Clinical risk factors for malignancy and overall survival in patients with pheochromocytomas and sympathetic paragangliomas: primary tumor size and primary tumor location as prognostic indicators.

CONTEXT: Pheochromocytomas and sympathetic paragangliomas are rare neuroendocrine tumors for which no precise histological or molecular markers have been identified to differentiate benign from malignant tumors. OBJECTIVE: The aim was to determine whether primary tumor location and size are associated with malignancy and decreased survival. DESIGN AND SETTING: We performed a retrospective chart review of patients with either pheochromocytoma or sympathetic paraganglioma. PATIENTS: The study group comprised 371 patients. MAIN OUTCOME MEASURES: Overall survival and disease-specific survival were analyzed according to tumor size and location. RESULTS: Sixty percent of patients with sympathetic paragangliomas and 25% of patients with pheochromocytomas had metastatic disease. Metastasis was more commonly associated with primary tumors located in the mediastinum (69%) and the infradiaphragmatic paraaortic area, including the organ of Zuckerkandl (66%). The primary tumor was larger in patients with metastases than in patients without metastatic disease (P < 0.0001). Patients with sympathetic paragangliomas had a shorter overall survival than patients with pheochromocytomas (P < 0.0001); increased tumor size was associated with shorter overall survival (P < 0.001). Patients with sympathetic paragangliomas were twice as likely to die of disease than patients with pheochromocytomas (hazard ratio = 1.93; 95% confidence interval = 1.20-3.12; P = 0.007). As per multivariate analysis, the location of the primary tumor was a stronger predictor of metastases than was the size of the primary tumor. CONCLUSIONS: The size and location of the primary tumor were significant clinical risk factors for metastasis and decreased overall survival duration. These findings delineate the follow-up and treatment for these tumors.

Regulated expression of GATA-6 transcription factor in gastric endocrine cells.

BACKGROUND & AIMS: GATA transcription factors may regulate gene expression in developing tissues, including gut epithelium. In the stomach, their expression has been linked to regulation of proton pump genes. However, GATA consensus sequences also occur in the promoter of the histidine decarboxylase gene, located in enterochrommafin-like cells. The aim of this study was to determine if GATA factors are located in gastric endocrine cells and to examine their expression during development and in response to changes in the gastric luminal environment. METHODS: Polymerase chain reaction cloning, Northern blot, and gel shift assays were used to examine GATA expression in gastric endocrine cells; changes in GATA messenger RNA during development and in response to fasting, feeding, and gastric achlorhydria were determined by Northern blot. RESULTS: GATA-6 was expressed strongly in rodent gastric endocrine cell fractions, in a human ECL cell tumor, and in an endocrine cell line (STC-1) derived from gut epithelium; proteins from STC-1 cells bound specifically to GATA consensus sequences in the human histidine decarboxylase promoter. GATA messenger RNA abundance was up-regulated during terminal differentiation of the rat stomach and on feeding after a fast. CONCLUSIONS: The GATA-6 transcription factor is expressed in gastric endocrine cells and is a potential regulator of gastric differentiation and of genes involved in the response to feeding.

pp60c-src enhances the acetylcholine receptor-dependent catecholamine release in vaccinia virus-infected bovine adrenal chromaffin cells.

Secretion of catecholamines by adrenal chromaffin cells is a highly regulated process that involves serine/threonine and tyrosine phosphorylations. The nonreceptor tyrosine kinase pp60c-src is expressed at high levels and localized to plasma membranes and secretory vesicle membranes in these cells, suggesting an interaction of this enzyme with components of the secretory process. To test the hypothesis that pp60c-src is involved in exocytosis, we transiently expressed exogenous c-src cDNA using a vaccinia virus vector in primary cultures of bovine adrenomedullary chromaffin cells. Chromaffin cells infected with a c-src recombinant virus restored the diminished secretory activity accompanying infection by wild type virus alone or a control recombinant virus. The level of enhanced catecholamine release correlated directly with the time and level of exogenous c-src expression. These results could not be attributed to differences in cytopathic effects of wild type versus recombinant viruses as assessed by cell viability assays, nor to differences in norepinephrine uptake or basal release, suggesting that pp60c-src is involved in stimulus-secretion coupling in infected cells. Surprisingly, exogenous expression of an enzymatically inactive mutant c-src also restored catecholamine release, indicating that regions of the introduced c-src protein other than the kinase domain may affect catecholamine release. Secretory activity was elevated by both forms of c-src in response to either nicotine or carbachol (which activate the nicotinic and the nicotinic/muscarinic receptors, respectively). In contrast, release of catecholamines upon membrane depolarization (as elicited by 55 mM K+) or by treatment with the calcium ionophore A23187 was unaffected by either vaccinia infection or increased levels of pp60c-src. These results suggest that pp60c-src affects secretory processes in vaccinia-infected cells that are activated through ligand-gated, but not voltage-gated, ion channels.

