Pediatric Adrenal Insufficiency: Challenges and Solutions

Authors Nisticò D , Bossini BBenvenuto SPellegrin MCTornese G

Received 29 October 2021

Accepted for publication 28 December 2021

Published 11 January 2022 Volume 2022:18 Pages 47—60

DOI https://doi.org/10.2147/TCRM.S294065

Checked for plagiarism Yes

Review by Single anonymous peer review

Peer reviewer comments 2

Editor who approved publication: Professor Garry Walsh

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Daniela Nisticò,1 Benedetta Bossini,1 Simone Benvenuto,1 Maria Chiara Pellegrin,1 Gianluca Tornese2

1University of Trieste, Trieste, Italy; 2Department of Pediatrics, Institute for Maternal and Child Health IRCCS Burlo Garofolo, Trieste, Italy

Correspondence: Gianluca Tornese
Department of Pediatrics, Institute for Maternal and Child Health IRCCS Burlo Garofolo, Via dell’Istria 65/1, Trieste, 34137, Italy
Tel +39 040 3785470
Email gianluca.tornese@burlo.trieste.it

Abstract: Adrenal insufficiency is an insidious diagnosis that can be initially misdiagnosed as other life-threatening endocrine conditions, as well as sepsis, metabolic disorders, or cardiovascular disease. In newborns, cortisol deficiency causes delayed bile acid synthesis and transport maturation, determining prolonged cholestatic jaundice. Subclinical adrenal insufficiency is a particular challenge for a pediatric endocrinologist, representing the preclinical stage of acute adrenal insufficiency. Although often included in the extensive work-up of an unwell child, a single cortisol value is usually difficult to interpret; therefore, in most cases, a dynamic test is required for diagnosis to assess the hypothalamic-pituitary-adrenal axis. Stimulation tests using corticotropin analogs are recommended as first-line for diagnosis. All patients with adrenal insufficiency need long-term glucocorticoid replacement therapy, and oral hydrocortisone is the first-choice replacement treatment in pediatric. However, children that experience low cortisol concentrations and symptoms of cortisol insufficiency can take advantage using a modified release hydrocortisone formulation. The acute adrenal crisis is a life-threatening condition in all ages, treatment is effective if administered promptly, and it must not be delayed for any reason.

Keywords: adrenal gland, primary adrenal insufficiency, central adrenal insufficiency, Addison disease, children, adrenal crisis, hydrocortisone

Introduction

Primary adrenal insufficiency (PAI) is a condition resulting from impaired steroid synthesis, adrenal destruction, or abnormal gland development affecting the adrenal cortex.1 Acquired primary adrenal insufficiency is termed Addison disease. Central adrenal insufficiency (CAI) is caused by an impaired production or release of adrenocorticotropic hormone (ACTH). It can originate either from a pituitary disease (secondary adrenal insufficiency) or arise from an impaired release of corticotropin-releasing hormone (CRH) from the hypothalamus (tertiary adrenal insufficiency). An underlying genetic cause should be investigated in every case of adrenal insufficiency (AI) presenting in the neonatal period or first few months of life, although AI is relatively rare at this age (1:5.000–10.000).2

Physiology of the Adrenal Gland

The adrenal cortex consists of three zones: the zona glomerulosa, the zona fasciculata, and the zona reticularis, responsible for aldosterone, cortisol, and androgens synthesis, respectively.3 Aldosterone production is under the control of the renin-angiotensin system, while cortisol is regulated by the hypothalamic-pituitary-adrenal axis (HPA).4 This explains why patients affected by CAI only manifest glucocorticoid deficiency while mineralocorticoid function is spared. CRH is secreted from the hypothalamic paraventricular nucleus into the hypophyseal-portal venous system in response to light, stress, and other inputs. It binds to a specific cell-surface receptor, the melanocortin 2 receptor, stimulating the release of preformed ACTH and the de novo transcription of the precursor molecule pro-opiomelanocortin (POMC). ACTH is derived from the cleavage of POMC by proprotein convertase-1.5–9 ACTH binds to steroidogenic cells of both the zona fasciculata and reticularis, activating adrenal steroidogenesis. It also has a trophic effect on adrenal tissue; therefore, ACTH deficiency determines adrenocortical atrophy and decreases the capacity to secrete glucocorticoids. Circulating cortisol is 75% bound to corticosteroid-binding protein, 15% to albumin, and 10% free. The endogenous production rate is estimated between 6 and 10 mg/m2/day, even though it depends on age, gender, and pubertal development. Glucocorticoids have multiple effects: they regulate immune, circulatory, and renal function, influence growth, development, energy and bone metabolism, and central nervous system activity. Several studies reported higher cortisol plasma concentrations in girls than in boys and younger children.3,4,8

Cortisol secretion follows a circadian and ultradian rhythm according to varying amplitudes of ACTH pulses. Pulses of ACTH and cortisol occur every 30–120 minutes, are highest at about the time of waking, and decline throughout the day, reaching a nadir overnight.3,8,9 This pattern can change in the presence of serious illness, major surgery, and sleep deprivation. During stressful situations, glucocorticoid secretion can increase up to 10-fold to enhance survival through increased cardiac contractility and cardiac output, sensitivity to catecholamines, work capacity of the skeletal muscles, and availability of energy stores.3

The interaction between the hypothalamus and the two endocrine glands is essential to maintain plasma cortisol homeostasis (Figure 1). Cortisol exerts double-negative feedback on the HPA axis. It acts on the hypothalamus and the corticotrophin cells of the anterior pituitary, reducing CRH and ACTH synthesis and release.6 ACTH inhibits its secretion through a feedback effect mediated at the level of the hypothalamus.3 Increased androgen production occurs in the case of cortisol biosynthesis enzymatic deficits.

Figure 1 The hypothalamic–pituitary–adrenal axis.

Primary Adrenal Insufficiency

PAI affects 10–15 per 100,000 individuals and recognizes different classes of genetic causes (Table 1). Congenital adrenal hyperplasia (CAH) is the main cause of PAI in the neonatal period, being included among the disorders of steroidogenesis secondary to deficits in enzymes. It has an autosomal recessive transmission.1,10,11 The estimated incidence ranges between 1:10,000 and 1:20,000 births. CAH phenotype depends on disease-causing mutations and residual enzyme activity. 21-hydroxylase deficiency (21OHD) accounts for more than 90% of cases, 21-hydroxylase converts cortisol and aldosterone precursors, respectively 17-hydroxyprogesterone (17-OHP) to 11-deoxycortisol and progesterone to deoxycortisone. Less frequent forms of CAH include 11 β -hydroxylase deficiency (11BOHD, 8% of cases), 17α-hydroxylase/17–20 lyase deficiency (17OHD), 3β-hydroxysteroid dehydrogenase deficiency (3BHDS), P450 oxidoreductase deficiency (PORD).12 Steroidogenesis may also be impaired by steroidogenic acute regulatory (StAR) protein deficiency, which is involved in cholesterol transport into mitochondria, or P450 cytochrome side-chain cleavage (P450scc) deficiency, that converts cholesterol into pregnenolone.12,13 Of these conditions, 21OHD and 11BOHD only affect adrenal steroidogenesis, whereas the other deficits also impact gonadal steroid production. In classic CAH, enzyme activity can be absent (salt-wasting form) or low (1–2% enzyme activity, simple virilizing form). The salt-wasting form is the most severe and affects 75% of patients with classic 21OHD.1,10,12,14 Non-classic CAH (NCCAH) is more prevalent than the classic form, in which there is 20–50% of residual enzymatic activity. Two-thirds of NCCAH individuals are compound heterozygotes with different CYP21A2 mutations in two different alleles (classic severe mutation plus mild mutation in two different alleles or homozygous with two mild mutations). Notably, 70% of NCCAH patients carry the point mutation Val281Leu.

