Intermittent Blurry Vision: An Unexpected Presentation of Cushing’s Syndrome Due to Primary Bilateral Macronodular Adrenal Hyperplasia (PBMAH)

Published: May 15, 2022 (see history)

DOI: 10.7759/cureus.25017

Cite this article as: Fernandez C, Bhatia S, Rucker A, et al. (May 15, 2022) Intermittent Blurry Vision: An Unexpected Presentation of Cushing’s Syndrome Due to Primary Bilateral Macronodular Adrenal Hyperplasia (PBMAH). Cureus 14(5): e25017. doi:10.7759/cureus.25017


Abstract

Cushing’s syndrome (CS) is an uncommon endocrine disorder resulting from prolonged exposure to elevated glucocorticoids, with 10-15 million annual cases per the American Association of Neurological Surgeons. Exogenous and endogenous causes can further be divided into adrenocorticotropic hormone (ACTH) dependent (i.e Cushing’s Disease) or ACTH independent. ACTH-independent CS can be caused by primary bilateral macronodular adrenal hyperplasia (PBMAH) representing less than 1% cases of CS. We report a case of a woman presenting with chronic resistant hypertension, episodic blurry vision, weight gain and wasting of extremities. She was diagnosed with Cushing’s syndrome due to PBMAH.

Our patient’s presentation was unusual as she presented at 40 years old, 10 years earlier than expected for PBMAH; and primarily with complaints of episodic blurry vision. Her symptoms also progressed rapidly as signs and symptoms largely presented over the course of 12 months, however responded well to surgical resection.

Introduction

Cushing’s syndrome (CS) is an uncommon endocrine disorder caused by prolonged exposure to elevated glucocorticoids [1]. There are exogenous or endogenous causes. The National Institute of Health’s (NIH) Genetic and Rare Diseases Information Center (GARD) estimated the prevalence of endogenous CS to be 1 in 26,000 [2]. According to a large study, the annual incidence of CS in individuals less than 65 years old was nearly 49 cases per million [3]. Cushing’s disease (CD), which is defined as Cushing’s syndrome caused by an adrenocorticotropic hormone (ACTH)-secreting pituitary tumor, accounts for approximately 80% of patients with CS; whereas ACTH-independent CS accounts for the remaining 20% [4]. Among the causes of pituitary ACTH-independent CS is bilateral macronodular adrenal hyperplasia which is rare, comprising less than 1% of patients with CS [5]. Herein is a case of rapid onset Cushing’s syndrome due to PBMAH initially presenting as episodes of bilateral blurry vision.

Case Presentation

The patient is a 40-year-old female with a past medical history of resistant hypertension (on four agents), and recently diagnosed type 2 diabetes mellitus (started on insulin regimen). Patient was recently seen by her primary care provider, with complaints of intermittent episodes of blurry vision going on for months.

As part of evaluation in December 2020, the patient underwent a renal ultrasound as part of evaluation by the primary physician for uncontrolled hypertension. The doppler incidentally showed an indeterminate hypoechoic mass on the right kidney and presumably located within the right adrenal gland, measuring 3.4 x 5.4 cm, without sonographic evidence of renal artery stenosis. The left kidney appeared normal. She was recommended to have further evaluation with contrast enhanced MR or CT with adrenal protocol.

In January 2021, the patient was sent from her PCP’s office to the ED as the patient was having blurred vision. She had a plain CT scan of the brain that was unremarkable. The patient’s systolic blood pressure was in the 160s-170s mm Hg upon arrival to ED compliance with home medications of 5mg of amlodipine daily, 25mg of metoprolol succinate daily, 100mg of losartan daily, and 25mg of hydrochlorothiazide daily. Physical exam reported obesity without evidence of abdominal striae. Blood work in the ED showed elevated blood glucose level over 600 (mg/dL) despite being on a regimen of lantus 60 units, metformin 1000mg twice a day, and semaglutide SQ weekly. Hemoglobin A1c was greater than 15.5%, and vitamin D was low (15.6 ng/mL). The morning ACTH was low (<5pg/mL) (nAM levels: 7.2 – 63.3 pg/mL), AM cortisol was high at 26.1 ug/ml (normal: 5.0 – 23.0 ug/mL), plasma aldosterone was normal at 4.2 ng/dL with a normal plasma renin at 1.96 (0.25 – 5.82 ng/mL/h). 24-hour urine free cortisol (UFC) was high at 1299.5 (4.0-50.0 mcg/24h). CT of the abdomen/pelvis with and without contrast showed low-attenuation masses (less than 5 Hounsfield units) present in both adrenal glands measuring 6.9 x 5.3 cm on the right and 4.5 x 3.9 cm on the left, and did not demonstrate significant arterial enhancement (Figure 1). MR imaging of the abdomen without and with contrast was also obtained and showed the same masses of the bilateral adrenal glands, with largest on the left measured 3.6 cm and largest on the right measured 3.7 cm, as well as mild fatty infiltration of the liver. General surgery and hematology/oncology were consulted and recommendations were made for outpatient follow-up with PCP and endocrinology.

