Characterization of C-Kit (CD117) Expression in Human Normal Pituitary Cells and Pituitary Adenomas

c-kit (CD117) is a tyrosine kinase receptor involved in the proliferation, differentiation, and secretory functions of various cells. In experimental animal models, c-kit has been detected in the pars intermedia of the normal pituitary gland and in alpha-melanocyte-stimulating-hormone-positive adenomas and it has been suggested that it plays a role in regulating adrenocorticotropic hormone (ACTH) secretion. To the best of our knowledge, the expression of c-kit in normal human pituitary cells and in pituitary adenomas has never been reported, so the possible biological role of this receptor in the control of pituitary hormone secretion remains unclear. The aim of this study was to evaluate the immunohistochemical expression of c-kit in normal human pituitary glands and in a series of 62 well-characterized pituitary adenomas. In normal adenohypophyses, several cells, mainly located in the central mucoid wedge, showed a c-kit immunoreactivity (IR). Double label immunostaining procedures showed that the c-kit-IR cells corresponded to ACTH cells. Out of 62 adenomas, 15 (24%) were c-kit-IR, including 7/16 (44%) ACTH cell, 3/7 (42%) null cell, 4/11 (36%) alpha-subunit cell, and 1/11 (10%) follicle-stimulating hormone-luteinizing hormone cell adenomas. By contrast, all ten prolactin cell and seven growth hormone cell adenomas were c-kit negative. These data suggest that, in normal conditions, c-kit may be involved in the pituitary-adrenal axis regulation.     

Vascular Endothelial Growth Factor (VEGF) Expression in Human Pituitary Adenomas and Carcinomas

Vascular endothelial growth factor (VEGF) is a key mediator of endothelial cell proliferation, angiogenesis, and vascular permeability. Little is known about its expression in human pituitary adenomas. We examined 148 human pituitary adenomas for VEGF protein expression by immunohistochemistry. The strongest immunoreactivity was present in GH adenomas, corticotroph, silent corticotroph, silent subtype 3, and nononcocytic null cell adenomas. GH adenomas treated with octreotide strained less intensely than did untreated tumors. Relatively weak staining was present in PRL, gonadotroph, thyrotroph, and oncocytic null cell adenomas in the same sections showed evidence of down-regulation of VEGF protein expression in adenomas. Pituitary carcinomas usually had stronger staining than adenomas. In situ hybridization studies with oligonucleotide probes showed positive staining in all groups with stronger staining in GH, ACTH, TSH, and gonadotroph adenomas and in pituitary carcinomas. These results indicate that VEGF expression is more prominent in certain adenoma subtypes, that decreased expression occurs in adenomas as compared to nontumorous pituitary and that carcinomas show increased VEGF expression relative to adenomas suggesting up-regulation of VEGF during pituitary tumor progression.     

The Immunophenotype of Pituitary Adenomas

Although the production of pituitary hormones by adenohypophysial tumors has been studied extensively, an examination of the immunophenotype of pituitary adenomas using a broad spectrum of antibodies has not been previously investigated. We studied 23 pituitary adenomas using a large panel of antibodies to determine if these tumors exhibited a common immunophenotype. Various neuroendocrine markers, synaptophysin, neuron-specific enolase (NSE), and the intermediate filament protein, low-mol-wt keratin were expressed in most examples. There was, however, differential expression of chromogranin A in that few prolactin (PRL) and adenocorticotrophic hormone (ACTH) adenomas stained positively, whereas all other adenoma subtypes were reactive. The ACTH adenomas had a unique profile with positive staining for galanin, neurophysin, vasopressin, and ubiquitin. These results indicate that (1) pituitary adenomas do not express a single “generic” immunophenotype; (2) synaptophysin is the most reliable and best broad spectrum marker for pituitary adenomas; (3) the neuroendocrine granule marker chromogranin A is useful in the identification of null cell adenoma, a tumor that usually does not stain for anterior pituitary tumors; and (4) among pituitary tumors, ACTH adenomas have a unique immunoprofile.     

