Identification of M2 macrophages in anterior pituitary glands of normal rats and rats with estrogen-induced prolactinoma.

Macrophages are present throughout the anterior pituitary gland. However, the features and function of macrophages in the gland are poorly understood. Recent studies have indicated that there are two main macrophage classes: M1 (classically activated) and M2 (alternatively activated). In this study, we examine whether both M1 and M2 macrophages are present in the anterior pituitary gland of rats. Our findings indicate that macrophages that are positive for CD68 (a pan-macrophage marker) were localized near capillaries in rat anterior pituitary gland. These macrophages were positive for iNOS or mannose receptor (MR), which are markers of M1 and M2 macrophages, respectively. To determine the morphological characteristics of M2 macrophages under pathological conditions, diethylstilbestrol (DES)-treated rats were used as an animal model of prolactinoma. After 2 weeks of DES treatment, a number of MR-immunopositive cells were present in the gland. Immunoelectron microscopy revealed that MR-immunopositive M2 macrophages had many small vesicles and moderately large vacuoles in cytoplasm. Phagosomes were sometimes present in cytoplasm. Interestingly, M2 macrophages in prolactinoma tissues did not usually exhibit distinct changes or differences during the normal, hyperplasia and adenoma stages. This study is the first to confirm that both M1 and M2 macrophages are present in the anterior pituitary gland of rats. Moreover, the number of M2 macrophages was greatly increased in rats with DES-induced prolactinoma. Future studies should attempt to characterize the functional role of M2 macrophages in the gland.

Temozolomide therapy in a man with an aggressive prolactin-secreting pituitary neoplasm: Morphological findings.

Administration of temozolomide to a 46-year-old man with an invasive aggressive prolactin (PRL)-secreting pituitary neoplasm resulted in improvement of the clinical condition and significant decrease of blood PRL levels. Histologic, immunohistochemical, and electron microscopic study demonstrated marked morphological differences in the tumor exposed to temozolomide compared with the unexposed tumor. Necrosis, hemorrhagic areas, accumulation of connective tissue, focal inflammatory infiltration, and neuronal transformation were seen. Immunohistochemical prognostic indicators showed a reduction in growth potential. Based on the clinical, laboratory, and morphological findings, we recommend temozolomide therapy in patients with pituitary tumors not responding adequately to other treatment options.

Pattern of FGF-2 isoform expression correlated with its biological action in experimental prolactinomas.

Fibroblast growth factor-2 (FGF-2) synthesized in the pituitary is involved in the formation and progression of pituitary tumors. The aim of this study was to analyze the pattern expression of two FGF-2 isoforms at different subcellular levels and to determine its correlation with prolactinoma development. Estrogen administration to male rats for 7, 20, and 60 days generated pituitary tumors, with lactotrophs being the prevalent cell type. Ultrastructural immunolabeling showed FGF-2 in the cytosolic and nuclear compartments of somatotrophs, lactotrophs and gonadotrophs, as well as in folliculo-stellate cells of normal rats. Estrogen stimulation increased FGF-2 immunoreactivity in various tumors and enhanced the expression of two FGF-2 isoforms, 18 and 22 kDa, as quantified by western blot. The 18 kDa isoform observed in cytosol extracts reached the highest levels after 60 days of hormonal stimulation and this was related to lactotroph proliferation. However, the 22 kDa FGF-2 isoform was only detected in the nuclear compartment and achieved the maximum expression at 7 days of estrogen treatment, without any correlation with lactotroph proliferation. These results suggest that the 18 kDa FGF-2 may play a role in the modulation of lactotroph proliferation in prolactinomas induced by estrogen. The overproduction of both FGF-2 isoforms appears to be implicated in autocrine-paracrine-intracrine mitogenic loops; this FGF-2 activity could lead to uncontrolled cell growth, angiogenesis, and tumor formation.

Giant invasive pituitary adenoma extending into the sphenoid sinus and nasopharynx: report of a case with intraoperative cytologic diagnosis.