Table 1 Causes of Primary Adrenal Insufficiency (PAI)

Central Adrenal Insufficiency

CAI incidence is estimated between 150 and 280 per million, and it should be suspected when mineralocorticoid function is preserved. When, rarely, isolated is due to iatrogenic HPA suppression secondary to prolonged glucocorticoid therapy or the removal of an ACTH- or cortisol-producing tumor (Cushing syndrome).15 Defects in POMC,16 characterized by red or auburn-haired children, pale skin (due to melanocyte stimulating hormone [MSH] – deficiency) and hyperphagia later in life, and in transcription factor TPIT,17 which regulates POMC synthesis in corticotrope cells, are the two leading genetic causes of isolated ACTH deficiency (Table 2). Mainly, it occurs as part of complex syndromes in which a combined multiple pituitary hormone deficiency (CMPD) is associated with craniofacial and midline defects, such as Prader-Willi syndrome, CHARGE syndrome, Pallister-Hall syndrome (anatomical pituitary abnormalities), white vanishing matter disease (progressive leukoencephalopathy).5 Individuals with an isolated pituitary deficiency, usually a growth hormone deficiency (GHD), may develop multiple pituitary hormone deficiencies over the years. Therefore, excluding a latent CAI at GHD onset and periodically monitoring of HPA axis is of utmost importance. Notably, cortisol reduction secondary to an increased basal metabolism when starting GHD or thyroxin substitutive therapy may unleash a misdiagnosed CAI. CMPD can be caused by several defective genes, such as GLI1, LHX3, LHX4, SOX2, SOX3, HESX1: in such cases, hypoglycemia or small penis with undescended testes may respectively suggest concomitant GH and gonadotropins deficits.18

Table 2 Causes of Central Adrenal Insufficiency (CAI)

Clinical Manifestations of Adrenal Insufficiency

AI is an insidious diagnosis presenting non-specific symptoms and may be mistaken with other life-threatening endocrine conditions (septic shock unresponsive to inotropes or recurrent sepsis, acute surgical abdomen).1,19 Children can be initially misdiagnosed as having sepsis, metabolic disorders, or cardiovascular disease, highlighting the need to consider adrenal dysfunction as a differential diagnosis for an unwell or deteriorating infant. With age-related items, clinical features depend on the type of AI (primary or central) and could manifest in an acute or chronic setting (Table 3).

Table 3 Features of Isolated Adrenal Insufficiency in Pediatric Age

Clinical signs of PAI are based on the deficiency of both gluco- and mineralocorticoids. Signs due to glucocorticoid deficiency are weakness, anorexia, and weight loss. Hypoglycemia with normal or low insulin levels is frequent and often severe in the pediatric population. Mineralocorticoid deficiency contributes to hyponatremia, hyperkalemia, acidosis, tachycardia, hypotension, and salt craving. The lack of glucocorticoid-negative feedback is responsible for the elevated ACTH levels. The high levels of ACTH and other POMC peptides, including the various forms of MSH, cause melanin hypersecretion, stimulating mucosal and cutaneous hyperpigmentation. Searching for an increased pigmentation may represent an essential diagnostic tool since all the other symptoms of PAI are non-specific. However, hyperpigmentation is variable, dependent on ethnic origin, and more prominent in skin exposed to sun and in extension surface of knees, elbows, and knuckles.15 In autoimmune PAI, vitiligo may be associated with hyperpigmentation.

In the classic CAH simple virilizing form, salt wasting is absent due to the presence of aldosterone production. In males, diagnosis typically occurs between 3 and 4 years of age with pubarche, accelerated growth velocity, and advanced bone age at presentation.1,10,12,14

NCCAH may occur in late childhood with signs of hyperandrogenism (premature pubarche, acne, adult apocrine odor, advanced bone age) or be asymptomatic. In adolescents and adult women, conditions of androgen excess (acne, oligomenorrhea, hirsutism) may underlie an NCCAH.20,21

The clinical presentation of CAI may be more complex when caused by an underlying central nervous system disease or by CMPD. In the case of a pituitary or hypothalamic tumor, patients may present headache, vomiting, visual disturbances, short stature, delayed or precocious puberty. In the case of CMPD, manifestations vary considerably and depend on the number and severity of the associated hormonal deficiencies. In CAI, aldosterone production is spared, which means that serum electrolytes are usually normal. However, cortisol contributes to regulating free water excretion, so patients with CAI are at risk for dilutional hyponatremia, with normal serum potassium levels. Since adrenal androgen secretion is under the control of ACTH, girls with ACTH deficiency may present light pubic hair. Patients with partial and isolated ACTH defects can be “asymptomatic”, and adrenal crisis appears during stress or in case of major illness (high fever, surgery).

The acute adrenal crisis is a life-threatening condition in all ages. Patients present with profound malaise, fatigue, nausea, vomiting, abdominal or flank pain, muscle pain or cramps, and dehydration, which lead to hypotension, shock, and metabolic acidosis. Hyponatremia and hyperkalemia are less common in CAI than in PAI, but possible in acute AI. Severe hypoglycemia causes weakness, pallor, sweatiness, and impaired cognitive function, including confusion, loss of consciousness, and coma. Immediate treatment is required (see below).

Children and adolescents affected by autoimmune primary adrenal insufficiency develop a chronic AI, with an insidious onset and slow progress to an acute adrenal crisis over months or even years. Initial symptoms are decreased appetite, anorexia, nausea, abdominal pain, unintentional weight loss, lethargy, headache, weakness, and fatigue, with prominent pain in the joints and muscles. Due to salt loss through the urine and the subsequent reduction in blood volume, blood pressure decreases, and orthostatic hypotension develops together with salt craving. An increased risk of infection in AI patients is reported only in those exposed to glucocorticoids. However, in APECED (Autoimmune Polyendocrinopathy-Candidiasis- Ectodermal-Dystrophy) patients, there is an increased risk of candidiasis and splenic atrophy increases the likelihood for severe infections.