Molecular Pathology of the Pituitary Adenomas

Molecular techniques have been applied to the study of pituitary adenomas by many investigators. We have used nonisotopicin situ hybridization (ISH) to analyze pituitary adenomas in our laboratory. These studies have provided insight into mRNA production by various adenomas, and have contributed toward our new proposed clinical and cytofunctional classification of pitutiary adenomas. Presented in part at the Molecular Endocrine Pathology Symposium at the International Academy of Pathology XXI International Congress, Budapest, Hungary, October 20–25, 1996.     

Assessment of Mitotic Activity in Pituitary Adenomas and Carcinomas

Assessment of mitotic activity represents one of the oldest and most routinely used histopathologic methods of evaluating the biological aggressiveness of human tumors. In the case of pituitary tumors, however, the relevance of this approach as a means of gaging tumor behavior remains ill-defined. In this article, the relationship between the mitotic index and biological aggressiveness of pituitary tumors was evaluated in a series of 54 pituitary adenomas and 6 primary pituitary carcinomas. All tumors were fully classified by immunohistochemistry and electron microscopy; adenomas were further stratified on the basis of their invasion status, the latter being defined as gross, operatively, or radiologically apparent infiltration of dura or bone. Mitotic figures were present in 11 tumors, 10 being either invasive adenomas or pituitary carcinomas. A significant association between the presence of mitotic figures and tumor behavior was noted, as evidenced by progressive increments in the proportion of cases expressing mitotic figures in the categories of noninvasive adenoma, invasive adenoma, and pituitary carcinoma (3.9, 21.4, and 66.7%, respectively; Fisher’s exact test, two-tailed,p<0.001). The mitotic index, however, appeared to be a less informative parameter, being extremely low in all cases (mean=0.016%±0.005 [±SEM]). Although the mean mitotic index in pituitary carcinomas (0.09%±0.035) was significantly higher than the mean mitotic index of either noninvasive adenomas (0.002%±0.002) or invasive adenomas (0.013%±0.005), no practical threshold value capable of distinguishing these three groups was evident. Comparison of the mitotic index with Ki-67 derived growth fractions in these tumors revealed a significant but weak linear correlation (r=0.41,p<0.01). These data suggest that when, mitotic figures are present, they do provide some indication of the behavior and invasive potential of pituitary tumors. For routine diagnostic purposes, however, the discriminating power of this parameter is somewhat limited, being superseded by alternative and more informative methods of growth fraction determination such as that provided by the Ki-67 immunolabeling.     

Silver-Staining Nucleolar Organizer Region Quantification in Pituitary Adenomas

Nucleolar organizer regions (NORs) are segments of DNA, encoding for ribosomal RNA. They are associated with argyrophilic proteins and, thus, they can be localized through silver staining. A correlation has been shown between the number, the size, or the intranuclear localization of AgNORs, and the proliferative activity of cells. The aim of this study was to examine numerous features of AgNORs in pituitary adenomas and to relate them to immunohistochemical typing of tumor. Histologic slides from 32 pituitary tumors and one normal pituitary were silver-stained and analyzed with a computerized system for microscopic image analysis, supported by an AgNORmeter95 program. All the tumors were also immunocyto chemically characterized. We have found that gonadotropinomas, when compared with plurihormonal adenomas, revealed a lower proportion of nuclei with a single AgNOR and a higher percentage of marginal dots. Recurrent adenomas, when compared with primary adenomas, showed a higher proportion of nuclei with three AgNOR dots, a larger total area of dots in the nuclei, and a higher standard deviation of the AgNOR dot area in the nucleus. Adenomas immunopositive for prolactin, when compared with immunonegative ones, showed a larger mean area of the AgNOR dot, a larger area of the biggest dot in the nucleus, and a higher proportion of nuclei within a single dot. These results suggest that the estimated parameters of AgNOR dots differ according to tumor aggressiveness and to the hormone immunopositivity of pituitary adenomas.     