CT-of-the-abdomen/pelvis-with-contrast-showing-low-attenuation-masses-present-in-both-adrenal-glands-measuring-6.9-x-5.3-cm-on-the-right-(dark-gray-arrow)-and-4.5-x-3.9-cm-on-the-left-(light-gray-arrow)
Figure 1: CT of the abdomen/pelvis with contrast showing low-attenuation masses present in both adrenal glands measuring 6.9 x 5.3 cm on the right (dark gray arrow) and 4.5 x 3.9 cm on the left (light gray arrow)

In early February 2021, the patient again presented to the ED complaining of recurrent episodes of bilateral blurry vision. Examination was unremarkable, including an ophthalmological exam with slit lamp exam. Blurred vision was suspected to be due to osmotic swelling in the setting of severe hyperglycemia as the patient had persistently uncontrolled blood sugars. Recommendations were for tighter control of blood glucose, and follow-up with primary care and ophthalmology.

Patient followed up with the endocrinologist in mid-February to which the patient reported first noticing a difference in her energy and changes to her weight around one year prior. She communicated a weight gain of 30 to 40 lbs over the past year. Patient had a reported history of gestational hypertension diagnosed five years ago when she gave birth to her daughter, which was steadily worsening over the past year. She reported intermittent myalgias and easy bruising. Patient had no family history or any apparent features to suggest multiple endocrine neoplasia (MEN) syndrome. Blood work revealed ACTH less than 1.5 pg/mL, AM cortisol was high at 24.5 mcg/dL, and normal aldosterone at 3.6 ng/dL, with normal renin and metanephrine levels. Physical examination revealed truncal obesity as well as a round face, cushingoid in appearance, and relatively thin extremities and abdominal striae.

She was then referred to a surgical specialist, and it was decided that she would undergo laparoscopic bilateral adrenalectomy due to severe Cushing’s syndrome. The surgical pathology report revealed macro-nodular cortical hyperplasia of both left and right adrenal gland masses with random endocrine atypia. The largest nodule on the left measured 4.5 cm and the largest nodule on the right measured 6.6 cm. Post-operatively she was started on hydrocortisone 20 mg every morning and 10 mg every evening, and fludrocortisone 0.1 mg twice a day as part of her steroid replacement regimen. Eventually she changed to hydrocortisone 10 mg three times a day and fludrocortisone 0.1 mg once a day. For her diabetes, her insulin glargine decreased from 60 units to 20 units. Amlodipine and hydrochlorothiazide were discontinued from her antihypertensive medications; she continued losartan and metoprolol. Follow up blood work showed stable electrolytes with potassium 4.2 mmol/L (3.5-5.2 mmol/L), sodium 137 mmol/L (134-144mmol/L), chloride 100 mmol/L (96-106 mmol/L), and carbon dioxide 23 mmol/L (20-29mmol/L).

Discussion

ACTH-independent Cushing’s syndrome due to bilateral cortisol-secreting nodules is rare, accounting for 2% of CS cases. The majority of causes include primary bilateral macronodular adrenal hyperplasia (PBMAH), primary pigmented nodular adrenocortical disease (PPNAD), and bilateral adrenocortical adenomas (BAA). In PBMAH, typically patients are diagnosed within the fifth or sixth decade of life [4]. The usual age of onset for PPNAD is within the first to third decade of life, with median age in the pediatric population at age 15 years [6]. BAA is such a rare entity that there exists little epidemiological data with less than 40 reported cases until 2019 [7]. A small subset of patients present with overt clinical symptoms of CS, as hypercortisolism often follows an insidious course that can delay diagnosis from years to decades, with one series reporting a diagnostic delay of approximately eight years [8]. Serum and urine hormone screening in the right clinical setting can provide clues to these endocrine disorders, however diagnosis of ACTH-independent CS often occurs incidentally wherein a radiographic study was done for reasons other than to identify adrenal disease [9]. CT or MRI alone are not able to differentiate these disease entities, requiring pathological examination for final determination [7]. Adrenal venous sampling (AVS) and I-6B-iodomethyl-19-norcholesterol (I-NP-59) can aid in identifying hormone-secreting status of each adrenal lesion, however usefulness is debated among experts [10-12].

In all cases the end goal is to normalize adrenocortical hormones, and PBMAH primarily involves surgical resection with exogenous hormone replacement. Bilateral adrenalectomy is generally the treatment of choice with overt Cushing syndrome regardless of cortisol level. These patients require lifelong steroid administration [9,13]. Another approach is unilateral adrenalectomy of the larger or more metabolically active gland, which can be identified after AVS or I-NP-59 testing. This has been proposed in order to preserve some autonomous hormonal production and prevent adrenal crisis, however remission rates of Cushing syndrome as high as 84% have been reported with eventual need for bilateral adrenalectomy [7,8,14]. Steroid enzyme inhibition to control cortisol secretion has been used as an adjunct before surgery. In some patients with identified aberrant adrenal hormone receptors, targeted pharmacological inhibition remains an alternative medical approach [8]. Despite these alternatives to surgery, surgical resection remains the optimal approach [1].

Conclusions

ACTH-independent Cushing’s syndrome due to PBMAH usually presents as an indolent course, with typical diagnosis in the fifth to sixth decade. As the use of imaging for other non-endocrine related investigations becomes more utilized, PBMAH being less of a rare entity. Clinical presentation usually dictates the timing of and type of surgical intervention. Although there are some reports of unilateral resection resulting in a cure, many of these cases eventually proceed to staged bilateral resection. Our patient’s presentation as her primary complaint was recurrent episodes of blurry vision that were suspected to be due to osmotic swelling because of her uncontrolled hyperglycemia. Her case was also unusual as she presented at 40 years old, an average of 10 years earlier than is typically diagnosed for PBMAH. Her symptoms also progressed rapidly over the course of 12 months with development of resistant hypertension and insulin-dependent diabetes requiring high basal insulin. Following surgical resection, her antihypertensive regimen was de-escalated and had significant reduction in insulin requirements, and was maintained on adrenocorticoid therapy.


