Chromatin texture analysis of cortical adrenal gland adenomas, including incidentalomas, and adjacent normal-appearing cortical tissue

OBJECTIVE: To describe, by morphometric and chromatin texture analysis, a series of adrenal gland lesions, including Cushing's and Conn's adenomas and incidentalomas. STUDY DESIGN: The material for the study consisted of five consecutive cases of incidentaloma, three cases of Conn's adenoma and three cases of Cushing's adenoma. Also included were five cases of adrenal carcinoma. Sections were stained according to the Feulgen procedure. Measurements were taken from the nodules and from two different zones, identified as outer and inner parts, of the normal-appearing adrenal cortex adjacent to the tumor. Data on approximately 50 nuclei were recorded for each of these three sites (tumor and outer and inner normal-appearing adrenal cortex). The nuclei were subjected to feature extraction and were analyzed by identification procedures--i.e., establishing nuclear and lesion signatures. RESULTS: The total optical density (OD) distributions of the nuclei from the normal-appearing adrenal cortex pointed to their diploid or near-diploid nature. In incidentalomas there was a very small increase in the number of nuclei, with increased total OD. In Conn's adenoma there was a noticeable but modest extension of the total OD distribution into the higher OD range. This trend continued for Cushing's adenoma. The pixel OD histograms for nuclei from normal-appearing tissue and from incidentalomas were hardly distinguishable. Starting with nuclei from Conn's adenoma, a shift toward lower pixel OD values began. The trend continued for nuclei from Cushing's adenoma and was very pronounced for nuclei from carcinoma. The nuclear signatures showed no appreciable difference between nuclei from normal-appearing cortex and from incidentaloma. Nuclei from Conn's adenoma were more similar to those from normal tissue in their signatures than nuclei from Cushing's adenoma. In fact, the nuclear signatures from Cushing's adenoma were almost identical to those of carcinoma. The lesion signatures for normal tissue, incidentaloma and Conn's adenoma confirmed the results seen in the nuclear signatures. There was a very modest increase in the number of nuclei with greater deviation from normal in incidentalomas, and the trend was more obvious in Conn's adenoma. However, in Cushing's adenoma there was a very substantial increase in the number of nuclei, with large deviations of their nuclear chromatin texture from normal. CONCLUSION: Computer-assisted analysis of nuclear characteristics proved useful in identifying and describing differences between groups of tumors arising in the adrenal cortex and highlighted the similarity between incidentalomas and adjacent normal-appearing cortex and between Cushing's adenoma and adrenal carcinoma.     

Measurement of urinary steroid profile in patients with adrenal tumor as a screening method for carcinoma

Results of measurement of urinary steroid metabolite profile using gas chromatographic analysis in eight patients with adrenocortical tumors, i.e. 3 adenomas with Cushing's Syndrome, one adenoma with virilization, one adenoma without clinical manifestations, one carcinoma with Cushing's syndrome and virilization, one carcinoma with Cushing's syndrome and feminization, and one carcinoma without endocrinological symptoms, are reported. A unique pattern dominated by 5 beta and 11 beta-hydroxy steroid metabolites was confirmed in five patients with Cushing's syndrome consisting of three cases with adenomas and two with carcinomas. Excessive 3 alpha, 17 alpha, 21-trihydroxy-5 beta-pregnan-20-one (tetrahydro-11-deoxycortisol, THS) and delta 5-pregnene-3 beta, 11 alpha, 20 alpha-triol (delta 5-pregnenetriol) values were found in all three carcinomas including a nonfunctional carcinoma. These findings would strongly suggest the tumor to be a carcinoma, although excessive excretion of THS and delta 5-pregnenetriol was detected in one patient with a large adenoma associated with virilization. One patient with carcinoma was responsive to ACTH stimulation while the remainder show almost no response to exogenous ACTH. Urinary steroid profiling using gas chromatographic analysis, especially the values for THS and delta 5-pregnenetriol, appears to be a useful method to use in detecting these steroid metabolic characteristics in patients with adrenocortical carcinoma.     