Ultrastructural Features of Apoptosis in Human Pituitary Adenomas

Although several recent studies deal with various molecular aspects of apoptosis, or programmed cell death, very little information is available on the ultrastructural changes associated with apoptosis in the adenohypophysis and its role in the regulation of pituitary adenoma growth and progression. This paper describes the distinct ultrastructural sequences that develop during the various phases of the apoptotic process. The study is based on the ultrastructural investigation of more than 8,000 surgically removed pituitary biopsies, which were examined by histology and immunocytochemistry for diagnostic purposes. No apoptosis was found in normal adenohypophysis and it is also a rare event in pituitary adenomas. When present, adenomatous adenohypophysial cells exhibit common and characteristic apoptotic changes. The ultrastructural alterations of membraneous organelles associated with apoptosis are similar to those previously reported in other tissues. It is noteworthy that apoptosis is clearly distinguishable from the ubiquitous dark cells denoting the common way of cell death. The findings suggest that apoptosis in pituitary adenomas is not a random event. Practically every specimen containing multiple apoptotic cells represents corticotroph adenoma. Occasional examples occur in lactotroph or gonadotroph adenomas. Although electron microscopic specimens are admittedly small, the large number of investigated cases gives credence to the observations.     

Ultrastructural Features of Apoptosis in Human Pituitary Adenomas

Although several recent studies deal with various molecular aspects of apoptosis, or programmed cell death, very little information is available on the ultrastructural changes associated with apoptosis in the adenohypophysis and its role in the regulation of pituitary adenoma growth and progression. This paper describes the distinct ultrastructural sequences that develop during the various phases of the apoptotic process. The study is based on the ultrastructural investigation of more than 8,000 surgically removed pituitary biopsies, which were examined by histology and immunocytochemistry for diagnostic purposes. No apoptosis was found in normal adenohypophysis and it is also a rare event in pituitary adenomas. When present, adenomatous adenohypophysial cells exhibit common and characteristic apoptotic changes. The ultrastructural alterations of membraneous organelles associated with apoptosis are similar to those previously reported in other tissues. It is noteworthy that apoptosis is clearly distinguishable from the ubiquitous dark cells denoting the common way of cell death. The findings suggest that apoptosis in pituitary adenomas is not a random event. Practically every specimen containing multiple apoptotic cells represents corticotroph adenoma. Occasional examples occur in lactotroph or gonadotroph adenomas. Although electron microscopic specimens are admittedly small, the large number of investigated cases gives credence to the observations.     

Immunohistochemical Heterogeneity Within Clinically Nonfunctioning Pituitary Adenomas

To investigate whether adenohypophysial hormone expression is heterogeneous within individual clinically nonfunctioning pituitary adenomas, immunohistochemical examinations were performed on tissues obtained by multiple sampling of 11 adenomas. Stained sections were assessed by morphometric image analysis as well as semiquantitative estimation under microscopy. All tumors except one were immunopositive for one or more gonadotropins. Results were divided into five grades based on the proportion of immunoreactive cells per section. Semiquantitative estimation showed only a one-grade difference among samples from the same tumor in four cases for FSHβ and in two cases for LHβ. These qualitative similarities between multiple samples were confirmed by morphometric image analysis. From the practical standpoint of making a diagnosis of nonfunctioning pituitary adenoma, it is not necessary to take into account immunohistochemical heterogeneity within an individual tumor, and immunohistochemical findings in a given sample obtained at surgery can be regarded as representative of the entire adenoma.     

Ultrastructural Morphology of Folliculo-Stellate Cells in Human Pituitary Adenomas

Fifty-six pituitary adenomas were studied by electron microscopy in a search for the presence of folliculo-stellate cells (FSCs) with the aim of evaluating their prevalence and ultrastructural morphology. FSCs were scattered in two adenomas (one oncocytoma and one densely granulated GH cell adenoma) and were numerous in a sparsely granulated GH cell adenoma; their overall prevalence was 5.4%. Ultrastructural examination of the three neoplasms revealed that FSCs were hypertrophic element with abundant cytoplasm and organelles (in contrast to FSCs of the normal pituitary) and no obvious signs of neoplastic transformation. Junctional complexes between FSCs were similar to those described in the normal gland. Numerous follicular structures were lined by FSCs. FSCs in pituitary adenomas are probably nonneoplastic, reactive cells showing signs of hyperactivity, similar to FSCs found during pituitary hypersecretion and in estrogen-induced tumor.     

