we are very concerned about similar suspicious cases in the clinic. On the other hand, we are going for following research for further downstream experiments to validate the molecular mechanism for secreting multiple hormones. Comments for the authors: Overall, I think this study is of broad interest given the rarity of this tumor type. My comments to the authors to improve the manuscript are as follows: 1. Given how rare the ACTH+CRH+ pheochromocytoma is, I think the study would be substantially strengthened if the authors could perform DNA sequencing (WGS or WES) and describe how, if at all, the genomic landscape differs from conventional pheochromocytoma. The frozen tissue of esPHEO_T1 and PHEO_T is unavailable and a few remaining for esPHEO_T2. For all rest of tissue samples, we supplemented with the whole-exome sequencing experiments for tumors (esPHEO_T2, esPHEO_T3) and controls (esPHEO_Adj) from the rare case with ectopic ACTH&CRH-secreting pheochromocytoma. (Page 13 line 352-358, Appendix 1—figure 7) 2. Can the authors comment on whether the hypothesis is whether the ACTH+CRH+ pheochromocytoma originates from a rare progenitor cell that is distinct from the chromaffin cell giving rise to pheochromocytoma? If so, can the authors stain a panel of normal adrenal glands with some of their marker genes to try and identify this cell in normal tissues? (Page 14 line 389-398) The RNA velocity estimation and pseudo-time analysis of different adrenal cell subtypes supported the sustentacular cells exhibiting stem-like properties. Although pheochromocyte was prior to ACTH&CRH secreting pheochromocyte in pseudotime order, the RNA velocity prediction of POMC+&CRH+ pheochromocytes might be under-estimated because the transcripts of POMC and CRH were all predicted as spliced ones. Based on the spliced vs. unspliced phase for CHGA, CHGB and TH it showed a clear more dynamics expression in POMC+&CRH+ pheochromocytes than in pheochromocytes. We assumed that ACTH&CRH secreting pheochromocyte have more hormone-producing functions, retain stem- and endocrine-differentiation ability. But further experiments are needed to validate our hypothesis. We thank the reviewer for raising good recommendations. We would like to test marker genes in normal tissues. But it is difficult to obtain normal adrenal glands in clinic. We searched POMC, CRH and GAL in Genotype-Tissue Expression Project (GTEx), which launched by the National Institutes of Health (NIH). GTEx has established a database (https://www.gtexportal.org/home/) to study genes in different normal tissues. The results, as shown in Author response images 1-3: POMC is over-expressed in pituitary, but expressed at a very low level in adrenal gland. CRH is overexpressed in brain-hypothalamus, but almost not expressed in adrenal gland. GAL is overexpressed in pituitary and brain-hypothalamus, but almost not expressed in adrenal gland. Author response image 1 Download asset Open asset Author response image 2 Download asset Open asset Author response image 3 Download asset Open asset 3. While the tumor type is interesting for its rarity, the analysis performed is quite standard and comes across as a bit superficial in parts. Although it is understandable that the authors have only one ACTH+CRH+ sample I think they can do more with the data and this would significantly strengthen the manuscript. For example, it would be interesting if the authors can point to specific master regulatory factors that drive the distinct programs in pheochromocytes vs. ACTH+CRH+ pheochromocytes. The immune microenvironment analysis, while inherently descriptive, is also somewhat superficial. Based on the routine differentially expressed genes analysis, we mainly found GAL co-expressed with POMC and CRH, could be a candidate marker to detect the rare ectopic ACTH+&CRH+ secreting pheochromocytes. As previous research reported, it might be involved in the regulation of the hypothalamic-pituitary-adrenal axis. (Page 7 line 175-182, Figure 3, Figure 4). Second, applied the SCENIC pipeline, we found an additional weak signal of transcription regulons for the DP cells (Page 6 line 153-157, Appendix 1—figure 4). It showed XPBP1 as the specific regulons for ACTH+&CRH+ pheochromocyte and adrenocortical cell type. Furthermore, RNA velocity analysis (Appendix 1—figure 5) demonstrated a clear more dynamics expression in POMC+&CRH+ pheochromocytes than in pheochromocytes. [Editors' note: further revisions were suggested prior to acceptance, as described below.] Reviewer #2: Although the authors have satisfactorily addressed most of my points, there are remaining concerns about RNA velocity data. Please cite any reference for the statement "For the high proportions of unspliced/spliced transcripts, stem-like characteristics of sustentacular cells were supported." Can global ratio of unspliced/spliced transcripts support stem-like characteristics? Please elaborate Figure 5 C-F. Currently, they don't seem to add any information. (Page 10 line 269-286, Figure 5 and its legend) We thank the reviewer for carefully reviewing and raising this concern about RNA velocity. We have revised our manuscript to add a paragraph and cite the appropriate references in the