ACTH sensitivity of isolated human pathological adrenocortical cells: variability of responses in aldosterone, corticosterone, deoxycorticosterone and cortisol production

In vitro aldosterone, deoxycorticosterone, corticosterone and cortisol production of human adrenocortical cells derived from adenomas (Conn's syndrome, Cushing's syndrome), from hyperplastic adrenals (Cushing's syndrome) and from adrenals surrounding aldosteronoma are described. Cells from adenomas causing either Cushing's syndrome or Conn's syndrome harboured the highest basal and ACTH-stimulated corticosteroid production. Adrenocortical cells derived from micronodular hyperplasia causing Cushing's syndrome and cells from cortisol producing adenoma displayed predominantly cortisol and corticosterone secretion both under basal conditions and following stimulation with ACTH. Aldosteronoma cells showed highly variable aldosterone, deoxycorticosterone, corticosterone and cortisol response to ACTH. However, in aldosteronoma cell suspensions, the basal and ACTH-stimulated ratios of aldosterone to cortisol were increased when compared to ratios of steroids produced by cells from other adrenal tissues. Chronic treatment with spironolactone of patients with Conn's syndrome before surgery was associated with a decreased ratio of aldosterone to corticosterone, revealing that 18-hydroxylase in aldosteronoma cells may be inhibited during long-term therapy. Non-tumorous cells isolated from adrenals surrounding aldosteronoma displayed less aldosterone prior to and after stimulation with ACTH than aldosteronoma cells.     

Fine needle aspiration diagnosis of hyperplastic and neoplastic follicular nodules of the thyroid. A morphometric study.

The differentiation of hyperplastic nodules, follicular adenomas and follicular carcinomas from fine needle aspiration (FNA) cytology smears may be difficult. To better define the diagnostic criteria, we studied the morphometric parameters of nuclear area (NA), nuclear:cytoplasmic ratio and nuclear roundness (NR) in single cells and cell aggregates. In addition, we quantitated the percentage of touching or overlapping nuclei (NO) and the percentage of extent of nuclear area of overlap (NAO) in cellular aggregates. We measured cellular samples from FNA aspirates obtained from 20 hyperplastic nodules, 21 follicular adenomas, 5 encapsulated follicular carcinomas and 22 invasive follicular carcinomas, all of which were subsequently confirmed by histologic examination. Cellular aggregates provided the maximum diagnostic information. Stepwise discriminant analysis revealed that nuclear size, nuclear roundness and the percentage NAO allow optimum differentiation of hyperplasia, adenomas and carcinomas. Clearly, all of the poorly differentiated carcinomas (large NA, low NR, high NO and NAO) could be reliably diagnosed. Discriminant analysis allowed the differentiation of carcinoma from adenoma in 20/22 carcinomas (91%) and all 21 adenomas (although 2 adenomas were called hyperplasias and 3 hyperplasias were called adenomas).     

Hyperparathyroidism associated with Cushing's syndrome due to an adrenal cortical adenoma

Two patients with the rare association of Cushing's syndrome and primary hyperparathyroidism are reported. Initially, both patients suffered from Cushing's syndrome due to adrenal cortical adenomas with typical features and laboratory findings. Five years after treatment of the Cushing's syndrome by removal of the tumor, asymptomatic mild hypercalcemia was incidentally noticed in both patients, which suggested the occurrence of primary hyperparathyroidism. An enlarged parathyroid gland was removed surgically in both cases and was histologically shown to be a parathyroid adenoma. The levels of serum calcium returned to normal after parathyroidectomy. Papillary adenocarcinoma of the thyroid in one patient and adenomatous goiter in the other were also incidentally detected at operation. These findings suggest that Cushing's syndrome resulting from an adrenal cortical adenoma may be another presentation of multiple endocrine neoplasia type I.     

