This paper highlights the problem of neuroendocrine tumours (NETs) with clinical symptoms of hypercorticism caused by hypersecretion of adrenocorticotropic hormone (ACTH) by tumour cells. In most cases (85%), the tumours were localized in the pituitary gland (Cushing's disease); 15% of the patients had an extrapituitary tumour that manifest as an ectopic ACTH secretion (EAS). Comparative analysis of clinical, hormonal, histological, and immunohistochemical characteristics of pituitary and extrapituitary ACTH-secreting NET was performed. It included 46 patients with CD and 38 ones exhibiting ectopic ACTH secretion (EAS). Results of the study suggest differences between CD and EAS in terms of the severity of clinical manifestations and duration of the disease. Hormonal studies showed that EAS unlike CD was associated with high plasma ACTH and cortisol levels, late-evening salivary cortisol and daily urinary free cortisol, the absence of a 60% or greater reduction of cortisol in the HDDST test, and the presence of a low (less than 2) ACTH gradient in response to desmopressin administration with catheterization of cavernous sinuses. The study of morphofunctional characteristics of the removed NET demonstrated the ability of both pituitary and extrapituitary NETs to express ACTH as well as GH, PRL, LH, and FSH. The angiogenic markers (CD31 and VEGF) were detected with equal frequency regardless of the NET localization. The histological structure of all corticotropinomas suggested their benign origin, but extrapituitary NETs were represented by different morphological types with varying malignancy, invasiveness, and metastatic properties. A higher cell proliferation potential (Ki-67) was documented for NET in patients presenting with an ectopic ACTH secretion compared to those having corticotropinomas.
NETs comprise a heterogeneous group of neoplasms originating from the neuroendocrine system. “Functioning” NETs that cause well apparent clinical symptoms due to hyperactivity of the involved hormones holds a special place in this group. CRH and ACTH-secreting tumours produce the clinical picture of hypercorticism. These tumours having a common origin in the cells of the diffuse neuroendocrine system, comparative analysis of the results of clinical, hormonal, histological, and immunohistochemical studies may contribute to the improvement of diagnostics of the disease and prognosis of remission after surgical intervention.
A total of 84 patients were presenting with clinically manifest of hypercorticism were enrolled in the study. Diagnosis was made based on the results of hormonal studies, functional tests, brain MRI, MCTC imaging of adrenals, lungs, and mediastinum, kidneys and appendix. Blood samples for hormonal analysis were taken at 8.00 and 23.00 to elucidate circadian rhythms. ACTH and cortisol levels in peripheral blood, late-night free salivary cortisol, and 24-hr urinary free cortisol were measured by electrochemiluminescence immunoassay using an automated Cobas 6-1 system (Roche).
The patients were divided into two groups depending on NET localization. Group 1 was composed of 46 patients with pituitary NET (Cushing’s disease, CD), and group 2 comprised 38 patients with extrapituitary NET (EAS). Group 1 consisted of 5 men and 41 women aged from 20 to 58 mean 39.5 +/− 10.2 years; duration of the disease 6.0 +/− 1.3 years. Group 2 included 14 men and 24 women aged from 21 to 70 mean 42 +/− 13 years; duration 2.89 +/− 1.5 years.
Forty practically healthy subjects (20 men and 20 women) at the age of 29.5 ± 11.2 years served as controls.
The removed tumours were fixed in a 10% neutral buffered formalin solution for 24 hours, dehydrated, compacted, and embedded in paraffin. Paraffin sections (5 mcm thick) were deparaffinized and stained with hematoxylin and eosin. Light microscopy was used to verify diagnosis of pituitary adenomas (basophilic, oxyphilic, chromophobic and mixed), carcinoid tumours (typical, atypical) of lungs, bronchi, kidneys, thymus, and small-cell lung carcinoma in accordance with the 1993 WHO histological classification of tumours (for pituitary adenomas), 2004 WHO classification (for ectopic ACTH-secreting lung and thymus tumours), and 2010 WHO classification (for ectopic ACTH-secreting appendix tumours).
