Thymic Neuroendocrine Tumor With Metastasis to the Breast Causing Ectopic Cushing’s Syndrome

Posted on April 9, 2025 by MaryO

Ectopic adrenocorticotropic hormone secretion (EAS) is responsible for approximately 10%–18% of Cushing’s syndrome cases. Thymic neuroendocrine tumors (NETs) comprise 5%–16% of EAS; therefore, they are very rare and the data about this particular tumors is scarce.

We present a case of a 34-year-old woman with a rapid onset of severe hypercortisolism in April 2016. After initial treatment with a steroid inhibitor (ketoconazole) and diagnostics including 68Ga DOTA-TATE PET/CT, it was shown to be caused by a small thymic NET.

After a successful surgery and the resolution of all symptoms, there was a recurrence after 5 years of observation caused by a metastasis to the breast, shown in the 68Ga DOTA-TATE PET/CT result and confirmed with a breast biopsy.

Treatment with a steroid inhibitor (metyrapone) and tumor resection were again curative. The last disease relapse appeared 7 years after the initial treatment, with severe hypercortisolism treated with osilodrostat. There was a local recurrence in the mediastinum, and a thoracoscopic surgery was performed with good clinical and biochemical effect.

The patient remains under careful follow-up. Our case stays in accordance with recent literature data, showing that patients with thymic NETs are younger than previously considered and that the severity of hypercortisolism does not correlate with the tumor size. The symptoms of EAS associated with thymic NET may develop rapidly and may be severe as in our case. Nuclear medicine improves the effectiveness of the tumor search, which is crucial in successful EAS therapy. Our case also underlines the need for lifelong monitoring of patients with thymic NETs and EAS.

1 Introduction

Ectopic adrenocorticotropic hormone secretion (EAS) represents between 9% and 18% of adrenocorticotropic hormone (ACTH)-dependent Cushing’s syndrome (CS) cases (1–3). The tumors secreting ACTH may occur in many locations and present with different histopathological differentiation, resulting in various clinical outcomes. In the past, most of the EAS cases were associated with small cell lung cancer, characterized by rapid tumor progression and unfavorable prognosis. Recently, well-differentiated neuroendocrine tumors (NETs) from the foregut prevail in the clinical series of EAS, with most common locations in the lungs, thymus, and pancreas (1).

EAS is often associated with severe hypercortisolism. Typical Cushing’s appearance may not be present due to the rapid onset of the disease. Patients with this type of hypercortisolism need urgent treatment because they have the highest mortality of all forms of CS (4). A retrospective review of 43 patients with EAS reported deaths in 27 patients (62.8%) and a median overall survival of 32.2 months. The leading causes of mortality were the progression of primary malignancies and systemic infections; two patients died from pulmonary embolism (5).

Prompt surgical removal of the tumor secreting ACTH is the mainstay of the therapy. However, finding the tumor causing EAS can be challenging due to its small size and variety of locations. Most authors recommend a combination of computed tomography (CT) scanning of the chest, abdomen, and pelvis, with additional magnetic resonance imaging (MRI) of the pituitary, as the first-line examinations (1, 6, 7). However, the sensitivity of standard imaging modalities is suboptimal (8). In the analysis of 231 patients with EAS, cross-sectional imaging revealed the source of ACTH in 52.4% of them at initial evaluation, and another 29% was found during follow-up or due to nuclear medicine functional imaging, while 18.6% remained occult (9). Nuclear medicine improves the sensitivity of conventional radiology in the case of EAS, with the use of 18-fluorodeoxyglucose (18F-FDG) positron emission tomography (PET)/CT (18F-FDG PET/CT) expected to be useful in identifying EAS tumors with high proliferative activity and 68gallium-labeled somatostatin analogues (68Ga DOTA-TATE) PET/CT with the potential to detect NETs. In the head-to-head comparison, the detection rate of the source of EAS was 75% for 68Ga DOTA-TATE and 60% for 18F-FDG PET/CT, while the highest sensitivity (90%) was achieved when both methods were combined (10).

Thymic NETs comprise 2%–5% of all thymic neoplasms and may cause some paraneoplastic syndromes, with the most frequent being myasthenia gravis, syndrome of inappropriate antidiuretic hormone secretion, and hypercortisolism (11). EAS associated with thymic NETs are rare, representing between 5% and 16% of EAS in published case series (1). Because of the rarity and heterogeneity of the disease, no evidence-based guidelines are available.

We present a case of a patient with thymic NET causing EAS, with metastasis to the breast after 5 years of post-surgical remission and another local recurrence 7 years after the first operation.

Our case is unique because thymic NETs causing EAS are known as an aggressive disease with a median recurrence time of 24 months after thymectomy (12). There are only a few cases described of metastases to the breast from thymic NETs causing EAS (13–16). Moreover, 68Ga-SSTR PET/CT was very helpful in detecting both primary and metastatic ectopic ACTH-secreting tumor, which underlines its role in the diagnostic workout of EAS.

2 Case description

A 32-year-old woman with no relevant medical history was admitted to the endocrinology department in April 2016 due to the rapid onset of symptoms: weight gain, hypertension, skin changes, and oligomenorrhoea.

The measurements at initial physical examination were as follows: body mass index (BMI)—29 kg/m2, blood pressure—180/90 mmHg, and heart rate—88/min. She had plethora, acne, moon face, buffalo hump, central obesity, many red striae in the abdominal area, and mild hirsutism. The baseline laboratory findings are presented in Table 1, with hypokalemia, diabetes, leukocytosis, high levels of serum cortisol, ACTH, and chromogranin A, and increased urine-free cortisol (UFC) secretion. There was no suppression of serum cortisol or UFC after a high-dose dexamethasone test. ACTH-dependent CS was diagnosed, and EAS was suspected. The patient’s family history was negative for endocrine diseases or genetic disorders.

Table 1

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Table 1. Laboratory results at diagnosis (April 2016).

The first-line cross-sectional imaging studies (chest, abdomen, and pelvis CT and MRI of the pituitary gland) did not reveal the source of ACTH. Only a symmetrical enlargement of adrenals was observed. 68Ga DOTA-TATE PET/CT revealed an oval lesion in the anterior mediastinum (1.9 × 1.3 cm) with a subtle overexpression of somatostatin receptors (SUV max. 2.8, Figures 1A, B). The chest MRI confirmed a mass 1.5 × 2.0 × 2.5 cm, with high T2-weighted signal and high contrast enhancement, suggestive of NET. The patient was given ketoconazole (600 mg daily), spironolactone, potassium supplementation, antihypertensive drugs, and thromboembolic prophylaxis. In June 2016, thoracoscopic removal of the mediastinal tumor was performed. In the histopathological examination, the tumor was encapsulated, without evidence of invasion, and no lymph node metastases were described. The immunophenotype of the tumor was as follows: CgA (+), Syn (+), CKAE1+E3 (+) “dot-like”, S100 (-), calcitonin (-), EMA (+/-), Ki67 3% to 4% in hot spots, no necrosis, mitotic index 0/10HPF with conclusion: thymic NET—typical carcinoid (low-grade). The presence of paraganglioma was also taken into consideration, as such cases were described (17). However, the significant reaction with cytokeratin and lack of S100 protein expression made this diagnosis less probable.

Figure 1

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Figure 1. 68Ga-DOTATATE PET/CT scans. (A, B) Before the first surgery (April 2016). (C, D) Before the second surgery (May 2021). (E, F) Before the third surgery (January 2023).

The postoperative morning serum cortisol concentration was below 5 µg/dL, indicating biochemical remission. The patient received hydrocortisone substitution for a month. The clinical signs of CS disappeared, and there was a normalization of UFC.

During 5 years of follow-up, the patient got pregnant and delivered a healthy child. Genetic counseling was performed, and no germline mutation of MEN1 gene was identified. Other clinical manifestations of MEN1 (like primary hyperparathyroidism and pituitary secreting tumors) were excluded.

In May 2021, the patient experienced a sudden recurrence of CS symptoms. The laboratory findings confirmed severe hypercortisolism (Table 2); therefore, treatment with steroid inhibitor metyrapone was administered. The patient tolerated only 750 mg daily; there were side effects (skin rash and tachycardia) with higher doses. The chest MRI revealed no recurrence in the location of the primary tumor, only a lesion in the right breast (1.2 × 1.0 × 1.1 cm) with atypical contrast enhancement. The 68Ga-DOTA-TATE PET/CT result showed a subtle overexpression of the tracer (SUV max 1.9) in the right breast (Figures 1C, D). Breast ultrasonography confirmed a hypoechogenic, hypervascular mass in the right breast, BIRADS 3/4, diagnosed as NET in the breast biopsy. The tumor was removed in July 2021 without complications. The histopathological samples were compared with the primary lesion, confirming the metastasis from thymic NET to the breast—tumor size 0.7 × 1.5 cm, clear surgical margins (8 mm) with Ki67 3% (NET G2), and no lymph node metastases. After the breast surgery, the cortisol levels normalized in blood and urine and the CS symptoms disappeared. 18F-FDG PET/CT and 68Ga-DOTA-TATE PET/CT were performed, showing no pathological increase of radiotracer uptake in post-operative locations or mediastinal lymph nodes. The patient consulted with the oncology team, and no adjuvant therapy was recommended.

Table 2

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Table 2. Laboratory results during 7 years of observation.

The next recurrence of the disease occurred in February 2023, with the symptoms developing suddenly during a very short period (1 to 2 weeks), additionally with significant mental deterioration (concentration disorders, anxiety, severe mood swing). The laboratory findings confirmed excessive hypercortisolism (Table 2). The patient was given osilodrostat (the initial dose was 20 mg daily but later reduced to 10 mg daily for 2 weeks until surgery) and symptomatic treatment with good clinical and biochemical effect. The 68Ga-DOTA-TATE PET/CT result showed a slightly increased uptake of the tracer in the left mediastinum, between cervical vessels, 0.9 × 1.2 cm (Figures 1E, F)—probably a local recurrence. Thoracotomy was performed in February 2023, with subsequent clinical and biochemical improvement (Table 2). In the histopathological examination, mediastinal NET G1 was diagnosed, without necrosis, mitotic activity 0/2 mm2, immunophenotype CgA (+), CD56 (+), Ki 67 1%, CK AE1/AE3 (+), CD117 (+), p40 (-), TdT (-), PAX8 (-), and the presence of tumor cell embolism in the vessels. One metastatic lesion was found in the pericardium (the maximal dimension of the tissue was 13 mm, resected radically). Two metastatic lesions in the fat tissue were found (one tissue fragment from the mediastinum, max. 16 mm diameter, and the second tissue fragment was surrounding the jugular vein, max. diameter up to 40 mm, both resected radically). Two of the 10 resected lymph nodes had metastatic lesions: one from the area of the jugular vein, diameter 11 mm, with capsular invasion, and the second lymph node N2R with capsular invasion, both resected radically. The symptoms of hypercortisolism disappeared, and the cortisol values were normalized after the operation. The patient is currently under careful monitoring, without signs of clinical or biochemical recurrence. 68Ga-DOTA-TATE PET/CT is performed every 6 months.

3 Discussion

Our case is representative for thymic NETs causing EAS presented in literature, but it also shows some distinct features, giving new insight into this rare condition.

In recent series, ACTH-secreting thymic NETs occurred often in young adults, like our patient. The typical age of presentation is 21–35 years in the largest case series, and 7.4% were children under 15 years (12, 13). In contrast, the former series of thymic NETs showed a peak incidence in the sixth decade of life (11).

ACTH-secreting thymic NETs show a slight male preponderance (58.6%); however, the patient’s gender does not seem to relate with the disease outcome (12). There was only an association between male sex and larger tumor size preoperatively as found in one case series (13).

Thymic NETs causing EAS are very rarely associated with MEN1; we have also excluded it in our patient. On the contrary, 30% of thymic NETs not associated with CS are found in patients with MEN1, mostly male smokers (18). It is not clear why thymic NETs with EAS are less likely caused by MEN1 gene mutation, but the possibility of this genetic predisposition should always be taken into consideration.

Thymic NETs associated with EAS are generally considered aggressive, presenting significant cellular atypia in the histopathological examination (19). However, the biology of the tumors is variable. In the histopathological examination of 92 thymic NETs secreting ACTH, the most common subtype was atypical NET (46.7%), while 30.4% of the cases were typical NETs and 21.7% were carcinomas, with the median Ki-67 10%, ranging from 1% to 40%. The median tumor size among 112 patients was 4.7 cm, ranging from 1 to 20 cm, and 55.7% of patients had metastases at presentation (12). It proves the significant heterogeneity of the disease.

Our patient had typical NET with small dimensions and localized disease at the time of diagnosis. Despite this, we observed aggressive Cushing’s syndrome with a short duration of symptoms and life-threatening hypokalemia. It has been observed that there is no correlation between tumor size and hormone levels (12). Thymic NETs associated with EAS are often large, which simplifies the diagnosis and localization. However, in the case of incidental sellar mass or small thymic tumor, the differential diagnosis might be difficult. The highest sensitivity in distinguishing thymic EAS from Cushing’s disease was documented in inferior petrosal sinus sampling and corticotropin-releasing hormone (CRH) stimulation test (12, 20).

In severe cases, when small ACTH-secreting NET needs to be found urgently, PET/CT is a very helpful diagnostic tool. In a prospective study comprising 20 patients with histologically proven EAS, the 68Ga-DOTATATE PET/CT result correctly identified the tumor in 75%, with SUV max. ranging from 1.4 to 20.7, while the 18F-FDG PET/CT findings had a slightly worse result (identified 60% tumors), with SUV max. ranging from 1.8 to 10.0. Those methods are believed to be complementary in case of localization and discrimination of EAS. The 68Ga-DOTATATE PET/CT result revealed tumor in six cases with a negative 18F-FDG PET/CT result, while the 18F-FDG PET/CT procedure was diagnostic in three cases with a negative 68Ga-DOTATATE uptake; the combined sensitivity of both methods was 90% (10). The typical first-line diagnostic modalities’ (CT and MRI) sensitivities range from 52% to 66% (9). Our case remains in accordance with those results, showing difficulties in localizing the ACTH source in first-line radiological methods and with 68Ga-DOTATATE PET/CT being the most useful diagnostic tool. It should also be noted that the 68Ga-DOTATATE uptake was only mildly elevated both in primary tumor and its recurrences despite excessive hormonal activity. We did not perform 18F-FDG PET/CT until second operation, as it was believed to be rather helpful in poorly differentiated tumors and 68Ga-DOTATATE PET/CT was diagnostic. Later, we performed it in search for other metastatic tumors, but the examination showed no tumor spread.