Morphometric image analysis of follicular lesions of the thyroid

OBJECTIVE: To determine the role of image morphometry in distinguishing various follicular lesions of the thyroid in cytologic smears. STUDY DESIGN: Archival fine needle aspiration smears of 10 cases each of follicular hyperplasia, follicular adenoma, follicular carcinoma and follicular variant of papillary carcinoma were used for the study. All cases were histopathologically proven. At least 100 random nuclei from each case were subjected to analysis with an image cytometer. Area, convex area, length, width, perimeter, convex perimeter and roundness of nuclei were measured using a 40 x objective (1 pixel = 0.446 micron). RESULTS: ANOVA showed that all the nuclear variables studied were significantly different (P < .05) in follicular hyperplasia as compared to follicular carcinoma and papillary carcinoma. All nuclear variables except roundness were also significantly different (P < .05) between follicular hyperplasia and follicular adenoma. However, between follicular adenoma, follicular carcinoma and papillary carcinoma there was considerable overlap of nuclear morphometric parameters. CONCLUSION: Image morphometry may help to distinguish nonneoplastic follicular lesions (hyperplasia) from neoplastic lesions (adenomas and carcinomas). However, to distinguish benign from malignant follicular lesions, image morphometry might not improve the accuracy of standard cytologic examination.     

Proliferating cell nuclear antigen (PCNA) expression in pituitary adenomas: relationship to the endocrine phenotype of adenoma

The expression of proliferating cell nuclear antigen (PCNA) correlates to cell proliferation and for this reason it is commonly considered as one of proliferation markers. Since proliferation rate is an important factor determining the tumor aggressiveness, the evaluation of PCNA index (the percentage of PCNA-immunopositive nuclei in the investigated tumor sample) is suggested as useful in predicting pituitary adenoma outcome. Seventy three unselected, surgically removed pituitary adenomas were immunostained with antibodies against the pituitary hormones or their subunits and against the proliferating cell nuclear antigen (PCNA). The highest PCNA index was found in ACTH-immunopositive tumors without the manifestation of the Cushing's disease ("silent" corticotropinomas). This value was significantly different in comparison to other adenoma subtypes including corticotropinomas manifesting themselves by Cushing's disease. The lowest PCNA index was noticed in monohormonal GH-secreting tumors. The adenomas which express more than one hormone (plurihormonal adenomas) seem to have a higher PCNA indices than monohormonal ones; the difference was significant in the case of mono- and plurihormonal prolactinomas. The recurrent tumors presented a higher mean PCNA index as compared to the primary tumors, although the difference was significant only in the case of prolactinomas. These findings suggest that the proliferative potential of pituitary adenomas is related to the tumor recurrence and hormone expression.     

Synthesis of 19-nor-aldosterone, 18-hydroxy-19-nor-corticosterone and 18,19-dihydroxycorticosterone in the human aldosterone-producing adenoma

The recently synthesized 18-C-steroid derivative, 19-nor-aldosterone(19-nor- aldo) and 18-hydroxy-19-nor-corticosterone(18-OH-19-nor-corticosterone) possess mineralocoroticoid and hypertensinogenic activity. They and an additional newly synthesized steriod, 18,19-dihydroxycorticosterone[18,19(OH)2-corticosterone], may play a role in the etiology and pathogenesis of disorders thought to be caused by steroids with mineralocorticoid and hypertensionogenic properties. In this study we provide evidence that 19-nor-aldo, 18-OH-19-nor-corticosterone and 18,19(OH)2-corticosterone are produced in vitro by aldosterone-producing adrenal adenomas and adenomas and adenoma of Cushing's syndrome. "silent" adrenal adenomas and the adjacent adrenal tissue. Measurable amounts of these steroids were found in the incubation fluids of adrenal tissues using specific RIAs performed after a sequence of HPLC systems. The rates of production of the three steroids were high in the aldosterone-producing adrenal adenomas and in adrenal hyperplasia compared with in either Cushing's adenoma or "silent" adenoma.     

Adrenocortical tumors

Hormonally active tumors of the adrenal cortex are either benign adenomas or adenocarcinomas. They may be located within the adrenal gland or as adrenal rests along the Wolffian tract. Hyperplastic cortical tissue without actual neoplastic formation is also capable of elaborating excessive cortical secretions.AT THE PRESENT STATE OF KNOWLEDGE, ANY ONE OR A COMBINATION OF THE FOLLOWING COMPOUNDS MAY BE ELABORATED IN A GIVEN CASE: the electrolytic, glucogenic, androgenic, or estrogenic corticosteroids. Whether or not Cushing's syndrome is primarily pituitary or adrenal in origin is still a matter of conjecture.     