Polyoma-induced neoplasms of the mouse adrenal medulla. Characterization of the tumors and establishment of cell lines.

BACKGROUND: Pheochromocytomas that are usually noradrenergic arise commonly in the adult rat adrenal medulla. The widely studied PC12 cell line, that is representative of these rat adrenal tumors, is also noradrenergic. The reasons for the absence of epinephrine production by most rat pheochromocytoma cells are unknown, and there are currently no adrenergic adrenal medullary cell lines. Pheochromocytomas are rare in mice. EXPERIMENTAL DESIGN: Tumors induced by polyoma virus in the adrenal medullas of postnatal mice were studied immunocytochemically for catecholamine biosynthetic enzymes in order to determine how their profiles of catecholamine production compared with those of rat pheochromocytomas. Clonal cell lines were established from a representative tumor and were evaluated for responsiveness to agents known to affect the development and function of normal and neoplastic rat chromaffin cells. RESULTS: Although adrenal medullary cells from normal rodents produce epinephrine before birth, polyoma-induced mouse adrenal tumor cells are immature or poorly differentiated. They synthesize norepinephrine, but not epinephrine, which during normal development is produced later than norepinephrine. They also produce relatively large quantities of dihydroxyphenylalanine, suggesting an abnormality of catecholamine biosynthesis such that tyrosine hydroxylase is not rate-limiting. Secretory granules are sparse, as demonstrated by electron microscopy or by staining for chromogranin A, and catecholamine stores are low. Further, the tumor cells appear to be phenotypically unstable, as judged from heterogeneous staining for tyrosine hydroxylase even in early passage, twice-cloned cell lines. Tumor cell morphology and catecholamine profiles appear to be unaffected or minimally affected by nerve growth factor, forskolin or dexamethasone, which are known to affect normal or neoplastic rat chromaffin cells. However, tumors formed after subcutaneous injection of cell lines into mice show up to a 10-fold increase in catecholamine stores, suggesting that the cells are subject to some forms of regulation. The cloned cell lines do not produce detectable polyoma virus, but express all three viral T antigens, including a characteristic, truncated form of large T. CONCLUSIONS: The findings suggest that the process of neoplastic transformation and/or the presence of polyoma virus T antigens results in suppression of the adrenergic phenotype in mouse adrenal chromaffin cells. T antigens might therefore be useful as tools for studying mechanisms that regulate the differentiation and maturation of chromaffin cells in normal and neoplastic states. Furthermore, although polyoma virus cannot be readily used to produce adrenergic cell lines from the mouse adrenal medulla, the lines that are produced might substitute for PC12 cells in some types of studies that require a mouse model.

Lambert-Eaton syndrome: antigen-antibody interaction and calcium current inhibition in chromaffin cells.

Plasma and IgG obtained from 10 Lambert-Eaton myasthenic syndrome (LES) patients (5 with carcinoma, 5 without associated cancer), 6 healthy subjects, and 1 patient with small-cell lung cancer (SCLC) were examined in their ability to recognize chromaffin cell antigens on Western blots. The pattern of antigen recognition was compared with the magnitude of inhibition of voltage-dependent calcium and sodium currents recorded with the patch-clamp technique from chromaffin cells. Eight of the 11 patients with LES and/or SCLC recognized plasma membrane proteins and 9 of the patients' IgG interacted with cytoplasmic antigens with no apparent pattern of antigen recognition between patients. Also, there was no obvious band pattern distinguishing patients with LES from those with LES and concurrent SCLC. Eighty percent of the LES patients' antibodies were capable of reducing the calcium current (ICa) in chromaffin cells. One of the novel findings of this study is that 30% of the patients had produced antibodies which were able to inhibit both calcium and sodium currents (INa). The heterogeneous response of the IgG on the Western blots does not appear to correlate with the efficacy of reducing the inward currents.