Forty six corticotropinomas and 30 ectopic ACTH-secreting tumours were studied immunohistochemically with the use of primary antibodies against ACTH, CRH, PRL, GH, LH, FSH, Ki-67, CD31, and VEGF. A sensitive complex of universal antibodies with biotin-free avidin-streptavidin-peroxidase system (Nichirei, Japan) was used as secondary antibodies and visualization system.
Tissues of 6 corticotropinomas and 3 tumours associated with ectopic ACTH secretion were examined using immunohistochemical double staining to identify two hormones at a time in a single section. For this purpose, an EnVision (Dako) G/2 Doublestain System, Rabbit/Mouse (DAB+/Permanent Red) was used.
Expression of ACTH, CRH, PRL, GH, LH, and FSH was assessed in 10 fields of view at ×400 as positive (staining of more than 10% tumour cells) or negative (staining of less than 10% cells). VEGF and CD31 expressions (10 fields of view, ×400) were described as positive and negative if more and fewer than 30% of the tumour cells were stained, respectively. Ki-67 labeling index (5 fields of view, ×400) was expressed as percentage of positively stained nuclei out of their total number in the tumour (
The tissue of thyroid papillary cancer was used as positive control for CD31 and VEGF and the lymph node tissue for Ki-67.
They were treated using Statsoft Statistica 8.0 for Windows, version 6. The normality of distribution was assessed by Shapiro-Wilk test. The data are presented as median ± SD. The Mann
All 84 patients were were presented with symptoms and clinical features of hypercorticism, such as central obesity, trophic skin lesions, arterial hypertension, cardiomyopathy, disturbed carbohydrate metabolism, secondary immunodeficiency, secondary hypogonadism, and systemic osteoporosis. Some differences in clinical symptoms were revealed between the groups (Table
Frequency of main symptoms and complaints in patients with CD and EAS.
Complaints/symptoms | % of the total number of patients with CD | % of the total number of patients with EAS |
---|---|---|
Overweight | 90.0 | 70.0 |
Underweight | 10.0 | 30.0 |
Melanoderma | 10.9 | 92.0 |
Myopathic syndrome | 30.0 | 42.9 |
Purple striae | 67.0 | 50.0 |
Skin impurity | 10.9 | 16.7 |
Excessive facial hair growth | 41.3 | 16.7 |
Hypokalemia | 38.0 | 73.0 |
Arterial hypertension | 82.6 | 72.2 |
Encephalopathy/depression | 64.0 | 54.0 |
Amenorrhea in women | 53.7 | 10.0 |
Reduced sexual potency in men | 40.0 | 30.0 |
Back pain | 57.0 | 38.9 |
Diabetes mellitus | 40.0 | 38.0 |
Systemic osteoporosis | 93.0 | 85.0 |
Nephrolithiasis/urolithiasis/chronic pyelonephritis | 65.0 | 46.0 |
It was shown that 3 patients with EAS (group 2) unlike those with CD (group 1) exhibited cyclic symptoms of hypercorticism and their fast worsening, frequent hypokalemia (70% of the cases), and skin hyperpigmentation (92%). Moreover, 4 patients of group 2 suffered metastatic lesions in the adjacent organs absent in group 1.
In our study, plasma ACTH and cortisol, salivary cortisol, and 24-hr urinary free cortisol levels were significantly elevated in the patients of both groups (Figure
Cortisol (pmol/L) and ACTH (pg/mL) levels during a 24-hour period in patients with Cushing’s disease and ectopic ACTH secretion compared with controls.
Free cortisol levels in 24-hr urine and saliva (measured at 23.00) in patients with Cushing’s disease and ectopic ACTH secretion compared with controls.
The dexamethasone suppression test (1 mg) yielded negative results in all the patients. The high-dose dexamethasone suppression test (HDDST, 8 mg) revealed differences in the regulation of the hypothalamo-pituitary-adrenal axis between the two groups. Specifically, 78% of the patients in group 1 experienced a 60% or greater decrease in the plasma cortisol level after dexamethasone administration. No similar drop was observed in group 2. It is concluded that HDDST is unsuitable for reliable differential diagnostics between CD and EAS.