In neonates, AI classically presents with failure to thrive and hypoglycemia, commonly severe and associated with seizures. The condition can be life-threatening and, if misdiagnosed, may result in coma and unexplained neonatal death. In newborns, cortisol deficiency causes delayed bile acid synthesis and transport maturation, determining prolonged cholestatic jaundice with persistently raised serum liver enzymes. The cholestasis can be resolved within ten weeks of correct treatment. StAR deficiency and P450scc cause salt-losing AI with female external genitalia in genetically male neonates.22 In the classic CAH salt-wasting form, the mineralocorticoid deficiency presents with the adrenal crisis at 10–20 days of life. Females show atypical genitalia with signs of virilization (clitoral enlargement, labial fusion, urogenital sinus), whereas males have normal-appearing genitalia, except for subtle signs as scrotal hyperpigmentation and enlarged phallus.1,10,12,14 Neonates with CMPD may display non-specific symptoms including hypoglycemia, lethargy, apnea, poor feeding, jaundice, seizures, hyponatremia without hyperkalemia, temperature and hemodynamic instability, recurrent sepsis, and poor weight gain. A male with hypogonadism may have undescended testes and micropenis. Infants with optic nerve hypoplasia or agenesis of the corpus callosum may present with nystagmus. Furthermore, infants with midline defects may have various neuro-psychological problems or sensorineural deafness.

Genetic Disorders and Other Conditions at Increased Risk for Adrenal Insufficiency

Among the cholesterol biosynthesis disorder, there is the Smith-Lemli-Opitz syndrome,23 where microcephaly, micrognathia, low-set posteriorly rotated ears, syndactyly of the second and third toes, and atypical genital may, although rarely, combine with AI; this autosomal recessive disorder is due to defective 7-dehydrocholesterol reductase so that elevated 7-dehydrocholesterol is diagnostic. In lysosomal acid lipase A deficiency,24 AI is due to calcification of the adrenal gland as a result of the accumulation of esterified lipids; in infantile form, that is Wolman disease, hepatosplenomegaly with hepatic fibrosis and malabsorption lead to death in the first year of life, if not treated with enzyme replacement therapy such as sebelipase alfa.25

Adrenal development may be impaired in X-linked congenital adrenal hypoplasia (AHC),13,26 a disorder caused by defective nuclear receptor DAX-1, presenting with salt-losing AI in infancy in approximately half of the cases, but also later in childhood or adolescence with two other key features such as hypogonadotropic hypogonadism and impaired spermatogenesis. Two syndromes combine adrenal hypoplasia with intrauterine growth restriction (IUGR): in IMAGe syndrome,27 caused by CDKN1C gain-of-function mutations, IUGR and AI present with metaphyseal dysplasia and genitourinary anomalies; MIRAGE syndrome28 is instead characterized by myelodysplasia, infections, genital abnormalities, and enteropathy, as a result of gain-of-function mutations in SAMD9, with elevated mortality rates.

In some other conditions, AI is due to ACTH resistance. Familial Glucocorticoid Deficiency type 1 (FGD1)13,29 and type 2 (FGD2)30 derive from defective ACTH receptor (MC2R) or its accessory protein MRAP, and both present with early glucocorticoid insufficiency (hypoglycemia, prolonged jaundice) and pronounced hyperpigmentation; there is usually an excellent response to cortisol replacement therapy, even though ACTH levels remain elevated.

In Allgrove or Triple-A Syndrome,13,31 defective Aladin protein (an acronym for alacrimia-achalasia-adrenal insufficiency) leads to primary ACTH-resistant adrenal insufficiency with achalasia and absent lacrimation, often combined with neurological dysfunction, either peripheral, central, or autonomic. It is an autosome recessive condition, phenotypically characterized by microcephaly, short stature, and skin hyperpigmentation.32,33

Among metabolic disorders associated with AI, Sphingosine-1-Phosphate Lyase (SGPL1) Deficiency34 is a sphingolipidosis with various features such as steroid-resistant nephrotic syndrome, primary hypothyroidism, undescended testes, neurological impairment, lymphopenia, ichthyosis; interestingly, in cases where nephrotic syndrome develops before AI, the latter may be masked by glucocorticoid treatment.

Adrenoleukodystrophy (ALD)35–37 is an X-linked recessive proximal disorder of beta-oxidation due to defective ABCD1, where the accumulation of very-long-chain fatty acids (VLCFA) affects in almost all cases adrenal gland among other tissues. Most patients present with progressive neurological impairment, but in some, AI is the only (approximately 10%) or first manifestation, so that every unexplained AI in boys should receive plasma VLCFA evaluation to diagnose ALD and reduce cerebral involvement through a low VLCFAs diet (Lorenzo’s oil) and allogeneic bone marrow transplantation. Early disease-modifying therapies have been developed. Gene therapy adds new functional copies of the ABCD1 gene in hematopoietic stem cells through a lentiviral vector reinfusing the modified cells in the patient’s bloodstream. Recent trials show encouraging results.38

In Zellweger syndrome, caused by mutations in peroxin genes (PEX), peroxisomes are absent, and disease presentation occurs in the neonatal period, with low survival rates after the first year of life. Finally, mitochondrial disorders have been described to occasionally develop AI: Pearson syndrome (sideroblastic anemia, pancreatic dysfunction), MELAS syndrome (encephalopathy with stroke-like episodes), and Kearns-Sayre syndrome (external ophthalmoplegia, heart block, retinal pigmentary changes) belong to this class.39

Autoimmune pathogenesis (Addison disease) accounts for approximately 15% of cases of primary AI in children, in contrast with adolescents and adults where it is the most common mechanism; half of these children present other glands involvement as well. Two syndromes recognize specific combinations: in Autoimmune Polyglandular Syndrome Type 1 (APS1, or APECED)40 defective autoimmune regulator AIRE causes AI, hypoparathyroidism, hypogonadism, malabsorption, chronic mucocutaneous candidiasis; APS2 usually present later in life (third-fourth decades) with AI, thyroiditis, and type 1 diabetes mellitus (T1DM). Antibodies against 21-hydroxylase enzyme are the hallmark of APS.