BACKGROUND: Invasive pituitary adenomas involving the skull base are difficult to distinguish from other, more aggressive tumors. Intraoperaive diagnoses are crucial for deciding the course of treatment. CASE: A large mass extending from the sella turcica to the sphenoid sinus and nasopharynx was identified in a 42-year-old male. Because of the lack of endocrine abnormalities and lack of an apparent rise in pituitary hormones, preoperative diagnoses included chordoma, chondrosarcoma, meningioma and pituitary adenoma. Tumor fragments were easily squeezed into a thin layer of cells for cytologic specimens. Uniform, round tumor cells were arranged in minimally cohesive cell sheets and possessed regular, ovoid nuclei with a fine chromatin pattern and granular cytoplasm with prominent Golgi areas. The cytologic features indicated a probable diagnosis of pituitary adenoma and excluded other possibilities. Immunohistochemical demonstration of prolactin and ultrastructural features established the final diagnosis of prolactinoma. With the administration of bromocriptine, a large reduction in tumor size occurred. As compared to frozen sections, cytologic preparations are more effective for the intraoperative diagnosis of pituitary adenomas. Such neoplasms should always be included in the differential diagnosis of tumors involving the skull base.

Treatments of multi-invasive giant prolactinoma.

We present a 68-year-old male patient with an exceptionally aggressive tumour which invaded to the skull base, cavernous sinus, nasopharynx, sphenoid sinus, pituitary fossa, bilateral parasellar regions, premedullary cistern, and left infratemporal fossa. Headache was the only symptom. The serum prolactin level was 95,973 ng/ml. The patient was treated by right subfrontal craniotomy with removal of the tumour. Because it did not respond well to surgical treatment and the electron micrograph showed abundant secretory granules in some parts of the specimen, post-operative radiotherapy and bromocriptine therapy were instituted. After combined therapies and a long-term follow-up, only little residual pituitary tumour was seen with serum prolactin progressively dropped to 717 ng/ml with no obvious symptoms. The histological findings, the ideal treatments and the clinical course of multi-invasive giant prolactinoma will be discussed.

Salmon roe-like amyloid deposition in a prolactinoma: a case report.

An unusual case of prolactin-producing adenoma with extensive amyloid deposition is reported to clarify its radiological, intraoperative, and light- and electron-microscopic findings. A 41-year-old female patient complained of amenorrhea persisting for 20 years. Magnetic resonance imaging (MRI) revealed a pituitary adenoma, which included low-intensity spots on T1- and T2-weighted images. Intraoperative examination found multiple small, yellowish, spherical masses resembling salmon roe within the adenoma. Light and electron microscopy revealed the presence of immunoreactive cells for prolactin intermingled with concentric lamellar bodies of radially arranged amyloid fibrils originating from the endoplasmic reticulum in prolactinoma cells. The extracellular lamellar amyloid deposits were apparently due to degradation of prolactin-producing cells, but the reason for the production and radially arranged accumulation of amyloid remains to be identified.

Prolactin-producing pituitary adenoma associated with prolactin cell hyperplasia.

A 24-yr-old woman with amenorrhea, galactorrhea, hyperprolactinemia, and sellar mass underwent transsphenoidal surgery. Histologic, immunohistochemical, and electron microscopic investigation revealed a well-differentiated, sparsely granulated prolactin (PRL) cell adenoma of the pituitary showing conclusive PRL immunoreactivity. In the nontumorous adenohypophysis PRL cell hyperplasia was noted. Marked differences were evident between the neoplastic and hyperplastic areas. The tumor consisted of sparsely granulated PRL cells immunoreactive only for PRL. As demonstrated by immunoelectron microscopy, the hyperplastic area comprised monohormonal sparsely granulated PRL cells as well as bihormonal mammosomatotrophs immunoreactive for both PRL and growth hormone. The MIB-1 index was higher whereas microvessel density was lower in the adenoma as compared with the hyperplastic area. In addition, the nontumorous area showed lymphocytic infiltration whereas inflammatory reaction was not seen in the adenoma. This case represents a rare association of a PRL cell adenoma and PRL cell hyperplasia. The fact that these two lesions were contiguous in the surgically removed material raises the possibility that hyperplasia can precede and transform into adenoma.