Many authors emphasized difficulties of differential diagnostics between Cushing’s disease and ectopic ACTH secretion [
We obtained blood samples from inferior petrosal sinuses of 46 patients for differential diagnostics between CD and EAS in the desmopressin stimulation test using the generally accepted criteria to evaluate the results [
Plasma ACTH gradient revealed by selective blood sampling from petrosal sinuses in the desmopressin stimulation test in patients with Cushing's disease and ectopic ACTH secretion.
ACTH gradient |
Cushing's disease |
Ectopic ACTH secretion |
---|---|---|
Baseline level | Above 2 | Below 2 |
After stimulation | Above 3 | Below 2 |
Intersinus gradient | Above 2.4 | Below 1.4 |
Dynamics of ACTH concentration based on selective blood sampling from petrosal sinuses in the desmopressin stimulation test in patients with Cushing’s disease and ectopic ACTH secretion.
Regarding Table
Histological data indicate that Cushing’s disease in all cases was due to benign pituitary tumours (Figure
Histological structure of corticotropinomas and ectopic ACTH-secreting tumours (hematoxylin and eosin stainings). (a) A fragment of basophilic corticotropinoma, ×20. (b) Oxyphilic cell hyperplasia in adenohypophysis, ×20. (c) Typical lung carcinoid, ×20. (d) Metastasis of atypical lung carcinoid to a mediastinal lymph node, ×10. (e) and (f) Appendiceal NET (G2), ×10, 25 mm in diameter, clinically manifested as cyclic Cushing’s syndrome with carcinoid crisis attacks between hypercortisolism cycles. (e) Invasion of the tumour into mesenteric fat. (f) NET metastasis to a mesenteric lymph node.
Thus, all corticotropinomas were benign neoplasms dominated by basophilic cell tumours in terms of tinctorial characteristics (52.2%). These neoplasms underwent secondary changes, such as necrosis, hemorrhage, and stromal oedema, besides angiomatosis and mitoses.
Postoperative material from 30 patients with ectopic ACTH secretion was available for morphological examination. Of these, 24 tumours were found in the bronchopulmonary system: 23 were lung carcinoids and 1 small-cell lung cancer. Four thymic tumours were represented by two atypical carcinoids, one typical carcinoid and one large-cell carcinoma. One tumour was typical renal carcinoid and one appendix neuroendocrine tumour (G2) (Figure
The study revealed ectopic ACTH-secreting tumours of different localization, morphological structure, and degree of malignancy (adenocarcinomas, thymic carcinomas, and small-cell lung cancer). Atypical lung carcinoids predominated. Metastases into various structures were documented in 4 cases. In contrast, all corticotropinomas were benign neoplasms with no signs of metastases or invasive growth.
Immunohistochemical reactions with antibodies against ACTH, PRL, GH, LH, and FSH were performed for comprehensive functional assessment in 46 corticotropinomas and 18 ACTH ectopic tumours (all EAS neoplasms were examined for CRH expression, none of them expressed FSH). Cytoplasmic expression of all hormones was measured (Figure
Distribution of pituitary and extrapituitary ACTH-secreting tumours based on the results of immunoexpression of tropic hormones.
Hormone expression |
Corticotropinomas, |
Ectopic ACTH-secreting tumours |
| |
---|---|---|---|---|
IHC characteristics | Morphology | |||
Monohormonal | ACTH, |
Total, |
3 AC/1 TC |
0.2 |
| ||||
Bihormonal | Total, |
Total, |
2 AC/2 TC/1 SCLC |
0.1 |
| ||||
Polyhormonal | Total, |
Total, |
2 AC |
0.2 |
Expression of pituitary tropic hormones in corticotropinomas and ectopic ACTH-secreting tumours. (a) Indirect immunoperoxidase staining of corticotropinoma cells with anti-ACTH antibodies, ×20. (b) Indirect double immunoperoxidase staining of corticotropinoma cells with anti-ACTH and anti-LH antibodies, ×20. (c) Indirect immunoperoxidase staining of atypical lung carcinoid cells with anti-ACTH antibodies, ×10. (d) Indirect double immunoperoxidase staining of atypical carcinoid cells with anti-ACTH and anti-CRH antibodies, ×20.