Apart from a genetic disorder, a strong link between autoimmune conditions and autoimmune primary AI has been established, with more than 50% of patients with the latter also having one or more other autoimmune endocrine disorders; on the other hand, only a few patients with T1DM or autoimmune thyroiditis or Graves’ disease develop AI. As an example, in a study of 629 patients with T1DM, only 11 (1.7%) presented 21-hydroxylase autoantibodies, with three of them having AI.41 Nevertheless, these patients are to be considered at increased risk for a condition that is potentially fatal yet easy to diagnose and treat; that is why it is reasonable to screen for autoimmune AI at least patients with T1DM, significantly if associated with DQ8 HLA combined with DRB*0404 HLA alleles, who have been observed to develop AI in 80% of cases if also 21-hydroxylase autoantibodies positive.42

Regarding immunological disruption, the link with celiac disease is instead well established: celiac patients have an 11-fold increased risk for AI, while in a study, 6 of 76 patients with AI had celiac disease, so that mutual evaluation should be granted in these patients.43,44

Subclinical Adrenal Insufficiency

Subclinical AI is a particularly insidious challenge for a pediatric endocrinologist. It represents the preclinical stage of Addison disease when 21-hydroxylase autoantibodies are already detectable but still absent from evident symptoms. 21-hydroxylase autoantibodies positivity carries a greater risk to develop overt AI in children than in adults: in a study, estimated risk was 100% in children versus 32% in adults on a medium six-year period of follow-up.45 As the adrenal crisis is a potentially lethal condition, it is essential to recognize and adequately manage subclinical AI.

Although asymptomatic by definition, subclinical AI may present with non-specific symptoms such as fatigue, lethargy, gastrointestinal symptoms (nausea, vomiting, diarrhea, constipation), hypotension; physical or psychosocial stresses may sometimes exacerbate these symptoms. When symptoms lack, subclinical AI may be identified thanks to the co-occurrence with other autoimmune endocrinopathies.46

21-hydroxylase autoantibodies titer is considered a marker of autoimmune activity and correlates with disease progression.47 Other reported risk factors for the disease evolution include young age, male sex, hypoparathyroidism or candidiasis coexistence, increased renin activity, or an altered synacthen test with normal baseline cortisol and ACTH.45 ACTH elevation has been reported as the best predictor of progression to the clinical stage in 2 years (94% sensitivity and 78% specificity).48

Management of patients with subclinical AI should include serum cortisol, ACTH, renin measurement, and a synacthen test. If normal, cortisol and ACTH should be repeated in 12–18 months, while synacthen test every two years. After synacthen test results are subnormal, cortisol and ACTH should be assessed every 6–9 months if ACTH remains in range or every six months if ACTH becomes elevated.49 In the latter case, therapy with hydrocortisone should be started.19 This strategy will prevent acute crises and possibly improve the quality of life in patients reporting non-specific symptoms.

Diagnosis

Laboratory evaluation of a stable patient with suspected AI should start with combined early morning (between 6 and 8 AM) serum cortisol and ACTH measurements (Figure 2).

Figure 2 Diagnostic algorithm for adrenal insufficiency.

Although often included in the extensive work-up of an unwell child, a single cortisol value is usually challenging to interpret: circadian cortisol rhythm is highly variable and morning peak is unpredictable; morning cortisol levels in children with diagnosed AI may range up to 706 nmol/L (97th percentile); several factors, such as exogenous estrogens, may alter total serum cortisol values by influencing the free cortisol to cortisol binding globulin or albumin-bound cortisol ratio.7

Significant variability is also observed depending on the specific type of cortisol assay; therefore, it is recommended to check the reference ranges with the laboratory. Mass spectrometry analysis and the new platform methods (Roche Diagnostics Elecsys Cortisol II)50 have more specificity because it detects lower cortisol concentrations than standard immunoassays.15 Low serum cortisol with normal or low ACTH levels is compatible with CAI. In such cases, morning serum cortisol levels below 3 µg/dL (83 nmol/L) best predict AI, while greater than 13 µg/dL (365 nmol/L) values tend to exclude it.51 This is why in most cases, a dynamic test is required for diagnosis and has been introduced to assess the hypothalamic-pituitary-adrenal (HPA) axis in case of intermediate values.5

The insulin tolerance test (ITT) is considered the gold standard for CAI diagnosis as hypoglycemia results in an excellent HPA axis activation; moreover, it allows simultaneous growth hormone evaluation in patients with suspected CPHD. Serum cortisol is measured at baseline and 15, 30, 45, 60, 90, and 120 minutes after intravenous administration of 0.1 UI/Kg regular insulin; the test is valid if serum glucose is reduced by 50% or below 2.2 mmol/L (40 mg/dL).52 CAI is diagnosed for a <20 µg/dL (550 nmol/L) cortisol value at its peak.15 Hypoglycemic seizures and hypokalemia (due to glucose infusion) are the main risks of this test so that it is contraindicated in case of a history of seizures or cardiovascular disease.

Glucagon stimulation test (GST, 30 µg/Kg up to 1 mg i.m. glucagon with cortisol measurements every 30 min for 180 min) allows both CAI and growth hormone deficiency evaluation as well but is characterized by frequent gastrointestinal side effects and poor specificity.8

Metyrapone is an 11-hydroxylase inhibitor, thereby decreasing cortisol synthesis and removing its negative feedback on ACTH release. Overnight metyrapone test is based on oral administration of 30 mg/Kg metyrapone at midnight, and 11-deoxycortisol measurement on the following morning: in case of CAI, its level will not reach 7 µg/dL (200 nmol/L). This test may, however, induce an adrenal crisis so that it is rarely performed.

Given their safety profile and accuracy, corticotropin analogs such as tetracosactrin (Synacthen®) or cosyntropin (Cortrosyn®) are recommended as first-line stimulation tests. Nevertheless, false-negative results are probable in the case of recent or moderate ACTH deficiency, which would not have induced adrenal atrophy. The standard dose short synacthen test (SDSST) is based on a 250 µg Synacthen vial administration with serum cortisol measurement at baseline and 30 and 60 minutes after. CAI is diagnosed if peak cortisol level is <16 µg/dL (440 nmol/L), or excluded if >39 µg/dL (1076 nmol/L). However, the cut-offs for both the new platform immunoassay and mass spectrometry serum cortisol assays are 13.5 to 14.9 mcg/dL (373 to 412 nmol/L).53 The 250 µg Synacthen dose is considered a supraphysiological stimulus since it is 500 times greater than the minimum ACTH dose reported to induce a maximal cortisol response (500 ng/1.73 m2). The low dose short synacthen test (LDSST) has been introduced as a more sensitive first-line test in children greater than two years.54 The recommended dose is 1 µg55, which is contained in 1 mL of the solution obtained by diluting a 250 µg vial into 250 mL saline. Serum cortisol level is then measured at baseline and after 30 minutes, resulting in diagnose of CAI if <16 µg/dL (440 nmol/L), otherwise ruling it out if >22 µg/dL (660 nmol/L). Using these thresholds, LDSST is more precise than SDSST in children, with an area under the ROC curve of 0.99 (95% CI 0.98–1.00).56 LDSST has not been validated in acutely ill patients, pituitary acute disorders or surgery or radiation therapy, and impaired sleep-wake cycle. Patients with an indeterminate LDSST result should be furtherly studied with ITT or metyrapone test.

Finally, the CRH test is based on 1 µg/Kg human CRH (Ferring®) administration and may differentiate secondary from tertiary AI, but its thresholds are still not precisely defined.57

Once CAI is diagnosed, other pituitary hormones should be assessed (prolactin, IGF1, LH, FSH, fT4, TSH), and an MRI of the pituitary region should be performed to exclude neoplastic or infiltrative processes.