The recommended treatment of thymic NETs regarded radically resectable is thymectomy by median sternotomy or thoracotomy and lymph node dissection (11, 21, 22). According to the last version of the ESMO Guidelines, available literature suggests no benefit from adjuvant therapy in ThCs. The majority of the authors of the Guidelines panel suggest individually discussing eventual postoperative therapies, including RT and/or systemic therapies, balancing the pros and cons only in selected patients with advanced stage R0 or R1-2 resection (22). Data on systemic therapies in thymic NETs are scarce; therefore, they should be discussed in a multidisciplinary expert team in case of morphologically progressive tumors, high tumor burden, or refractory hormonal syndromes. Somatostatin analogs are recommended as the first-line systemic therapy in typical carcinoids (22). We considered the adjuvant therapy with somatostatin analogs; however, due to the low uptake in PET examination and complete resolution of symptoms as well as the radical type of surgical removal, we did not decide to initiate such therapy. Other systemic treatment options include everolimus (second line in typical carcinoids or first line in atypical carcinoids), chemotherapy, peptide receptor radionuclide therapy (PRRT), and interferon-α (22, 23). There is also data on the benefits of combining long-acting lanreotide with temozolomide in progressive thymic NETs (24).

Due to the variable availability of steroid inhibitors during the course of the disease, our patient received three different preparations at each disease relapse. Both ketoconazole and osilodrostat were well tolerated and reduced the hypercortisolism within a few days, but metyrapone caused significant side effects (see below—”Patient’s perspective”), and it was not possible to normalize the cortisol values with this steroid inhibitor. It is worth noting that when using the most recent steroid inhibitor—osilodrostat—we initiated the therapy with a high dose without a previous dose titration. This strategy might be used in the case of severe hypercortisolism and proved effective and safe in our patient (25).

Most commonly, metastases from thymic NET producing ACTH are localized in lymph nodes, bone, lung, pleura, and, less commonly, liver and parotid gland (13). There are very few cases of EAS-related thymic NETs with breast metastases described in the literature, with some histopathological variability (one case related to atypical carcinoid, another to combined large-cell neuroendocrine carcinoma and atypical carcinoid, and third case of neuroendocrine carcinoma). All of them were female patients between 24 and 36 years of age, with mediastinal lymph nodes metastases at the time of presentation; one also had distant metastases to the bones (13–15). Contrary to the reported cases, our patient had typical carcinoid (confirmed by three independent pathologists from different centers) but similarly presented with severe hypercortisolism. It suggests that there is no connection between tumor differentiation and the severity of hypercortisolism. Interestingly, in a review of 661 patients with metastatic NETs from Sweden, there were 20 patients with NETs and breast metastases, and among them only one case of thymic NET (Ki 67 12%), but without EAS. A total of 11 patients with breast metastases had a primary tumor in the small intestine and eight in the lung (16).

Our case underlines the necessity of long-term follow-up in EAS, as the recurrences occurred 5 and 7 years after the initial successful treatment. According to guidelines, follow-up after treatment of thymic NETs should be life-long (22).

The strength of our report is the presentation of a thymic NET with metastasis to the breast, diagnosed and treated with many currently available tools and with a long period of follow-up. The limitation is the low number of other similar cases to compare, which is a consequence of the rarity of this disease.

In conclusion, our case proves that thymic NETs with EAS might present in young patients with well-differentiated character in histopathological examination and severe, life-threatening hypercortisolism despite the small size of the primary lesion. 68Ga-DOTATATE PET/CT is a very helpful tool to localize the tumor. Finally, life-long follow-up should be performed despite complete remission after surgery.

4 Patient’s perspective

The first symptoms that I observed were face edema and mood changes. I rapidly lost muscle mass (approximately 6 kg in 2 weeks), and I was not able to climb stairs, especially with my child’s pram. The most difficult to accept were changes in my appearances—hirsutism, losing hair, changes of my facial features. My sense of pain (for example, during medical procedures) was diminished. Other disruptive symptoms were intensive sweating, increased appetite, thirst, brain fog, and digestive problems. At every relapse, the disease manifestations were fluctuating, all of them intensifying at the same time, which was very difficult for me. Also stress evoked disease symptoms. I experienced a strange feeling of warm during cortisol outbursts.

As for the treatment, I did not tolerate metyrapone well. I had skin rash, anxiety attacks with heart palpitations, and a metallic taste in my mouth. Other drugs (ketoconazole, osilodrostat) were better for me.

After operations of the relapses, the symptoms diminished very quickly, especially the most difficult ones. My blood pressure and glycemia normalized within a few days. Other manifestations, like loss of hair or skin changes, persisted up to 3 months.

Data availability statement

The datasets presented in this article are not readily available because the data are potentially identifiable. Requests to access the datasets should be directed to Aleksandra Zdrojowy-Wełna, aleksandra.zdrojowy-welna@umw.edu.pl.

Ethics statement

This study was exempt from ethical approval procedures being a case report of a single patient who has voluntarily provided oral and written consent to participate in the study and to have her case published for the sake of helping us better understand the clinical picture and the course of thymic neuroendocrine tumors with EAS and share it with the medical community for awareness about it. Written informed consent was obtained from the participant/patient(s) for the publication of this case report.

Author contributions

AZ-W: Conceptualization, Data curation, Investigation, Methodology, Software, Writing – original draft. MB: Conceptualization, Supervision, Writing – review & editing. JS: Data curation, Investigation, Methodology, Writing – review & editing. AJ-P: Data curation, Investigation, Writing – review & editing. JK-P: Conceptualization, Data curation, Investigation, Methodology, Supervision, Writing – original draft.

Funding

The author(s) declare that no financial support was received for the research, authorship, and/or publication of this article.

Acknowledgments

We would like to thank Prof. Barbara Górnicka and Prof. Michał Jeleń for their collaboration throughout the patient’s treatment.

Conflict of interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

The handling editor AJ declared a past co-authorship with the author MB.

The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision.

Publisher’s note

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.

 

Supplementary material

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fonc.2025.1492187/full#supplementary-material

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Keywords: ectopic Cushing`s syndrome, thymic neuroendocrine tumor, thymic NET, ectopic ACTH secretion, case report

Citation: Zdrojowy-Wełna A, Bolanowski M, Syrycka J, Jawiarczyk-Przybyłowska A and Kuliczkowska-Płaksej J (2025) Case Report: Thymic neuroendocrine tumor with metastasis to the breast causing ectopic Cushing’s syndrome. Front. Oncol. 15:1492187. doi: 10.3389/fonc.2025.1492187

Received: 11 September 2024; Accepted: 31 January 2025;
Published: 25 February 2025.

Edited by:

Aleksandra Gilis-Januszewska, Jagiellonian University Medical College, Poland

Reviewed by:

Piero Ferolla, Umbria Regional Cancer Network, Italy
Lukasz Dzialach, Warsaw Medical University, Poland

Copyright © 2025 Zdrojowy-Wełna, Bolanowski, Syrycka, Jawiarczyk-Przybyłowska and Kuliczkowska-Płaksej. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

*Correspondence: Aleksandra Zdrojowy-Wełna, aleksandra.zdrojowy-welna@umw.edu.pl

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Ectopic Adrenocorticotrophic Hormone Syndrome in a 10-Year-Old Girl With a Thymic Neuroendocrine Tumor

Posted on November 29, 2024 by MaryO

Abstract

Background

Thymic neuroendocrine tumor as a cause of Cushing syndrome is extremely rare in children.

Case presentation

We report a case of a 10-year-old girl who presented with typical symptoms and signs of hypercortisolemia, including bone fractures, growth retardation, and kidney stones. The patient was managed with oral ketoconazole, during which she experienced adrenal insufficiency, possibly due to either cyclic adrenocorticotropic hormone (ACTH) secretion or concurrent COVID-19 infection. The patient underwent a diagnostic work-up which indicated the possibility of an ACTH-secreting pituitary neuroendocrine tumor. However, after a transsphenoidal surgery, the diagnosis was not confirmed on histopathological examination. Subsequent bilateral inferior petrosal sinus sampling showed strong indications of the presence of ectopic ACTH syndrome. Detailed rereading of functional imaging studies, including 18F-FDG PET/MRI and 68Ga DOTATOC PET/CT, ultimately identified a small lesion in the thymus. The patient underwent videothoracoscopic thymectomy that confirmed a neuroendocrine tumor with ACTH positivity on histopathological examination.

Conclusion

This case presents some unique challenges related to the diagnosis, management, and treatment of thymic neuroendocrine tumor in a child. We can conclude that ketoconazole treatment was effective in managing hypercortisolemia in our patient. Further, a combination of functional imaging studies can be a useful tool in locating the source of ectopic ACTH secretion. Lastly, in cases of discrepancy in the results of stimulation tests, bilateral inferior petrosal sinus sampling is highly recommended to differentiate between Cushing disease and ectopic ACTH syndrome.

Peer Review reports

Background

In children above seven years of age, the majority of pediatric Cushing syndrome (CS) cases are caused by a pituitary neuroendocrine tumors (PitNET). However, a differential diagnosis of hypercortisolemia in children is often challenging concerning the interpretation of stimulation tests and the fact that up to 50% of PitNET may not be detected on magnetic resonance imaging (MRI) [1]. An ectopic adrenocorticotropic hormone (ACTH) syndrome (EAS) is extremely rare in children. Its diagnosis is often missed or confused with Cushing disease (CD) [2]. Most ACTH-secreting tumors originate from bronchial or thymic neuroendocrine tumors (NETs), or less commonly, from NETs in other locations. To diagnose EAS, specific functional imaging studies are often indicated to elucidate the source of ACTH production.

Pharmacotherapy may be used before surgery to control hypercortisolemia and its symptoms/signs, or in patients in whom the source of hypercortisolism has not been found (e.g., EAS), or surgery failed. Ketoconazole or metyrapone, as adrenal steroidogenesis blockers, were found to be very efficient, although they exhibit side effects [3].

Furthermore, cyclic secretion of ACTH followed by fluctuating plasma cortisol levels is extremely rare in children, including those with EAS [4, 5]. Therefore, in cyclic EAS, the use of steroid inhibitors or acute illness or trauma can be associated with adrenal insufficiency, which can be life-threatening. Here we describe the clinical features, laboratory and radiological investigations, results, management, and clinical outcome of a 10-year-old girl with a thymic NET presenting with ACTH secretion.

Case presentation

A 10-year-old girl was acutely admitted to our university hospital for evaluation of facial edema and macroscopic hematuria in May 2021. A day before admission, she presented to the emergency room for dysuria, pollakiuria, nausea, and pain in her right lower back. Over the past year she had experienced excessive weight gain with increased appetite and growth retardation (Fig. 1). Her height over three years had shifted from the 34th to the 13th centile (Fig. 1). Her parents noticed facial changes, pubic hair development, increased irritability, and moodiness.

Fig. 1

figure 1

Body weight, body height, and body mass index development of the case patient. The black arrow indicates the first presentation, the blue arrow indicates the start of ketoconazole treatment and the yellow arrow indicates the time of thymectomy. Mid-parental height is indicated by the green line

At admission, she was found to have a moon face with a plethora, few acne spots on forehead, as well as facial puffiness. In contrast to slim extremities, an abnormal fat accumulation was observed in the abdomen. Purple striae were present on abdomen and thighs. She did not present with any bruising, proximal myopathy, or edema. On physical examination, she was prepubertal, height was 135 cm (13th centile), and weight was 37 kg (69th centile) with a BMI of 20.4 kg/m2 (90th centile). She developed persistent hypertension. Her past medical history was uneventful except for two fractures of her upper left extremity after minimal trips one and three years ago, both treated with a caste. Apart from hypothyroidism on the maternal side, there was no history of endocrine abnormalities or tumors in the family.

In the emergency room, the patient was started on sulfonamide, pain medication, and intravenous (IV) fluids. Her hypertensive crises were treated orally with angiotensin-converting enzyme inhibitor or with a combination of adrenergic antagonists and serotonin agonists administered IV. Hypokalemia had initially been treated with IV infusion and then with oral potassium supplements. A low serum phosphate concentration required IV management. The initial investigation carried out in the emergency room found hematuria with trace proteinuria. Kidney ultrasound showed a 5 mm stone in her right ureter with a 20 mm hydronephrosis. She did not pass any kidney stones, however, fine white sand urine analysis reported 100% brushite stone.

Hypercortisolemia was confirmed by repeatedly increased 24-hour urinary free cortisol (UFC), (5011.9 nmol/day, normal range 79.0-590.0 nmol/day). Her midnight cortisol levels were elevated (961 nmol/l, normal range 68.2–537 nmol/l). There was no suppression of serum cortisol after 1 mg overnight dexamethasone suppression test (DST) or after low-dose DST (LDDST). An increased morning plasma ACTH (30.9 pmol/l, normal range 1.6–13.9 pmol/) suggested ACTH-dependent hypercortisolemia. There was no evidence of a PitNET on a 1T contrast-enhanced MRI. The high-dose DST (HDDST) did not induce cortisol suppression (cortisol 1112 nmol/l at 23:00, cortisol 1338 nmol/l at 8:00). Apart from the kidney stone, a contrast-enhanced computed tomography (CT) of her neck, chest, and abdomen/pelvis did not detect any lesion. Various tumor markers were negative and the concentration of chromogranin A was also normal.

A corticotropin-releasing hormone (CRH) stimulation test induced an increase in serum cortisol by 32% at 30 min and ACTH concentration by 67% at 15 min (Table 1). A 3T contrast-enhanced MRI scan of the brain identified a 3 × 2 mm lesion in the lateral right side of the pituitary gland (Fig. 2). An investigation of other pituitary hormones was unremarkable. Apart from low serum potassium (minimal level of 2.8 mmol/l; normal range 3.3–4.7 mmol/l) and phosphate (0.94 mmol/l; normal range 1.28–1.82 mmol/l) concentrations, electrolytes were normal. The bone mineral density assessed by whole dual-energy X-ray absorptiometry was normal.

