Immunohistological study on the ACTH producing islet cell carcinoma of the pancreas.

ACTH producing cells in islet cell carcinoma associated with Cushing's syndrome were studied by an improved immunoperoxidase as well as immunofluorescence technique. The cells which contained ACTH antigen were distributed throughout the tumor tissue of both primary and metastatic lesions in rather small numbers. In the small cancer nests they were present in an irregular or mosaic pattern and, on the contrary, they tend to line along the margin in the larger nests. The intracytoplasmic localization of the ACTH antigen was characteristically demonstrated in the infranuclear area of the tumor cells, closely simulating that of the argentaffin granules of the enterochromaffin cells. The electron microscopic observation identified membrane enclosed, cored granules, 200-300 nm in diameter, present in clusters in the infranuclear area of some tumor cells, which were considered corresponding well to the ACTH positive cells in immune staining.     

IMMUNOHISTOLOGICAL STUDY ON THE ACTH PRODUCING ISLET CELL CARCINOMA OF THE PANCREAS

ACTH producing cells In islet cell carcinoma associated with Cushing's syndrome were studied by an improved immunoperoxidase as well as immunofluorescence technique. The cells which contained ACTH antigen were distributed throughout the tumor tissue of both primary and metastatic lesions in rather small numbers. In the small cancer nests they were present in an irregular or mosaic pattern and, on the contrary, they tend to line along the margin in the larger nests. The intracytoplasmlc localization of the ACTH antigen was characteristically demonstrated in the intranuclear area of the tumor cells, closely simulating that of the argentaffin granules of the entero-chromaffin cells. The electron microscopic observation identified membrane enclosed, cored granules,200–300 nm in diameter, present in clusters in the intranuclear area of some tumor cells, which were considered corresponding well to the ACTH positive cells in immune staining.     

Cushing syndrome-associated pheochromocytoma and adrenal carcinoma. An immunohistological investigation.

Seven adrenal carcinomas and seventeen pheochromocytomas (PHs), two of which were clinically associated with a Cushing's syndrome and one associated with multiple endocrine neoplasia Type II (MEN-II), were investigated immunohistologically with a panel of antibodies against intermediate filament proteins, a proliferation-associated nuclear antigen (Ki-67), neuroendocrine tumor markers, and different hormones on paraffin-embedded tissue sections and, from 19 cases, also on frozen tissue sections. Synaptophysin proved to be the most reliable tumor cell marker on both snap-frozen and paraffin-embedded tissue but, like antibodies against NSE, yielded unspecific stainings in the carcinomas. The two Cushing-associated pheochromocytomas (CaPH) showed the same immunohistological profile as the other PHs, except one chromogranin-negative tumor. Five PHs showed weak reactivity for calcitonin, one for serotonin, and two for a-HCG in small amounts. All PHs lacked other hormone expression, including ACTH. The average growth fraction was small (2.2%) in 13 cases, but 80% of the tumor cells were proliferating in one case of CaPH. Adrenal carcinomas showed only weak or no expression of keratin in one case, a homogenous or droplet, non-filamentous cytoplasmic staining with antibodies against neurofilament in frozen tissue section, and they were completely chromogranin-negative. The average growth fraction was 7.6% in 5 cases.     

Multiple hormone production in an oat cell carcinoma of the larynx

A 60-year-old white woman with laryngeal oat cell carcinoma is described. She was a heavy smoker who had been treated seven years earlier with 5,000 rads for a well differentiated squamous cell carcinoma metastatic to a left submandibular lymph node. She presented this time with a two month history of hoarseness and tumor of the supraglottic larynx was found. There was clinical and chemical evidence of an ectopic ACTH syndrome. The histology and fine structure of the tumor were typical of oat cell carcinoma. Immunoreactive ACTH, GRP, NSE, Beta-endorphin, calcitonin, and keratin were found in the cytoplasm of the tumor cells by indirect immunoperoxidase techniques. We could find no previously reported case of laryngeal oat cell carcinoma with ectopic ACTH syndrome or cytoplasmic localization of polypeptides.     