Primary adrenal insufficiency (PAI) should be suspected in case of low serum cortisol with elevated ACTH levels. When hypocortisolemia has been confirmed, ACTH levels >66 pmol/L or greater than twice the upper limit best predict PAI. Nevertheless, a confirmatory dynamic test is always recommended for diagnosis.19 Given the comparable accuracy between standard and low dose SST reported in these patients, SDSST is recommended as the most feasible test.58 Moreover, suspected PAI cases should receive plasma renin activity or direct renin and aldosterone assessment to evaluate mineralocorticoid deficiency.

Etiologic work-up of confirmed PAI should start from 21-hydroxylase antibodies assessment: if positive, differential diagnosis will include Addison disease and APS1 or APS2. Adrenal autoantibody negative patients should instead be screened for CAH by measuring 17-hydroxyprogesterone, ALD (if young male) by assessing VLCFA, and tuberculosis if endemic; adrenal glands imaging will complete the work-up in order to exclude infection, hemorrhage, or tumor.6

While universal newborn screening is already implemented for CAH in many countries, allowing a timely replacement therapy, basal salivary cortisol, and salivary cortisone measurements could improve CAI screening in the future: this technique is simple, cost-effective, and independent of binding proteins.15

Treatment

All patients with adrenal insufficiency need long-term glucocorticoid replacement therapy. Individuals with PAI also require mineralocorticoids replacement, together with salt intake as required (Table 4). Otherwise, guidelines do not recommend androgen replacement.5,9,19

Table 4 Management of Adrenal Insufficiency (AI)

Oral hydrocortisone is the first-choice replacement treatment in children due to its short half-life, rapid peak in plasma concentration, lower potency, and fewer adverse effects than prednisolone and dexamethasone.5,8 Based on endogenous production, dosing replacement regimens vary from 7.5 to 15 mg/m2/day, divided into two, three, or four doses.19 The first and largest dose should be taken at awakening, the next in the early afternoon to avoid sleep disturbances. Small and frequent dosing mimic the physiological rhythm of cortisol secretion, but high peak cortisol levels after drug assumption and prolonged periods of hypocortisolemia between doses are described.8,9 Some children experience low cortisol concentrations and symptoms of cortisol insufficiency (eg, fatigue, nausea, headache) despite modifications in dosing. This cohort of patients can take advantage of using a modified-release hydrocortisone formulation, such as Chronocort® and Plenadren®. Plenadren®, approved for adults, consists of a coating of hydrocortisone released rapidly, followed by a slow release of hydrocortisone from the tablet center. It is available as 5 and 20 mg tablets. Park et al demonstrate smoother cortisol profiles and normal growth and weight gain patterns using Plenadren® in children.59 In a few cases, the continuous subcutaneous infusion of hydrocortisone using insulin pump technology proved to be a feasible, well-tolerated and safe option for selected patients with poor response to conventional therapy.19

Monitoring glucocorticoid therapy is based on growth, weight gain, and well-being. Cortisol measurements are usually not useful, apart from cases when a discrepancy between daily doses and patient symptoms exists.15 The concomitant use of hydrocortisone and CYP3A4 inducers, such as Rifampicin, Phenytoin, Carbamazepine, requires an increased dose of glucocorticoids. Conversely, the inhibition of CYP3A4 impairs hydrocortisone metabolism.5

Mineralocorticoid replacement is unnecessary if the patient has a normal renin-angiotensin-aldosterone axis and, hence, normal aldosterone secretion, as well as in CAI. By contrast, patients with PAI and confirmed aldosterone deficiency need fludrocortisone at the dosage of 0.1–0.2 mg/day when given together with hydrocortisone, which has some mineralocorticoid activity. When using other synthetic glucocorticoids for replacement, higher fludrocortisone doses may be needed. Infants younger than one year should also be supplemented with sodium chloride due to their relatively low dietary sodium intake and relative renal resistance to mineralocorticoids. The dose is approximately 1 gram (17 mEq) daily.19

Surgery and anesthesia increase the glucocorticoid requirement during the pre-, intra-, and post-operative periods (Table 4). All children with AI should receive an intravenous dose of hydrocortisone at induction (2 mg/kg for minor or major surgery under general anesthesia). For minor procedures or sedation, the child should receive a double morning dose of hydrocortisone orally.60

Adrenal crisis is a life-threatening condition, treatment is effective if administered promptly, and it must not be delayed for any reason. Hydrocortisone should be administered as soon as possible with an intravenous bolus of 4 mg/kg followed by a continuous infusion of 2 mg/kg/day until stabilization. In the alternative, it can be administered as a bolus every four hours intravenous or intramuscular. In difficult peripheral venous access, the intramuscular route must be used as the first choice. In order to counteract hypotension, a bolus of normal saline 0.9% should be given at a dose of 20 mL/kg; it can repeat up to a total of 60 mL/kg within one hour for shock. If there is hypoglycemia, 10% dextrose at a 5 mL/kg dose should be administered.5,19,61,62

Patients with AI require additional doses of glucocorticoids in case of physiologic stress such as illness or surgical procedures to avoid an adrenal crisis. Home management of illness with a fever (> 38°C), vomiting or diarrhea, is based on the increase from two to three times the usual dose orally. If the child is unable to tolerate oral therapy, intramuscular injection of hydrocortisone should be administered (Table 4).

Education for caregivers and patients (if adolescent) is crucial to prevent adrenal crisis. They should recognize signs and symptoms of adrenal crisis and should receive a steroid emergency card with the sick day rules. Prescribing doctors should provide for additional oral glucocorticoids and adequate training in hydrocortisone emergency self-injection.

Abbreviations

AI, adrenal insufficiency; PAI, primary adrenal insufficiency; CAI, central adrenal insufficiency; HPA, hypothalamic-pituitary-adrenal axis; CRH, corticotropin-releasing hormone; ACTH, adrenocorticotropic hormone; POMC, pro-opiomelanocortin; CAH, congenital adrenal hyperplasia; STAR, steroidogenic acute regulatory; 21OHD, 21-hydroxylase deficiency; 11BOHD, 11-B-hydroxylase deficiency; P450scc, P450 cytochrome side-chain cleavage deficiency; 17-OHP, 17-hydroxyprogesterone; NCCAH, non-classic congenital adrenal hyperplasia; ALD, adrenoleukodystrophy; VLCFA, very long-chain fatty acids; CMPD, combined multiple pituitary hormone deficiency; GHD, growth hormone deficiency; MSH, melanocyte stimulating hormone; IUGR, intrauterine growth restriction; APS1, autoimmune polyglandular syndrome type 1; SDSST, standard dose short synacthen test; LDSST, low dose short synacthen test.