Fig. 2

figure 2

Coronal and sagittal 3T contrast-enhanced brain MRI scans. A suspected 3 × 2 mm lesion in the lateral right side of the pituitary gland (yellow arrows)

The patient was presented at the multidisciplinary tumor board and it was decided that she undergoes transsphenoidal surgery for the pituitary lesion. No PitNET was detected on histopathological examination and no favorable biochemical changes were noted after surgery. After the patient recovered from surgery, subsequent bilateral inferior petrosal sinus sampling (BIPSS) confirmed EAS as the maximum ratio of central to peripheral ACTH concentrations was only 1.7. During the investigation for tumor localization, she was started on ketoconazole treatment (300 mg/day) to alleviate symptoms and signs of hypercortisolism. Treatment with ketoconazole had a beneficial effect on patient health (Fig. 1). There was a weight loss of 2 kg in a month, a disappearance of facial plethora, and a decrease in vigorous appetite. Her liver function tests remained within the normal range.

Table 1 Result of corticotropin-releasing hormone stimulation test

The 24-hour UFC excretion normalized three weeks after ketoconazole initiation. However, six weeks after continuing ketoconazole therapy (400 mg/day), the patient complained of nausea, vomiting, and diarrhea. She was found to have adrenal insufficiency with a low morning serum cortisol of 10.70 nmol/l (normal range 68.2–537 nmol/l) and salivary cortisol concentrations < 1.5 nmol/l (normal range 1.7–29 nmol/l). She was also found to be positive for COVID-19 infection. Ketoconazole treatment was stopped and our patient was educated to take stress steroids in case of persisting or worsening symptoms. Her clinical status gradually improved and steroids were not required.

Meanwhile, whole-body fluorine-18 fluorodeoxyglucose positron emission tomography (18F-FDG PET)/MRI was performed with no obvious hypermetabolic lesion suspicious of a tumor. No obvious accumulation was detected on 68Ga-DOTATOC PET/CT images (Fig. 3). However, a subsequent careful and detailed re-review of the images detected a discrete lesion on 18F-FDG PET/MRI and 68Ga-DOTATOC PET/CT scans in the left anterior mediastinum, in the thymus (Fig. 4).

Fig. 3

figure 3

18F-FDG PET/MRI (A) and 68Ga-DOTATOC (B) PET/CT scans. Whole body MIP reconstructions. Subtle correspondent focal hyperactivity in the left mediastinum (black arrow). The 18F-FDG PET/MRI image courtesy of Prof. Jiri Ferda, MD, PhD, Clinic of the Imaging Methods, University Hospital Plzen, Czech Republic

Fig. 4

figure 4

Axial slices of PET/MRI (A–C) and 68Ga-DOTATOC (D–F) PET/CT scans. Subtle correspondent focal hyperactivity in the left mediastinum (white arrow). No obvious finding on MRI (C) and CT (F) scans. The FDG PET/MRI image courtesy of Prof. Jiri Ferda, MD, PhD, Clinic of the Imaging Methods, University Hospital Plzen, Czech Republic

Three weeks after the episode of adrenal insufficiency and being off ketoconazole treatment, our patient´s pre-surgery laboratory tests showed slightly low morning cortisol 132 nmol/l with surprisingly normal ACTH 2.96 pmol/l (normal range 1.6–13.9 pmol/). Given the upcoming surgery, she was initiated on a maintenance dose of hydrocortisone (15 mg daily = 12.5 mg/m2/day). Further improvement of cushingoid characteristics (improvement of facial plethora and moon face, weight loss) was noticed. Our patient underwent videothoracoscopic surgery, and a hyperplastic thymus of 80 × 70 × 15 mm with a 4 mm nodule was successfully removed. Tumor immunohistochemistry was positive for ACTH, chromogranin A, CD56, and synaptophysin. Histopathological findings were consistent with a well-differentiated NET grade 1. A subsequent genetic screening did not detect any pathogenic variant in the MEN1 gene.

After surgery, hydrocortisone was switched to a stress dose and gradually decreased to a maintenance dose. Antihypertensive medication was stopped and further weight loss was observed after thymectomy. Within a few weeks after the thoracic surgery, the patient entered puberty, her mood improved significantly, and potassium supplements were stopped. Finally, hydrocortisone treatment was stopped ten months after thymectomy.

Discussion and conclusions

The case presented here demonstrates a particularly challenging work-up of the pediatric patient with the diagnosis of CS caused by EAS due to thymic NET. Differentiating CD and EAS can sometimes be difficult, including the use of various laboratory and stimulation tests and their interpretation, as well as proper, often challenging, reading of functional imaging modalities, especially if a discrete lesion is present at an unusual location [1]. When using established criteria for Cushing disease (for the CRH test an increase of cortisol and/or ACTH by ≥ 20% or ≥ 35%, respectively, and a ≥ 50% suppression of cortisol for the HDDST) our patient presented discordant results. The CRH stimulation test induced an increase in cortisol by 32% and ACTH by 67% and the 3T MRI pointed to the right-side pituitary lesion, both to yield false positive results. The HDDST, on the other hand, did not induce cortisol suppression and was against characteristic findings for CD. We did not proceed with desmopressin testing, which also induces an excess ACTH and cortisol response in CD patients and has rarely been used in pediatric patients, except in those with extremely difficult venous access [6]. Recently published articles investigated the reliability of CRH stimulation tests and HDDST and both concluded that the CRH test has greater specificity than HDDST [7, 8]. Elenius et al. suggested optimal response criteria as a ≥ 40% increase of ACTH and/or cortisol (cortisol as the most specific measure of CD) during the CRH test and a ≥ 69% suppression of serum cortisol during HDDST [7]. Using these criteria, the CD would be excluded in our patient. To demonstrate that the proposed thresholds for the test interpretation widely differ, Detomas et al. proposed a ≥ 12% cortisol increase and ≥ 31% ACTH increase during the CRH test to confirm CD [8].

The fact that up to 50% of PitNET may not be detected on MRI [1] and that more than 20% of patients with EAS are reported to have pituitary incidentalomas [9] makes MRI somewhat unreliable in differentiating CD and EAS. However, finally, well-established and generally reliable BIPSS in our patient supported the diagnosis of EAS. Thus, BIPSS is considered a gold standard to differentiate between CD and EAS; however, it can still provide false negative results in cyclic CS if performed in the trough phase [10] or in vascular anomalies or false positive results as in a recent case of orbital EAS [11].

In children, the presence of thymus tissue may be misinterpreted as normal. Among other reports of thymic NET [12], Hanson et al. reported a case of a prepubertal boy in whom a small thymic NET was initially treated as normal thymus tissue on CT [13]. In our case, initially, the lesion was not detected on the 18F-FDG and 68Ga-DOTATOC PET scans. A small thymic NET was visible only after a detailed and careful re-reading of both PET scans. Although somatostatin receptor (SSR) PET imaging may be helpful in identifying ectopic CRH- or ACTH-producing tumors, there are still some limitations [13]. For example, in the study by Wannachalee et al., 68Ga-DOTATATE identified suspected primary lesions causing ECS in 65% of patients with previously occult tumors and was therefore concluded as a sensitive method for primary as well as metastatic tumors [14]. In our patient, the final correct diagnosis was based on the results of both PET scans. This is in full support of the article published by Liu et al. who concluded that 18F-FDG and SSR PET scans are complementary in determining the proper localization of ectopic ACTH production [15]. Additionally, it is worth noting that not all NETs stain positively for ACTH which may present a burden in its identification.

To control hypercortisolemia, both ketoconazole and metyrapone were considered in our patient. Due to the side effects of metyrapone on blood pressure, ketoconazole was started as a preferred option in our pediatric patient. A retrospective multicenter study concluded that ketoconazole treatment is effective with acceptable side effects, with no fatal hepatitis and adrenal insufficiency in 5.4% of patients [3]. During ketoconazole treatment, our patient developed adrenal insufficiency; however, it is impossible to conclude whether this was solely due to ketoconazole treatment or whether an ongoing COVID-19 infection contributed to the adrenal insufficiency or whether this was caused by a phase of lower or no ACTH secretion from the tumor often seen in patients with cyclic ACTH secretion. The patient’s cyclic ACTH secretion is highly probable since her morning cortisol was slightly lower and ACTH was normal, even after being off ketoconazole treatment for 3 weeks.

When retrospectively and carefully reviewing all approaches to the diagnostic and management care of our pediatric patient, it would be essential to proceed to BIPSS before any pituitary surgery, especially when obtaining discrepant results from stimulation tests, as well as detecting a discrete pituitary lesion (≤ 6 mm) as recommended by the current guidelines [16]. This was our first experience using ketoconazole in a young child, and although this treatment was associated with very good outcomes in treating hypercortisolemia, close monitoring, and family education on signs and symptoms of adrenal insufficiency are essential to recognizing adrenal insufficiency promptly in any patient with EAS, especially those presenting also with some other comorbidities or stress, here COVID-19 infection.

In conclusion, the pediatric patient here presenting with EAS caused by thymic NET needs very careful assessment including whether cyclic CS is present, the outline of a good management plan to use all tests appropriately and in the correct sequence, monitoring carefully for any signs or symptoms of adrenal insufficiency, and apply appropriate imaging studies, with experienced radiologists providing accurate readings. Furthermore, ketoconazole treatment was found to be effective in reducing the symptoms and signs of CS in this pediatric patient. Finally, due to the rarity of this disease and the challenging work-up, we suggest that a multidisciplinary team of experienced physicians in CS management is highly recommended.

Data availability

No datasets were generated or analysed during the current study.

Abbreviations

ACTH:
Adrenocorticotrophic hormone
BIPSS:
Bilateral inferior petrosal sinus sampling
CD:
Cushing disease
CRH:
Corticotropin-releasing hormone
CS:
Cushing syndrome
CT:
Computed tomography
DST:
Dexamethasone suppression test
EAS:
Ectopic adrenocorticotropic hormone syndrome
18F-FDG PET:
Fluorine-18 fluorodeoxyglucose positron emission tomography
HDDST:
High-dose dexamethasone suppression test
IV:
Intravenous
LDDST:
Low-dose dexamethasone suppression test
NET:
Neuroendocrine tumor
PitNET:
Pituitary neuroendocrine tumor
UFC:
Urinary free cortisol

References

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Acknowledgements

The authors thank all the colleagues from the Thomayer University Hospital and Military University Hospital who were involved in the inpatient care of this patient.

Funding

This work was supported by the Charles University research program Cooperatio Pediatrics, Charles University, Third Faculty of Medicine, Prague.

Author information

Authors and Affiliations

  1. Department of Children and Adolescents, Third Faculty of Medicine, Charles University, University Hospital Kralovske Vinohrady, Šrobárova 50, Prague, 100 34, Czech Republic

    Irena Aldhoon-Hainerová

  2. Department of Pediatrics, Thomayer University Hospital, Prague, Czech Republic

    Irena Aldhoon-Hainerová

  3. Department of Medicine, Military University Hospital, Prague, Czech Republic

    Mikuláš Kosák

  4. Third Department of Medicine, First Faculty of Medicine, Charles University, Prague, Czech Republic

    Michal Kršek

  5. Institute of Nuclear Medicine, First Faculty of Medicine, Charles University, General University Hospital, Prague, Czech Republic

    David Zogala

  6. Developmental Endocrinology, Metabolism, Genetics and Endocrine Oncology Affinity Group, Eunice Kennedy Shriver NICHD, NIH, Bethesda, MD, USA

    Karel Pacak

Contributions

All authors made individual contributions to the authorship. IAH, MK, MK, and DZ were involved in the diagnosis and management of this patient. DZ was responsible for the patient´s imaging studies. IAH wrote the first draft of the manuscript. KP revised the manuscript critically. All authors reviewed and approved the final draft.

Corresponding author

Correspondence to Irena Aldhoon-Hainerová.

Ethics declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

Signed informed consent was obtained from the patient and the patient´s parents for the publication of this case report and accompanying images.

Competing interests

The authors declare no competing interests.

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Insights on Diagnosing and Managing Cushing’s Syndrome

Posted on August 18, 2024 by MaryO

Cushing’s syndrome, or endogenous hypercortisolemia, is a rare condition that both general practice clinicians and endocrinologists should be prepared to diagnose and treat. Including both the pituitary and adrenal forms of the disease, the Endocrine Society estimates that the disorder affects 10 to 15 people per million every year in the United States. It is more common in women and occurs most often in people between the ages of 20 and 50.

Even though Cushing’s remains a rare disease, cortisol recently made waves at the American Diabetes Association 84th Scientific Session. A highlight of the meeting was the initial presentation of data from the CATALYST trial, which assessed the prevalence of hypercortisolism in patients with difficult-to-control type 2 diabetes (A1c 7.5+).

CATALYST is a prospective, Phase 4 study with two parts. In the prevalence phase, 24% of 1,055 enrolled patients had hypercortisolism, defined as an overnight dexamethasone suppression test (ODST) value greater than 1.8 µg/dL and dexamethasone levels greater than 140 µg/dL. Results of CATALYST’s randomized treatment phase are expected in late 2024.

Elena Christofides, MD, FACE, founder of Endocrinology Associates, Inc., in Columbus, OH, believes the CATALYST results will be a wake-up call for both physicians and patients seeking to advocate for their own health. “This means that nearly 1 in 4 patients with type 2 diabetes have some other underlying hormonal/endocrine dysfunction as the reason for their diabetes, or significant contribution to their diabetes, and they should all be screened,” she said. “All providers need to get comfortable with diagnosing and treating hypercortisolemia, and you need to do it quickly because patients are going to pay attention as well.”

In Dr. Christofides’ experience, patients who suspect they have a hormonal issue may start with their primary care provider or they may self-refer to an endocrinologist. “A lot of Cushing’s patients are getting diagnosed and treated in primary care, which is completely appropriate. But I’ve also met endocrinologists who are uncomfortable diagnosing and managing Cushing’s because it is so rare,” she said. “The important thing is that the physician is comfortable with Cushing’s or is willing to put in the work get comfortable with it.”

According to Dr. Christofides, the widespread popular belief that “adrenal fatigue” is causing millions of Americans to feel sick, tired, and debilitated may be creating barriers to care for people who may actually have Cushing’s. “As physicians, we know that adrenal fatigue doesn’t exist, but we should still be receptive to seeing patients who raise that as a concern,” said Dr. Christofides. “We need to acknowledsalige their lived experience as being very real and it can be any number of diseases causing very real symptoms. If we don’t see these patients, real cases of hypercortisolemia could be left undiagnosed and untreated.”

Dr. Christofides, who also serves as a MedCentral Editor-at-Large, said she reminds colleagues that overnight dexamethasone suppression test (ODST) should always be the first test when you suspect Cushing’s. “While technically a screening test, the ODST can almost be considered diagnostic, depending on how abnormal the result is,” she noted. “But I always recommend that you do the ODST, the ACTH, a.m. cortisol, and the DHEAS levels at the same time because it allows you to differentiate more quickly between pituitary and adrenal problems.”