References

  1. Nieman LK: Recent updates on the diagnosis and management of Cushing’s syndrome. Endocrinol Metab (Seoul). 2018, 33:139-46. 10.3803/EnM.2018.33.2.139
  2. Rare Disease Database: Cushing Syndrome. (2021). Accessed: 12/17/2021: https://rarediseases.org/rare-diseases/cushing-syndrome/.
  3. Broder MS, Neary MP, Chang E, Cherepanov D, Ludlam WH: Incidence of Cushing’s syndrome and Cushing’s disease in commercially-insured patients <65 years old in the United States. Pituitary. 2015, 18:283-9. 10.1007/s11102-014-0569-6
  4. Lacroix A, Feelders RA, Stratakis CA, Nieman LK: Cushing’s syndrome. Lancet. 2015, 386:913-27. 10.1016/S0140-6736(14)61375-1
  5. Tokumoto M, Onoda N, Tauchi Y, et al.: A case of adrenocoricotrophic hormone -independent bilateral adrenocortical macronodular hyperplasia concomitant with primary aldosteronism. BMC Surg. 2017, 17:97. 10.1186/s12893-017-0293-z
  6. Stratakis CA: Cushing syndrome caused by adrenocortical tumors and hyperplasias (corticotropin- independent Cushing syndrome). Endocr Dev. 2008, 13:117-32. 10.1159/000134829
  7. Gu YL, Gu WJ, Dou JT, et al.: Bilateral adrenocortical adenomas causing adrenocorticotropic hormone-independent Cushing’s syndrome: a case report and review of the literature. World J Clin Cases. 2019, 7:961-71. 10.12998/wjcc.v7.i8.961
  8. Lacroix A: ACTH-independent macronodular adrenal hyperplasia. Best Pract Res Clin Endocrinol Metab. 2009, 23:245-59. 10.1016/j.beem.2008.10.011
  9. Sweeney AT, Srivoleti P, Blake MA: Management of the patient with incidental bilateral adrenal nodules. J Clin Transl Endocrinol Case Rep. 2021, 20:100082. 10.1016/j.jecr.2021.100082
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From https://www.cureus.com/articles/90069-intermittent-blurry-vision-an-unexpected-presentation-of-cushings-syndrome-due-to-primary-bilateral-macronodular-adrenal-hyperplasia-pbmah

Simultaneous Pituitary and Adrenal Adenomas in a Patient with Non ACTH Dependent Cushing Syndrome

Highlights

Cushing syndrome (CS) is a rare disorder with a variety of underlying etiologies.

CS is expected to affect 0.2 to 5 people per million per year.

Adrenal-dependent CS is an uncommon variant of CS.

This study reports a rare occurrence of pituitary and adrenal adenoma with CS.

Abstract

Introduction

Cushing syndrome is a rare disorder with a variety of underlying etiologies, that can be exogenous or endogenous (adrenocorticotropic hormone (ACTH)-dependent or ACTH-independent). The current study aims to report a case of ACTH-independent Cushing syndrome with adrenal adenoma and nonfunctioning pituitary adenoma.

Case report

A 37–year–old female presented with amenorrhea for the last year, associated with weight gain. She had a moon face, buffalo hump, and central obesity. A 24-hour urine collection for cortisol was performed, revealing elevated cortisol. Cortisol level was non-suppressed after administering dexamethasone. MRI of the pituitary revealed a pituitary microadenoma, and the CT scan of the abdomen with adrenal protocol revealed a left adrenal adenoma.

Discussion

Early diagnosis may be postponed due to the variety of clinical presentations and the referral of patients to different subspecialists based on their dominant symptoms (gynecological, dermatological, cardiovascular, psychiatric); it is, therefore, critical to consider the entire clinical presentation for correct diagnosis.

Conclusion

Due to the diversity in the presentation of CS, an accurate clinical, physical and endocrine examination is always recommended.

Keywords

Cushing syndrome
Cushing’s disease
Adrenal adenoma
Pituitary adenoma
Urine free cortisol

1. Introduction

Cushing syndrome (CS) is a collection of clinical manifestations caused by an excess of glucocorticoids [1]. CS is a rare disorder with a variety of underlying etiologies that can be exogenous due to continuous corticosteroid therapy for any underlying inflammatory illness or endogenous due to either adrenocorticotropic hormone (ACTH)-dependent or ACTH-independent [2][3]. Cushing syndrome is expected to affect 0.2 to 5 people per million per year. Around 10% of such cases involve children [4][5]. ACTH-dependent glucocorticoid excess owing to pituitary adenoma accounts for the majority (60–70%) of endogenous CS, with primary adrenal causes accounting for only 20–30% and ectopic ACTH-secreting tumors accounting for the remaining 5–10% [6]. Adrenal-dependent CS is an uncommon variant of CS caused mostly by benign (90%) or malignant (8%) adrenal tumors or, less frequently, bilateral micronodular (1%) or macronodular (1%) adrenal hyperplasia [7].

The current study aims to report a case of ACTH-independent Cushing syndrome with adrenal adenoma and nonfunctioning pituitary adenoma. The report has been arranged in line with SCARE guidelines and includes a brief literature review [8].