Take Home Messages

  1. In neonates and infants CAH is the commonest cause of PAI, causing almost 71.8% of cases.
  2. Adrenoleukodystrophy should be considered in any male with hypoadrenalism.
  3. Unexplained hyponatremia, hyperpigmentation and the loss of pubic and axillary hair should raise the suspicion of AI.
  4. Adrenal insufficiency can present with non-specific clinical features; therefore a single cortisol measurement should be included in the biochemical work-up of an unwell child.
  5. Patients and parents should be well-trained in adrenal crisis recognition and management.

Disclosure

The authors report no conflicts of interest in this work.

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Unique Cell in Rare Tumor Tied to Ectopic Cushing’s

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., 2015Newell-Price et al., 2006Lindholm et al., 2001Steffensen et al., 2010Bolland et al., 2011Valassi 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, 2010Ejaz et al., 2011Ballav 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, 2010Isidori et al., 2006Ilias et al., 2005Aniszewski et al., 2001). Pheochromocytoma, a catecholamine-producing tumor, becomes even rarer when it is capable of both secreting ACTH and CRH (Lenders et al., 2005Zelinka et al., 2007). By 2020, only two cases with pheochromocytoma secreted both ACTH and CRH were reported (Elliott et al., 2021O’Brien et al., 1992Jessop 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., 2012Ilias et al., 2005Lenders et al., 2014Lase 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., 2009Tammela and Sage, 2020Kolodziejczyk et al., 2015Patel et al., 2014Tirosh et al., 2016bTirosh et al., 2016aPuram et al., 2017Venteicher et al., 2017Young et al., 2018Bernard et al., 2019Segerstolpe et al., 2016Reichert 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., 2018Plasschaert 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, 2018Stubbington 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.

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 1Appendix 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).

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., 1988McKnight et al., 1992Murakami et al., 1989Hooi 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., 1998Petrunak 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., 2007Fitzgerald et al., 2009Poli et al., 2019Scriba et al., 2020).

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 3DAppendix 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 3EAppendix 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).

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).

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.

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.

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 8ASupplementary 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.

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., 2016Nölting 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, 1976Burnstock, 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, 1983Apergis-Schoute et al., 2019Svensson 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, 1995Lloyd 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., 2007Fitzgerald et al., 2009Poli et al., 2019Scriba 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., 1989Bernton et al., 1987), and induce the activation of the HPA axis (Gisslinger et al., 1993Fukata et al., 1994Kakucska et al., 1993Murakami 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., 2018Thome 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., 2003Tan et al., 2001Tan et al., 2002Lenz et al., 2004Li et al., 2003Seddon et al., 2003), and disruption of the IL-7R signaling pathway may lead to skewed T cell distribution and cause immunodeficiency (Maraskovsky et al., 1996Kaech et al., 2003Carini 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

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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

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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

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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., 2018Stuart 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., 2019Appendix 1—figure 2).

Clustering analysis and cell phenotype recognition

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Seurat (Butler et al., 2018Stuart 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

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The cell-type-specific genes were identified by running Seurat (Butler et al., 2018Stuart 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

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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

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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

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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

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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

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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

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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

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

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

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

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

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

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

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

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

Immunofluorescence co-staining for POMC&CRH and POMC&TH on two serial biopsies from tumor specimen esPHEO_T3.

The magnification is 10× (top) and 40× (bottom). Red rectangular indicates the magnified area of the location, as shown in Figure 3E.

Data availability

The raw data of scRNA-seq sequencing reads generated in this study were deposited in The National Genomics Data Center (NGDC, https://bigd.big.ac.cn/) under the accession number: PRJCA003766.

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    https://doi.org/10.1530/EJE-11-0272

Decision letter

  1. Murim Choi
    Reviewing Editor; Seoul National University, Republic of Korea
  2. Mone Zaidi
    Senior Editor; Icahn School of Medicine at Mount Sinai, United States
  3. 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

Author response image 2

Author response image 3

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

  1. 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

  2. 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

  3. 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

    ORCID icon “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

    1. Institute of Basic Medical Sciences Chinese Academy of Medical Sciences, School of Basic Medicine Peking Union Medical College, Beijing, China
    2. 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

    ORCID icon “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

  1. Mone Zaidi, Icahn School of Medicine at Mount Sinai, United States

Reviewing Editor

  1. Murim Choi, Seoul National University, Republic of Korea

Reviewer

  1. Murim Choi, Seoul National University, Republic of Korea

Publication history

  1. Received: March 16, 2021
  2. Accepted: December 13, 2021
  3. Accepted Manuscript published: December 14, 2021 (version 1)
  4. Accepted Manuscript updated: December 15, 2021 (version 2)
  5. 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

Korlym: Failure to Show a Reasonable Expectation of Success Dooms Obviousness Allegations

In Teva Pharmaceuticals USA, Inc. v. Corcept Therapeutics, Inc.,1 the Federal Circuit affirmed the obviousness analysis performed by the Patent Trial and Appeal Board (“PTAB”), which found that Corcept’s patent for methods of treating Cushing’s disease by co-administering two different types of drugs with a specific range of dosing amounts was not obvious—even where the prior art directed one to combine the two—because there was no reasonable expectation of success for the specific dose claimed in the patent.

Background

The patent relates to methods for treating Cushing’s syndrome by co-administering mifepristone and a strong CYP3A inhibitor. Cushing’s syndrome is a metabolic disorder caused by excess cortisol.,2 Mifepristone was recognized in the prior art as a potential treatment for Cushing’s syndrome in the 1980’s.,3 Decades later, Corcept sponsored the first major clinical trial of mifepristone in patients with Cushing’s syndrome, in which participants were dosed with 300 to 1200 mg per day of mifepristone. Thereafter, Corcept filed a New Drug Application (“NDA”) with the U.S. Food and Drug Administration (“FDA”) to seek marketing approval for Korlym®, a 300 mg mifepristone tablet to control “hypercalcemia secondary to hypercortisolism” in patients with Cushing’s syndrome.,4

The FDA approved the NDA, including the prescribing information contained in the label.5 The label “recommended [a] starting dose [of] 300 mg once daily” and allowed for a dosage increase “in 300 mg increments to a maximum of 1200 mg once daily.”6 The label specifically warned against using mifepristone “with strong CYP3A inhibitors” and limited the dose to “300 mg per day when used with strong CYP3A inhibitors.”7

However, when it approved the NDA, the FDA issued several post market requirements, one of which was that Corcept must conduct a drug-drug interaction study with mifepristone and a strong CYP3A inhibitor.8 A memo from the Office of Clinical Pharmacology was provided to Corcept by the FDA (“the Lee memo”), which explained that “[t]he degree of change in exposure of mifepristone when co-administered with strong CYP3A inhibitors is unknown” and “may present a safety risk.”9 The concern was that without the required study the patients with Cushing’s syndrome that take strong inhibitors may be unable to use mifepristone.10

