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.     

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.     

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.     

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。     

Incidence,Pathology, and Recurrence of Pituitary Adenomas: Study of 647 Unselected Surgical Cases

The incidence of various types of unselected pituitary adenomas based on correlation of pathologic and clinical data was assessed. We investigated 647 cases of unselected pituitary adenomas, which were surgically removed between 1980 and 1993. All cases were examined by immunohistochemistry and electron microscopy. The mean age of patients was 44.0 years with 40.0 years for women (55.2%) and 49.1 years for men (44.8%). Age distribution indicated a remarkable sex difference: 52.4% of women and 26.8% of men were between 21 and 40 years at the time of surgery. Based on immunohistochemistry and electron microscopy, prolactin (PRL) cell adenomas represented 26.3% of tumors, growth hormone (GH) cell adenomas 12.5%, adrenocorticotrophic hormone (ACTH) cell adenomas 12.4%, oncocytomas 12.4%, and gonadotroph cell adenomas 9.4%. Seventy-three percent of the prolactinomas occurred in women and 73.8% of the oncocytomas were found in men. The incidence of pediatrics pituitary adenomas was 4.6%. All 647 cases were followed up; the mean follow-up period was 96.6 months. In 40 patients (6.2%), the adenoma recurred. Recurrence was common in functioning ACTH cell adenomas (8 cases: 9.5%) followed by silent adenomas (7 cases: 25.9%). Recurrence was noted after 2–96 months (average 28.7 months) following surgery. The shortest remission period was found in a patient with oncocytoma followed by a patient with prolactinoma.     

伽玛刀治疗垂体瘤疗效进一步分析

分析γ-刀治疗垂体腺瘤共87例,并随访1月～2年,总成功率为90.8％,其中显效率47.1％,有效率43.7％;无效率9.2％。比较术前后及阶段间瘤体大小变化,显示疗效与术后随访时间有关(P<0.05),与瘤体大小无关(P>0.05)。认为γ-刀治疗效果有重要临床价值。     

Pituitary Hormone mRNA in Null Cell Adenomas and Oncocytomas by In Situ Hybridization Comparison with Immunohistochemical and Clinical Data

Null cell adenomas and oncocytomas are clinically inactive adenomas of the pituitary gland. They do not show any significant hormone content detectable by immunohistochemistry. This study aimed at demonstrating mRNAs for all main pituitary hormones in 32 null cell adenomas and 31 oncocytomas by non-isotopic in situ hybridization using digoxigenin-labeled oligonucleotide probes. The results were compared with immunohistochemical and clinical data. Immunohistochemistry (ABC method) was done with monoclonal antibodies against PRL, GH, FSH, LH, TSH, ACTH, alpha-subunit, and Ki-67 (mib-1). The signals for hormone production were detected in both adenoma types in a range from 42% for GH in oncocytomas to 78% for beta-FSH in null cell adenomas. However, these signals are apparently not effective on hormone production, as was shown by almost negative immunostaining. Owing to the simultaneous detection of at least two mRNAs in 78% of null cell adenomas and in 94% of oncocytomas, we assume that both tumor types originate from pluripotential precursor cells that are capable of producing various hormones. According to our data, it is unlikely that the signals influence the clinical behavior.     

Vasculature in Nontumorous Hypophyses,Pituitary Adenomas,and Carcinomas: A Quantitative Morphologic Study

Vascular supply is essential for tumor proliferation and metastasis formation. Correlation was noted between vascular density and tumor size as well as metastases in several tumor types. The aim of the present study was to assess vascular density in nontumorous hypophyses, pituitary adenomas, primary pituitary carcinomas, and carcinomas metastatic to the pituitary. Twenty nontumorous hypophyses, 87 endocrinologically active or inactive pituitary adenomas, 8 primary pituitary carcinomas, 8 metastatic carcinomas, and 10 randomly selected noninvasive and 6 invasive adenomas were included in the study. Tissues were fixed in formalin, embedded in paraffin, cut, stained with hematoxylin and eosin, PAS, and immunostained for adenohypophysial hormones as well as Factor VIII-related antigen using the streptavidin-biotin-peroxidase complex method Four counts were performed: percentage of capillary area, number of vessels per field, percentage of endothelial cells, and number of endothelial cells per field. The results show that pituitary adenomas have significantly lower vascular densities as compared to nontumorous adenohypophyses. Prolactin-producing adenomas removed from untreated patients have the highest counts and growth hormone-producing adenomas the lowest counts. However, the observed differences among adenoma types are not of statistical significance. No differences are noted between noninvasive and invasive tumors. Primary pituitary carcinomas show no significant increase in vascular densities. Some metastatic tumors exhibit high vascularity. It can be concluded that pituitary adenomas have a limited capacity to induce angiogenesis. Lack of significant angiogenesis may play a role in the slow pace of pituitary tumor growth and rarity of metastases.     

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.     

Matrix Metalloproteinases and Tissue Inhibitors of Metalloproteinases in Pituitary Adenomas: Possible Markers of Neuroendocrine Cells

Matrix metalloproteinases (MMPs) are involved in remodeling processes and have been immunocytochemically localized in some endocrine glands and their tumors. Using anterior pituitary gland and pituitary adenomas, immunocytochemical localization of MMP-2 (gelatinase-A),-9 (gelatinase-B), tissue inhibitor of metalloproteinase (TIMP)-1 and-2 was performed. Normal anterior-pituitary cells all contain MMPs and lesser amount of TIMPs, whereas far fewer MMPs and TIMPs are identified in anterior pituitary adenomas. There is no correlation between pituitary hormone and MMPs-TIMPs localization, thus MMP-TIMP homeostasis may not be involved in hormone synthesis and secretion of anterior pituitary cells and their adenomas. Because MMPs and TIMPs are more abundantly and specifically localized in pituitary cells and their adenomas, MMPs and TIMPs may be included as markers for endocrine cells, including anterior-pituitary cells.     