2. Case report

2.1. Patient’s information

A 37–year–old female presented with amenorrhea for the last year, associated with weight gain. She denied having polyuria, polydipsia, headaches, visual changes, dizziness, dryness of the skin, cold intolerance, or constipation. She had no history of chronic disease and denied using steroids. She visited an internist, a general surgeon, and a gynecologist and was treated for hypothyroidism. She was put on Thyroxin 100 μg daily, and oral contraceptive pills were given for her menstrual problems. Last time, the patient was referred to an endocrinology clinic, and they reviewed the clinical and physical examinations.

2.2. Clinical examination

She had a moon face, buffalo hump, central obesity, pink striae over her abdomen, and proximal weakness of the upper limbs. After reviewing the history and clinical examination, CS was suspected.

2.3. Diagnostic assessment

Because the thyroid function test revealed low thyroid-stimulating hormone (TSH), free T3, and freeT4, the patient was sent for a magnetic resonance imaging (MRI) of the pituitary, which revealed a pituitary microadenoma (7 ∗ 6 ∗ 5) mm (Fig. 1). Since the patient was taking thyroxin and oral contraceptive pills, the investigations were postponed for another six weeks due to the contraceptive pills’ influence on the results of the hormonal assessment for CS. After six weeks of no medication, a 24-hour urinary free cortisol (UFC) was performed three times, revealing elevated cortisol levels (1238, 1100, and 1248) nmol (normal range, 100–400) nmol. A dexamethasone suppression test was done (after administering dexamethasone tab 1 mg at 11 p.m., serum cortisol was measured at 9 a.m.). The morning serum cortisol level was 620 nmol (non-suppressed), which normally should be less than 50 nmol. The ACTH level was below 1 pg/mL.

Fig. 1

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Fig. 1. Contrast enhanced T1W weighted MRI (coronal section) showing small 7 mm hypo-enhanced microadenoma (yellow arrow) in right side of pituitary gland with mild superior bulge.

Based on these findings, ACTH independent CS was suspected. The computerized tomography (CT) scan of the abdomen with adrenal protocol revealed a left adrenal adenoma (33 mm × 25 mm) without features of malignancy (Fig. 2).

Fig. 2

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Fig. 2. Computed tomography scan of the abdomen with IV contrast, coronal section, showing 33 mm × 25 mm lobulated enhanced left adrenal tumor (yellow arrow), showing absolute washout on dynamic adrenal CT protocol, consistent with adrenal adenoma.

2.4. Therapeutic intervention

The patient was referred to the urologist clinic for left adrenalectomy after preparation for surgery and perioperative hormonal management. She underwent laparoscopic adrenalectomy and remained in the hospital for two days. The histopathology results supported the diagnosis of adrenal adenoma.

2.5. Follow-up

She was released home after two days on oral hydrocortisone 20 mg in the morning and 10 mg in the afternoon. After one month of follow-up, serum cortisol was 36 nmol, with the resolution of some features such as weight reduction (3 kg) and skin color (pink striae became white).

3. Discussion

Cushing’s syndrome is a serious and well-known medical condition that results from persistent exposure of the body to excessive glucocorticoids, either from endogenous or, most frequently, exogenous sources [9]. The average age of diagnosis is 41.4 years, with a female-to-male ratio of 3:1 [10]. ACTH-dependent CS accounts for almost 80% of endogenous CS, while ACTH-independent CS accounts for nearly 20% [10]. This potentially fatal condition is accompanied by several comorbidities, including hypertension, diabetes, coagulopathy, cardiovascular disease, infections, and fractures [11]. Exogenous CS, also known as iatrogenic CS, is more prevalent than endogenous CS and is caused by the injection of supraphysiologic glucocorticoid dosages [12]. ACTH-independent CS is induced by uncontrolled cortisol release from an adrenal gland lesion, most often an adenoma, adrenocortical cancer, or, in rare cases, ACTH-independent macronodular adrenal hyperplasia or primary pigmented nodular adrenal disease [13].

The majority of data suggests that early diagnosis is critical for reducing morbidity and mortality. Detection is based on clinical suspicion initially, followed by biochemical confirmation [14]. The clinical manifestation of CS varies depending on the severity and duration of glucocorticoid excess [14]. Some individuals may manifest varying symptoms and signs because of a rhythmic change in cortisol secretion, resulting in cyclical CS [15]. The classical symptoms of CS include weight gain, hirsutism, striae, plethora, hypertension, ecchymosis, lethargy, monthly irregularities, diminished libido, and proximal myopathy [16]. Neurobehavioral presentations include anxiety, sadness, mood swings, and memory loss [17]. Less commonly presented features include headaches, acne, edema, abdominal pain, backache, recurrent infection, female baldness, dorsal fat pad, frank diabetes, electrocardiographic abnormalities suggestive of cardiac hypertrophy, osteoporotic fractures, and cardiovascular disease from accelerated atherosclerosis [10]. The current case presented with amenorrhea, weight gain, moon face, buffalo hump, and skin discoloration of the abdomen.

Similar to the current case, early diagnosis may be postponed due to the variety of clinical presentations and the referral of patients to different subspecialists based on their dominant symptoms (gynecological, dermatological, cardiovascular, psychiatric); it is, therefore, critical to consider the entire clinical presentation for correct diagnosis [18]. Weight gain may be less apparent in children, but there is frequently an arrest in growth with a fall in height percentile and a delay in puberty [19].