Dr. Christofides does see a place for 24-hour urine collection and salivary cortisol testing at times when diagnosing and monitoring patients with Cushing’s. “The 24-hour urine is only positive in ACTH-driven Cushing’s, so an abnormal result can help you identify the source, but too many physicians erroneously believe you can’t have Cushing’s if the 24-hour urine is normal,” she explained. “Surgeons tend to want this test before they operate and it’s a good benchmark for resolution of pituitary disease.” She reserves salivary cortisol testing for cases when the patient’s ODST is negative, but she suspects Cushing’s may be either nascent or cyclical.

Surgical resection has long been considered first-line treatment in both the pituitary and adrenal forms of Cushing’s. For example, data shared from Massachusetts General Hospital showed that nearly 90% of patients with microadenomas did not relapse within a 30-year period. A recent study found an overall recurrence rate of about 25% within a 10-year period. When reoperation is necessary, remission is achieved in up to 80% of patients.

As new medications for Cushing’s syndrome have become available, Dr. Christofides said she favors medical intervention prior to surgery. “The best part about medical therapy is you can easily stop it if you’re wrong,” she noted. “I would argue that every patient with confirmed Cushing’s deserves nonsurgical medical management prior to a consideration of surgery to improve their comorbidities and surgical risk management, and give time to have a proper informed consent discussion.”

In general, medications to treat Cushing’s disease rely on either cortisol production blockade or receptor blockade, said Dr. Christofides. Medications that directly limit cortisol production include ketoconazole, osilodrostat (Isturisa), mitotane (Lysodren), levoketoconazole (Recorlev), and metyrapone (Metopirone). Mifepristone (Korlym, Mifeprex) is approved for people with Cushing’s who also have type 2 diabetes to block the effects of cortisol. Mifepristone does not lower the amount of cortisol the body makes but limits its effects. Pasireotide (Signifor) lowers the amount of ACTH from the tumor. Cabergoline is sometimes used off-label in the US for the same purpose.

Following surgery, people with Cushing’s need replacement steroids until their adrenal function resumes, when replacement steroids must be tapered. But Dr. Christofides said she believes that all physicians who prescribe steroids should have a clear understanding of when and how to taper patients off steroids.

“Steroid dosing for therapeutic purposes is cumulative in terms of body exposure and the risk of needing to taper. A single 2-week dose of steroids in a year does not require a taper,” she said. “It’s patients who are getting repeated doses of more than 10 mg of prednisone equivalent per day for 2 or more weeks multiple times per year who are at risk of adrenal failure without tapering.”

Physicians often underestimate how long a safe, comfortable taper can take, per Dr. Christofides. “It takes 6 to 9 months for the adrenals to wake up so if you’re using high-dose steroids more frequently, that will cause the patient to need more steroids more frequently,” she explained. “If you’re treating an illness that responds to steroids and you stop them without tapering, the patient’s disease will flare, and then a month from then to 6 weeks from then you’ll be giving them steroids again, engendering a dependence on steroids by doing so.”

When developing a steroid taper plan for postoperative individuals with Cushing’s (and others), Dr. Christofides suggests basing it on the fact that 5 mg of prednisone or its equivalent is the physiologic dose. “Reduce the dose by 5 mg per month until you get to the last 5 mg, and then you’re going to reduce it by 1 mg monthly until done,” she said. “If a patient has difficulty during that last phase, consider a switch to hydrocortisone because a 1 mg reduction of hydrocortisone at a time may be easier to tolerate.”

Prednisone, hydrocortisone, and the other steroids have different half-lives, so you’ll need to plan accordingly, adds Dr. Christofides. “If you do a slower taper using hydrocortisone, the patient might feel worse than with prednisone unless you prescribe it BID.” She suggests thinking of the daily prednisone equivalent of hydrocortisone as 30 mg to allow for divided dosing, rather than the straight 20 mg/day conversion often used.

What happens after a patient’s Cushing’s has been successfully treated? Cushing’s is a chronic disease, even in remission, Dr. Christofides emphasized. “Once you have achieved remission, my general follow-up is to schedule visits every 6 months to a year with scans and labs, always with the instruction if the patient feels symptomatic, they should come in sooner,” she said.

More on Cushing’s diagnosis and therapies.

https://www.medcentral.com/endocrinology/cushings-syndrome-a-clinical-update

Filed under: adrenal, adrenal crisis, Cushing's, Diagnostic Testing, pituitary, Treatments | Tagged: , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , | Leave a comment »

Hormones and High Blood Pressure: Study Reveals Endocrine Culprits and Targeted Treatments

Posted on October 20, 2023 by MaryO

In a recent study published in Hypertension Research, scientists examine the endocrine causes of hypertension (HTN) and investigate the efficacy of treatments to alleviate HTN.

 

What is HTN?

About 30% of the global population is affected by HTN. HTN is a modifiable cardiovascular (CV) risk factor that is associated with a significant number of deaths worldwide.

There are two types of HTN known as primary and secondary HTN. As compared to primary HTN, secondary HTN causes greater morbidity and mortality.

The most common endocrine causes of HTN include primary aldosteronism (PA), paragangliomas and pheochromocytomas (PGL), Cushing’s syndrome (CS), and acromegaly. Other causes include congenital adrenal hyperplasia, mineralocorticoid excess, cortisol resistance, Liddle syndrome, Gordon syndrome, and thyroid and parathyroid dysfunction.

What is PA?

PA is the most common endocrine cause of hypertension, which is associated with excessive aldosterone secretion by the adrenal gland and low renin secretion. It is difficult to estimate the true prevalence of PA due to the complexity of its diagnosis.

Typically, the plasma aldosterone-to-renin ratio (ARR) is measured to diagnose PA. The diagnosis of PA can also be confirmed using other diagnostic tools like chemiluminescent enzyme immunoassays (CLEIAs) and radio immune assay (RIA).

Continuous aldosterone secretion is associated with organ damage due to chronic activation of the mineralocorticoid (MR) receptor in many organs, including fibroblasts and cardiomyocytes. An elevated level of aldosterone causes diastolic dysfunction, endothelial dysfunction, left ventricular hypertrophy, and arterial stiffness.

Increased aldosterone secretion also leads to obstructive sleep apnea and increases the risk of osteoporosis. This is why individuals with PA are at a higher risk of cardiovascular events (CVDs), including heart failure, myocardial infarction, coronary artery disease, and atrial fibrillation.

PA is treated by focusing on normalizing potassium and optimizing HTN and aldosterone secretion. Unilateral adrenalectomy is a surgical procedure proposed to treat PA.

Young patients who are willing to stop medication are recommended surgical treatment. The most common pharmaceutical treatment for PA includes mineralocorticoid receptor antagonists such as spironolactone and eplerenone.

Pheochromocytomas and paragangliomas

PGL are tumors that develop at the thoracic-abdominal-pelvic sympathetic ganglia, which are present along the spine, as well as in the parasympathetic ganglia located at the base of the skull. The incidence rate of PGL is about 0.6 for every 100,000 individuals each year. PGL tumors synthesize excessive catecholamines (CTN), which induce HTN.

Some of the common symptoms linked to HTN associated with PGL are palpitations, sweating, and headache. PGL can be diagnosed by determining metanephrines (MN) levels, which are degraded products of CTN. Bio-imaging tools also play an important role in confirming the diagnosis of PGL.

Excessive secretion of CTN increases the risk of CVDs, including Takotsubo adrenergic heart disease, ventricular or supraventricular rhythm disorders, hypertrophic obstructive or ischaemic cardiomyopathy, myocarditis, and hemorrhagic stroke. Excessive CTN secretion also causes left ventricular systolic and diastolic dysfunction.

Typically, PGL treatment is associated with surgical procedures. Two weeks before the surgery, patients are treated with alpha-blockers. For these patients, beta-blockers are not used as the first line of treatment without prior use of alpha-adrenergic receptors.

Patients with high CTN secretion are treated with metyrosine, as this can inhibit tyrosine hydroxylase. Hydroxylase converts tyrosine into dihydroxyphenylalanine, which is related to CTN synthesis.

What is CS?

CS, which arises due to persistent exposure to glucocorticoids, is a rare disease with an incidence rate of one in five million individuals each year. The most common symptoms of CS include weight gain, purple stretch marks, muscle weakness, acne, and hirsutism. A high cortisol level causes cardiovascular complications such as HTN, hypercholesterolemia, and diabetes.

CS is diagnosed based on the presence of two or more biomarkers that can be identified through pathological tests, such as salivary nocturnal cortisol, 24-hour urinary-free cortisol, and dexamethasone suppression tests.

CS is treated through surgical procedures based on the detected lesions. Patients with severe CS are treated with steroidogenic inhibitors, such as metyrapone, ketoconazole, osilodrostat, and mitotane. Pituitary radiotherapy and bilateral adrenalectomy are performed when other treatments are not effective.

Acromegaly

Acromegaly arises due to chronic exposure to growth hormone (GH), leading to excessive insulin-like growth factor 1 (IGF1) synthesis. This condition has a relatively higher incidence rate of 3.8 million person-years. Clinical symptoms of acromegaly include thickened lips, widened nose, a rectangular face, prominent cheekbones, soft tissue overgrowth, or skeletal deformities.

Prolonged exposure to GH leads to increased water and sodium retention, insulin resistance, reduced glucose uptake, and increased systemic vascular resistance. These conditions increase the risk of HTN and diabetes in patients with acromegaly. Acromegalic patients are also at a higher risk of cancer, particularly those affecting the thyroid and colon.

Acromegaly is diagnosed using the IGF1 assay, which determines IGF1 levels in serum. After confirming the presence of high IGF1 levels, a GH suppression test must be performed to confirm the diagnosis. Bioimaging is also conducted to locate adenoma.

Acromegaly is commonly treated through surgical procedures. Patients who refuse this line of treatment are treated with somatostatin receptor ligands, growth hormone receptor antagonists, dopaminergic agonists, or radiotherapy.

Journal reference:
  • De Freminville, J., Amar, L., & Azizi, M. (2023) Endocrine causes of hypertension: Literature review and practical approach. Hypertension Research; 1-14. doi:10.1038/s41440-023-01461-1

From https://www.news-medical.net/news/20231015/Hormones-and-high-blood-pressure-Study-reveals-endocrine-culprits-and-targeted-treatments.aspx

Filed under: adrenal, Cushing's, pituitary, Rare Diseases, symptoms, Treatments | Tagged: , , , , , , , , , , , , | Leave a comment »

Development of Human Pituitary Neuroendocrine Tumor Organoids to Facilitate Effective Targeted Treatments of Cushing’s Disease

Posted on November 7, 2022 by MaryO

Abstract

(1) Background: Cushing’s disease (CD) is a serious endocrine disorder caused by an adrenocorticotropic hormone (ACTH)-secreting pituitary neuroendocrine tumor (PitNET) that stimulates the adrenal glands to overproduce cortisol. Chronic exposure to excess cortisol has detrimental effects on health, including increased stroke rates, diabetes, obesity, cognitive impairment, anxiety, depression, and death. The first-line treatment for CD is pituitary surgery. Current surgical remission rates reported in only 56% of patients depending on several criteria. The lack of specificity, poor tolerability, and low efficacy of the subsequent second-line medical therapies make CD a medical therapeutic challenge. One major limitation that hinders the development of specific medical therapies is the lack of relevant human model systems that recapitulate the cellular composition of PitNET microenvironment.
(2) Methods: human pituitary tumor tissue was harvested during transsphenoidal surgery from CD patients to generate organoids (hPITOs).
(3) Results: hPITOs generated from corticotroph, lactotroph, gonadotroph, and somatotroph tumors exhibited morphological diversity among the organoid lines between individual patients and amongst subtypes. The similarity in cell lineages between the organoid line and the patient’s tumor was validated by comparing the neuropathology report to the expression pattern of PitNET specific markers, using spectral flow cytometry and exome sequencing. A high-throughput drug screen demonstrated patient-specific drug responses of hPITOs amongst each tumor subtype. Generation of induced pluripotent stem cells (iPSCs) from a CD patient carrying germline mutation CDH23 exhibited dysregulated cell lineage commitment.
(4) Conclusions: The human pituitary neuroendocrine tumor organoids represent a novel approach in how we model complex pathologies in CD patients, which will enable effective personalized medicine for these patients.

1. Introduction

Cushing’s disease (CD) is a serious endocrine disorder caused by an adrenocorticotropic hormone (ACTH)-secreting pituitary neuroendocrine tumor (PitNET) that stimulates the adrenal glands to overproduce cortisol [1,2,3,4]. The WHO renamed pituitary adenomas as PitNETs [5]. While PitNETs have been defined as benign, implying that these tumors cause a disease that is not life threatening or harmful to health, in fact chronic exposure to excess cortisol has wide-ranging and detrimental effects on health. Hypercortisolism causes increased stroke rates, diabetes, obesity, depression, anxiety, and a three-fold increase in the risk of death from cardiovascular disease and cancer [4,6,7,8].
The first-line treatment for CD is pituitary surgery, which is followed by disease recurrence in 50% of patients during the 10-year follow-up period after surgery in the hands of an experienced surgeon [9,10,11]. Studies have demonstrated that surgical failures and recurrences of CD are common, and despite multiple treatments, biochemical control is not achieved in approximately 30% of patients. This suggests that in routine clinical practice, initial and long-term disease remission is not achieved in a substantial number of CD patients [7,12]. Hence, medical therapy is often considered in the following situations: when surgery is contraindicated or fails to achieve remission, or when recurrence occurs after apparent surgical remission. While stereotactic radiosurgery treats incompletely resected or recurrent PitNETs, the main drawbacks include the longer time to remission (12–60 months) and the risk of hypopituitarism [3,13,14]. There is an inverse relationship between disease duration and reversibility of complications associated with the disease, thus emphasizing the importance of identifying an effective medical strategy to rapidly normalize cortisol production by targeting the pituitary adenoma [4,7,12]. Unfortunately, the lack of current standard of care treatments with low efficacy and tolerability makes CD a medical therapeutic challenge.
The overall goal of medical therapy for CD is to target the signaling mechanisms to lower cortisol levels in the body [15,16]. The drugs offered for treatment of CD vary in the mechanism of action, safety, tolerability, route of administration, and drug–drug interactions [15,16]. In the era of precision medicine [17], where it is imperative to identify effective therapies early, there is an urgent need to accelerate the identification of therapies targeted to the ACTH-secreting pituitary tumor which are tailored for each individual patient. The absence of preclinical models that replicate the complexity of the PitNET microenvironment has prevented us from acquiring the knowledge to advance clinical care by implementing therapies specifically targeting the tumor, which would have a higher efficacy and tolerability for CD patients. In this instance, organoids can replicate much of the complexity of an tumor. An “organoid” is defined as a three-dimensional cell structure, grown from primary cells of dissociated pituitary tumors in Matrigel matrix, which proliferate, and differentiate in three dimensions, eventually replicating key biological properties of the tissue [18]. While pituitary cell lines predominantly represent hormonal lineages, these cultures do not reproduce the primary pituitary tissue because of the tumor transformation and non-physiological 2D culture conditions [19,20,21]. Pituitary tissue-derived organoids have been generated from mouse models [22,23]. While several human and rat pituitary spheroid/aggregate/tumoroid models have been reported, these cultures consist of poorly differentiated cells with high replicative potential which can affect drug response and produce data that poorly translate to the clinic [24,25]. In this study, we developed an organoid model derived from human PitNETs that replicated much of the cellular complexity and function of the patient’s tumor. Organoids derived from corticotroph PitNETs retained the genetic alterations of the patient’s primary tissue.