Cushing’s Syndrome in a Child with Pancreatic Acinar Cell Carcinoma

A case of pancreatic acinar cell tumor (ACC) is presented in a 10-year-old boy. The tumor manifested clinically with Cushing’s syndrome, high serum adrenocorticotropic hormone (ACTH) and cortisol concentrations. In addition, excessive serum levels of alpha-fetoprotein (AFP) were detected. Surgical resection was not possible due to retroperitoneal invasion. Biopsy of the mass showed a solid, poorly differentiated ACC of the pancreas. Periodic acid Schiff positive cytoplasmic granules, trypsinogen, keratins, alpha-1-antitrypsin, and AFP were identified in the tumor cells. Electron microscopy demonstrated zymogen granules as well as isolated dense core granules. Using immunochemiluminometric assay, a high quantity of ACTH was found in the fresh frozen tumor extract. ACTH, chromogranin A, and corticotropin-releasing factor were identified only in a few cells by immunohistochemistry. Combined radiochemotherapy was temporarily effective in reducing the tumor mass and serum AFP. Serum ACTH and cortisol levels dropped progressively and definitively to normal values after chemotherapy, and the Cushing’s syndrome subsided. Two years later, the patient died with metastatic disease. The presented case of ACC is interesting due to high serum AFP values and ectopic ACTH secretion resulting in Cushing’s syndrome. Andrea Luczay is working now at the Second Department of Pediatrics, Semmelweis University, Budapest, Hungary.     

ACTH-producing pheochromocytoma.

A benign adrenal medullary tumor that secreted adrenocorticotropic hormone (ACTH) was associated with bilateral adrenocortical hyperplasia and clinically evident Cushing syndrome. The clinical and chemical features were those usually associated with pituitary Cushing disease, including partial suppression of urinary OH steroids after administration of 8 mg of dexamethasone. The fractionization of the tumor's ACTH revealed 70% little "biologically active" ACTH, which is usually found in this concentration only in pituitary tissue.     

MULTIPLE HORMONE PRODUCTION IN AN OAT CELL CARCINOMA OF THE LARYNX

A 60-year-old white woman with laryngeal oat cell carcinoma is described. She was a heavy smoker who had been treated seven years earlier with 5,000 rads for a well differentiated squamous cell carcinoma metastatic to a left submandibular lymph node. She presented this time with a two month history of hoarseness and tumor of the supraglottic larynx was found. There was clinical and chemical evidence of an ectopic ACTH syndrome. The histology and fine structure of the tumor were typical of oat cell carcinoma. Immuno-reactive ACTH, GRP, NSE, Beta-endorphin, calcitonin, and keratin were found in the cytoplasm of the tumor cells by indirect immunoperoxidase techniques. We could find no previously reported case of laryngeal oat cell carcinoma with ectopic ACTH syndrome or cytoplasmic localization of polypetides. ACTA PATHOL. JPN. 35: 915–923, 1985.     

Galectin-3 expression in functioning and silent ACTH-Producing adenomas

Galectin-3 (Gal-3), a β galactoside-binding protein, has been implicated in a variety of biological functions including cell growth, differentiation, tumor cell adhesion, angiogenesis, tumor progression, and metastasis. We recently reported that Gal-3 was expressed in a subset of normal pituitary cells and tumors including PRL, ACTH, and in folliculostellate (FS) cells and tumors [1,2] and that Gal-3 had an important regulatory role in pituitary cell proliferation [1]. We further investigated the expression of Gal-3 protein in ACTH- and PRL-producing tumors and the expression of various galectin mRNAs by RT-PCR in pituitary adenomas and normal pituitary. Most silent ACTH subtypes 1 and 2 adenomas were negative or only focally positive for Gal-3 expression compared to functioning ACTH tumors from patients with Cushing’s disease and Nelson’s syndrome. In the normal pituitary, Gal-3 was expressed in less than 1% of the basophil-invading cells (ACTH cells present in the posterior pituitary) and in a subset of the anterior lobe ACTH-positive cells. RT-PCR analyses showed that many members of the galectin family including galectins 1, 2, 3, 4, 5, 6, 7, 8, and 9 were expressed in normal pituitary and in functioning ACTH- and PRL-producing tumors. These results indicate that Gal-3 is associated with functioning ACTH and PRL tumors and is expressed infrequently in silent ACTH adenomas, suggesting that Gal-3 protein and/or gene is altered in non-functioning ACTH tumors. The use of ACTH and Gal-3 immunostaining should help in the diagnosis of silent ACTH adenomas.     