Medical management of pituitary adenomas: structural and ultrastructural changes.

The morphology of the various pituitary cell types is highly dynamic and allows recognition of many cellular functions. Most pituitary cells show morphologic changes that reflect stimulation or inhibition by hormones. Drugs have also been shown to alter the morphology of several pituitary tumor types, allowing a measure of therapeutic efficiency and a careful dissection of the mechanisms of action of various medical therapies. In some cases, these morphologic alterations can evoke diagnostic problems.

Secretory granules of pituitary adenomas: quantitative study of hormonal antigenicity.

Ultrastructurally, the antigenicity of major pituitary hormones in secretory granules was quantitatively investigated in five growth hormone (GH)-secreting adenomas, five prolactin (PRL)-secreting adenomas and eight clinically non-functioning (CN-F) adenomas. Sparsely granulated cells with a few or several small secretory granules (60-100 nm) exhibiting little or only weak antigenicity of various biochemically unrelated hormones were commonly observed in CN-F adenomas and occasionally in GH- and PRL-secreting adenomas. GH- or PRL-secreting adenomas consisted of many densely granulated cells with medium-sized (200-250 nm) or large (over 250 nm) secretory granules and a few or several sparsely granulated cells with small secretory granules. The densely granulated cells showed intense GH or PRL antigenicity and slight to moderate antigenicity for other hormones in large secretory granules and little or only weak antigenicity for various hormones including GH or PRL in small secretory granules. Their secretory granules larger than 160 nm or 140 nm significantly exhibited intense GH or PRL antigenicity (Fisher's exact test; P < 0.05 and < 0.01, respectively). Two CN-F adenomas showed sparsely and densely granulated cells as well as intermediate cells. The densely granulated cells closely resembled GH-secreting cells. The intermediate cells simultaneously included small and medium-sized or large secretory granules exhibiting little/slight and intense GH-antigenicity, respectively. This study indicates that sparsely granulated cells of different categories showing slight antigenicity for various hormones, antigenically share the same origin, and that their hormonality, single or multiple, may be selectively activated in the developmental course of secretory granules.

Intracellular distribution of GABA in the rat anterior pituitary. An electron microscopic autoradiographic study.

We studied the internalization and intracellular distribution of [3H] GABA in rat anterior pituitary cells. Electron microscopic autoradiography of anterior pituitary fragments or dispersed pituitary cells incubated with [3H] GABA showed that lactotrophs and, to a lesser extent, somatotrophs were the only cells that contained radioactive grains. Grain density analysis performed on dispersed pituitary cells after a pulse-chase experiment (10 min pulse and then change to a medium without radioactive GABA for various periods up to 2 h) revealed that GABA internalized by lactotrophs was distributed in various intracellular membranous organelles. Of the cell compartments examined, plasma membrane, Golgi apparatus, mitochondria and secretory granules had different time-dependent labeling patterns. The highest grain density values were associated with plasma membrane (at the first chase time) and the Golgi apparatus. Mitochondria and secretory granules also showed significant grain density values. A similar pattern of distribution was observed when fragments of prolactin-secreting pituitary adenomas were incubated with [3H] GABA. These results provide morphological data on the cellular specificity and intracellular distribution of GABA in anterior pituitary cells.

Functional membrane changes due to tumor induction in rat pituitary cell cultures.