Six bi- and polyhormonal corticotropinomas were double labeled with antibodies against pituitary tropic hormones using an immunohistochemical method. The same technique was employed to stain 3 ectopic ACTH-secreting tumours with anti-ACTH and anti-CRH antibodies. This procedure was performed to confirm expression of two hormones in a single tumour section and to identify hormone-synthesizing cells (Figures
Immunohistochemical staining revealed the expression of Ki-67 in tumour cell nuclei (Figures
Expression of Ki-67, CD31 in corticotropinomas and ectopic ACTH-secreting tumours. (a) Indirect anti-Ki-67 immunoperoxidase staining of corticotropinoma cell nuclei, ×40. (b) Indirect anti-Ki-67 immunoperoxidase staining of the cell nuclei in atypical lung carcinoid, ×40. (c) Indirect anti-CD31 immunoperoxidase staining of vascular endothelium in corticotropinomas, ×10. (d) Indirect anti-CD31 immunoperoxidase staining of vascular endothelium in typical lung carcinoid, ×10.
Expression of Ki-67 was demonstrated in 45 (97.8%) corticotropinomas. The labeling index of this marker varied from 0.07% to 0.46% (median 0.14%). Ki-67 expression was detected in all ectopic tumours, with a median labeling index of 1.3% (maximum 2.5%, minimum 0.5%) for typical carcinoids (
Expression of the angiogenesis marker CD31 was documented in 24 corticotropinomas (52.2%) and VEGF in 28 adenomas (60.9%). CD31 was detected in EAS tumours: one SCLC, 5 AC (two metastatic), and 2 TC. VEGF was present in 2 AC (without metastases) and 1 TC. Statistical analysis of CD31 (
Thus, immunohistochemical studies showed that ACTH-expressing monohormonal tumours (69.6%) prevailed among the corticotropinomas, the rest of them (30.4%) expressed other pituitary tropic hormones, besides ACTH determining the clinical presentation of the disease. EAS tumours (33.3%) were shown to synthesize either ACTH or CRH responsible for the development of hypercorticism or coexpress ACTH and CRH (
Most corticotropinomas and ectopic ACTH-secreting tumours expressed CD31 and VEGF. There was no significant difference in the expression rate of these markers between the two types of tumours.
This comparative study demonstrated differences in the clinical presentation of Cushing’s disease and ectopic ACTH secretion. For example, ectopic ACTH secretion is more frequently associated with hypokalemia (73%), melanoderma (92.9%), and pronounced myopathic syndrome (42.9%). These findings agree with the literature data [
For example, blood ACTH level in patients with ectopic ACTH secretion was significantly higher than in Cushing’s disease. The daily rhythm of cortisol and ACTH was preserved differently in the two groups. It was virtually absent in patients with ectopic ACTH secretion but persisted in Cushing’s disease even if it was less apparent than in the control group. Probably, the hypothalamo-pituitary axis of some CD patients remains sensitive to hypothalamic and suprahypothalamic regulators of circadian rhythms of these hormones.
Many authors emphasize difficulties encountered in differential diagnostics of ectopic ACTH secretion and Cushing’s disease. Selective blood sampling from inferior petrosal sinuses after CRH or desmopressin stimulation has recently been proposed as the most reliable method for differential diagnostics of ectopic ACTH secretion and Cushing’s disease [
In CD patients, all tumours had a structure of pituitary adenomas. Eight cases (17.4%) had in addition fragments of adenohypophyseal hyperplasia. EAS tumours included carcinoids affecting different organs and SCLC characterized by higher malignant potential compared with corticotropinomas; they showed an infiltrative growth pattern and caused metastatic lesions in various tissues.