Pituitary Carcinomas

Pituitary carcinomas are defined by their metastatic growth. Most of them also invade into surrounding tissues. They should be classified by the site of their metastases (cerebrospinal, systemic, or combined) and by the presumable cell type of origin, respectively with the hormone being demonstrable by immunohistochemistry (adrenocorticotrophic hormone [ACTH], prolactin [PRL], growth hormone [GH], hormone-negative). Pituitary carcinomas develop from invasive adenomas. Nearly all tumors had been treated by surgery or X-ray before they metastasized. Since 1976, 37 cases demonstrated with modern methods were reported: 23 had metastasized into the brain or meninges, 10 showed extracerebral metastases, and 4 showed both types of metastases. In our collection of pituitary tumors, three carcinomas (0.13%) were identified: two with systemic metastases (one ACTH secreting and one PRL secreting) and one with meningeal dissemination and ACTH production. The diagnosis of pituitary carcinomas should be based on four criteria: a demonstrable metastasis, identification of the primary tumor as a pituitary tumor, similarity between the structure and immunohistological marker expression of metastsis and primary tumor, and exclusion of an alternative primary tumor.     

The corticotroph of the rat adenohypophysis: A comparative study

The immunological and cytological specificity of antiserum to adrenocorticotrophic hormone was established and the corticotroph defined at the light microscopic level by immunocytochemistry. Our observations substantiated recent reports by several investigators. By comparing thin methacrylate sections with adjacent 1 μ-methacrylate sections immunochemically stained with anti-ACTH, the ACTH cell was identified at the ultrastructural level and its morphology studied. Using the criteria established at light and electron microscopic levels, the corticotroph was readily found in tissue which had been fixed in paraformaldehyde-osmium mixture and embedded in epoxy resin. Ultrastructural changes in this cell in response to experimentally designed alterations in the pituitary ACTH levels confirmed this identification. The ACTH cell was likewise identified in glutaraldehyde-fixed material. Thyrotrophs were tentatively defined. Several ultrastructural studies responsible for conflicting reports in the literature were investigated. It was concluded that the corticotroph is a unique cell type and that granule size and morphology remain important criteria for adenohypophyseal cell identification regardless of the fixation used. The need for a definitive immunocytochemical study of thyrotrophs at the ultrastructural level was recognized.     

Immunohistochemical and immunoelectron-microscopic study of pituitary adenomas associated with Cushing''s disease. A report of 13 cases.

Thirteen pituitary adenomas were removed from patients with Cushing's disease by the transphenoidal route. All cases demonstrated a typical histochemical and ultrastructural pattern. Immunocytochemical study by means of the immunoperoxidase technique and light or electron microscopy demonstrated 1-24/1-39 adrenocorticotropic hormone (ACTH) in all cases, lipotropin/melanotropin (beta-LPH/beta-MSH) in 10 cases, beta-endorphin in 8 cases, and an absence of calcitonin in all cases. In addition, in 2 cases tumor tissue contained a few antiprolactin immunoreactive cells. These ACTH, beta-LPH, and beta-endorphin immunoreactivities may reflect either the peptides themselves or their precursors or intermediate products. The authors also suggest a possible intermediate-lobe-like processing of beta-LPH leading to beta-endorphin production, which may act on PRL cells. In addition, no positive arguments for the existence of a common precursor for calcitonin and ACTH could be provided from this study.     