The diagnosis and confirmation of the etiology can be difficult and time-consuming, requiring a variety of laboratory testing and imaging studies [20]. According to endocrine society guidelines, the initial assessment of CS must include one or more of the three following tests: 24-hour UFC measurement; evaluation of the diurnal variation of cortisol secretion by assessing the midnight serum or salivary cortisol level; and a low-dose dexamethasone suppression test, typically the 1 mg overnight test [21]. Although UFC has sufficient sensitivity and specificity, it does not function well in milder cases of Cushing’s syndrome [22]. In CS patients, the typical circadian rhythm of cortisol secretion is disrupted, and a high late-night cortisol serum level is the earliest and most sensitive diagnostic indicator of the condition [23]. In the current case, the UFC was elevated, and cortisol was unsuppressed after administration of dexamethasone.

All patients with CS should have a high-resolution pituitary MRI with a gadolinium-based contrast agent to prove the existence or absence of a pituitary lesion and to identify the source of ACTH between pituitary adenomas and ectopic lesions [24]. Adrenal CT scan is the imaging modality of choice for preoperatively localizing and subtyping adrenocortical lesions in ACTH-independent Cushing’s syndrome [9]. MRI of the pituitary gland of the current case showed a microadenoma and a CT scan of the adrenals showed left adrenal adenoma.

Surgical resection of the origin of the ACTH or glucocorticoid excess (pituitary adenoma, nonpituitary tumor-secreting ACTH, or adrenal tumor) is still the first-line treatment of all forms of CS because it leaves normal adjacent structures and results in prompt remission and inevitable recovery of regular adrenal function [12][25]. Laparoscopic (retroperitoneal or transperitoneal) adrenalectomy has become the gold standard technique for adrenal adenomas since it is associated with fewer postoperative morbidity, hospitalization, and expense when compared to open adrenalectomy [17]. In refractory cases, or when a patient is not a good candidate for surgery, cortisol-lowering medication may be employed [26]. The current case underwent left adrenalectomy.

Symptoms of CS, such as central obesity, muscular wasting or weakness, acne, hirsutism, and purple striae generally improve first and may subside gradually over a few months or even a year; nevertheless, these symptoms may remain in 10–30% of patients [27]. Glucocorticoid replacement is essential after adrenal-sparing curative surgery until the pituitary-adrenal function returns, which might take up to two years, especially if adrenal adenomas have been resected [25]. Chronic glucocorticoid excess causes lots of new co-morbidities, lowering the quality of life and increasing mortality. The most common causes of mortality in CS are cardiovascular disease and infections [28]. After one month of follow-up, serum cortisol was 36 nmol, and several features, such as weight loss (3 kg) and skin color, were resolved (pink striae became white).

In conclusion, the coexistence of adrenal adenoma and pituitary adenoma with CS is a rare possibility. Due to the diversity in the presentation of CS, an accurate clinical, physical and endocrine examination is always recommended. Laparoscopic adrenalectomy is the gold standard for treating adrenal adenoma.

Consent

Written informed consent was obtained from the patient’s family for publication of this case report and accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal on request.

Provenance and peer review

Not commissioned, externally peer-reviewed.

Ethical approval

Approval is not necessary for case report (till 3 cases in single report) in our locality.

The family gave consent for the publication of the report.

Funding

None.

Guarantor

Fahmi Hussein Kakamad, Fahmi.hussein@univsul.edu.iq.

Research registration number

Not applicable.

CRediT authorship contribution statement

Abdulwahid M. Salh: major contribution of the idea, literature review, final approval of the manuscript.

Rawa Bapir: Surgeon performing the operation, final approval of the manuscript.

Fahmi H. Kakamad: Writing the manuscript, literature review, final approval of the manuscript.

Soran H. Tahir, Fattah H. Fattah, Aras Gh. Mahmood, Rawezh Q. Salih, Shaho F. Ahmed: literature review, final approval of the manuscript.

Declaration of competing interest

None to be declared.

References

A Case of Acute Exacerbation of Chronic Hepatitis C During the Course of Adrenal Cushing’s Syndrome

https://doi.org/10.1002/ccr3.5337

Abstract

A 50-year-old woman with adrenal Cushing’s syndrome and chronic hepatitis C developed an acute exacerbation of chronic hepatitis C before adrenectomy. After administration of glecaprevir/pibrentasvir was started, her transaminase levels normalized promptly and a rapid virological response also was achieved. Laparoscopic left adrenectomy was then performed safely.

1 INTRODUCTION

Reports of reactivation of hepatitis C virus (HCV) and acute exacerbation of chronic hepatitis C associated with immunosuppressive therapy and cancer drug therapy are rarer than for hepatitis B virus (HBV) but have been made occasionally. In HBV infection, viral reactivation and acute hepatitis caused by an excess of endogenous cortisol due to Cushing’s syndrome have been reported, but no acute exacerbation of chronic hepatitis C has been reported so far. Here, we report a case of acute exacerbation of chronic hepatitis C during the course of adrenal Cushing’s syndrome.