Immunohistochemical studies using antibodies against pituitary tropic hormones showed that the number of ACTH and CRH-secreting cells in pituitary and extrapituitary tumours is much greater than the number of cells expressing other hormones. Anti-ACTH and anti-CRF antibodies staining density was higher than in the studies with antibodies against GH, PRL, LH, and FSH. These findings are in agreement with those reported by other authors [
We estimated the malignancy potential of ACTH-secreting pituitary tumours and extrapituitary NE neoplasms by studying expression of factors controlling cell proliferation (Ki-67) and angiogenesis (CD31 and VEGF).
We documented expression of Ki-67 by dividing cells in all phases of the cell cycle; it was present in the cell nuclei of practically all tumours excepting one corticotropinomas. Analysis of Ki-67 labeling indices in corticotropinomas and ectopic tumours revealed significant difference in the expression of this marker (
Neoangiogenesis in a tumour is an important precondition for neoplasm growth and spread due to activation of proangiogenic growth factors (with VEGF being the key one), suppression of inhibitors of angiogenesis (angiostatin, thrombospondins, etc.), and interaction of these factors with tumour stroma. Metalloproteinases (collagenases) produced in the stroma destroy its structure thereby promoting formation of new vessels and tumour progression. Activity of metalloproteinase inhibitors decreases. CD31 is an adhesion molecule of epithelial cells and platelets that can be used to label tumour vascular endothelium and serves as an indicator of angiogenic activity. Our study revealed positive expression of CD31 in 24 (52.2%) corticotropinomas, SCLC, and 4 atypical carcinoids (with mediastinal lymph node metastasis in SCLC and two AC) that reflected the angiogenic activity of the tumours. Turner et al. [
The intensity of neoangiogenesis is related to VEGF [
To sum up, the Ki-67 labeling index was the only marker of differentiation between malignant potentials of corticotropinomas and ectopic ACTH-secreting tumours in our study. These neoplasms had similar characteristics of cellular adhesion and angiogenesis regardless of their histological structure.
Comprehensive comparative analysis of clinical, hormonal, histological, and immunohistochemical studies of pituitary and extrapituitary ACTH-secreting NET revealed a number of similar and different features. The clinical picture of hypercorticism due to EAS tumours, unlike CD, is characterized by rapid deterioration of specific symptoms of the disease including marked hypokalemia, skin hyperpigmentation, high evening plasma and salivary ACTH and cortisol levels as well as daily urinary free cortisol, the absence of a 60% or greater reduction of cortisol in the HDDST test, and the presence of a low (less than 2) cortisol gradient in response to desmopressin administration with catheterization of cavernous sinuses.
Histological and immunohistochemical investigations of pituitary and extrapituitary ACTH-secreting NET also demonstrated similar and different features. Similarity between corticotropinomas and ectopic ACTH-secreting tumours included their ability to express not only ACTH (or CRH in EAS neoplasms), but also two or more pituitary tropic hormones (GH, PRL, LH, and FSH) and proangiogenic markers (CD31 and VEGF), with equal frequency. Pituitary and extrapituitary ACTH-secreting tumours differed in histological structure and malignant potential. All corticotropinomas were benign neoplasms, whereas ectopic tumours were different in terms of morphological structure and degree of malignancy (AC, TC, SCLC, and large-cell NE carcinoma); some of them possessed metastatic activity. Cellular proliferation (Ki-67 labeling index) was especially pronounced in ectopic ACTH-secreting tumours.
Adrenocorticotropic hormone
Atypical carcinoid
Ectopic ACTH secretion
Cushing’s disease
Immunohistochemical study
Corticotropin-releasing hormone
Luteinizing hormone
Small-cell lung cancer
Neuroendocrine tumour
Prolactin
Growth hormone
Typical carcinoid
Follicle-stimulating hormone
Marker of proliferative activity
Angiogenic marker
Vascular endothelial growthfactor.