MALIGNANT PHEOCHROMOCYTOMA WITH ACTH PRODUCTION

An autopsy case of a 54-y-ear-old woman with malignant pheochromocytoma and ectopic ACTH production was reported. Noradrenaline was increased in 24 hour urine and in the blood sample from the left adrenal vein. Hormone assay studies of the tumor tissue and plasma revealed abnormally high levels of ACTH. Formaldehyde fume induced fluorescence method demonstrated biogenic amine in the tumor cytoplasm. Electron microscopic examinations also disclosed numerous neurosecretory granules in the tumor cytoplasm. These findings confirmed that this pleomorphic carcinoma of the left adrenal gland was one of the APUDoma originating from the adrenal medulla, so-called pheochromocytoma. ACTA PATHOL. JPN. 34 : 1403–1410, 1984.     

Insights into the Infiltrative Behavior of Adamantinomatous Craniopharyngioma in a New Xenotransplant Mouse Model

Adamantinomatous craniopharyngiomas (adaCP) cause hypothalamic pituitary dysfunction. Elucidation of pathomechanisms underlying tumor progression is essential for the development of targeted chemotherapeutic treatment options. In order to study the mechanisms of tumor outgrowth, we implanted human primary adaCP tissue from three different surgical specimens stereotactically into the brain of immunodeficient mice (n = 20). Three months after tumor inoculation, magnetic resonance imaging and histology confirmed tumor engraftment in all 20 mice (100%) that obtained tissue transplants. The lesions invaded adjoining brain tissue with micro finger‐shaped protrusions. Immunohistochemical comparison of the primary tumor and xenotransplants revealed a similar amount of proliferation (Mib‐1) and cytokeratin expression pattern (KL‐1). Whole tumor reconstruction using serial sections confirmed whirl‐like cell clusters with nuclear β‐catenin accumulations at the tumor brain border. These whirls were surrounded by a belt of Claudin‐1 expressing cells, showed an activated epidermal growth factor receptor (EGFR) and distinct CD133 as well as p21^WAF1/Cip1 positivity, indicating a tumor stem cell phenotype. Consistent with our previous in vitro studies, intracranial xenotransplants of adaCP confirmed cells with nuclear β‐catenin and activated EGFR being the driving force of tumor outgrowth. This model provides the possibility to study in vivo tumor cell migration and to test novel treatment regimens targeting this tumor stem cell niche.     

FatalAspergillus fumigatus septicemia in a patient with an ectopic ACTH-producing neuroendocrine pancreatic tumor

The case of a 71-yr-old Caucasian patient who suffered from an adrenocorticotropic hormone (ACTH)-producing tumor and developed a fulminantAspergillus fumigatus septicemia is reported. The patient presented initially with a recent onset of hyperglycemia and had excessive plasma levels of cortisol and ACTH. A computer-assisted tomography scan showed a tumor associated within the head of the pancreas. During his hospital course, he developed a rapid progressive fatalAspergillus fumigatus pneumonia and an upper gastrointestinal bleeding. At necropsy multiple abscesses in the brain, lungs, heart, kidneys, small bowel and mesentery were present as a result of Aspergillus septicemia. A neuroendocrine pancreatic tumor, 8 cm in diameter, was found in the head of the pancreas without any signs of malignancy. The tumor showed immunohistochemical and electron microscopic evidence of ACTH expression, but of no other pituitary or pancreatic hormones. This case demonstrates the fulminant progress of a septicemia based on an immunologically compromised patient because of ectopic Cushing’s syndrome. Early diagnosis of ectopic ACTH syndromes is essential for adequate therapy in order to prevent complications and fatal infections.     

Correlation of alkaline phosphatase staining of cortisol-producing adrenocortical tumors with dexamethasone suppression and ACTH stimulation.