2. Materials and Methods

2.1. Generation and Culture of Human Pituitary Neuroendocrine Tumor (PitNET) Organoids

Patients with planned transsphenoidal surgery for pituitary tumors were identified in the outpatient neurosurgery clinics. Tissues were collected under the St. Joseph’s Hospital and Barrow Neurological Institute Biobank collection protocol PHXA-05TS038 and collection of outcomes data protocol PHXA-0004-72-29, with the approval of the Institutional Review Board (IRB) and patient consent. Samples were de-identified and shipped to the Zavros laboratory (University of Arizona) for processing.
Pituitary tumor tissue was collected in Serum-Free Defined Medium (SFDM) supplemented with ROCK inhibitor (Y27632, 10 µM), L-glutamine (2 mM), A83-01 (activin receptor-like kinase (Alk) 4/5/7 inhibitor, 0.5 mM), penicillin/streptavidin (1%), kanamycin (1%), amphotericin/gentamycin (0.2%), CHIR-98014 (4 mM), and thiazovivin (TZV, 2.5 mM). Tissues that contained red blood cells were incubated with Red Blood Cell (RBC) Lysis Buffer according to the manufacturer’s protocol (Thermo Fisher Scientific, San Fransisco, CA, USA). Tissues were dissected into small pieces, transferred to digestion buffer (DMEM/F12 supplemented with 0.4% collagenase 2, 0.1% hyaluronic acid, 0.03% trypsin-EDTA) and incubated for 5–10 min at 37 °C with gentle shaking. Tissue was further incubated with Accutase™ (Thermo Fisher Scientific) for 5 min at 37 °C. Enzymatically dissociated cells were pelleted and washed in DPBS supplemented with antibiotics at a 400 relative centrifugal force (RCF) for 5 min. Dissociated adenoma cells were resuspended in Matrigel™, and Matrigel™ domes containing the cells were then plated in culture dishes and overlaid with pituitary growth media (Supplemental Table S1). The culture was maintained at 37 °C at a relative humidity of 95% and 5% CO2. Organoid growth medium was replenished every 3–4 days and passaged after 15 days in culture.

2.2. Generation of Induced Pluripotent Stem Cells (iPSCs)

Induced pluripotent stem cell lines (iPSC lines) were generated from control individuals (no reported disease) or CD patients according to published protocols by the University of Arizona iPSC Core [26]. All human iPSC lines were tested and found to be negative for mycoplasma contamination using the Mycoalert Mycoplasma testing kits (LT07-318, Lonza), and no karyotype abnormalities were found (KaryoStat+, Thermo).

2.3. Pituitary Organoids Generated from iPSCs

Six well culture plates were coated with 2 mL/well 0.67% Matrigel (diluted in E8 media, UA iPSC core, 151169-01) and incubated at 37 °C at a relative humidity of 95% and 5% CO2 overnight. The iPSC lines were reprogrammed from the blood of either a healthy donor (JCAZ001) or a CD patient (iPSC7 and iPSC1063) at the University of Arizona iPSC Core. Passage 12 iPSCs were plated onto the coated plates and incubated at 37 °C at a relative humidity of 95% and 5% CO2. At 70% confluency, cells were passaged to freshly coated 24 well plates at a ratio of 1:8 and grown to 85–90% confluency before beginning the directed differentiation schedule. From days 0 to 3, cells were cultured in E6 media supplemented with 1% penicillin/streptomycin, 10 μM SB431542, and 5 ng/mL BMP4. BMP4 was withdrawn from the culture at day 3. Starting on day 4, the cells were cultured in E6 media, supplemented with 10 μM SB431542, 30 ng/mL human recombinant SHH, 100 ng/mL FGF8b, 10 ng/mL FGF18, and 50 ng/mL FGF10. Fifteen days after culture, the cells were harvested in cold E6 media by pipetting and resuspended in Matrigel™ (20,000 cells/50 mL Matrigel™). Matrigel™ domes containing the cells were plated in culture dishes and overlaid with differentiation media containing E6 media which was supplemented with 10 μM Y-27632, 30 ng/mL human recombinant SHH, 100 ng/mL FGF8b, 10 ng/mL FGF18, and 50 ng/mL FGF10 (Supplemental Table S2). Organoids were cultured for a further 15 days at 37 °C at a relative humidity of 95% and 5% CO2.

2.4. Spectral Flow Cytometry (Cytek™ Aurora)

The multicolor flow cytometry panel was designed using the Cytek® Full Spectrum Viewer online tool to calculate the similarity index (Supplemental Figure S1). The organoids were harvested in cold SFDM media and centrifuged at 400× g for 5 min. Supernatant was discarded and organoids were dissociated to single cells using Accutase® (Thermo Fisher Scientific 00-4555-56). The enzymatic reaction was stopped using prewarmed DPBS, and cells were then centrifuged at 400× g for 5 min and incubated with fluorochrome-conjugated/unconjugated primary surface or cytoplasmic antibodies (Supplemental Figure S1) at 4 °C for 30 min. Cells were then washed with Cell Staining Buffer (BioLegend # 420-201) and incubated with secondary antibodies (Supplemental Figure S1) at 4 °C for 30 min. Cells were fixed using Cytofix/Cytoperm™ Fixation/Permeabilization Solution (BD Biosciences # 554714) at 4 °C for 20 min, followed by washing with Fixation/Permeabilization wash buffer. Cells were labeled with fluorochrome-conjugated/unconjugated intracellular primary antibodies (Supplemental Figure S1) at 4 °C for 30 min, then washed and incubated with secondary antibodies at 4 °C for 30 min. Cells were resuspended in cell staining buffer and fluorescence and measured using the Cytek Aurora 5 Laser Spectral Flow Cytometer. An unstained cell sample was fixed and used as a reference control. UltraComp eBeads™, Compensation Beads (Thermo Fisher Scientific # 01-2222-42) were stained with the individual antibodies and used as single stain controls for compensation and gating. Data were acquired using the Cytek™ Aurora and analyzed using Cytobank software (Beckman Coulter, Indianapolis, IN, USA).

2.5. Whole Mount Immunofluorescence

Organoids were immunostained using published protocols by our laboratory [27,28,29]. Proliferation was measured by using 5-ethynyl-2′-deoxyuridine (EdU) incorporation according to the Manufacturer’s protocol (Click-IT EdU Alexa Fluor 555 Imaging Kit, Thermo Fisher Scientific C10338). Co-staining was performed by blocking fixed organoids with 2% donkey serum (Jackson Immuno Research, # 017-000-121) diluted in 0.01% PBST for 1hr at room temperature. Organoids were then incubated overnight at 4 °C with primary antibodies, followed by secondary antibodies and Hoechst (Thermo Fisher Scientific H1399, 1:1000 in 0.01% PBST) for 1 h at room temperature. Human specific primary antibodies used included: rabbit anti-ACTH (Thermo Fisher Scientific 701293, 1:250), rabbit anti-Synaptophysin (Thermo Fisher Scientific PA5-27286, 1:100), species PIT1 (Thermo Fisher Scientific PA5-98650, 1:50), rabbit anti-LH (Thermo Fisher Scientific PA5-102674, 1:100), mouse anti-FSH (Thermo Fisher Scientific MIF2709, 1:100), mouse anti-PRL (Thermo Fisher Scientific CF500720, 1:100), Alexa Flour conjugated GH (NB500-364AF647, 1:100), and mouse anti-CAM5.2 (SIGMA 452M-95, 1:250). The secondary antibodies used included Alexa Fluor 488 Donkey Anti Rabbit IgG (H+L) (Thermo Fisher Scientific A21206, 1:100) or Alexa Fluor 647 Donkey Anti Mouse IgG (H+L) (Thermo Fisher Scientific A31571, 1:100). Organoids were visualized and images were acquired by confocal microscopy using the Nikon CrestV2 Spinning Disk (Nikon, Melville, NY, USA). Fluorescence intensity and percentage of EdU positive cells of total cells, were calculated using Nikon Elements Software (Version 5.21.05, Nikon, Melville, NY, USA).

2.6. Nuclear Morphometric Analysis (NMA)

Nuclear Morphometric Analysis (NMA) using treated organoids was performed based on a published protocol that measures cell viability based on the changes in nuclear morphology of the cells, using nuclear stain Hoechst or DAPI [30]. Images of organoid nuclei were analyzed using the ImageJ Nuclear Irregularity Index (NII) plugin for key parameters, which included cell area, radius ratio, area box, aspect, and roundness. Using the published spreadsheet template [30], the NII of each cell was calculated with the following formula: NII = Aspect − Area Box + Radius Ratio + Roundness. The area vs. NII of vehicle-treated cells were plotted as a scatter plot using the template, and was considered as the normal cell nuclei. The same plots were generated for each condition, and the NII and area of treated cells were compared to the normal nuclei, and classified as one of the following NMA populations: Normal (N; similar area and NII), Mitotic (S; similar area, slightly higher NII), Irregular (I; similar area, high NII), Small Regular (SR; apoptotic, low area and NII), Senescent (LR; high area, low NII), Small Irregular (SI; low area, high NII), or Large Irregular (LI; high area, high NII). Cells classified as SR exhibited early stages of apoptosis, and cells classified as either I, SI, or LI exhibited significant nuclear damage. The percentage of cells in each NII classification category were calculated and plotted as a histogram using GraphPad Prism.

2.7. ELISA

Concentration of secreted ACTH in conditioned media that was collected from organoid cultures was measured using the Human ACTH ELISA Kit (Novus Biologicals, NBP2-66401), according to the manufacturer’s protocol. The enzyme–substrate reaction was measured spectrophotometrically (BioTek Gen5 Micro Plate Reader Version 3.11, Santa Clara, CA, USA) at a wavelength of 450 nm, and the ACTH concentration (pg/mL) was interpolated by a standard curve with a 4-parameter logistic regression analysis, using GraphPad Prism (Version 9.2.0, San Diego, CA, USA).

2.8. Drug Assay

Patient adenoma-derived pituitary organoids were grown in 96-well plates and treated with 147 small molecules taken from the NCI AOD9 compound library for 72 h. (https://dtp.cancer.gov/organization/dscb/obtaining/available_plates.html (accessed on 22 August 2021)). Drugs were diluted from 10 mM DMSO stock plates into 100 M DMSO working stocks with a final concentration of 1μM. All vehicle controls were treated with 0.1% DMSO. Organoid proliferation was measured using a CellTiter 96® AQueous One Solution Cell Proliferation Assay kit (MTS, Promega, G3582, Madison, WI, USA) according to the manufacturer’s instruction. Organoid death was calculated based on the absorbance readings at 490 nm, collected from the MTS assay relative to the vehicle controls. Drug screens were performed with biological replicates in the same screen. Drugs were selected based on their ability to target key signaling pathways as well as clinical relevance to the treatment. Drug sensitivity is represented by cell viability, and is significant at <0.5 suppressive effect of the drugs. The percent of cell viability relative to the vehicle control was calculated. Correlation coefficients across each organoid were calculated using the Pearson method to assess confidence in replication. The variance component was detected for each drug across all organoids. A random effect model was run with a single random factor for each drug, and estimated variance was calculated by rejecting the null hypothesis that variation was not present among samples. The drug responses were grouped by variance factor, into large (vc > 100), median (100 > vc > 50), and small (vc < 50). A heatmap was used to display the differential responses in cell viability for the drugs.
Drugs that clustered together and showed response within corticotrophs were investigated further based on their mode of action. Pathways (Kegg and Reactome) and gene ontology mapping were conducted for the genes that were being targeted by the drugs, in order to evaluate the key responses in cellular processes. A network was constructed in Cytoscape v 3.8.2 (San Diego, CA, USA) for the purpose of association between the drugs and genes.

2.9. Drug Dose Responses

Organoids were grown in Matrigel™ domes within 96-well round-bottom culture plates. Recombinant human SHH was removed from the pituitary organoid growth media, 24 h prior to drug treatment. Organoids were treated with either vehicle (DMSO), cabergoline (Selleckchem S5842), ketoconazole (Selleckchem S1353), roscovitine (Selleckchem S1153), GANT61 (Stemcell Technologies 73692), pasireotide (TargetMol TP2207), mifeprostone (Selleckchem S2606), etomidate (Selleckchem S1329), mitotane (Selleckchem S1732), metyropane (Selleckchem S5416), or osilodrostat (Selleckchem S7456) at concentrations of 0, 1, 10, 100, 1000, and 10,000 nM, for 72 h. The percentage of cell viability was measured using an MTS assay (Promega G3580). Absorbance was measured at 490 nm and normalized to the vehicle. Concentrations were plotted in a logarithmic scale, and a nonlinear dose response curve regression was calculated using GraphPad Prism. An IC50 value for each drug treatment was determined based on the dose response curve, using GraphPad Prism analysis software.