Membrane functions in tumorous cells are different from those in healthy cells. The aim of the present study was to investigate the changes in pituitary cell membrane functions and hormone secretion after tumor induction in vivo and in vitro. Prolactinomas were induced in vivo in female Wistar rats with estrone acetate. Normal anterior pituitaries and prolactinomas of female Wistar rats were dissociated enzymatically and mechanically, then cultured on collagen-treated plastic dishes. Some normal anterior pituitary cultures were treated with benz(c)acridines as tumorigenic agents in vitro. Intracellular 3',5'-cyclic-adenosine monophosphate (cAMP) levels were determined by a competitive binding technique, membrane fluidity was assayed by fluorescence anisotropy, and ATP-ase activities were estimated via ATP loss. The results indicated decreased membrane fluidity in tumorous cell cultures. However, in vitro benz(c)acridine treatment exerted more pronounced effects than those observed after in vivo estrone treatment. The ATP-ase activities were highly increased in benz(c)acridine-treated cells and in estrogen-induced prolactinoma cells, more strongly so in the former ones. The intracellular cAMP levels were higher than normal in both of them. The results concerning the ACTH, alpha-MSH, PRL and GH levels of normal and tumorous cell cultures were published in our previous study. Our findings show that the tumorous transformation of pituitary cells can cause significant changes in functional membrane parameters and hormone secretion. Decreased membrane fluidity was accompanied by an increased exocytosis (hormone release) and adenylate cyclase activity in tumorous cells.

Characterization of spherical amyloid protein from a prolactin-producing pituitary adenoma.

Prolactin (PRL)-producing pituitary adenomas are in some cases associated with deposition of abundant spherical amyloid; however, the origin of the amyloid has not been established. In this report, a PRL-producing pituitary adenoma composed almost entirely of spherical amyloid was analyzed biochemically. The tumor was removed surgically from a 56-year-old man. Immunohistochemical analysis revealed that residual tumor cells were strongly positive for PRL, while the spherical amyloid was not. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a band of approximately 4 kDa associated with the amyloid, which was not present in a nonamyloid producing prolactinoma. The 4-kDa band is similar in size to other known amyloidogenic peptides. Immunoblot analysis of the tumor material using polyclonal anti-human PRL antibodies revealed a small amount of normal-sized PRL; however, the abundant 4-kDa band was nonimmunoreactive. Amino acid sequencing showed that this peptide represents the first 34 amino acids of the intact PRL protein with a predicted size of 4313 Da. The presence of a small amount of normal-sized PRL in this tumor, as well as elevated circulating levels of PRL implies that intact PRL is being abnormally processed in the formation of spherical amyloid.

Human prolactinomas in cell culture: structure-function correlation and indication of early relapse.

Cell cultures of 7 micro- and 16 macroprolactinomas obtained from twenty-three patients (10 males and 13 females) by transsphenoidal adenomectomies were found to be capable of secreting hormones in vitro. Prolactin levels in the culture media were measured during the culture periods and showed that the high PRL levels in culture media may indicate early relapse. Morphological features of both micro- and macroprolactinomas in vitro resembled those of the original tumors. Most macroprolactinomas harbored densely-granulated cells, while most microprolactinomas sparsely-granulated cells. The cell culture techniques combined with morphological studies of prolactinomas allow further investigation of structure-function correlation in human prolactinomas.

Usefulness of electron microscopy in the diagnosis of "small" round cell tumors of the sinonasal region.