The histochemical activity of alkaline phosphatase (Al-Pase), the induction of which is one of the effects of ACTH on the adrenocortical cells, was examined in 10 adrenocortical tumors causing Cushing's syndrome and in 65 adrenal cortices. All of the compact cells in every gland, and almost all, about half, or a small proportion in the four tumors showed Al-Pase activity. These tumors decreased in steroidogenesis after the administration of dexamethasone. No compact cells exhibited the activity in six tumors, none of which was "dexamethasone-suppressible". Three of the seven attached glands examined were halfway between those of typical Cushing's syndrome and those of other than Cushing's syndrome from the viewpoint of compact/clear cell morphology. All of the tumors that had Al-Pase-positive clear cells increased in steroidogenesis after ACTH administration. These results suggested that Al-Pase activity of tumor cells was also ACTH effect and that a decrease in steroidogenesis of tumors after dexamethasone administration was due not to fluctuations but to suppression of intrinsic ACTH.     

Adrenocorticotropic hormone cells and immunoreactive beta-endorphin cells in the human pituitary gland: normal and pathologic conditions studied by the peroxidase-labeled antibody method.

The comparative immunohistochemical localization of adrenocorticotropic hormone (ACTH) and beta-endorphin-like immunoreactivity (referred to as beta-endorphin) was studied in 16 human anterior pituitary glands from 6 normal adults, 4 normal fetuses, 1 stillborn infant, 2 anencephalic infants, 1 adult with Crooke's hyaline degeneration, and 2 patients with pituitary adenomas associated with Cushing's syndrome. Mirror sections were used in order to enable precise comparison of the same cells on the two consecutive tissue sections. In these normal and pathologic human anterior pituitary glands, ACTH and beta-endorphin were mostly localized in the same cells. In the normal adult pituitary glands, however, some ACTH cells were negative for beta-endorphin; and in one of the pituitary adenomas, some tumor cells were positive for only one hormone (beta-endorphin). These data suggest concomitant production of ACTH and beta-endorphin in the same cells and support the production of precursor molecules for these two hormones. The significance of ACTH-positive, beta-endorphin-negative normal cells and beta-endorphin-positive, ACTH-negative tumor cells is also discussed.     

Oat cell carcinoma of the urinary bladder with ectopic adrenocorticotropic hormone production

A case of primary oat cell carcinoma of the urinary bladder with ectopic adrenocorticotropic hormone (ACTH) secretion in a 55-year-old woman is reported. Cystoscopy revealed a large necrotic tumor in the bladder; the tumor was partially electroresected. At autopsy, a large residual tumor infiltrating the wall of the bladder was found. It had metastasized to the lymph nodes, left ovary, liver, spinal canal, bone marrow, and hypophysis. Bilateral adrenocortical hyperplasia was found. Microscopically, the tumor was identical morphologically to oat cell carcinoma, and the cells contained argyrophilic hormone granules and immunohistochemically demonstrable ACTH. Apparently, oat cell carcinomas with endocrinologic activity have not been reported previously in the urinary bladder.     

Pituitary tumorigenesis and hPit-1 cells.

Despite decades of clinical data verifying the success of therapeutic approaches to human pituitary tumors, a significant number of tumors progress and can be life-threatening. The development of better therapeutic strategies for pituitary tumors is complicated by the relative scarcity of human pituitary material for basic experimentation. Human pituitary tissue was used to derive cell cultures, and a cell line, hPIT-1. Molecular and functional analyses were used to further characterize the cells as human pituitary explants in vitro. Functional analyses of the cell cultures indicated that the cells were tumorigenic and of human folliculostellate origin. hPit-1 cells revealed numerous abnormalities of ploidy. Molecular analyses indicated the absence of expression of the following pituitary hormones or hormone subunits by this culture: growth hormone, prolactin, ACTH, FSHbeta, LHbeta, THbeta, and p-glycoprotein. By contrast, the cells expressed uniformly high levels of human follistatin mRNA. Finally, the cells are moderately tumorigenic in immune-deficient mice. Although the precise molecular genetic mechanisms for tumorigenesis in the established cell culture are unknown, the cells serve as a future resource in the study of pituitary tumor initiation, progression, and response to therapy.