2.10. Calculation of Area under the Curve (AUC)

AUC (area under the curve) was determined by plotting the normalized % cell viability versus transformed concentration of the drugs, using a trapezoidal approximation for the area [31]. The formula was based on splitting the curve into trapezoids with bases equal to the % viability (V) and height equal to the interval length (difference in concentrations (C), and then summing the areas of each trapezoid:

∑n0(Vn+Vn−1)2∗(Cn−Cn−1)

2.11. Quantitative RT PCR (qRT-PCR)

RNA was collected from patient-derived organoid cultures using the RNeasy Mini Kit (Qiagen). cDNA was generated from the extracted RNA, and then pre-amplified using TaqMan PreAmp Master Mix (Thermo Fisher Scientific 391128). The primers used were human-specific GAPDH (Thermo Fisher Scientific, Applied Biosystems Hs02786624_g1), NR5A1 (SF1) (Thermo Fisher Scientific, Hs00610436_m1), PIT1 (Thermo Fisher Scientific, Hs00230821_m1), TPit (Thermo Fisher Scientific, Hs00193027), and POMC (Thermo Fisher Scientific, Hs01596743_m1). Each PCR reaction was performed using a final volume of 20 µL, composed of 20X TaqMan Expression Assay primers, 2X TaqMan Universal Master Mix (Applied Biosystems, TaqMan® Gene Expression Systems), and a cDNA template. Amplification of each PCR reaction was conducted in a StepOne™ Real-Time PCR System (Applied Biosystems, Foster City, CA, USA), using the following PCR conditions: 2 min at 50 °C, 10 min at 95 °C, denaturing for 15 s at 95 °C, and annealing/extending for 1 min at 60 °C, for a total of 40 cycles. Relative fold change was calculated using the 2 − ∆∆Ct method [32], where CT = threshold cycle. Results were analyzed as the average fold change in gene expression compared to the control, and GAPDH served as an internal control.

2.12. Whole Exome Sequencing

WES was performed by the University of Arizona Center for Applied Genetics and Genomic Medicine. Isolated DNA from patient adenoma tissue will be quantified using the Qubit quantitation system with standard curve, as per the supplier protocol (Thermo Fisher Scientific). All samples were further tested for quality using the Fragment Analyzer (Advanced Analytical), following the manufacturer-recommended protocols. Whole exome sequencing (WES) was performed by array capture and approximately 60 Mb of exome target sequence, using the SureSelectXT Human All Exon V6 enrichment (Agilent) or equivalent (which one was used). All exome library builds were quantified via qPCR and subsequently sequenced to a minimum 20X coverage, using paired-end chemistry on the Illumina NovaSeq platform. Whole exome sequencing (WES) was performed by hybridization capture of approx. 35 Mb of the exome target sequence, using the Swift Exome Hyb Panel (Swift Biosciences 83216). All exome library builds were quantified via qPCR and subsequently sequenced to a minimum 20X coverage, using paired-end chemistry on the Illumina NextSeq500 or NovaSeq platform (Illumina). DNA reads were trimmed, filtered by quality scores and aligned to the human genome (hg38) with Burrows–Wheeler Aligner with default parameters. Picard (http://broadinstitute.github.io/picard (accessed on 22 December 2021)) was used to mark duplicates. Germline single nucleotide variants (SNV) were called using the Genome Analysis Tool Kit (GATK), using the given guidelines. Mutations were annotated using ANNOVAR for coding sequences. Variants that passed the quality filter were further investigated for similarity. Concordance between tissue and organoids was calculated using Jaccard similarity index (Jij = Mij/(Mi + Mj − Mij) where Mi is the number of variants in tissues, Mj is the number of variants in organoids, and Mij is the number of identical variants in both tissue and organoid.

2.13. Single Cell RNA Sequencing (scRNA-Seq)

Cultures were collected on day 15 of the pituitary directed differentiation schedule, and cells were dissociated into a single-cell suspension using Cell Dissociation Buffer (Thermo Fisher Scientific 13151014). Cells (15,000 cells/sample) were resuspended in the sample buffer (BD Biosciences 65000062), filtered using cell strainer (40 microns), and loaded into a BD Rhapsody cartridge (BD Biosciences 400000847) for single-cell transcriptome isolation. Based on the BD Rhapsody system whole-transcriptome analysis for single-cell whole-transcriptome analysis, microbead-captured single-cell transcriptomes were used to prepare a cDNA library. Briefly, double-stranded cDNA was first generated from the microbead-captured single-cell transcriptome in several steps, including reverse transcription, second-strand synthesis, end preparation, adapter ligation, and whole-transcriptome amplification (WTA). Then, the final cDNA library was generated from double-stranded full-length cDNA by random priming amplification using a BD Rhapsody cDNA Kit (BD Biosciences, 633773), as well as the BD Rhapsody Targeted mRNA and WTA Amplification Kit (BD Biosciences, 633801). The library was sequenced in PE150 mode (paired-end with 150-bp reads) on NovaSeq6000 System (Illumina). A total of 80,000 reads were demultiplexed, trimmed, mapped to the GRCh38 annotation, and quantified using the whole transcriptome analysis pipeline (BD Rhapsody™ WTA Analysis Pipeline v1.10 rev6, San Jose, CA, USA) on the Seven Bridges Genomics platform (https://igor.sbgenomics.com (accessed on 4 April 2022)), prior to clustering analysis in Seurat. For QC and filtration, read counting and unique molecular identifier (UMI) counting were the principal gene expression quantification schemes used in this single-cell RNA-sequencing (scRNA-seq) analysis. The low-quality cells, empty droplets, cell doublets, or multiplets were excluded based on unique feature count (less than 200 or larger than 2500), as they may often exhibit either an aberrantly high gene count or very few genes. Additionally, the mitochondrial QC metrics were calculated, and the cells with >5% mitochondrial counts were filtered out, as the percentage of counts originating from a set of low-quality or dying cells often exhibit extensive mitochondrial contamination. After the removal of unwanted cells from the single cell dataset, the global-scaling normalization method LogNormalize was employed. This method normalizes the feature expression measurements for each cell by the total expression, multiplies this by a scale factor (10,000), and log-transforms the result. The molecules per gene per cell, based on RSEC error correction (RSEC_MolsPerCell file) matrix files from iPSCctrl and iPSCCDH23 samples, were imported into Seurat v4, merged, and processed (as stated above) for UMAP reduction, cluster identification, and differential marker assessment using the FindAllMarkers function within Seurat.

2.14. Statistical Analyses

Sample size was based on assessment of power analysis using SigmaStat software. Data collected from each study from at least 4 in vitro technical replicates were analyzed by obtaining the mean ± standard error of the mean (SEM), unless otherwise stated. The significance of the results was then tested using commercially available software (GraphPad Prism, GraphPad software, San Diego, CA, USA).

3. Results

3.1. Generation and Validation of Human PitNET Tissue Derived Organoids

Human PitNET tissue was harvested during endoscopic transsphenoidal pituitary surgery from 35 patients in order to generate organoids. These cultures are referred to as human PitNET tissue derived organoids (hPITOs). Supplementary Table S3 summarizes the neuropathology reports and clinical diagnosis from these cases. In summary, 12 corticotroph (functional, CD), and 3 silent corticotroph tumors (nonfunctional tumors), 9 gonadotroph tumors, 8 lactotroph tumors, and 3 somatotroph tumors (acromegaly) were used to generate hPITOs (Supplementary Table S3).
Bright-field microscopy images of hPITOs that were generated from corticotroph adenomas from patients diagnosed with CD (Figure 1a–e). Silent/nonfunctioning tumors (Figure 1f,g) revealed morphological diversity among the organoid lines between individual patients and amongst subtypes. Confocal microscopy was used to capture a z-stack through the hPITO38, immunofluorescently stained for CAM5.2 (red), ACTH (green), and Hoechst (nuclear staining, blue) and emphasizes the 3D cellular structure of the hPITOs (Supplemental Video S1). Lactotroph, gonadotroph, and somatotroph adenomas were used to generate hPITOs, and showed the same morphological divergence amongst subtypes and between each patient line (Supplemental Figure S2). Proliferation was measured within the cultures using 5-ethynyl-2′-deoxyuridine (EdU) uptake and showed that the percentage of EdU+ve cells/total Hoechst+ve nuclei directly correlated with the pathology MIB-1 (Ki67) score (red, R2 = 0.9256) (Figure 1a–g, Supplemental Figure S2). ACTH concentration, which was measured by ELISA using organoid conditioned culture media collected from each hPITO line, showed the highest expression in the corticotroph adenoma organoids generated from CD patients (Figure 1h).
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Figure 1. Morphology and function of corticotroph hPITOs. (a–g) Brightfield images, immunofluorescence staining using antibodies specific for CAM5.2 (red), ACTH (green), and EdU (magenta, inset) of organoid cultures generated from patients with Cushing’s disease (hPITOs 1, 7, 10, 33, 35) or nonfunctional corticotroph adenomas (hPITO8, 12). Quantification of %EdU positive cells/total cell number is shown and compared to the Ki67 score given in the pathology report (Supplemental Table S3). An ELISA was performed using conditioned media collected from (h) corticotroph hPITO cultures and (i) lactotroph, somatotroph, and gonadotroph hPITO cultures for the measurement of ACTH secretion (pg/mL).

3.2. Characterization of Cell Lineages in Pituitary Adenoma-Derived Organoids by Spectral Cytek™ Aurora Analysis

In order to validate the similarity in cell lineages identified between the organoid line and the patient’s tumor, we compared the immunohistochemistry from the neuropathology report (Supplemental Table S3) to the expression pattern of pituitary adenoma-specific markers, which were measured using Cytek™ Aurora spectral flow cytometry (Figure 2). The location of cells that are found in each cluster based on the highly expressed antigens are shown in the representative tSNE (viSNE) maps (Figure 2a). Compared to nonfunctional adenoma-derived hPITOs, organoids derived from corticotroph adenomas of CD patients highly expressed proliferating (Ki67+) T-Pit+ ACTH cells (Figure 2a). Interestingly, there was an increase in SOX2+ cells within the total cell population, associated with Crooke’s cell adenoma hPITOs (Figure 2a). Within the total cell population, cell clusters expressing CD45 and vimentin were also measured (Figure 2a). Data for the analysis of corticotroph hPITOs, derived from CD patients and individuals with nonfunctional adenomas, were summarized in a heatmap for each subtype organoid line based on quantified cell abundance (percent of total cells) using spectral flow cytometry (Figure 2b).
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Figure 2. Cell heterogeneity of corticotroph hPITOs. (a) viSNE maps define spatially distinct cell populations using pituitary specific cell lineage, stem cell, and transcription factor markers. Cell populations were quantified in organoids generated from CD patients with corticotroph adenomas (sparsely granulated and Crooke’s cell adenoma) or patients with nonfunctional corticotroph adenomas. (b) Quantification of the abundance of cells expressing pituitary specific markers as a percent total. viSNE maps define spatially distinct cell populations in organoid cultures generated from CD patient with (c) corticotroph adenoma (hPITO37, Crooke’s cell adenoma) and adjacent normal tissue (hPITO37N), or (d) sparsely granulated corticotroph adenomas (hPITO38) and adjacent normal tissue (hPITO38N).
Organoid cultures derived from pituitary adenomas (hPITO37 and hPITO38) were compared to organoids derived from adjacent normal pituitary tissue (hPITO37N and hPITO38N) (Figure 2c,d). While Pit1 lineages including cells expressing GH and PRL, as well as SF1 lineages expressing FSH and LH, were detected in the hPITO37N and hPITO38N organoid cultures, these cell populations were significantly reduced within the patient’s matched adenoma tissue (Figure 2c,d). Overall, hPITOs derived from CD patients expressed increased stem and progenitor cell markers, including CXCR4, SOX2, and CD133 (Figure 2). Collectively, our findings of the characterization of the hPITO cultures support our prediction that this in vitro model recapitulates much of the patient’s adenoma pathophysiology.