2 CASE REPORT

A woman in her 50s underwent a CT scan at a nearby hospital to investigate treatment-resistant hypertension and was found to have a left adrenal mass. Her blood tests showed low ACTH and HCV antibody positivity, and she was referred to our hospital because she was suspected of having Cushing’s syndrome and chronic hepatitis C. There is nothing special to note about her medical or family history. She had never smoked and drank very little. Her physical findings on admission were 164.5 cm tall, 92.6 kg in weight, and a BMI of 34.2 kg/m2. Her blood pressure was 179 / 73 mmHg, pulse 64 /min (rhythmic), body temperature 36.8°C, and respiratory rate 12 /min. She had findings of central obesity, moon face, buffalo hump, and red skin stretch marks. Her blood test findings (Table 1) showed an increase in ALT, HCV antibody positivity, and an HCV RNA concentration of 4.1 log IU/mL. The virus was genotype 2. Cortisol was within the reference range, but ACTH was as low, less than 1.5 pg/mL. Her bedtime cortisol level was 7.07 μg/dL, which was above her reference of 5 μg/dL, suggesting the loss of diurnal variation in cortisol secretion. Testing showed the amount of cortisol by 24-hour urine collection was 62.1 μg/day, and this level of cortisol secretion was maintained. In an overnight low-dose dexamethasone suppression test, cortisol after loading was 6.61 μg/dL, which exceeded 5 μg/dL, suggesting that cortisol was autonomously secreted. Her contrast-enhanced CT scan (Figure 1) revealed a tumor with a major axis of about 30 mm in her left adrenal gland. MRI scans showed mild hyperintensity in the “in phase” (Figure 2A) and decreased signal in the “out of phase” (Figure 2B), suggesting her adrenal mass was an adenoma. Based on the above test results, she was diagnosed with chronic hepatitis C and adrenal Cushing’s syndrome. She agreed to receive treatment with direct acting antiviral agents (DAAs) after resection of the left adrenal tumor. However, two months later, she had liver dysfunction with AST 116 U/L and ALT 213 U/L (Figure 3). HBV DNA was undetectable at the time of liver injury, but the HCV RNA concentration increased to 6.4 logIU/mL. Therefore, an acute exacerbation of chronic hepatitis C was suspected, and a percutaneous liver biopsy was performed. The biopsy revealed an inflammatory cell infiltration, mostly composed of lymphocytes and plasma cells and mainly in the portal vein area (Figure 4). Fibrosis and interface hepatitis were also observed, and spotty necrosis was evident in the hepatic lobule. No clear fat deposits were found in the hepatocytes, ruling out NASH or NAFLD. According to the New Inuyama classification, hepatitis equivalent to A2-3/F1-2 was considered. Because HBV DNA was not detected, no new drug was used, and no cause of liver damage, such as biliary atresia, was found; the patient was diagnosed with liver damage due to reactivation of HCV, with acute exacerbation of chronic hepatitis C. The treatment policy was changed, in order to treat hepatitis C before the left adrenal resection, and administration of glecaprevir/pibrentasvir was started. A blood test two weeks after the start of treatment confirmed normalization of AST and ALT, and a rapid virological response was achieved (Figure 3). Subsequently, HCV RNA remained negative, no liver damage was observed, and laparoscopic left adrenectomy was safely performed nine months after the initial diagnosis. The pathological findings were adrenal adenoma, and no atrophy was observed in the attached normal adrenal cortical gland. After the operation, hypertension improved and weight loss was obtained (92.6 kg (BMI: 34.2 kg/m2) before the operation, but 77.0 kg (BMI: 28.5 kg/m2) one year after the operation). ACTH increased, and the adrenal Cushing’s syndrome was considered to have been cured. Regarding HCV infection, the sustained virological response has been maintained to date, more than 2 years after the completion of DAA therapy, and the follow-up continues.

TABLE 1. Laboratory data on admission
Hematology Chemistry
WBC 6100 /μL TP 8.2 g/dL DHEA-S 48 /μL
RBC 526 x 104 /μL Alb 3.4 g/dL PRA 0.7 ng/mL/h
Hb 15.8 g/dL T-Bil 0.3 mg/dL ALD 189 pg/mL
Ht 49.1 % AST 33 U/L
PLT 25.5 x 104 /μL ALT 46 U/L Serological tests
LDH 201 U/L CRP <0.10 mg/dL
ALP 292 U/L HBsAg (-)
γ-GTP 77 U/L anti-HBs (-)
Coagulation BUN 13 mg/dL anti-HBc (+)
PT 126.1 % Cr 0.63 mg/dL HBeAg (-)
APTT 27.5 sec HbA1c 6.2 % anti-HBe (+)
Cortisol 7.46 μg/dL anti-HCV (+)
ACTH <1.5 pg/mL
FBS 82 mg/dL Genetic tests
Na 138 mmol/L HBV DNA Undetectable
Cl 105 mmol/L HCV RNA 4.1 LogIU/Ml
K 3.6 mmol/L HCV genotype 2
Ca 9.0 mg/dL
  • Abbreviations: Hematology: WBC, white blood cells; RBC, red blood cells; Hb, hemoglobin; Ht, hematocrit; PLT, platelets.
  • Coagulation: PT, prothrombin time; APTT, activated partial thromboplastin time.
  • Chemistry: TP, total protein; Alb, albumin; T-Bil, total bilirubin; AST, aspartate transaminase; ALT, alanine aminotransferase; LDH, lactate dehydrogenase; ALP, alkaline phosphatase; γGTP, γ-glutamyl transpeptidase; BUN, blood urea nitrogen; Cr, creatinine; HbA1c, Hemoglobin A1c; FBS, fasting blood sugar; Na, sodium; Cl, chlorine; K, potassium; Ca, calcium; DHEA-S, dehydroepiandrosterone sulfate; PRA, plasma renin activity; ALD, aldosterone.
  • Serological tests: CRP, C-reactive protein; HBsAg, hepatitis B surface antigen; anti-HBs, hepatitis B surface antibody; anti-HBc, hepatitis B core antibody; HBeAg, hepatitis B e antigen; anti-HBe, hepatitis B e antibody; anti-HCV, hepatitis C virus antibody.
  • Genetic tests: HBV DNA, hepatitis B virus deoxyribonucleic acid; HCV RNA, hepatitis C virus ribonucleic acid.