Effects of adrenal medulla grafts on plasma catecholamines and rotational behavior.

The mechanisms by which adrenal medulla grafts influence the function of host brains in animal models of Parkinson's disease are unclear. To explore this issue, fragments of adrenal medulla or sciatic nerve were transplanted into the lateral ventricle of bilaterally adrenalectomized (ADX) or sham-ADX rats with unilateral 6-hydroxydopamine lesions of the substantia nigra. Additional control group received sham-transplantation surgery. Behavioral effects of these procedures were tested following administration of apomorphine, amphetamine, or nicotine. Plasma catecholamines were measured before and after transplantation surgery. In both ADX and sham-ADX rats, adrenal medulla grafts produced greater decreases in apomorphine-induced rotational behavior than did sciatic nerve grafts or sham-transplanted groups. Decreases in rotation were smaller in ADX than in sham-ADX animals, regardless of graft treatment. Plasma catecholamines increased after transplantation surgery in each of the sham-ADX groups, regardless of graft type. Increases in plasma dopamine concentrations were associated with decreases in rotational behavior. Five months after transplantation, grafted chromaffin cells demonstrated catecholamine fluorescence, tyrosine hydroxylase (TH) and chromogranin A immunoreactivities, and expression of TH mRNA. It is concluded that adrenal medulla grafts produce decreases in apomorphine-induced rotation through a combination of two independent effects. One is a specific effect of adrenal medulla grafts. The second is a nonspecific effect that requires an intact adrenal gland and may be related to increases in plasma catecholamine concentrations.

Circulating gastrin, endocrine cells, histamine content, and histidine decarboxylase activity in atrophic gastritis.

Thirty-five patients with fundic atrophic gastritis and achlorhydria were classified in two groups according to the presence or absence of fundic argyrophil, mostly enterochromaffinlike cell hyperplasia. Among the biologic and histologic parameters studied, the hyperplasic group differed only by a circulating hypergastrinemia and an antral G-cell hyperplasia. The histamine content, the histidine decarboxylase activity, and the mast cell number of fundic biopsies were determined in 10 controls, 16 of the preceding patients (11 with and 5 without fundic argyrophil-cell hyperplasia), and 5 patients with fundic atrophic gastritis and neither achlorhydria nor hyperplasia. Histamine content and histidine decarboxylase activity were increased only in the hyperplasic group despite an unchanged mast cell number. For all fundic biopsies the argyrophil-cell density was positively related to the histamine content. Finally, the argyrophil-cell hyperplasia occurring in fundic atrophic gastritis with achlorhydria is associated not with the gastritis intensity, as assessed by histologic and secretory criteria, but with a circulating hypergastrinemia and an increase of both fundic histamine content and histidine decarboxylase activity.

Demonstration of neuroendocrine cells in ovarian mucinous tumors.

The frequency of argyrophil cells in mucinous cystadenocarcinomas, borderline tumors (MBT) and cystadenomas was 29.8% (14 of 47), 46.7% (7 of 15) and 11.1% (2 of 18), respectively. These were statistically higher than the frequencies in 17 clear cell carcinomas, 43 serous cystadenocarcinomas, and 24 metastatic carcinomas. Immunoreactive cells for serotonin, somatostatin, gastrin, pancreatic polypeptide, growth hormone-releasing hormone, metenkephalin, neuron-specific enolase, and chromogranin-A were detected in almost all these cases with argyrophil cells. However, immunoreactivities for glucagon, vasoactive intestinal polypeptide, and adrenocorticotropic hormone were negative in ovarian mucinous tumors. Immunohistochemical multiplicity of neurohormones was remarkable in 15 MBT (including 5 müllerian and 10 intestinal MBT) and it was not related to the number of argyrophil cells per unit tumor cells. Individual hormones demonstrated here seemed to be present in different cells, but certain cells were immunoreactive for both gastrin and somatostatin by double immunostaining. Based on the high frequency of endocrine cells, borderline tumors seemed to be unique in the spectrum of mucinous ovarian tumors.