3.3. Inherent Patient Differences to Drug Response Is Reflected in the Organoid Culture

Tumor recurrence can occur in as many as 30–50% of CD patients after successful surgical treatment [10,33,34]. Unfortunately, bilateral adrenalectomy is the chosen surgical treatment for patients with persistent CD [35]. Bilateral adrenalectomy leads to the increased risk for development of Nelson’s syndrome (progressive hyperpigmentation due to ACTH secretion and expansion of the residual pituitary tumor). Although the risk of developing Nelson’s syndrome following adrenalectomy can be reduced by 50% with stereotactic radiotherapy [35], there is a need to develop medical therapies that directly target the pituitary adenoma. Thus, we established a high-throughput drug screening assay using patient-derived PitNET organoids. After 72 h of treatment, cell viability was measured using an MTS assay, and data were represented as a heatmap whereby blue indicated higher cell death, and red suggested higher cell viability. The replicates behaved consistently with the drug response, with correlation scores of >0.8 for these samples (Figure 3a). We estimated the variance component for each drug across all organoids. Variation among samples was found to be significant (p ≤ 0.05) for each of the 83 drugs. The drug responses were grouped by variance factor into large, median, and small. The larger the variance, the more variable the drug response was across the organoids. We noted a set of drugs that showed a significant differential response across the functional corticotroph organoids. Unsupervised clustering of drug responses across organoids shows a pattern that relates to our statistically calculated results (Figure 3a,c), and the replicates for each independent organoid cluster together. The drugs with higher variance components across all the functional corticotrophs cluster together as a group (Figure 3a). These drugs show cell viability of 10% to 60% across different organoids. Analyzing the pattern more closely, we observe that, within a pathologically defined group, there was a differential organoid response to drugs as well as inherent patient differences to drugs within this group. Figure 3 demonstrates a variation in drug responsiveness amongst the organoid lines generated from individual patients. Importantly, there was further divergence in drug responsiveness amongst the individual organoid lines within each pathologically defined corticotroph subtype. These data clearly demonstrate that the inherent patient difference to drug response which is often observed among CD patients is reflected in the organoid culture.
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Figure 3. Drug screen using hPITOs generated from CD patients. (a) High-throughput drug screening of hPITOs reveals sensitivities to a range of therapeutic agents. Cell viability with high values (indicating resistance) are depicted in red, and low values (indicating sensitivity) are in blue in the clustered heatmap. (b,c) Clusters showing response to therapeutic agents with the most variance across the organoids. (d) Network of drugs from the clusters b and c and their gene targets, showing their participation in signaling pathways and cellular processes.
Drugs that clustered together and showed correlated responses were investigated further for their mode of action based on target genes (Figure 3d). The genes were analyzed for their associations in cellular pathways and gene ontology functional processes. Identified drug–gene pairs were interconnected by cellular pathways that are known to regulate cell cycle, WNT signaling, hedgehog signaling, and neuroactive ligand-receptor interaction signaling pathways (Figure 3d). These identified genes are also known to be influenced by multiple cellular functions, such as cytokine–cytokine receptor interactions and Notch signaling. Proteosome 20S subunit genes PSMAs/PSMBs and the HDAC gene family are involved in many cellular functions. The ephrin receptors (EPHs), adrenoceptor alpha receptors (ADRs), dopamine receptors (DRDs), and the 5-hydroxytryptamine serotonin receptors (HTRs) gene families influence neuronal functions and are targeted by multiple drugs in our focused cluster. These data reveal potential therapeutic pathways for CD patients.
Divergent half maximal inhibitory concentration (IC50) values, as documented by an MTS cell viability assay, were observed in response to drug treatment among hPITOs lines 28, 33, 34, 35, and 37. Note that a shift of the curve to the right indicates a higher IC50 (i.e., more resistant to that drug). Cell viability assays were normalized to vehicle-treated controls in order to ensure that toxicity was specific to the drug effects (Figure 4). Dose response curves for organoid 33 and organoid 34 showed better responses at lower doses for cabergoline compared to Metyrapone and osilodrostat, but different for organoid 35, where Metyrapone and osilodrostat gave better responses than Cabergoline (Figure 4a–h). For the drugs mifepristone and GANT61, 33 and 34 had the same level of response to both the drugs. However, when the two organoid responses were compared, 34 had a better response than 33 (Figure 4a–h). Similar divergent drug responses were observed in hPITO lines 37 and 38 (Figure 4i,k). However, organoids generated from adjacent normal pituitary tissue from patients 37 and 38 were nonresponsive to the same standard of care of investigational drugs for CD (Figure 4j,l). These data were consistent with observation made in the drug screen (Figure 3a–c), and demonstrate that there was an inherent difference to drug response within the organoid cultures of the same corticotroph subtype.
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Figure 4. Drug dose responses by hPITOs generated from CD patients. Dose responses to mifepristone, GANT61, cabergoline, and osilodrostat. (a,e) hPITO28, (b,f) hPITO33, (c,g) hPITO34, and (d,h) hPITO35. Dose responses to cabergoline, ketoconazole, roscovitine, GANT61, pasireotide, mifepristone, etomidate, mitotane, metyrapone, and osilodrostat in (i) hPITO37, (j) organoids generated from adjacent normal pituitary tissue (hPITO37N), (k) hPITO38, (l) hPITO38N, and (m) hPITO39. (n) IC50 and integrated area under the curve in response to mifepristone, ketoconazole, and pasireotide using hPITO39 cultures. Nuclear morphometric analysis of hPITO39 cultures in response to (o,p) vehicle, (q,r) mifepristone, (s,t) pasireotide, and (u,v) ketoconazole. Morphometric classification of NII was based on the normal (N), small (S), small regular (SR), short irregular (SI), large regular (LR), large irregular (LI), and irregular (I) nuclear morphology. Representative Hoechst staining of organoids in response to drug treatments for the calculation of the nuclear irregularity index (NII) are shown in the insets in (p,r,t,v).
In addition to cell viability, Nuclear Morphometric Analysis (NMA) using treated organoids was performed based on a published protocol that measures cell viability according to the changes in nuclear morphology of the cells, using nuclear stain Hoechst or DAPI [30]. Nuclear Irregularity Index (NII) was measured based on the quantification of the morphometric changes in the nuclei in response to the standard-of-care drugs mifepristone, pasireotide, and ketoconazole in hPITO39 (Figure 4o–v). The area vs. NII of vehicle-treated cells were plotted as a scatter plot using the template, and considered as the normal cell nuclei (Figure 4o). The same plots were generated for mifepristone (Figure 4q), pasireotide (Figure 4s), and ketoconazole (Figure 4u). The NII and area of treated cells were compared to those of the normal nuclei, and classified as one of the following NMA populations: Normal (N; similar area and NII), Mitotic (S; similar area, slightly higher NII), Irregular (I; similar area, high NII), Small Regular (SR; apoptotic, low area and NII), Senescent (LR; high area, low NII), Small Irregular (SI; low area, high NII), or Large Irregular (LI; high area, high NII) (Figure 4p,r,t,v). Cells classified as SR exhibited early stages of apoptosis, and cells classified as either I, SI, or LI exhibited significant nuclear damage. Data showed that mifepristone induced significant apoptosis in hPITO39 cultures (Figure 4r), compared to responses to pasireotide (Figure 4t) and ketoconazole (Figure 4v). These responses were consistent with the IC50 and the total area under the curve in response to drugs (Figure 4m,n). Measurement of NII is an approach which may be used to confirm potential drug targets identified from the drug screen.

3.4. Organoid Responsiveness to Pasireotide Correlates with SSTR2 and SSTR5 Expression

Organoid lines hPITO28, 31, 33, 34, and 35 exhibited divergent IC50 values in response to SSTR agonist pasireotide (Figure 5a). hPITO34 was the most responsive to pasireotide, with a low IC50 value of 6.1 nM (Figure 5a). Organoid lines hPITO33 and hPITO35 were the least responsive, with IC50 values of 1.2 µM and 1 µM, respectively, in response to pasireotide (Figure 5a). The expression of SSTR subtypes 1–5 among the different organoid lines were measured by qRT-PCR and IHC (Figure 5b). One of the least responsive organoid lines, hPITO28, exhibited lower differential expression in SSTR2 and SSTR5 compared to the highly responsive hPITO34 line (Figure 5a,b). Gene expression levels of SSTR2 and SSTR5 within hPITO28 and 34 correlated with protein levels within the patient’s tumor tissue (Figure 5c–f). Given the greater binding affinity for SSTR5 compared to SSTR2 by pasireotide, these data were consistent with greater responsiveness to the drug by hPITO34 in comparison to hPITO28 (Figure 5a,c–f). The expression of SSTR subtypes 2 and 5 within the organoid cultures correlated with the expression patterns of the patient’s tumor tissues (Figure 5a,c–f).
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Figure 5. SSTR1-5 expression in hPITOs and patient’s PitNET tissue. (a) Dose response of hPITO28, 31, 33, 34, and 35 lines to pasireotide. (b) Differential expression of SSTR subtypes 1–5 (SSTR1, SSTR2, SSTR3, SSTR4, SSTR5) in hPITO28, hPITO31, hPITO33, hPITO34, and hPITO35. Immunohistochemistry of (c,e) SSTR2 and (d,f) SSTR5 expression in patient PitNET tissue (Pt28 and Pt34), from which hPITO28 and 34 were generated.

3.5. Organoids Derived from Pituitary Corticotroph Adenomas Retain the Genetic Alterations of the Patient’s Primary Tumor

In order to identify the genetic features of the organoids derived from pituitary adenomas of CD patients, we performed whole-exome sequencing (WES) of hPITOs and the corresponding primary adenoma tissues. We performed WES analysis of each hPITO line, and compared the results with those for the corresponding primary adenoma tissues. We showed the concordance rate of exonic variants between the primary tumor tissues obtained from CD patients and the corresponding organoid line. We identified, on average, approximately 5000 mutations across each of the 14 paired samples of organoids and tissues. For the variants detected, all seven pairs showed a Jaccard index ranging from 0.5 to 0.8. Out of seven pairs, five (hPITO24, 25, 28 and 35) pairs had a Jaccard score of 0.8, while hPITO33 and 34 pairs had 0.7, and hPITO1 had 0.5. In order to investigate the similarity across the SNV (single nucleotide variation) sites, we calculated the Jaccard index of exon sites for synonymous and non-synonymous events, and found scores for all pairs ranging from 0.8 to 0.9. Furthermore, for only non-synonymous events, Jaccard scores also ranged from 0.8 to 0.9, except for hPITO1, which showed overall lower concordance, and had a score of 0.4 to 0.5. Figure 6 shows non-synonymous mutations found in organoid and tissue pairs for some of the key genes that are known to be involved in pituitary adenoma disease. Concordance indices between organoids and the matched patient’s adenoma tissues is reported in Figure 6. Therefore, WES data demonstrated that organoids derived from pituitary corticotroph adenomas retained the genetic alterations of the patient’s primary tumor tissue.
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Figure 6. Genomic landscape of hPITOs recapitulates genetic alterations commonly found PitNETs. Overview of single nucleotide variation events detected in hPITOs in genes commonly altered in PitNETs. The mutation frequency across the organoid population is depicted on the right. Color coding of the figure shows that organoid lines are derived from the same patient tumor tissue. ORG: organoid line, TIS: matched patient’s PitNET tissue.

3.6. IPSC Pituitary Organoids Generated from a CD Patients Expressing Familial Mutations Reveal Corticotroph Adenoma Pathology In Vitro

Extensive research has revealed the role of somatic and germline mutations in the development of CD adenomas [36,37]. Pituitary organoids were developed from iPSCs generated from the PBMCs of CD patients and carrying germline mutations that were identified by WES (Supplemental Figure S4). Chromosomal aberrations were not found when comparing against the reference dataset in the iPSCs generated from the CD patients (Supplemental Figure S3a,b). PBMCs isolated from patients diagnosed with CD were analyzed by WES in order to determine the expression of germline mutations. WES revealed the expression of a more recently identified gene predisposing patients to CD, namely cadherin-related 23 [38] (Supplemental Figure S5).
Pituitary organoids were then developed from iPSCs which were generated from the PBMCs of patients with CD (iPSCCDH23 and iPSCMEN1) and a healthy individual (iPSCctrl). Expression of PIT1 (pituitary-specific positive transcription factor 1), ACTH (adrenocorticotropic hormone), GH (growth hormone), FSH (follicle-stimulating hormone), LH (luteinizing hormone), PRL (prolactin), and synaptophysin (synaptophysin) with co-stain Hoechst (nuclei, blue) was measured by immunofluorescence, using chamber slides collected at 15 of the differentiation schedules (Supplemental Figure S6). While pituitary tissue that was differentiated from iPSCctrl expressed all major hormone-producing cell lineages (Supplemental Figure S6a), there was a significant increase in the expression of ACTH and synaptophysin, with a concomitant loss of PIT1, GH, FSH, LH, and PRL in iPSCsMEN1 (Supplemental Figure S6b,c). Interestingly, iPSCCDH23 cultures exhibited a significant increase in the expression of ACTH, GH, LH, and synaptophysin, with a concomitant loss of PIT1, FSH, and PRL (Supplemental Figure S6b,c). Immunofluorescence of iPSCs collected on the fourth day of the differentiation schedule revealed no expression of PIT1, ACTH, GH, FSH, LH, or PRL in (data not shown). Compared to control lines, iPSC lines expressing mutated CDH23 secreted significantly greater concentrations of ACTH earlier in the differentiation schedule (Supplemental Figure S7a). The upregulated expression of pituitary corticotroph adenoma-specific markers in iPSCCDH23 and iPSCMEN1 demonstrates that the iPSC-derived organoids represented the pathology of corticotroph adenomas in vitro.

3.7. ScRNA-seq Reveals the Existence of Unique Proliferative Cell Populations in iPSCCDH23 Cultures When Compared to iPSCsctrl

Using Seurat to identify cell clusters, as well as Uniform Manifold Approximation and Projection 9UMAP, clustering analysis identified 16 distinct cell populations/clusters consisting of known marker genes. Clusters 1, 5, and 7 of the iPSCsCDH23 were distinct from the iPSCctrl cultures (Figure 7a,b). Pituitary stem cells were characterized in iPSCctrl and iPSCCDH23 cultures (Figure 7b). Clusters 1 and 5 expressed markers consistent with the corticotroph subtype cell lineage (Figure 5c). Markers of dysregulated cell cycles and increased proliferation were identified in cell cluster 7 (Figure 7c). Expression of the E2 factor (E2F) family of transcription factors, which are downstream effectors of the retinoblastoma (RB) protein pathway and play a crucial role in cell division control, were identified in distinct cell cluster 7, which was identified within the iPSCCDH23 cultures (Figure 7c). Stem cell markers were also upregulated in cell cluster 7, and identified within the iPSCCDH23 cultures (Figure 7c). Using Cytobank software to analyze organoids collected 30 days post-differentiation, cells were gated on live CK20 positive singlets, and 9000 events per sample were analyzed by the viSNE algorithm. ViSNE plots are shown in two dimensions with axes identified by tSNE- 1 and tSNE-2, and each dot representing a single cell positioned in the multidimensional space (Figure 7d). Individual flow cytometry standard files were concatenated into single flow cytometry standard files, from which 12 spatially distinct populations were identified (Figure 7e). Overlaying cell populations identified by traditional gating strategies onto viSNE plots identified unique cell populations within the iPSCCDH23 cultures (Figure 7e). There were distinct cell populations between the iPSCctrl and iPSCCDH23 organoids, in addition to expression of hormone and cell lineage markers such as ACTH, TPit, PRL, and PIT1 (Figure 7e). The cell populations that exhibited high expression of Ki67 within the iPSCctrl organoid cultures included SOX2+ and PIT1+ populations (Figure 7f). The highly proliferating cell populations within the iPSCCDH23 organoid cultures included those that expressed CD90+/VIM+/CXCR4+ (mesenchymal stem cells), CXCR4+/SOX2+ (stem cells), TPit+ (corticotroph cell lineage), CD133+/CD31+ (endothelial progenitor cells), and CK20+/VIM+/CXCR4+ (hybrid epithelial-mesenchymal stem cells) (Figure 7f). Overall, the iPSCCDH23 organoids were significantly more proliferative compared to the iPSCctrl cultures (Figure 7f). Immunofluorescence staining of iPSCCDH23 organoids revealed increased mRNA expression of TPit and POMC, which correlated with increased ACTH protein compared to iPSCsctrl (Supplemental Figure S6). As shown in Supplemental Figure S6b,c, iPSCCDH23 cultures also exhibited a significant increase in the expression of GH and LH (Supplemental Figure S6b,c).
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Figure 7. Single cell analysis of iPSCctrl and iPSCCDH23 cultures 15 and 30 days post-directed differentiation. (a) UMAP plots showing identified cell clusters 0–16 in iPSCctrl and iPSCCDH23 cultures 15 days post-directed differentiation. (b) Violin plots of representative identified markers of the corticotroph cell lineage, where 2 subpopulations were observed among iPSCctrl and iPSCCDH23 cultures. Arrows highlight clusters 1, 5, and 7. (c) Violin plots showing expression of genes representative of stem cells, Wnt, NOTCH, Hh and SST signaling, anterior pituitary (corticotroph) cell lineage, and cell cycle in clusters 1, 5, and 7 of iPSCCDH23 cultures. Plot width: cell number, plot height: gene expression. (d) viSNE maps showing concatenated flow cytometry standard files for both samples and iPSCctrl and iPSCCDH23 organoids 30 days post-directed differentiation. (e) Overlay of manually gated cell populations onto viSNE plots. (f) Fluorescent intensity of Ki67 of viSNE maps for both samples and iPSCctrl and iPSCCDH23 organoids. iPSCctrl = 22518 events; iPSCCDH23 = 17542 events.
Collectively, Figure 7 demonstrates that the development of pituitary organoids generated from iPSCs of CD patients may reveal the existence of cell populations which, potentially, contribute to the support of adenoma growth and progression, as well as an expansion of stem and progenitor cells that may be the targets for tumor recurrence.