Details are in the caption following the image

Contrast-enhanced CT examination. Contrast-enhanced CT examination revealed a tumor (arrow) with a major axis of about 30 mm in the left adrenal gland

Details are in the caption following the image

MRI image of the adrenal lesion. MRI showed mild hyperintensity in the “in phase” (A) and decreased signal in the “out of phase” (B), suggesting adrenocortical adenoma (arrow)

Details are in the caption following the image

Changes in serum transaminase and HCV RNA levels. All showed rapid improvement by administration of direct acting antivirals. ALT: alanine aminotransferase, AST: aspartate transaminase, HCV RNA: hepatitis C virus ribonucleic acid

Details are in the caption following the image

Pathological findings of tissues obtained by percutaneous liver biopsy. Infiltration of inflammatory cells, which was mostly composed of lymphocytes and plasma cells and a small number of neutrophils, was observed mainly in the portal vein area. This was accompanied by fibrous enlargement and interface hepatitis. Although the arrangement of hepatocytes was maintained in the hepatic lobule, spotty necrosis was observed in some parts. No clear fat deposits were found in the hepatocytes, and NASH or NAFLD was a negative finding. According to the New Inuyama classification, hepatitis equivalent to A2-3/F1-2 was considered (a; ×100, b; ×200, scale bar = 500 µm)

3 DISCUSSION

Reactivation of HBV can cause serious liver damage. Therefore, it is recommended to check the HBV infection status before starting anticancer chemotherapy or immunotherapy and to continue monitoring for the presence or absence of reactivation thereafter.12 On the other hand, there are fewer reports of the reactivation of HCV, and many aspects of the pathophysiology of HCV reactivation remain unclear. In this case, it is possible that chronic hepatitis C was acutely exacerbated due to endogenous cortisol secretion in Cushing’s syndrome. Although the definition of HCV reactivation has not been defined, several studies35 have defined an increase of HCVRNA of 1.0 log IU/ml or more as HCV reactivation. In addition, the definition of acute exacerbation of chronic hepatitis C is that ALT increases to more than three times the upper limit of the reference range.346 Mahale et al. reported a retrospective study in which acute exacerbation of chronic hepatitis C due to cancer medication was seen in 11% of 308 patients.3 Torres et al. also reported that, in a prospective study of 100 patients with cancer medication, HCV reactivation was found in 23%.4 Given these reports, HCV reactivation potentially could occur quite frequently. However, Torres et al. reported that only 10% of all patients had acute exacerbations, none of which led to liver failure.4 Such data suggest that HCV reactivation may often be overlooked in actual cases without aggravation. Thus, the frequency of aggravation due to hepatitis virus reactivation is thought to be lower for HCV than for HBV. However, there are some reports of deaths from acute exacerbation of chronic hepatitis C.710 In addition, if severe hepatitis develops following viral reactivation, mortality rates have been reported to be similar for HBV and HCV.811 Thus, reactivation of HCV is considered to be a pathological condition that requires caution, similar to HBV. Torres et al. reported that administration of rituximab or corticosteroids is a significant independent risk factor.4 In addition, there are reports of acute exacerbation of chronic hepatitis C due to corticosteroids administered as antiemetics and as immunosuppressive therapy.1214 Therefore, excess cortisol can reactivate not only HBV but also HCV. The mechanism by which HCV is reactivated with cortisol is assumed to be decreased cell-mediated immunity due to rapid apoptosis of circulating T cells caused by glucocorticoids,4 enhancement of HCV infectivity by upregulation of viral receptor expression on the hepatocyte surface,15 and enhanced viral replication.16 In addition, there is a report that genotype 2 is more common in cases with acute exacerbation of chronic hepatitis C,413 which is consistent with this case.

Regarding HBV reactivation due to Cushing’s syndrome, three cases of acute exacerbation of chronic hepatitis B have been reported.1719 It is believed that Cushing’s syndrome caused a decrease in cell-mediated immunity and humoral immunity due to an endogenous excess of cortisol, resulting in an acute exacerbation of chronic hepatitis B.13 As described above, because an excess of cortisol can cause reactivation of HCV, it is considered that a decrease in immunocompetence due to Cushing’s syndrome, which is an excess of endogenous cortisol, can also cause reactivation of HCV and acute exacerbation of chronic hepatitis. However, as far as we can determine, no cases of Cushing’s syndrome causing HCV reactivation or acute exacerbation of chronic hepatitis C have been reported and similar cases may be latent. Among the reports of acute exacerbation of hepatitis B due to adrenal Cushing’s syndrome, there is a case in which the liver damage and viral load were improved only by adrenalectomy.17 Therefore, it is also possible that hepatitis C was improved by adrenal resection in this case. However, general anesthesia associated with adrenalectomy and the use of various drugs used for postoperative physical management should be avoided, if possible, in situations where some severe liver damage is present. In addition, reactivation of immunity due to rapid depletion of glucocorticoid, following resection of an adrenal tumor, may lead to exacerbation of liver damage. In this case, the amount of HCV and hepatic transaminase levels were improved rapidly by glecaprevir/pibrentasvir treatment, and the operation could be performed safely. If Cushing’s syndrome is complicated by an acute exacerbation of hepatitis C, clinicians should consider including treatment strategies such as in this case. Summarizing the above, when liver damage appears in HCV-infected patients with Cushing’s syndrome, it will be necessary to distinguish the acute exacerbation and reactivation of chronic hepatitis C. Treatment with DAAs may then be considered to be effective for reactivation of HCV and acute exacerbation of chronic hepatitis.