Fine-needle aspiration cytology of pheochromocytoma-ganglioneuroma of the organ of Zuckerkandl.

Fine-needle aspiration (FNA) cytologic and immunocytochemical findings of a rare combined pheochromocytoma-ganglioneuroma developing in a 48-yr-old Japanese man in the organ of Zuckerkandl are described. This is the first report of a combined pheochromocytoma-ganglioneuroma of the organ of Zuckerkandl. FNA cytology showed typical cytologic findings of these two components similar to those described individually in fine-needle aspirates of these neoplasms. The neoplastic cells showed positive reactions for vasoactive intestinal polypeptide, neuron-specific enolase, and S-100.

The distribution of duodenal G cells in duodenal ulcer patients.

The number of G cells is evaluated in biopsy specimens of fundic, antral and duodenal mucosa from the bulb, second and third parts in 10 patients with duodenal ulcer, and compared with that observed in 6 normal controls. G cells are absent in fundic mucosa but in the antrum their number in duodenal ulcer patients does not differ from that of controls and is strictly related to the histological pattern of the mucosa. In the second and third duodenum of duodenal ulcer patients the number of G cells is significantly higher in comparison with controls, while in the bulb the two groups do not differ significantly. Moreover, when different duodenal portions are compared no differences in the number of G cells are observed in the duodenal ulcer group; while in controls the bulbar number of G cells is higher in comparison with second and third duodenum.

Histopathology, cytology and cytochemistry of pheochromocytomas and paragangliomas including chemodectomas.

The results of histopathological, histochemical and ultrastructural investigations on pheochromocytomas and paragangliomas have been reported. These results allowed the functional identification of the cell types composing many of such tumours. Moreover, comparison of these data with clinico-pathologic findings outlined the advantages and limits of cytologic studies for understanding the natural history of pheochromocytomas and paragangliomas and improving our diagnostic and prognostic criteria.

Morphological studies on the paraneuron in spontaneously hypertensive rats.

Some of the paraneuronic cells in SHR in the prehypertensive stage were studied and compared to cells of the control. In the adrenal medulla, the area of the norepinephrine storing cell islets of SHR was about twice the size. The number of both the norepinephrine storing granules and vesicles in the cytoplasm was increased. In the anterior pituitary, percentage of the ACTH producing cell of SHR was increased. In the enterochromaffin cell, the average number of argyrophilic cells through the gut was larger in SHR. In the mast cells, an increased number of intrathyroidal mast cells was observed. These paraneuronic cells in SHR were considered to be hyperplastic and/or overactive from early stage after birth.

Gastric enterochromaffin-like cell hyperplasia and neoplasia in the rat: an indirect effect of the histamine H2-receptor antagonist, BL-6341.

Oral administration of BL-6341 hydrochloride, a long-acting histamine H2-receptor antagonist, to rats for 2 years at doses of 10, 55 or 300 mg/kg/day resulted in several changes in the fundic (oxyntic) mucosa of the glandular stomach. The most significant alteration was a proliferation of argyrophil endocrine cells that was demonstrated to be enterochromaffin-like (ECL) cells. The ECL cell proliferation consisted of a continuum of changes involving diffuse hyperplasia, focal adenomatous hyperplasia, and carcinoid tumor formation at the highest dose level of 300 mg/kg. At 55 mg/kg only ECL cell hyperplasia occurred, and at the low dose of 10 mg/kg there were no remarkable proliferative changes. The reference compound, cimetidine (950 mg/kg), produced a degree of ECL cell proliferation that was slightly less, but not significantly different than, that observed with 55 mg/kg of BL-6341. Dose-related elevations of serum gastrin were observed with BL-6341, while cimetidine produced hypergastrinemia that was generally intermediate between that produced by the middle and low doses of BL-6341. The hypergastrinemia resulted from the pharmacologic inhibition of acid secretion, which is the negative feedback mechanism controlling the production of gastrin. Only the 300 mg/kg dose of BL-6341 produced a significant, sustained (24 hours) hypergastrinemia and carcinoid tumors. The chronic, sustained hypergastrinemia was considered to be the primary cause of the ECL cell carcinoid neoplasia. All genetic toxicology tests performed with BL-6341 were negative. It was concluded that the demonstrated hypergastrinemia represents an indirect, hormonal, epigenetic mechanism of tumorigenesis.