4. Discussion

Our studies demonstrate the development of organoids generated from human PitNETs (hPITOs) can potentially be used to screen for the sensitivity and efficacy of responses to targeted therapies for CD patients that either fail to achieve remission or exhibit recurrence of disease after surgery. In addition, we have documented that induced pluripotent stem cells (iPSCs) generated from a CD patient expressing germline mutation CDH23 (iPSCCDH23) reveals the disease pathogenesis under directed differentiation. Many early in vitro experiments have used pituitary cell lines, spheroids, aggregates, and/or tumoroids that do not replicate the primary PitNET microenvironment [19,20,21], and lack a multicellular identity [39,40]. The development of PitNET tissue-generated organoids is limited to the use of transgenic mouse models as the source [22,23,41]. The recent organoid cultures reported by Nys et al. [42] have been generated from single stem cells isolated from PitNET tissue, and are claimed to be true organoids due to their clonality. However, multicellular complexity was not validated by the protein expression or hormone secretion from pituitary cell lineages in these cultures [42]. According to the National Cancer Institute (NCI, NIH), an ‘organoid’ is defined as “a tiny, 3-dimensional mass of tissue that is made by growing stem cells (cells from which other types of cells develop) in the laboratory” [43]. The hPITOs reported here begin from single and/or 3–4 cell clusters dissociated from the PitNET tissue that harbors the stem cells. Supplemental Video S2 demonstrates a process of ‘budding,’ as well as lumen formation as organoids grow and differentiate. We document differentiation and function by comprehensive spectral flow cytometry, ELISA, and response to standard of care drugs. The growth of PitNET organoids reported in the current study is consistent with that of gastrointestinal tissue derived cultures that begin from cell clusters, crypts, or glands [27,44,45].
Our studies report a PitNET tissue organoid culture with a multicellular identity consisting of differentiated cell lineages, stem/progenitor cells, and immune and stromal cell compartments, which replicates much of the patient’s own adenoma pathology, functionality, and complexity. We have also demonstrated that iPSCs, derived from the blood of a CD patient, can be directly differentiated into pituitary organoids that resemble similar characteristics to the tumor tissue. Many investigators have proposed the use of organoids in personalized medicine, but have focused these efforts on targeted treatment of cancers [27,46,47,48]. The findings reported in these studies are the first to implement this approach for the potential treatment of PitNETs. Collectively, we have developed a relevant human in vitro approach to potentially advance our knowledge as well as our approach to studies in the field of pituitary tumor research. Both the hPITOs and the iPSCCDH23 may be implemented in studies that strive to (1) define the molecular and cellular events that are crucial for the development of PitNETs leading to CD, and (2) accelerate the identification of effective targeted therapies for patients with CD.
While published studies have advanced our understanding of the molecular mechanisms of the pathogenesis of corticotroph adenomas and elucidated candidate therapeutic targets for CD, these reports fall short of directly informing clinical decisions for patient treatment. Using organoids to screen potential drugs and compounds can potentially improve therapeutic accuracy. Figure 3 demonstrated a variation in drug responsiveness amongst the organoid lines generated from individual patients. Importantly, there was further divergence in drug responsiveness amongst the individual organoid lines within each pathologically defined corticotroph subtype. For example, hPITOs generated from patients with sparsely granulated corticotroph adenomas (hPIT0s 10, 25, 34, 35) and Crooke’s cell adenomas (hPITOs 7, 33) showed variable responses regardless of similar pathologically defined subtypes. In addition, the response of the tumor cells within the organoids to the standard of care drugs that directly target the pituitary in the body, including mifepristone and cabergoline, was only 50% in hPITO34 and hPITO35, and almost 0% in the other lines, including hPITO7, 10, and 25. These data clearly demonstrate that the inherent patient difference to drug response that is often observed among CD patients is reflected in the organoid culture. This culture system may be an approach that will provide functional data revealing actionable treatment options for each patient. Patient-derived organoids from several tumors have served as a platform for testing the efficacy of anticancer drugs and predicting responses to targeted therapies in individual patients [27,46,48,49,50]. An example of the use of organoids in identifying drug responsiveness within an endocrine gland is that of papillary thyroid cancer [51]. Organoids developed from PTC patients were used as a preclinical model for studying responsiveness to anticancer drugs in a personalized approach [51]. However, our study is the first report of the use of hPITOs for drug screening. Connecting genetic and drug sensitivity data will further categorize corticotroph subtypes associated with CD. WES analysis of each hPITO line was compared to the results for the corresponding primary adenoma tissues. We showed the concordance rate of exonic variants between the primary tumor tissues obtained from CD patients and the corresponding organoid line. On average, approximately 80% of the variants observed in the CD patients’ adenoma tissues were retained in the corresponding hPITOs.
Pituitary organoids were also developed from iPSCs generated from PBMCs of a CD patient expressing a germline genetic alteration in cadherin-related 23 CDH23 (iPSCCDH23), a CD patient expressing an MEN1 mutation (iPSCMEN1), and a healthy individual (iPSCctrl). Foundational studies performed by investigators at the genome level have revealed significant knowledge regarding the pathophysiology of CD [36,37,52,53]. In some instances, CD is a manifestation of genetic mutation syndromes that include multiple endocrine neoplasia type 1 (MEN1), familial isolated pituitary adenoma (FIPA), and Carney complex [54,55]. CDH23 syndrome is clinically associated with the development of Usher syndrome, deafness, and vestibular dysfunction [56]. Several mutations in CDH23 are associated with inherited hearing loss and blindness [57]. However, none of the variants found in this study were linked to any symptoms of deafness or blindness. A possible explanation is that deafness-related CDH23 mutations are caused by either homozygous or compound heterozygous mutations [57]. In a study that linked mutations in CDH23 with familial and sporadic pituitary adenomas, it was suggested that these genetic alterations could play important roles in the pathogenesis of CD [38]. Genomic screening in a total of 12 families with familial PitNETs, 125 individuals with sporadic pituitary tumors, and 260 control individuals showed that 33% of the families with familial pituitary tumors and 12% of individuals with sporadic pituitary tumors expressed functional or pathogenic CDH23 variants [38]. Consistent with the expected pathology and function of a PitNET from a patient with CD, iPSCCDH23 organoids exhibited hypersecretion of ACTH, and expression of transcription factors and cell markers were reported in the pathology report for corticotroph PitNETs. Collectively, these findings warrant further investigation to determine whether carriers of CDH23 mutations are at a high risk of developing CD and/or hearing loss. Specifically, clinical investigation is required to determine whether pituitary MRI scans should be adopted in the screening of CDH23-related diseases, including Usher syndrome and age-related hearing loss.
Pituitary organoids generated from iPSCs of a CD patient revealed the existence of cell populations that potentially contribute to the support of PitNET growth and disease progression, as well as an expansion of stem and progenitor cells that may be the targets for tumor recurrence. Organoids derived from both pituitary adenomas and iPSCs exhibited increased expression of stem cell and progenitor markers at both the protein and transcriptomic levels. Unique clusters that were proliferative in the iPSCCDH23 organoids expressed a hybrid pituitary cell population which was in an epithelial/mesenchymal state (CK20+/VIM+/CXCR4+/Ki67+). In support of our findings, a similar report of a hybrid epithelial/mesenchymal pituitary cell has been made as part of the normal developmental stages of the human fetal pituitary [58]. Previous studies have suggested that pituitary stem cells undergo an EMT-like process during cell migration and differentiation [59,60,61]. Consistent with our findings are extensive studies using single cells isolated from human pituitary adenomas to show increased expression of stem cell markers SOX2 and CXCR4 [22,23,41,62,63]. Within the clusters identified in the iPSCCDH23 culture were cell populations expressing stem cell markers, including SOX2, NESTIN, CXCR4, KLF4, and CD34. The same iPSCCDH23 cell clusters, 4, 8, 9, and 11, co-expressed upregulated genes of NOTCH, Hedgehog, WNT, and TGFβ signaling, which are pivotal not only in pituitary tumorigenesis and pituitary embryonic development, but also in ‘tumor stemness’ [22,23,41,62,63,64]. We also noted that clusters of cell populations 5 and 14 unique within the iPSCCDH23 cultures expressed upregulated genes which were indicative of high proliferation. We observed upregulated expression of the E2F family of transcription factors (E2Fs) E2F1 and E2F7. These findings are of significance, given that there is evidence to show that upregulation of E2Fs is fundamental for tumorigenesis, metastasis, drug resistance, and recurrence [65]. Within the pituitary adenoma microenvironment, whether these stem cells directly differentiate into pituitary tumors or support the growth of the adenoma is largely unknown. In addition, whether pituitary stem cell populations become activated in response to injury is also understudied. Although the role of stem cells has been identified using a mouse model through implantation of the cells within the right forebrain [66], the identification of pituitary tumor-initiating stem cells using in vivo orthotopic transplantation models is impossible in mice. Pituitary tumors harboring the stem cells may require engraftment within the environment from which the cells are derived in order to enable growth and differentiation of the tumor. However, it is technically impossible to implant cells orthotopically in the murine pituitary. The pituitary tumor organoid cultures presented in these studies may offer an approach by which isolation, identification, and characterization of this stem cell population is possible. Therefore, we would gain knowledge on the mechanisms of pituitary tumor pathogenesis and reveal potential novel targets for therapeutic interventions by using the iPSC generated pituitary organoid culture.
PitNETs associated with the development of CD cause serious morbidity due to chronic cortisol exposure that dysregulates almost every organ system in the body. Overall, existing medical therapies remain suboptimal, with negative impact on health and quality of life, including considerable risk of therapy resistance and tumor recurrence. To date, little is known about the pathogenesis of PitNETs. Here, we present a human organoid-based approach that will allow us to acquire knowledge of the mechanisms underlying pituitary tumorigenesis. Such an approach is essential to identify targeted treatments and improve clinical management of patients with CD.

5. Conclusions

Cushing’s disease (CD) is a serious endocrine disorder caused by an adrenocorticotropic hormone (ACTH)-secreting pituitary neuroendocrine tumor (PitNET), which stimulates the adrenal glands to overproduce cortisol. The absence of preclinical models that replicate the PitNET microenvironment has prevented us from acquiring the knowledge to identify therapies that can be targeted to the tumor with a higher efficacy and tolerability for patients. Our studies demonstrate the development of organoids generated from human PitNETs or induced pluripotent stem cells as an essential approach to identifying targeted therapy methods for CD patients.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/cells11213344/s1, Figure S1: Antibodies used and Cytek® Full Spectrum Viewer showing calculated similarity indices; Figure S2: Morphology and proliferation of lactotroph, somatotroph, and gonadotroph hPITOs; Table S1: Pituitary Growth Media; Table S2: Components used for pituitary organoids generated from iPSCs; Table S3: clinical characteristics of pituitary adenoma samples used for the generation of organoids; Table S4: Average correlation of replicates reported in Figure 3; Table S5: pituitary cell lineage or stem cell markers used in the scRNA-seq analysis; Video S1: hPITO38 EdU ACTH 3.

Author Contributions

Conceptualization, Y.Z.; methodology, J.C., Y.Z., J.M.C., B.N.S., S.M. and K.W.P.; software, J.C., Y.Z., J.M.C., S.M., Y.C., P.M. and R.P.; validation, Y.Z., J.C., J.M.C., A.S.L., K.C.J.Y. and R.P.; formal analysis, J.C., Y.Z., J.M.C., R.P., Y.C., S.M. and P.M.; investigation, Y.Z.; resources, Y.Z., J.C., J.E., C.A.T., B.H. and A.S.L.; data curation, J.C., Y.Z., J.M.C., R.P. and S.M.; writing—original draft preparation, Y.Z., J.C, S.M., J.M.C., Y.C., B.H. and R.P.; writing—review and editing, Y.Z., J.C., J.M.C., A.S.L., K.C.J.Y., S.M., J.E., C.A.T., K.W.P., B.H., Y.C., P.M., B.N.S. and R.P.; visualization, Y.Z., J.C., J.M.C., A.S.L., K.C.J.Y. and R.P.; supervision, Y.Z.; project administration, Y.Z.; funding acquisition, Y.Z. All authors have read and agreed to the published version of the manuscript.

Funding

This research was supported by the Department of Cellular and Molecular Medicine (University of Arizona College of Medicine) startup funds (Zavros). This research study was also partly supported by the National Cancer Institute of the National Institutes of Health under award number P30 CA023074 (Sweasy).

Institutional Review Board Statement

The study was conducted in accordance with the Declaration of Helsinki and approved by the Institutional Review Board of St. Joseph’s Hospital and Barrow Neurological Institute Biobank collection protocol PHXA-05TS038, and collection of outcomes data protocol PHXA-0004-72-29, and patient consent (protocol date of approval).

Informed Consent Statement

Written informed consent was obtained from all subjects involved in the study.

Data Availability Statement

The datasets generated during the analysis of the present study are available in the ReDATA repository, https://doi.org/10.25422/azu.data.19755244.v1. The datasets generated in the current study are also available from the corresponding author on reasonable request. All data generated or analyzed during this study are included in this published article (and its Supplementary Information Files).

Acknowledgments

We acknowledge the technical support of Maga Sanchez in the Tissue Acquisition and Cellular/Molecular Analysis Shared Resource (TACMASR University of Arizona Cancer Center) for assistance with embedding and sectioning of organoids. We would also like to acknowledge Patty Jansma (Marley Imaging Core, University Arizona) and, Douglas W Cromey (TACMASR imaging, University of Arizona Cancer Center) for assistance in microscopy. The authors thank the patients who consented to donate pituitary tumor tissues and blood for the development of the organoids. Without their willingness to participate in the study, this work would not be possible.

Conflicts of Interest

The authors declare no conflict of interest.

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