4 CONCLUSION

We report a case of chronic hepatitis C with acute exacerbation during the course of Cushing’s syndrome. At the time of cancer drug therapy and in the state of endogenous and extrinsic corticosteroid excess, it is necessary to pay attention not only to acute exacerbation of chronic hepatitis B but also to hepatitis C.

ACKNOWLEDGEMENTS

All authors would like to thank the patient and his family for allowing this case study.

CONFLICT OF INTEREST

The authors have no conflict of interests.

AUTHOR CONTRIBUTIONS

TO and KM were collected and analyzed the data and wrote and edited the manuscript. KH, ST, HO, KT, KM, and JK were involved in the patient’s care and provided advice on the preparation of this case report.

ETHICAL APPROVAL

This study complied with the standards of the Declaration of Helsinki and the current ethical guidelines.

CONSENT

Written informed consent was obtained from the patient to publish this report in accordance with the journal’s patient consent policy.

From https://onlinelibrary.wiley.com/doi/10.1002/ccr3.5337

Post-Operative Cushing Syndrome Care

Justine Herndon, PA-C, and Irina Bancos, MD, on Post-Operative Cushing Syndrome Care

– Curative procedures led to widespread resolution or improvement of hyperglycemia

by Scott Harris , Contributing Writer, MedPage Today January 18, 2022

In a recent study, two-thirds of people with Cushing syndrome (CS) saw resolved or improved hyperglycemia after a curative procedure, with close post-operative monitoring an important component of the process.

Among 174 patients with CS included in the longitudinal cohort study (pituitary in 106, ectopic in 25, adrenal in 43), median baseline HbA1c was 6.9%. Of these, 41 patients were not on any therapy for hyperglycemia, 93 (52%) took oral medications, and 64 (37%) were on insulin.

At the end of the period following CS remission (median 10.5 months), 37 (21%) patients had resolution of hyperglycemia, 82 (47%) demonstrated improvement, and 55 (32%) had no change or worsened hyperglycemia. Also at the end of follow-up, HbA1c had fallen 0.84% (P<0.0001), with daily insulin dose decreasing by a mean of 30 units (P<0.0001).

Justine Herndon, PA-C, and Irina Bancos, MD, both endocrinology researchers with Mayo Clinic in Minnesota, served as co-authors of the report, which was published in the Journal of the Endocrine Society. Here they discuss the study and its findings with MedPage Today. The exchange has been edited for length and clarity.

What was the study’s main objective?

Herndon: As both a hospital diabetes provider and clinic pituitary/gonadal/adrenal provider, I often hear questions from colleagues about how to manage a patient’s diabetes post-operatively after cure from CS. While clinical experience has been helpful in guiding these discussions, the literature offered a paucity of data on diabetes/hyperglycemia specifically after surgery. There was also a lack of data on specific subgroups of CS, whether by sub-type or severity.

Therefore, we felt it was important to see what our past patient experiences showed in terms of changes in laboratory data, medications, and which patients were more likely to see improvement in their diabetes/hyperglycemia. The overall goal was to help clinicians provide appropriate patient education and care following a curative procedure.

In addition to its primary findings, the study also identified several factors associated with resolution or improvement of hyperglycemia. What were these factors?

Bancos: Both clinical and biochemical severity of CS, as well as Cushing subtype, were associated with improvement. We calculated severity based on symptoms and presence of comorbidities, and we calculated biochemical severity based on hormonal measurements. As clinical and biochemical scores were strongly correlated, we chose only one (biochemical) for multivariable analysis.

In the multivariable analysis of biochemical severity of Cushing, subtype of Cushing, and subtype of hyperglycemia, we found that patients with a severe biochemical severity score were 2.4 fold more likely to see improved hyperglycemia than people with a moderate or mild severity score (OR 2.4 (95% CI 1.1-4.9). We also found that patients with the nonadrenal CS subtype were 2.9 fold more likely to see improved hyperglycemia when compared to people with adrenal CS (OR of 2.9 (95% CI 1.3-6.4).

The type of hyperglycemia (diabetes versus prediabetes) was not found to be significant.

Did anything surprise you about the study results?

Herndon: I was surprised to see improvement in hyperglycemia in patients who were still on steroids, as you would expect the steroids to still have an impact. This shows how much a CS curative procedure truly leads to changes in the comorbidities that were a result of the underlying disease.

Also, I was surprised that the type of hyperglycemia was not a predictor of improvement after cure, although it was quite close. We also had a few patients whose hyperglycemia worsened, and we could not find a specific factor that predicted which patients did not improve.

What are the study’s implications for clinicians who treat people with CS?

Bancos: We think our study shows the clear need for closer follow-up — more frequently than the typical three-to-six months for diabetes. This can be accomplished through review of more than just HbA1c, such as reviewing blood glucose logbooks, asking about hypoglycemia symptoms, and so forth.

Patients with severe CS who are being treated with insulin or hypoglycemic medications are especially likely to decrease their medications to avoid hypoglycemia during postoperative period.

Read the study here.

Bancos reported advisory board participation and/or consulting with Strongbridge, Sparrow Pharmaceutics, Adrenas Therapeutics, and HRA Pharma outside the submitted work. Herndon did not disclose any relevant financial relationships with industry.

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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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

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