Computerized morphometric study on fine needle aspirates of cellular follicular lesions of the thyroid

OBJECTIVE: To investigate the value of computerized nuclear morphometry in the differential diagnosis of cellular follicular lesions of the thyroid cytologically diagnosed on fine needle aspiration (FNA) smears. STUDY DESIGN: Sixty cases of FNA thyroid smears were cytologically diagnosed and classified as follows: 30 cases of follicular carcinoma, 20 cases of cellular hyperplastic nodules and 10 cases of follicular adenoma. Using an image analysis system, two morphometric variables, nuclear area and major axis length of the nucleus, were measured for each case. RESULTS: For both nuclear morphometric variables, statistical differences were found between carcinomas and hyperplastic nodules as well as between carcinomas and adenomas. No statistical differences were found between the nuclear variables in either hyperplastic nodules or adenomas. CONCLUSION: The results confirm the aim of our study, to establish nuclear morphometry by computerized image analysis as an additional tool in the differential diagnosis of thyroid follicular lesions cytologically diagnosed on FNA smears.     

MEN I gene mutations in sporadic adrenal adenomas

Loss of heterozygosity (LOH) on chromosome 11q13 occurs in about 20% of sporadic adrenal neoplasms. Adrenal lesions, mostly benign, occur in up to 40% of patients from MEN I kindreds. The MEN I gene, positioned on 11q13, has been considered a primary candidate gene in these lesions. We studied a group of 15 patients with sporadic adrenal adenoma, and 1 patient with multinodular hyperplasia. Of the 16 patients, 4 had incidentally discovered masses, 5 had Conn's syndrome, 6 suffered from Cushing's syndrome, and 9 had high sex hormone production. Studies with the markers D11S480, PYGM, D11S449, and D11S987 in 13 patients (12 of whom were from our group of 16) revealed 4 losses of heterozygosity on D11 S480 on 11q13, but the deletion did not affect the MEN I gene in any case. We present complete direct DNA sequencing data of the menin gene in 14 sporadic adrenal adenomas and one with adrenal hyperplasia. We identified one heterozygous missense mutation, T552S, in a hormonally inactive adrenal adenoma. One base exchange was identified close to the intron-exon boundary in intron 9 of a nodular adrenal hyperplasia. mRNA expression studies found that MEN I was transcribed in all 13 samples analyzed. In summary, our study identified the second patient with sporadic benign adrenal tumor presenting a menin gene mutation. Our complete direct sequencing approach adds evidence that menin gene mutations may account only for a minority of benign adrenal tumors if at all. Another tumor-suppressor gene inactivated in sporadic adrenal neoplasms may be located on chromosome 11q13.     

Preclinical Cushing's syndrome resulting from black adrenal adenoma. A case report

A 59-year-old Japanese woman, admitted for the treatment of diabetes mellitus and hypertension, was incidentally discovered to have a solid mass of 1.4 cm in diameter by CT scan with the attenuation value of 38 Hounsfield units, relatively higher for ordinary adrenal adenomas. Magnetic resonance imaging revealed no reduction of signal intensity on opposite-phase image on T1-weighted sequence. Adrenal scintigraphy imaging with 131I-adosterol did not show any uptake of the isotope in the area corresponding to both adrenals. Although she had no characteristic feature of overt Cushing's syndrome, her serum cortisol level was not suppressed after an overnight dexamethasone administration. She was diagnosed as having preclinical Cushing's syndrome. Left adrenalectomy was performed, revealing the well-circumscribed black tumor, mainly consisted of compact cell, in which cytoplasm was filled with numerous granules pigmented with dark to golden brown colors on hematoxylin-eosin staining. These findings suggested that her incidentaloma was a black adrenal adenoma. Production of steroid hormones was confirmed by immunohistochemical analysis of steroidogenic enzymes and by measurement of the tissue contents of hormones, whose levels were comparable with those in adenomas of overt Cushing's syndrome. This is the first case report of preclinical Cushing's syndrome resulting from black adrenal adenoma.     