Octreotide Effect on Growth Hormone and Somatostatin Subtype 2 Receptor mRNAs of the Human Pituitary Somatotroph Adenomas

Octreotide, a somatostatin analog used to treat acromegalic patients harboring a pituitary tumor, acts via somatostatin subtype 2 receptor (SSTR2) and causes significant decrease of circulating GH levels and sometimes mild to moderate tumor shrinkage. To further elucidate the mechanism of octreotide action, we studied GH and SSTR2 mRNAs by in situ hybridization in densely and sparsely granulated somatotroph adenomas removed by surgery from 14 treated and 14 untreated patients. Only in densely granulated adenomas were the GH and SSTR2 mRNA signals mildly decreased relative to untreated matched adenomas. The decrease of GH mRNA in densely granulated somatotroph adenomas suggests that they may have a more favorable response to octreotide therapy than sparsely granulated tumors.     

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.     

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.     

Amyloid Deposits in Pituitaries and Pituitary Adenomas: Immunohistochemistry and In Situ Hybridization

The patterns of deposition and immunoreactivity of interstitial amyloid were studied in 11 pituitary glands obtained at autopsy and 9 surgically resected pituitary adenomas using Congo red staining and a panel of antisera directed against 5 major amyloid fibril proteins and all pituitary hormones. The deposition pattern of amyloid in pituitary glands differed from that in adenomas but all amyloid deposits showed an immunostaining with anti-amyloid λ-light chain. The remaining antisera were immunonegative.In situ hybridization using an oligodeoxyribonucleotide-probe complementary to the mRNA coding for the constant region of human λ-light chain yielded no hybridization signals in the pituitaries or pituitary adenomas, excluding local synthesis and secretion of immunoglobulins. Since no case studied suffered from generalized Aλ-amyloidosis and adsorption of immunoglobulins to the unknown amyloid fribril protein of the pituitary seems to be unlikely, crossreaction of the polyclonal antisera with an undefined antigen is probable. The similar immunostaining properties of amyloid deposits in “normal” pituitaries and pituitary adenomas suggest they both originate from the same precursor protein.     

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.     

经鼻蝶人路神经内镜下垂体腺瘤切除术进展

目的：探讨神经内镜下经鼻蝶人路在垂体腺瘤切除术中的应用。方法：在Pubmed、中国知网等数据库查阅有关经鼻蝶入路神经内镜下垂体腺瘤切除术研究的文献,汇总分析。结果：经鼻蝶入路神经内镜下垂体腺瘤切除术分为标准入路和扩大入路。通过扩大入路,可以切除侵犯海绵窦、鞍上和斜坡的垂体腺瘤。经鼻蝶入路神经内镜垂体腺瘤切除术的并发症主要有脑脊液鼻漏、尿崩和出血等。结论：经鼻蝶入路神经内镜下垂体腺瘤切除术具有损伤小、暴露充分、肿瘤全切率高及术后并发症少等优点,是垂体腺瘤切除的理想术式。     

A Serum-Free System for Primary Cultures of Human Pituitary Adenomas

We report the successful use of a serum-free culture system for primary cultures of human pituitary adenomas. The system utilizes histiotypic suspension culture with low protein-binding membrane inserts that enable cells to retain their three-dimensional tissue configuration, closely mimicking the growth pattern in vivo. A serum-free defined medium was developed with CMRL-1969 (Connaught, Willowdale, Ontario, Canada) supplemented with 0.375% albumin bovine Fraction V, 5 μg/mL insulin, 5 μg/mL transferrin, 5 ng/mL sodium selenite, 30 μg/mL putrescine, 6.85 × 10⁻¹¹ M hydrocortisone, and 3.7 × 10⁻¹¹ M tri-iodothyronine (T₃). We analyzed eight surgically resected human pituitary adenomas. Basal pituitary hormone secretion measured by radioimmunoassay of pituitary hormones was compared with hormone hypersecretion in vivo and with control cells of the same tumors cultured in CMRL-1969 with 10% fetal calf serum. The light microscopic, immunocytochemical, and ultrastructural morphology of cells cultured in this serum-free histiotypic system was compared with cells cultured in serum-supplemented media and with cells cultured on collagen-coated plastic; all cultured cells were compared with the morphology of surgically resected tissues of the same specimens. Basal pituitary hormone secretion during 24-hour incubations correlated with the clinical patterns of hormone excess; the data were similar in serum-enriched and serum-free cultures, however, hormone secretion decreased less rapidly in the serum-free cultures. Cells maintained in the histiotypic culture system closely resembled the corresponding surgically resected tumor using the morphologic parameters and were better preserved than those plated in collagen-coated plastic wells. This comparative study indicates that this serum-free histiotypic culture system provides an ideal method of examining pituitary adenomas in vitro without altering the profile of hormone secretion and cell morphology documented in vivo. This system can be used to examine the production and effects of a wide range of hormones and growth factors that have been implicated as causative agents in pituitary tumorigenesis.