Immunoexpression of Androgen Receptor in the Nontumorous Pituitary and in Adenomas

Little information is available regarding androgen receptor immunoexpression (AR) in the normal and neoplastic human pituitary. Available experimental data links it to primarily gonadotroph cells. We undertook an immunohistochemical study of 41 autopsy-derived normal glands from patients of both sexes and all ages as well as 79 fully characterized pituitary adenomas of all types, the focus being upon AR expression in normal and neoplastic gonadotrophs. Nuclear AR immunoreactivity was noted in gonadotrophs and other normal adeno- and neurohypophysial cells. In addition to its presence in 74% of gonadotroph and 55% of null cell adenomas, lesser proportions of other adenoma types (adrenocorticotropic hormone 50%, prolactin 38%, growth hormone 33%) also exhibited AR immunoreactivity. No staining of thyroid-stimulating hormone adenomas was noted. The physiologic significance of our findings remains to be explored. The literature regarding AR expression in animal and human pituitaries is reviewed.     

PTTG is a Secretory Protein in Human Pituitary Adenomas and in Mouse Pituitary Tumor Cell Lines

The pituitary tumor-transforming gene (PTTG) is a homolog of yeast Securin, which arrests the activation of Separin to induce sister chromatid separation in the transition from metaphase to anaphase. Pituitary tumor-transforming gene is also known to induce angiogenesis during pituitary tumorigenesis. It has not been clarified whether PTTG functions as a cytoplasmic or a nuclear protein. Our immunohistochemical study indicated that PTTG is localized in the cytoplasm of pituitary tumor cells. In the present study, confocal laser scanning microscopy (CLSM) analysis of human pituitary adenomas and immunoelectron microscopy of the mouse pituitary cell line, AtT-20, demonstrated the localization of PTTG in the Golgi apparatus and vesicles. Secreted PTTG was detected by immunoblotting from culture medium of mouse pituitary tumor cell lines. Our results suggested that PTTG is a secretory protein produced by pituitary tumor cells. In addition, PTTG may exert autocrine and/or paracrine functions as a newly proposed important pathway for the action of PTTG.     

Ultrastructural Markers in the Pathologic Diagnosis of Pituitary Adenomas

The functional-morphologic classification of pituitary adenomas increased the number of distinguishable morphologic entities from 3 (acidophilic, basophilic, chromophobic) to 15. Identification of tumor types requires histology as well as im-munohistochemistry and electron microscopy. Electron microscopy has a pivotal role in the recognition and separation of entities with overlapping immunohistochemical profiles. Electron microscopic diagnosis is facilitated considerably by structural markers present in the majority of adenoma types. Two groups of ultrastructural markers are discussed. Organelle markers are represented by specific morphology/arrangement of ubiquitous cytoplasmic constituents (rough endoplasmic reticulum, Golgi apparatus, mitochondria). Special markers are usually restricted to a particular pituitary cell type, its tumors, or both (filamentous aggregates, particular morphology of secretory granules, etc). Evaluation of general ultrastructural features and identification of structural markers permit conclusive diagnosis in the majority of pituitary adenomas. Cell derivation remains uncertain in many so-called clinically nonfunctioning adenomas with no or insufficient markers and poorly developed cytoplasmic organization associated with low endocrine activity.     

Immunocytochemical Investigations on the Vascularization of Pituitary Adenomas

Blood vessels within pituitary adenomas were visualized using the immunocytochemical reaction for Factor VIII (von Willebrand Factor), a specific marker of the vascular endothelium. The number of immunopositive vascular profiles were counted and expressed as a mean number per one microscopic field. The results were related to the type of adenoma, established on the basis of immunocytochemical investigation using the antibodies against pituitary hormones or α-subunit (α-SU). It was found that the richest vascularization occurred in adenomas expressing follicle-stimulating hormone (FSH). The possible role of FSH in pituitary angiogenesis is discussed.     