Mucinous cystadenocarcinoma of the pancreas. Morphologic and immunocytochemical observations.

Twenty mucinous cystadenocarcinomas of the pancreas, most of which occurred in the tail of the pancreas in middle-aged women, were examined histologically and by immunohistochemical stains. Thirteen tumors displayed a marked histological heterogeneity and expressed intestinal differentiation as shown by the colonic appearance of the glands both at the light- and electron-microscopic levels. Other intestinal features included varying numbers of goblet cells, argyrophil and argentaffin cells, and even Paneth cells. By immunohistochemistry, endocrine cells were present in 13 of the 20 tumors (65%) and were more numerous in the poorly differentiated than in the well-differentiated epithelial component of the tumors. Serotonin-containing cells were the most common endocrine cells, followed by somatostatin-containing cells and cells that showed immunoreactivity for pancreatic polypeptide and gastrin. However, none of the patients had clinical manifestations of carcinoid, somatostatinoma, or the Zollinger-Ellison syndrome. The findings support the hypothesis that mucinous cystadenocarcinomas of the pancreas arise from an "endodermal stem cell" that differentiates into cells with intestinal phenotypes.

Hyperplastic lesions of the gastrointestinal endocrine cells.

A substantial body of knowledge is presently available on the morphologic, histochemical, ultrastructural, and functional characteristics of both the normal endocrine cell population of the gut and their related endocrine tumors. In contrast to this, we have only recently begun to recognize the existence of hyperplastic proliferations of various endocrine cell types, and information is therefore steadily accumulating on the morphologic criteria for their recognition, their clinicopathologic correlates and the clinical relevance of this morphologic finding. Hyperplastic proliferations of various endocrine cell types most often develop as a secondary phenomenon in a variety of clinical situations, and may modify the clinical course of the associated condition in a manner that underscores the functional interrelationships these endocrine cells have not only with each other but with other cell types as well. However, similar proliferations may also occur as a primary event (e.g. primary antral G-cell hyperplasia) and give rise to clinical and biochemical features attributable to the overproduction of their specific hormonal product (e.g. Zollinger-Ellison Syndrome, type I). This communication provides a broad overview of the current state of our knowledge of hyperplastic lesions of a variety of gut endocrine cell types in humans, their pathophysiologic significance, their relationship (if any) to the subsequent development of endocrine tumors (i.e. the hyperplasia-neoplasia sequence), and the utility of certain experimental models for the study of such proliferations in a variety of animal species.

The relationship of endocrine cells, dysplasia and carcinoembryonic antigen in Barrett's mucosa to adenocarcinoma of the oesophagus.

This study was undertaken to assess the prevalence and characteristic hormonal profile of endocrine cells in Barrett's mucosa and to determine to what extent this profile was shared by endocrine cells of adenocarcinomas arising therefrom. In addition, lower oesophageal carcinomas, not associated with columnar metaplasia, were examined to see if they exhibited a different hormonal profile. The patients studied comprised 43 who had had multiple oesophageal biopsies. 35 who had had oesophagogastric resection for adenocarcinoma arising in Barrett's mucosa and 26 in whom the resection showed no metaplastic epithelium adjacent to tumour. Argyrophil cells were present in 90% of biopsies and resections of Barrett's mucosa combined, irrespective of the histological type of metaplastic epithelium. By immunocytochemistry the most frequently identified substance in mucosal endocrine cells was serotonin (82%) followed by somatostatin (54%), secretin (22%) and pancreatic polypeptide (17%). Gastrin, bombesin, cholecystokinin, ACTH and substance P were not identified in metaplastic mucosa in any case. The difference in expression of serotonin by endocrine cells of tumours arising in Barrett's mucosa (31%) and those not (3.8%) was statistically significant (P less than 0.0186). Carcinoembryonic antigen (CEA) was demonstrated in 60% of oesophageal carcinomas, both endocrine positive and endocrine negative. Focal CEA expression was seen in 4.6% of biopsies and 14% of Barrett's mucosa adjacent to tumour. These results indicate a higher prevalence of endocrine cells in Barrett's mucosa than hitherto documented and suggest that serotonin may be a useful marker in distinguishing between primary oesophageal and putative gastric cancers at the gastro-oesophageal junction. The identification of CEA in oesophageal columnar epithelium is of little value in predicting the development of malignancy.