The sinonasal region is known to harbor several types of tumors that belong to the general category of "small" round cell tumors and offer considerable diagnostic challenges. This study evaluated 33 cases of such tumors by electron microscopy to characterize their ultrastructural features in conjunction with immunohistochemistry, in an attempt to define diagnostic criteria of various types. Electron microscopy was useful in the proper classification of tumors in 27 cases: esthesioneuroblastoma (EN), 12; undifferentiated carcinoma, 6; melanoma, 3; lymphoma, 3; melanotic neuroectodermal tumor, 1; rhabdomyosarcoma, 1; and pituitary adenoma, 1. In the remaining six cases, the ultrastructural features were those of poorly differentiated carcinomas. They usually exhibited some epithelial characteristics as well as neuroendocrine features by immunohistochemistry and electron microscopy. These tumors could be best described as poorly differentiated neuro-endocrine carcinomas (malignant neuroepitheliomas). The most controversial diagnostic problems existed between the tumors categorized as esthesioneuroblastomas and neuroendocrine (NE) carcinomas. Esthesioneuroblastomas were characterized by uniform round nucleated cells with variable amounts of dendritic processes containing numerous dense core granules ranging from 150 to 350 nm in the perikarya and dendritic processes. Dendritic processes contained longitudinally arranged neural tubules and revealed an occasional synaptic junction. In three of the 12 cases of EN, cells with the appearance of sustentacular cells were recognized by electron microscopy. The NE carcinomas usually consisted of closely packed round cells with scanty cytoplasm that lacked any feature of neuroblastic cells. The tumor cells in this category often were epithelioid in appearance and exhibited a varying degree of cytokeratin positivity. Neuron-specific enolase was also positive in all cases, further suggesting their neuroepithelial nature. The greatest difference between EN and NE carcinomas was the absence of sustentacular cells in NE carcinomas. Immunohistochemical and electron microscopic studies are essential in the differential diagnosis of EN and NE carcinomas, because their microscopic appearance is very similar. The study indicates that EM is useful in the diagnostic categorization of sinonasal tumors of uncertain nature, particularly when it is used in conjunction with immunohistochemistry.

Effects of bromocriptine on staining indices of Ki-67 and proliferating cell nuclear antigen, and nucleolar organizer region number in pituitary adenomas.

The effects of bromocriptine (CB-154) on the proliferative capacities of prolactinoma and somatotropinoma were investigated by immunocytochemical staining indices of proliferating cell nuclear antigen (PCNA) and Ki-67 (with MIB-1 antibody), and silver staining of nucleolar organizer region (NOR) number in histological sections. Patients with prolactinoma and somatotropinoma were divided into two groups: no preoperative treatment (control group), and treated with CB-154 for 2 weeks before adenomectomy (CB-154 group). The prolactinoma CB-154 group showed a significantly lower PCNA staining index (n = 6, 13.1 +/- 2.0%) and Ki-67 staining index (n = 6, 0.2 +/- 0.03%) than the control group (n = 4, 27.1 +/- 2.1%; n = 8, 1.9 +/- 0.5%; respectively) (p < 0.01). The somatotropinoma CB-154 group showed a significantly lower Ki-67 staining index (n = 5, 0.7 +/- 0.07%) than the control group (n = 11, 1.2 +/- 0.2%) (p < 0.05), but there was no significant difference in PCNA staining index (control: n = 5, 19.1 +/- 2.8% vs. CB-154: n = 5, 20.2 +/- 1.4%). However, variable intensities of PCNA staining between the cells were observed, resulting in an extraordinarily high staining index. NOR numbers did not vary significantly between the two prolactinoma groups (control: n = 4, 2.0 +/- 0.3 vs. CB-154: n = 6, 1.7 +/- 0.1) and two somatotropinoma groups (control: n = 5, 1.3 +/- 0.1 vs. CB-154: n = 5, 1.4 +/- 0.2).(ABSTRACT TRUNCATED AT 250 WORDS)

Heterogeneous expression of two somatostatin receptor subtypes in pituitary tumors.

The expression of two somatostatin receptor subtypes, SSTR1 and SSTR2, was studied in 27 pituitary tumors and 1 chronic lymphocytic leukemia pituitary infiltrate. Solution hybridization techniques were used for RNA analysis. SSTR1 and SSTR2 were, respectively, expressed in 3 of 7 and 9 of 10 GH-secreting tumors, 1 of 9 and 5 of 9 nonfunctioning tumors, 4 of 5 and 0 of 5 of prolactinomas, and 1 of 3 and 0 of 3 ACTH-secreting tumors. The chronic lymphocytic leukemia infiltrate expressed the SSTR2 subtype. No correlation was detected among tumor size, level of hormonal hypersecretion, and somatostatin receptor expression status. Two acromegalic patients who responded to octreotide therapy exclusively expressed the SSTR2 subtype in their tumors. The results indicate that two SSTR subtypes are heterogeneously expressed in different pituitary adenoma cell types.