垂体腺瘤101例免疫组织化学研究

组织病理学习惯上将垂体腺瘤分为嗜酸性、嗜碱性、嫌色性,往往不能反映肿瘤的功能和性质。应用免疫组织化学方法对101例垂体腺瘤进行检测,按照激素在腺瘤内的定位,将其划分为单一激素分泌性腺瘤、多激素分泌性腺瘤和免疫组化阴性腺瘤,从而使形态和机能密切联系在一起。     

Utility of Pit-1 Immunostaining in Distinguishing Pituitary Adenomas of Primitive Differentiation from Null Cell Adenomas

Pit-1 immunostaining is not routinely used in the characterization of pituitary adenomas, and its utility in distinguishing adenomas dedicated towards the lactotroph, somatotroph, and thyrotroph lineage from null cell adenomas warrants further evaluation. Pituitary adenomas that were negative for expression of a basic panel of hormonal markers (ACTH, prolactin, and growth hormone) were further evaluated for TSH, SF-1, and Pit-1 expression using a tissue microarray. Among the 147 identified pituitary adenomas that were negative for ACTH, prolactin, growth hormone, and TSH, expression of SF-1 was present in 68 cases (46%). Of the remaining 72 cases with sufficient tissue for further analysis, four were Pit-1 positive (6% of the adenomas negative for ACTH, prolactin, growth hormone, TSH, and SF-1); the remaining 68 were potentially null cell adenomas. Two of the Pit-1-positive adenomas displayed a paranuclear CAM 5.2 staining pattern suggestive of a sparsely granulated somatotroph adenoma; however, only one case contained fibrous bodies within a majority of the adenoma cells. Our data suggests that Pit-1 can be utilized as a second tier immunostain in cases of clinically non-functioning adenomas that are immunonegative for ACTH, prolactin, growth hormone, TSH, and SF-1 in order to further segregate rare cases of Pit-1-positive adenomas from null cell adenomas. Pit-1 immunostaining can recognize rare cases of sparsely granulated somatotroph adenomas that appear immunonegative for growth hormone, as well as rare cases of other Pit-1-positive adenomas that are negative for Pit-1 lineage hormones. Overall, pituitary adenomas of the Pit-1 lineage that do not produce prolactin, growth hormone, or TSH are rare, with only four cases identified in the current study.     

Proliferation Markers and EGF in ACTH-Secreting Adenomas and Carcinomas of the Pituitary

Pituitary carcinomas are only defined by their metastatic growth, which may be intracranial or systemic. To establish further morphological and immunohistochemical differences between pituitary carcinomas and adenomas, 19 ACTH-secreting adenomas (10 non invasive and 9 invasive) and 2 ACTH-secreting carcinomas with their metastases were studied for expression of the intermediate filaments keratin and vimentin and the tumor-associated antigens Ki67, proliferating cell nuclear antigen (PCNA), epidermal growth factor (EGF), cathepsin D, p53, and carcinoembryonic antigen (CEA). Immunohistochemistry was performed using avidin-biotin techniques on formalin-fixed, paraffin-embedded tissue. With the exception of one noninvasive pituitary adenoma, one carcinoma, and the metastases, all tumors contained keratin; none contained vimentin. All tumors stained negative for CEA and p53. Eleven (58.5%) adenomas and both pituitary carcinomas contained Ki67-positive nuclei; 14 (74%) adenomas and one carcinoma revealed PCNA. No correlation was found between the two markers. Seven (38%) adenomas showed a labeling index <1 % for cathepsin D, whereas none of the carcinomas or metastases did so. EGF was found in 7 (38%) adenomas and in both carcinomas. A tendency to a higher rate of EGF positivity in the invasive adenomas was observed. The metastases showed a higher labeling index, and far more intense staining results for Ki67, PCNA, and EGF than the primary tumor. The metastases also had a higher proliferation rate and growth factor content than the carcinoma itself.