Corcept conducted the study requested in the Lee memo.11 Based on the resulting data, Corcept sought a patent claiming a method of treating Cushing’s syndrome by co-administering mifepristone and a strong CYP3A4 inhibitor, which is the patent at issue here.12 Claim 1, which is representative of the claims, reads:

A method of treating Cushing’s syndrome in a patient who is taking an original once-daily dose of 1200 mg or 900 mg per day of mifepristone, comprising the steps of:

reducing the original once-daily dose to an adjusted once-daily dose of 600 mg mifepristone,

administering the adjusted once-daily dose of 600 mg mifepristone and a strong CYP3A inhibitor to the patient,

wherein said strong CYP3A inhibitor is selected from the group consisting of ketoconazole, itraconazole, nefazodone, ritonavir, nelfmavir, indinavir, boceprevir, clarithromycin, conivaptan, lopinavir, posaconazole, saquinavir, telaprevir, cobicistat, troleandomycin, tipranivir, paritaprevir, and voriconazole.13

Procedural Posture

In 2018 Corcept brought suit against Teva in the District of New Jersey alleging that Teva’s proposed generic infringed the patent, among others.14 Teva then sought post-grant review of the patent’s claims at the PTAB, arguing that the claims would have been obvious over the Korlym® label and the Lee memo, optionally in combination with FDA guidance on drug-drug interaction studies.15

The PTAB instituted review, “construed the claims to require safe administration of mifepristone,” and found that Teva failed to meet its burden of showing that a “skilled artisan would have had a reasonable expectation of success for safe co-administration of more than 300 mg of mifepristone with a strong CYP3A inhibitor.”16 Thus, the PTAB concluded that Teva failed to prove obviousness.17

Teva’s Arguments on Appeal

Teva argued to the Federal Circuit that the PTAB committed two legal errors in finding that Teva did not prove obviousness: (1) it “required precise predictability, rather than a reasonable expectation of success in achieving the claimed invention,” and (2) it found that Teva “failed to prove the general working conditions disclosed in the prior art encompassed the claimed invention” instead of applying the Federal Circuit’s “prior-art-range precedents.”18

The Federal Circuit Panel, consisting of Chief Judge Moore and Judges Newman and Reyna, rejected both of Teva’s arguments.19

The Panel determined that the PTAB “did not err by requiring Teva to show a reasonable expectation of success for a specific mifepristone dosage.”

In discussing the proper standard for evaluating a reasonable expectation of success, the Panel cited prior Federal Circuit decisions explaining that the analysis “must be tied to the scope of the claimed invention.”20 It noted that because the claims of the patent require administration of a specific dosage of mifepristone, the PTAB was required to frame its analysis around that specific dosage.21 The Panel emphasized that Teva was not “required to prove a skilled artisan would have precisely predicted safe co-administration of 600 mg of mifepristone” because “[a]bsolute predictability is not required.”22 Teva was, however, required “to prove a reasonable expectation of success in achieving the specific invention claimed, a 600 mg dosage.”23

The Panel explained that the PTAB found that Teva failed to prove a reasonable expectation of success.24 Based on the prior art, a skilled artisan would not have reasonably “expected co-administration of more than 300 mg of mifepristone with strong CYP3A inhibitor to be a safe treatment of Cushing’s syndrome or related symptoms in patients.”25 Moreover, the PTAB found that a skilled artisan “would have had no expectation as to whether co-administering dosages of mifepristone above the 300 mg/day threshold” would be successful.26 Thus, because there was no expectation of success for any dosage over 300 mg, the PTAB concluded that there could not have been an expectation of success for the specific dosage of 600 mg per day.27 The Panel found that this analysis by the PTAB was correct under Federal Circuit precedent, and that “[n]othing about this analysis required precise predictability, only a reasonable expectation of success tied to the claimed invention.”28

The Panel decided that the PTAB did not err in finding that “the prior art ranges do not overlap with the claimed range”

The Panel next considered the applicability of the Federal Circuit’s precedent concerning claimed ranges that overlap with those disclosed in the prior art.29 The PTAB refused to apply that line of cases, finding that “Teva had failed to prove the general working conditions disclosed in the prior art encompass the claimed invention.”30

The Panel noted a Federal Circuit decision that “where the general conditions of a claim are disclosed in the prior art, it is not inventive to discover the optimum or workable ranges by routine experimentation.”31 In other words, “a prima facie case of obviousness typically exists when the ranges of a claimed composition overlap the ranges disclosed in the prior art.”32 “But overlap is not strictly necessary for a conclusion of obviousness” and can exist even where the ranges are “close enough” that a skilled artisan would expect them to exhibit similar properties.33

Here, the Panel explained that “[s]ubstantial evidence supports the [PTAB’s] finding that the general working conditions disclosed in the prior art did not encompass the claimed invention, i.e., there was no overlap in ranges.”34 The Korlym® label warned against taking mifepristone with a strong CYP3A inhibitor altogether, and stated that anyone nonetheless combining the two should take a maximum of 300 mg/day of mifepristone.35 This 300 mg/day cap was also repeated in other industry publications.36 The PTAB found that “the prior art capped the range of co-administration dosages at 300 mg per day.”37 The Panel agreed with this finding, concluding that the claimed range was not disclosed in the prior art.38

Teva attempted to argue that the claimed range overlaps with monotherapy dosages—which were dosages of mifepristone alone—in the prior art.39 However, because “monotherapy dosages alone cannot create an overlap with the claimed range, which is limited to co-administering mifepristone with a strong CYP3A inhibitor,” the PTAB had to determine “whether a skilled artisan would have expected “monotherapy and co-administration dosages to behave similarly.”40 As the Panel had already concluded in its reasonable expectation of success analysis, a “skilled artisan would have no such expectation.”41

Conclusion

Although Teva argued that this was an “uncommonly clear-cut obviousness case” where the prior art discloses “the problem, . . . the solution, . . . and the way to find the solution,” the Panel disagreed, explaining that: “At best, the prior art directed a skilled artisan to try combing the Korlym Label, Lee, and the FDA guidance. But without showing a reasonable expectation of success, Teva did not prove obviousness.”42 Thus, the Panel’s decision helps to clarify that evaluating obviousness based on ranges disclosed in the prior art is a fact-specific analysis, in which bright lines should not be drawn.