Tumors of the respiratory tract observed at the German Primate Center, 1978–1994

Abstract: Eight spontaneous pulmonary tumors (four bronchiolar tubular adenomas, two bronchiolar adenocarcinomas, two squamous-cell carcinomas) occurred in a total of 54 adult tree shrews (Tupaia belangeri) of the GPC colonies between 1978 and 1994. The adenomas and adenocarcinomas consisted of tubularly or trabecularly arranged cuboidal to cylindrical cells interspersed with some PAS-positive goblet cells, thus resembling the epithelial lining of respiratory bronchioles of tree shrews. The two squamous-cell carcinomas probably originated from the pulmonary alveoles. Three more pulmonary tumors (one small-cell carcinoma, one bronchial adenoma, one squamous-cell carcinoma) developed in 409 adult callitrichids of the GPC colonies during the same period, and one more bronchial adenoma was observed in a common marmoset (Callithrix jacchus) of another colony located in Göttingen. With regard to the adenomas and squamous-cell carcinomas, a similar cellular origin with the tree shrews is assumed. The small-cell carcinoma possibly developed from the bronchial epithelium, provided a pathogenesis parallel to that of human small-cell carcinoma is suggested. Four of the tree shrew pulmonary adenomas/adenocarcinomas and the small-cell Ca were macroscopically visible as yellowish-grey nodules of 1 mm × 1 mm to 15 mm × 15 mm diameter, predominantly involving the main lobes (2 × right main lobes, 2 × left main lobes, 1 × all lobes). The pulmonary tumors of the other animals were below macroscopical detectability.     

A possible association between aldosterone response to vasopressin and circadian change of aldosterone in the patients with aldosterone-producing adenoma

Vasopressin was reported to stimulate secretion of both cortisol and aldosterone through eutopic V1a receptors in adrenal gland. Recently, adrenal hyper-responsiveness of plasma cortisol to vasopressin with eutopic overexpession of V1a receptors has been reported in Cushing's syndrome, such as a majority of cases of ACTH-independent macronodular adrenal hyperplasia and some cases of Cushing's adenomas. There were a few reports regarding the aldosterone response to vasopressin in aldosterone-producing adenoma. The aim of our study was to investigate the aldosterone response to vasopressin and its pathophysiological roles in the patients with aldosterone-producing adenoma. Vasopressin-loading test was performed in 10 patients with aldosterone-producing adenoma, and in 16 patients with non-functioning adrenal tumors. The roles of the aldosterone response to vasopressin were analyzed in terms of hormonal secretion and the expression of V1a receptor mRNA on the operated adrenal gland in aldosterone-producing adenoma. We found that (1) a varying aldosterone response to vasopressin was observed, (2) absolute response of plasma aldosterone in aldosterone-producing adenoma was significantly higher than that in non-functioning tumor, (3) aldosterone response rate to vasopressin was significantly and negatively correlated with the decline rate (%) in plasma aldosterone from morning to evening in aldosterone-producing adenoma, (4) V1a receptor mRNA was expressed at various values in aldosterone-producing adenoma, and (5) surgical removal of aldosterone-producing adenoma eliminated the aldosterone response to vasopressin observed in patients with aldosterone-producing adenoma. These findings indicated that vasopressin might be involved in the coordination of aldosterone secretion through eutopic expression of V1a receptor in aldosterone-producing adenoma.     

Ultrastructural pathology of the adrenal gland in Cushing's syndrome

The ultrastructural pathology of the adrenal glands was studied in fifteen cases of Cushing's syndrome. Some specific features correlated with the pathological aspects of adrenals were found. In the hyperplastic adrenal cortex the cytoplasms contained a rich smooth endoplasmic reticulum and many mitochondria. Increased lipid-pigment complexes were found especially in the compact cells. In adenomas, the clear cells showed large lipid vacuoles; the compact cells presented anisomorphous mitochondria, a well developed smooth endoplasmic reticulum and many pigment bodies. The nuclei of adenomatous cells were irregular, with deep invaginations. In adrenal carcinomas, the pleomorphism of nuclei, nucleoli, mitochondria, and smooth endoplasmic reticulum was more obvious. Absolutely reliable characteristics proving malignancy at ultrastructural level do not, however, exist. The steroidogenic activity of both hyperplastic and tumoral adrenal glands can be assessed using the agranular endoplasmic reticulum and the mitochondria as functional parameters.     