updated revision. Previously study had observed that the unspliced transcripts were enriched in genes involved in DNA binding and RNA processing in hematopoietic stem cells [1]. And Schwann cell precursors, which can differentiate into chromaffin cells, also had positive unspliced-spliced phase portrait [2]. Therefore, we claimed that, as for the high proportions of unspliced/spliced transcripts, stem-like characteristics of sustentacular cells were supported. We remove Figure 5 C-D, as the reviewer mentioned, because they don't seem to add any valuable information. Besides, we added more description about the results for new Figure 5 C-D (old Figure 5 E-F) in Page 10 line 282-288, which showed estimated pseudo-time grounded on transcriptional dynamics and velocity streamlines accounting for speed and direction of motion. These results indicated that medullary cells are earlier than cortical cells (new Figure 5C). From velocity streamlines (new Figure 5D), we found the four adrenal cell subtypes, that is, POMC+&CRH+ pheochromocytes, pheochromocytes adrenocortical cells, and sustentacular cells, were independent respectively and not directed toward other cell types. Reviewer #3: In the revised manuscript Zhang et al. have included additional data and analyses including more exhaustive QC, RNA velocity analysis, regulome analysis, and have performed WES of the ACTH/CRH-secreting pheochromocytoma. They have generally addressed my technical concerns from the prior review. I maintain that the analysis remains somewhat superficial and descriptive in parts and this may be somewhat of a missed opportunity to more deeply explore the underlying biology of this unique case, understanding the caveats of its rarity. Nonetheless, I think a description of this tumor at single-cell resolution and availability of the dataset is of value to the scientific community. However, I would like to see a more careful analysis of the WES data prior to publication. I do not see any basic metrics (mutation rate etc.), description of pathogenicity filtering/annotation, or copy number analysis. The mutations shown are primarily missense and I do not really see any obvious driver genes – how many of these are putative driver vs. passenger mutations? ACAN is mentioned, but what is its significance, if any? The somatic landscape should be discussed in comparison to typical phenochromocytomas and adrenocortical carcinomas, which have been more extensively sequenced. If there is no obvious genetic driver of this ACTH/CRH-secreting phenochromocytoma, that should be stated. If the claim is that ACAN alterations are somehow related to this tumor type, that needs to be substantiated. Or if the implication is that ACAN is a passenger alteration, that needs to be stated explicitly also. (Page 13 line 359-378; Page 21 line 587-597; Supplementary File 4) We thank the reviewer for carefully reviewing and raising concerns about our WES analysis. We supplemented the variants filtering criteria in Page 21 line 587-597, and further discussed the WES results in Page 13 line 359-378. Besides, the germline and somatic mutations were listed in Supplementary File 4 including detailed annotations. Genetic mutations of phaeochromocytoma and paraganglioma are mainly classified into two major clusters, that is, pseudo hypoxic pathway and kinase signaling pathways [3-4]. We did not find any gene mutations or copy number variations that were related to these two major clusters. We only identified 1 shared somatic variant of ACAN mutation (c.5951T>A:p.L1984Q) comparing variants in tumor samples to controls. ACAN, encoding a major component of the extracellular matrix, is a member of the aggrecan/versican proteoglycan family. Mutations of ACAN were reported related to steroid levels [5]. It is well-established that circulating steroid levels are linked to inflammatory diseases such as arthritis, because arthritis as well as most autoimmune disorders result from a combination of several predisposing factors including the stress response system such as the hypothalamic-pituitary-adrenocortical axis [6]. But no direct evidence related to ACAN for phaeochromocytoma. Therefore, no obvious genetic driver was found to explain the rare case of ACTH/CRH-secreting phaeochromocytoma. Further investigations would be needed to uncover the relation between ACAN to phaeochromocytoma. References: [1]. Bowman TV, McCooey AJ, Merchant AA, Ramos CA, Fonseca P, Poindexter A, Bradfute SB, Oliveira DM, Green R, Zheng Y, Jackson KA, Chambers SM, McKinney-Freeman SL, Norwood KG, Darlington G, Gunaratne PH, Steffen D, Goodell MA. Differential mRNA processing in hematopoietic stem cells. Stem Cells. 2006. Mar;24(3):662-70. [2]. La Manno G., Soldatov R., Zeisel A., Braun E., Hochgerner H., Petukhov V., Lidschreiber K., Kastriti M.E., Lönnerberg P., Furlan A. RNA velocity of single cells. Nature. 2018 560:494-498. [3] Pillai S, Gopalan V, Smith RA, Lam AK. Updates on the genetics and the clinical impacts on phaeochromocytoma and paraganglioma in the new era. Crit Rev Oncol Hematol. 2016. Apr;100:190-208. [4] Nölting S, Grossman AB. Signaling pathways in pheochromocytomas and paragangliomas: prospects for future therapies. Endocr Pathol. 