1 Teva Pharm. USA, Inc. v. Corcept Therapeutics, Inc., No. 21-1360, slip op. (Fed. Cir. Dec. 7, 2021).
2 Id. at 2.
3 Id.
4 Id. at 2-3.
5 Id. at 3.
6 Id.
7 Id. at 3-4.
8 Id. at 3.
9 Id.
10 Id.
11 Id. at 4.
12 Id.
13 Id. at 3.
14 Corcept Therapeutics, Inc. v. Teva Pharmaceuticals USA, Inc., No. 18-3632 (D.N.J.).
15 Teva Pharmaceuticals USA, Inc. v. Corcept Therapeutics, Inc., PGR2019-00048, 2020 WL 6809812 (P.T.A.B. Nov. 18, 2020) (Final Decision).
16 Id. (emphasis added).
17 Id.
18 Teva Pharm. USA, Inc. v. Corcept Therapeutics, Inc., No. 21-1360, slip op. at 5 (Fed. Cir. Dec. 7, 2021)
19 See generally id.
20 Id. at 6 (citing Allergan, Inc. v. Apotex Inc., 753 F.3d 952, 966 (Fed. Cir. 2014); Intelligent Bio-Sys., Inc. v. Illumina Cambridge Ltd., 821 F.3d 1359, 1366 (Fed. Cir. 2016)).
21 Id.
22 Id.
23 Id.
24 Id.
25 Id. at 6-7 (citing Final Decision at *22).
26 Id. at 7.
27 Id.
28 Id.
29 Id. at 8.
30 Id.
31 Id. (citing E.I. DuPont de Nemours & Co. v. Synvina C. V., 904 F.3d 996, 1006 (Fed. Cir. 2018)).
32 Id. at 8-9.
33 Id. at 9.
34 Id.
35 Id.
36 Id.
37 Id.
38 Id.
39 Id.
40 Id. at 9-10.
41 Id. at 10.
42 Id.

‘Benign’ Adrenal Gland Tumors Might Cause Harm to Millions

Millions of people are at increased risk of type 2 diabetes and high blood pressure and don’t even know it, due to a hidden hormone problem in their bodies.

As many as 1 in 10 people have a non-cancerous tumor on one or both of their adrenal glands that could cause the gland to produce excess amounts of the stress hormone cortisol.

Up to now, doctors have thought that these tumors had little impact on your health.

But a new study out of Britain has found that up to half of people with these adrenal tumors are secreting enough excess cortisol to raise their risk of diabetes and high blood pressure.

Nearly 1.3 million adults in the United Kingdom alone could suffer from this disorder, which is called Mild Autonomous Cortisol Secretion (MACS), the researchers said.

Anyone found with one of these adrenal tumors should be screened to see if their health is at risk, said senior researcher Dr. Wiebke Arlt, director of the University of Birmingham Institute of Metabolism and Systems Research in England.

“People who are found to have an adrenal tumor should undergo assessment for cortisol excess and if they are found to suffer from cortisol overproduction they should be regularly screened for type 2 diabetes and hypertension and receive treatment if appropriate,” Arlt said.

These tumors are usually discovered during imaging scans of the abdomen to treat other illnesses, said Dr. André Lacroix, an endocrinologist at the University of Montreal Hospital Center, who wrote an editorial accompanying the study. Both were published Jan. 4 in the Annals of Internal Medicine.

Adrenal glands primarily produce the hormone adrenaline, but they are also responsible for the production of a number of other hormones, including cortisol, Lacroix said.

Cortisol is called the “fight-or-flight” hormone, and can cause blood sugar levels to rise and blood pressure to surge — usually in response to some perceived bodily threat.

Previous studies had indicated that about 1 in 3 adrenal tumors secrete excess cortisol, and an even lower number caused cortisol levels to rise so high that they affected health, researchers said in background notes.

But this new study of more than 1,300 people with adrenal tumors found that previous estimates were wrong.

About half of these patients had excess cortisol due to their adrenal tumors. Further, more than 15% had levels high enough to impact their health, compared to those with truly benign tumors.

MACS patients were more likely to be diagnosed with high blood pressure, and were as much as twice as likely to be on three or more blood pressure medications.

They also were more likely to have type 2 diabetes, and were twice as likely to require insulin to manage their blood sugar, the study found.

“This study clearly shows that mild cortisol production is more frequent than we thought before, and that the more cortisol you produce, the more likely to you are to have consequences such as diabetes and hypertension,” Lacroix said.

About 70% of people with MACS were women, and most were of postmenopausal age, the researchers said.

“Adrenal tumor-related cortisol excess is an important previously overlooked health issue that particularly affects women after the menopause,” Arlt said.

Lacroix agreed that guidelines should be changed so that people with adrenal tumors are regularly screened.

“Everybody who is found to have an adrenal nodule larger than 1 centimeter needs to be screened to see if they’re producing excess hormone or not,” he said. “That’s very clear.”

A number of medications can reduce cortisol overproduction or block cortisol action, if an adrenal tumor is found to be causing an excess of hormone.

People with severe cortisol excess can even have one of their two adrenal glands removed if necessary, Lacroix said.

“It is quite possible to live completely normally with one adrenal gland,” he said.

More information

The Cleveland Clinic has more about adrenal tumors.

SOURCES: Wiebke Arlt, MD, DSc, director, Institute of Metabolism and Systems Research, University of Birmingham, U.K.; André Lacroix, MD, endocrinologist, University of Montreal Hospital Center; Annals of Internal Medicine, Jan. 4, 2022

From https://consumer.healthday.com/1-4-benign-adrenal-gland-tumors-might-cause-harm-to-millions-2656172346.html

FDA Approval for Endogenous Cushing’s Syndrome Drug Recorlev

Ahead of its New Year’s Day decision deadline at the FDA, Xeris Biopharma has snagged an approval for Recorlev, a drug formerly known as levoketoconazole.

Based on results from phase 3 studies called SONICS and LOGICS, the FDA approved the drug for adults with Cushing’s syndrome. Xeris picked up Recorlev earlier this year in its acquisition of rare disease biotech Strongbridge Biopharma. It’s planning to launch in the first quarter of 2022.

Recorlev’s approval covers the treatment of endogenous hypercortisolemia in adults with Cushing’s syndrome who aren’t eligible for surgery or haven’t responded to surgery.

Endogenous Cushing’s disease is caused by a benign tumor in the pituitary gland that prompts the body to produce elevated levels of cortisol, which over time triggers a range of devastating physical and emotional symptoms for patients.

 

In the SONICS study, the drug significantly cut and normalized mean urinary free cortisol concentrations without a dose increase, according to the company. The LOGICS trial confirmed the drug’s efficacy and safety, Xeris says.

Cushion’s is a potentially fatal endocrine disease, and patients often experience years of symptoms before an accurate diagnosis, the company says. After a diagnosis, they’re presented with limited effective treatment options.

Following the approval, the company’s “experienced endocrinology-focused commercial organization can begin rapidly working to help address the needs of Cushing’s syndrome patients in the U.S. who are treated with prescription therapy,” Xeris CEO Paul R. Edick said in a statement.

Aside from its forthcoming Recorlev launch, Xeris markets Gvoke for severe hypoglycemia and Keveyis for primary periodic paralysis.

Back in October, the company partnered up with Merck to help reformulate some of the New Jersey pharma giant’s monoclonal antibody drugs.

From https://www.fiercepharma.com/pharma/xeris-biopharma-scores-fda-approval-for-endogenous-cushing-s-syndrome-drug-recorlev

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