The number of nucleoli in benign and malignant thyroid lesions: a useful diagnostic sign in cytological preparations

The slides of fine needle aspiration cytology specimens from 99 cases of cold thyroid nodules with known histology were reviewed and the number of nucleoli per nucleus counted and correlated with the different histopathological groups. Significant differences were observed between benign and malignant thyroid lesions in the number of nucleoli in the cytological material. Lower values were present in nodular goitres and follicular adenomas compared to carcinomas. In benign lesions the majority of nuclei contained one nucleolus and nuclei with two, three or more nucleoli were less frequent than in follicular, papillary, medullary and anaplastic carcinomas. Only one case of follicular adenoma had cells containing three or more nucleoli compared to more than half the cases of follicular carcinoma.     

Immunolocalization of urotensin II and its receptor in human adrenal tumors and attached non-neoplastic adrenal tissues

Urotensin II (UII), first identified from goby urophysis, is a potent vasoactive peptide hormone and an endogenous ligand for an orphan G protein-coupled receptor GPR14, now named urotensin II receptor (UT-R). In addition to its vascular actions, UII has been shown to have mitogenic effects on tumor growth and some regulatory effects on adrenal steroidogenesis. In the present study, we examined expression of UII and UT-R in human adrenal tumors and attached non-neoplastic adrenal tissues by immunohistochemistry. Both UII and UT-R were immunolocalized in tumor cells of all adrenal tumors examined: 8 cases of cortisol-producing adenomas, 8 cases of aldosterone-producing adenomas, 2 cases of non-functioning adenomas, 17 cases of adrenocortical carcinomas, and 8 cases of pheochromocytomas. In attached adrenals, immunoreactivity for UII was detected in medulla, but much weaker in the cortex than in cortical tumors, suggesting that expression of UII was up-regulated in neoplastic adrenocortical tissues. No significant differences were found in the degree of immunoreactivity for UT-R between the tumors and the attached adrenal tissues. The present study showed that both UII and UT-R were expressed in the adrenal tumors and attached non-neoplastic adrenal tissues, and suggests possible roles of UII and UT-R in tumor growth and/or secretory activities of these tumors.     

Renin-angiotensin system and plasma aldosterone in Cushing's syndrome

The renin-angiotensin system was studied in eight patients with Cushing's syndrome (four with adrenal adenoma and four with adrenal hyperplasia) and in five normal controls. Basal plasma renin activity (PRA) and aldosterone concentration (PAC) were similar in supine position among Cushing's syndrome due to adrenal adenoma (PRA; 1.0 +/- 0.3 ng/ml/h, PAC; 7.4 +/- 1.0 ng/dl, mean +/- SE), those due to adrenal hyperplasia (1.0 +/- 0.2, 6.9 +/- 0.8) and the controls (0.8 +/- 0.1, 6.4 +/- 0.4). The PRA after furosemide (1 mg/kg i.v.) and 120 min. upright posture stimulation was similar among Cushing's syndrome due to adrenal adenoma (2.2 +/- 0.7 ng/ml/h), those due to adrenal hyperplasia (2.6 +/- 1.7) and the controls (2.5 +/- 1.2). However, the PAC response after the stimulation in Cushing's syndrome due to adrenal hyperplasia (7.1 +/- 1.2 ng/dl) was significantly lower than that in the controls (17.5 +/- 2.1) (p less than 0.01), although there was no significant difference between the PAC response in Cushing's syndrome due to adrenal adenoma (12.6 +/- 1.0) and the controls. These results indicate that PAC response to furosemide and upright pasture stimulation might be suppressed in Cushing's syndrome due to adrenal hyperplasia.