2012. Mar;23(1):21-33. [5] Yousri NA, Fakhro KA, Robay A, Rodriguez-Flores JL, Mohney RP, Zeriri H, Odeh T, Kader SA, Aldous EK, Thareja G, Kumar M, Al-Shakaki A, Chidiac OM, Mohamoud YA, Mezey JG, Malek JA, Crystal RG, Suhre K. Whole-exome sequencing identifies common and rare variant metabolic QTLs in a Middle Eastern population. Nat Commun. 2018 Jan 23;9(1):333. [6]. Cutolo M, Sulli A, Pizzorni C, Craviotto C, Straub RH. Hypothalamic-pituitary-adrenocortical and gonadal functions in rheumatoid arthritis. Ann N Y Acad Sci. 2003 May;992:107-17. https://doi.org/10.7554/eLife.68436.sa2 Article and author information Author details Xuebin Zhang Department of Urology, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, China Contribution Data curation, Formal analysis, Investigation, Methodology, Resources, Software, Writing – original draft, Writing – review and editing Contributed equally with Penghu Lian and Mingming Su Competing interests No competing interests declared Penghu Lian Department of Urology, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, China Contribution Formal analysis, Investigation, Methodology, Project administration, Resources, Software, Validation, Visualization, Writing – original draft, Writing – review and editing Contributed equally with Xuebin Zhang and Mingming Su Competing interests No competing interests declared Mingming Su Institute of Basic Medical Sciences Chinese Academy of Medical Sciences, School of Basic Medicine Peking Union Medical College, Beijing, China Contribution Data curation, Formal analysis, Investigation, Methodology, Resources, Software, Validation, Visualization, Writing – original draft, Writing – review and editing Contributed equally with Xuebin Zhang and Penghu Lian Competing interests No competing interests declared "This ORCID iD identifies the author of this article:"0000-0002-1393-0800 Zhigang Ji Department of Urology, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, China Contribution Data curation, Investigation, Methodology, Visualization, Writing – review and editing Competing interests No competing interests declared Jianhua Deng Department of Urology, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, China Contribution Data curation, Investigation, Methodology, Writing – review and editing Competing interests No competing interests declared Guoyang Zheng Department of Urology, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, China Contribution Data curation, Investigation, Writing – review and editing Competing interests No competing interests declared Wenda Wang Department of Urology, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, China Contribution Data curation, Investigation, Writing – review and editing Competing interests No competing interests declared Xinyu Ren Department of Pathology, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, China Contribution Data curation, Visualization Competing interests No competing interests declared Taijiao Jiang Institute of Basic Medical Sciences Chinese Academy of Medical Sciences, School of Basic Medicine Peking Union Medical College, Beijing, China Suzhou Institute of Systems Medicine, Jiangsu, China Contribution Conceptualization, Funding acquisition, Project administration, Supervision, Writing – review and editing Competing interests No competing interests declared Peng Zhang Beijing Key Laboratory for Genetics of Birth Defects, Beijing Pediatric Research Institute, Beijing Children's Hospital, Capital Medical University, National Center for Children's Health, Beijing, China Contribution Investigation, Methodology, Supervision, Validation, Writing – original draft, Writing – review and editing For correspondence zhangpengdyx@163.com Competing interests No competing interests declared "This ORCID iD identifies the author of this article:"0000-0002-6218-1885 Hanzhong Li Department of Urology, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, China Contribution Conceptualization, Funding acquisition, Project administration, Supervision, Writing – review and editing For correspondence lihzh@pumch.cn Competing interests No competing interests declared Funding Chinese Academy of Medical Sciences (2017-I2M-1-001) Hanzhong Li Chinese Academy of Medical Sciences (2021-I2M-1-051) Taijiao Jiang Chinese Academy of Medical Sciences (2021-I2M-1-001) Taijiao Jiang The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication. Acknowledgements This work was supported by CAMS Innovation Funds for Medical Sciences (CIFMS), which were 2017-I2M-1-001, 2021-I2M-1-051 and 2021-I2M-1-001. Ethics Specimen collection was obtained after appropriate research consents (and assents when applicable) and was approved (protocol number: S-K431) by the Institutional Review Board, Peking Union Medical College Hospital. All information obtained was protected and de-identified. Senior Editor Mone Zaidi, Icahn School of Medicine at Mount Sinai, United States Reviewing Editor Murim Choi, Seoul National University, Republic of Korea Reviewer Murim Choi, Seoul National University, Republic of Korea Publication history Received: March 16, 2021 Accepted: December 13, 2021 Accepted Manuscript published: December 14, 2021 (version 1) Accepted Manuscript updated: December 15, 2021 (version 2) Version of Record published: December 31, 2021 (version 3) Copyright © 2021, Zhang et al. This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited. from https://elifesciences.org/articles/68436