Activation and hyperplasia of gastrin and enterochromaffin-like cells in the stomach.

The stomach is rich in endocrine cells, most of which are still unidentified with respect to the peptide hormones they produce. The endocrine cell populations in the antrum usually differ from those in the oxyntic mucosa. Gastrin cells are found in the antrum and respond readily to stimuli from the gastric lumen, such as changes in the pH and the presence of food. In order to study the functional control of the antral gastrin cell, rats were subjected to different kinds of surgery. The serum gastrin concentrations in the various experimental groups were measured 8-10 weeks after the operations. Elevated antral pH raised the serum gastrin concentration. The combination of elevated antral pH and the passage of food over the pyloric glands produced gastrin cell hyperplasia. The operation that was most effective in inducing gastrin cell hyperplasia was removal of the acid-producing part of the stomach. Interestingly, gastrin cell hyperplasia was seen also after bilateral truncal vagotomy, indicating that an intact vagal innervation is not essential for the development of gastrin cell hyperplasia. Enterochromaffin-like (ECL) cells are endocrine/paracrine cells that are numerous in the acid-producing part of the stomach in many species. In the rat, they occur predominantly in the basal half of the oxyntic mucosa and produce and store histamine. The ECL cells have an unknown function and do not seem to respond to stimuli from the gastric lumen. They are activated by circulating gastrin and by vagal excitation. Gastrin mobilises histamine from these cells and activates the histamine-forming enzyme, histidine decarboxylase. Long-term hypergastrinaemia produces diffuse ECL cell hyperplasia, whereas hypogastrinaemia (following removal of the endogenous stores of gastrin by antrectomy) reduces the ECL cell number. Portacaval shunt brings about a marked increase in the number of ECL cells through an unknown mechanism. Also neuronal stimuli are important for the trophic control of the ECL cells. Studies of unilaterally vagotomised rats showed reduced weight and thickness of the oxyntic mucosa as well as a markedly reduced number of ECL cells on the denervated side. Gastric carcinoids in man are rare tumours predominantly made up of ECL cells. The incidence of such tumours is increased in patients with hypergastrinaemia (pernicious anaemia, Zollinger-Ellison syndrome). A diffuse ECL cell hyperplasia is a common finding in such patients, which is in keeping with the known gastrin sensitivity of the normal ECL cell in the rat.

Argyrophil cells in Brenner tumors: histochemical and immunohistochemical analysis.

Argyrophil cells were identified by the single-impregnation Grimelius technique in 11 of 28 (39%) Brenner tumors, accounting for less than 1% of the tumor cell population in all the cases. All tumors with argyrophil cells were stained to demonstrate calcitonin, somatostatin, gastrin, adrenocorticotropic hormone, neurotensin, insulin, glucagon, and serotonin; and four of them (three benign and one borderline) were also stained for chromogranins with the monoclonal antibody LK2H10. Serotonin was present in nine of the 11 cases with argyrophil cells. Neurotensin and somatostatin were found in one borderline tumor, which also contained serotonin. Chromogranin reactivity was demonstrated in all four cases in which it was examined. Ultrastructural examination of one tumor revealed that the argyrophil cells contained secretory granules, 80 nm in diameter, and had elongated cytoplasmic processes that extended between the more numerous nonargyrophil tumor cells. The argyrophil cells of Brenner tumors are similar to those of urothelium in the frequency with which they are immunoreactive for serotonin and the rarity with which they are reactive for peptide hormones. These cells differ from those of mucinous ovarian tumors, which often contain both serotonin and peptide hormones. The findings of this study lend additional support to the close similarity of the epithelial components of Brenner tumors and urothelium.