Aggressive small cell tumor of the skull base.

A 40-year-old Black man presenting with increasing nasal discharge of bloody, mucoid pus as well as nasal obstruction over a 2-month period is described. Magnetic resonance imaging of the skull showed a tumor eroding through the skull base into the clivus and extending into the sphenoid sinus. Endoscopy of the sphenoid sinus demonstrated a polypoid mass extending into the posterior choanae. The lesion was partially resected. Histologic evaluation showed a cellular small blue cell tumor punctuated by bland, epithelial-lined microcysts. Electron microscopy revealed epithelial cells with abundant rough endoplasmic reticulum and electron-dense membrane-bound endocrine granules, some undergoing misplaced exocytosis. Immunohistochemical evaluation demonstrated cytoplasmic reactivity for neuron-specific enolase, synaptophysin, and prolactin. Stains for leukocyte common antigen, HMB-45, desmin, cytokeratin, chromogranin, and the remaining spectrum of pituitary hormones including growth hormone, corticotropin, luteinizing hormone, follicle-stimulating hormone, and thyrotrophic hormone were negative. In contrast, the epithelium lining the cysts was cytokeratin positive and synaptophysin negative. This ostensibly small cell tumor therefore represented a remarkably extensive and aggressive prolactin cell adenoma with unusual light microscopic features. Characterization of the lesion required electron microscopy and further confirmation by immunocytology. The distinction of pituitary adenomas and particularly of prolactin cell tumors from other adenoma types and from other small cell lesions markedly affects therapy and patient prognosis.

Ultrastructural study of a pituitary adenoma (prolactinoma) within the clivus bone using immunoelectron microscopy.

In a case of a pituitary adenoma in the clivus bone in a 71-year-old man, ultrastructural investigation using conventional aldehyde-fixed, epoxy-embedded tissue revealed the tumor to be composed of cells with euchromatic nuclei, dense nucleoli, abundant rough endoplasmic reticulum, spherical secretory granules, and granule extrusion at the lateral cell surface, all of which suggest a prolactin-producing adenoma. Using a protein A-gold immunolabeling technique on snap-frozen tissue subsequently fixed in a mild fixative and embedded in a hydrophilic resin, the presence of prolactin immunoreactivity within secretory granules at the ultrastructural level was demonstrated. This case represented the first use of protein A-gold immunolabeling at the electron microscopic level for diagnostic purposes at our institution and exemplifies the value of this technique when the need for diagnostic immunoelectron microscopy is not anticipated. Because this tumor arose in an unusual location, ultrastructural study, including immunoelectron microscopy, not only confirmed the light microscopic diagnosis of pituitary adenoma, but further allowed subclassification of the tumor.

[Inventions for preservation of hormonal function in long-term culture of human functioning pituitary adenoma].

This study was designed to establish in vitro model systems in human hormone-producing pituitary adenomas that are analogous to the in vivo cellular environment. Mechanically dispersed cells composed of single cells and aggregates from 6 pituitary adenomas (3 GH producing adenomas and 3 prolactinomas) were cultured on microporous membrane cell culture inserts (Millicell-CM) coated with Basement Membrane Matrigel for up to 6 months. Growth hormone or prolactin in the medium was measured during the culture, and morphological feature in vitro was also compared with that of the original tumor at intervals. Not only single cells but also large aggregated cells which usually float in the medium when seeded on conventional plastic, were flattened and firmly attached to coated microporous membrane under the control of medium volume in culture. In both type adenomas, especially prolactinomas, surviving aggregated adenoma cells revealed preserved hormone activity and no dedifferentiation of cell characteristics after 6 months in culture. Particularly during the first 2 months in culture, close similarity existed between in vivo and in vitro conditions with regard to cell morphology and hormone release. These results indicate that this new culture method may further aid the investigation of in vitro cellular structure and function in human pituitary adenomas under conditions which closely mimic the in vivo cellular environment.