This month marks a little over one year since the first surge of COVID-19 across the United States. April is also Adrenal Insufficiency Awareness month, a good time to review the data on how COVID-19 infection can impact the adrenal glands. The adrenal glands make hormones to help regulate blood pressure and the ability to respond to stress. The hormones include steroids such as glucocorticoid (cortisol), mineralocorticoid (aldosterone), and forms of adrenaline known as catecholamines (norepinephrine, epinephrine, and dopamine). The activity of the adrenal gland is controlled through its relationship with the pituitary gland (the master regulator of hormones in the body). Some common adrenal diseases include the following: Addison’s Disease (where the body attacks the adrenal glands making them dysfunctional) Hyperaldosteronism Cushing’s Syndrome Pheochromocytoma Adrenal Nodules/Masses (termed incidentaloma) Congenital adrenal hyperplasia COVID-19 was found in the adrenal and pituitary glands of some patients who succumbed to the illness, suggesting that these organs might be among the targets for infection. One of the first highly effective therapies for COVID-19 infection was the use of IV steroid (dexamethasone) supplementation in hospitalized patients in patients requiring oxygen. A focused search of COVID-19-related health literature shows 85 peer-reviewed papers that have been published in medical literature specifically on the adrenal gland and COVID-19. This literature focuses on three phases of COVID infection that may impact the adrenal gland: the acute active infection phase, the immediate post-infection phase, and the long-term recovery phase. Medical research has identified that during the acute active infection, the adrenal system is one of the most heavily affected organ systems in the body in patients who have COVID-19 infection requiring hospitalization. In these cases, supplementation with the steroid dexamethasone serves as one of the most powerful lifesaving treatments. Concern has also been raised regarding the period of time just after the acute infection phase – particularly, the development of adrenal insufficiency following cases of COVID-19 hospitalizations. Additionally, some professional societies recommend that for patients who have adrenal insufficiency and are on adrenal replacement therapy, they be monitored closely post-COVID-19 vaccine for the development of stress-induced adrenal insufficiency. In mild-to-moderate COVID-19 cases, there does not seem to be an effect on adrenaline-related hormones (norepinephrine, epinephrine, dopamine). However, in cases of severe COVID-19 infection triggering the development of shock, patients will need supplementation with an infusion of catecholamines and a hormone called vasopressin to maintain their blood pressure. Finally, some studies have addressed the concern of adrenal insufficiency during the long-term recovery phase. Dr Sara Bedrose, adrenal endocrine specialist at Baylor College of Medicine, indicates that studies which included adrenal function in COVID survivors showed a large percentage of patients with suboptimal cortisol secretion during what is called ACTH stimulation testing. Results indicated that most of those cases had central adrenal insufficiency. It was concluded that adrenal insufficiency might be among the long-term consequences of COVID-19 and it seemed to be secondary to pituitary gland inflammation (called hypophysitis) or due to direct hypothalamic damage. Long-term follow-up of COVID 19 survivors will be necessary to exclude a gradual and late-onset adrenal insufficiency. Some patients who have COVID-19 will experience prolonged symptoms. To understand what is happening to them, patients may question whether or not they have a phenomenon called adrenal fatigue. This is a natural question to ask, especially after having such a severe health condition. A tremendous amount of resources are being developed to investigate the source and treatment of the symptoms, and this work has only just begun. However, adrenal fatigue is not a real medical diagnosis. It’s a term to describe a group of signs and symptoms that arise due to underactive adrenal glands. Current scientific data indicate that adrenal fatigue is not in and of itself a medical disease – although a variety of over-the-counter supplements and compounded medications may be advocated for in treatment by alternative medicine/naturopathic practitioners. My takeaway is that we have learned a great deal about the effects COVID-19 infection has on the adrenal glands. Long-term COVID-19 remains an area to be explored – especially in regards to how it may affect the adrenal glands. -By Dr. James Suliburk, associate professor of surgery in the Division of Surgical Oncology and section chief of endocrine surgery for the Thyroid and Parathyroid Center at Baylor College of Medicine From https://blogs.bcm.edu/2021/04/22/how-does-covid-19-impact-the-adrenal-gland/