Vasoactive intestinal peptide in the human pituitary gland and adenomas. An immunocytochemical study

Recent evidence indicates that vasoactive intestinal peptide (VIP) may be involved in normal pituitary function. Immunocytochemistry was used to localize VIP in human biopsied pituitary adenomas and postmortem anterior pituitary glands. Paraffin sections were immunostained for VIP with the avidin-biotin-peroxidase technique. Strong VIP-like immunoreactivity (VIP-LI) was observed in 16 of 17 prolactinomas, 12 of 14 growth hormone-secreting tumors associated with acromegaly, four of 12 ACTH-secreting tumors, and 14 of 18 nonfunctioning pituitary adenomas. In most cases, VIP was colocalized with the classical pituitary hormones. Six of the 18 nonfunctioning tumors had no demonstrable hormone immunoreactivity; five of these stained strongly for VIP, whereas one was negative. Of 18 normal anterior pituitaries, 12 showed strong diffuse staining for VIP throughout the gland. One pituitary with VIP-LI came from an individual who had undergone pituitary stalk transection. Double-immunoenzyme labeling and immunoelectron microscopy demonstrated VIP-LI in many lactotrophs, scattered thyrotrophs, corticotrophs, and in an occasional gonadotroph. These results suggest the following: 1) VIP is present in more than one cell type in normal and adenomatous human pituitaries; and 2) VIP may be involved in the function and development of pituitary tumors.     

Molecular cytogenetics of chromosome 11 in pituitary adenomas: a comparison of fluorescence in situ hybridization and DNA ploidy study

Chromosome 11 abnormalities were detected by fluorescence in situ hybridization (FISH) technique and compared with DNA ploidy in 24 surgically removed pituitary adenomas. The tumors were diagnosed and classified by histology, electron microscopy, and pituitary hormone immunocytochemistry. They included 2 densely granulated somatotroph (DG-SM) and 4 sparsely granulated somatotroph (SG-SM) adenomas, 3 SG lactotroph (LT), 2 mixed somatotroph-lactotroph (SM-LT), 4 functioning corticotroph (CRT), 1 silent CRT subtype 1, 1 thyrotroph, 1 mixed thyrotroph-somatotroph, 2 gonadotrophs, and 4 null cell adenomas. FISH analysis with an alpha-satellite DNA probe specific for chromosome 11 showed numerical abnormalities in 16 functioning (94%) and 5 nonfunctioning (71%) adenomas. Ten functioning tumors showed aneuploid histograms, whereas the remaining and all nonfunctioning adenomas were diploid. Aberrant chromosome 11 signals were noted mostly in aneuploid adenomas involving 17% to 100% of their cell population. The severity of chromosome 11 aberrations in adenomas containing extra copies often correlated with a higher DNA index (DI). Monosomy 11 as dominant aberration was noted in a mixed SM-LT and to a lesser degree in 3 CRT adenomas involving 21% to 97% of their cell population. Two of these CRT adenomas were associated with normal DI, whereas the remaining third showed a high DI, indicating increased copy number of chromosomes other than of chromosome 11. In conclusion, chromosome 11 abnormalities are common in all types of pituitary adenomas, occurring more frequently in functioning tumors. Specific numerical abnormalities, such as monosomy and trisomy, tend to be associated with certain adenoma types, whereas tumors with extra chromosome 11 copies often exhibit aneuploid histograms.     

Detection of inhibin α and βA subunits (inhibin A, B, activin A,AB) in the human pituitary gland and in pituitary adenomas by immunohistochemistry and nonradioisotopicin situ hybridization

Inhibin and activin are gonadal hormones produced in human ovaries. They are known to act on anterior pituitary cells to regulate the synthesis and secretion of follicle-stimulating hormone (FSH). The purpose of the present study was to determine the localization of inhibin and activin subunits α and βA as endocrine markers in the human normal pituitary gland and pituitary adenomas, using immunohistochemistry andin situ hybridization (ISH) methods. Pituitary tissues from surgical and autopsy materials were fixed in 10% formalin and embedded in paraffin. Five normal pituitary glands and 79 pituitary adenomas were immunostained with the avidin-biotin peroxidase complex (ABC) method using polyclonal antibodies against inhibin and activin subunits α and βA. The other antibodies against anterior pituitary hormones used in this study were as follows: antigrowth hormone (anti-GH), antiprolactin (anti-PRL), antiadrenocorticotropic hormone (anti-ACTH), anti-FSHβ, antilutenizing hormone (anti-LH) β, antithyroid-stimulating hormone (anti-TSH) β, and antiglycoprotein α-subunit (anti-α-SU). We analyzed gene expressions of subunits α and βA by nonradioisotopic ISH in pituitary adenomas. In the normal human pituitary glands, inhibin and activin subunits α and βA immunoreactivities were found diffusely in the cytoplasm of anterior pituitary cells. The percentage of subunit α-immunopositive cells was 40% of the anterior pituitary cells. Subunit βA immunoreactivities were observed in about 15% of the anterior pituitary cells. By the double-staining method, subunit α immunoreactivity was detected in all types of anterior pituitary cells, and it was colocalized most frequently with GH and α-SU-positive cells. Subunit βA immunoreactivity was colocalized predominantly with PRL, FSH-β, LH-β, and α-SU. Among the 79 adenomas, 75 cases (94.9%) were positive for subunit α, and 50 cases (63.3%) were positive for subunit βA. Subunit βA was positive in tumor cells with the following incidences: GH adenomas, 3 of 14 (21.4%); PRL adenomas, 5 of 8 (62.5%); ACTH adenomas, 6 of 6 (100%); TSH adenomas, 7 of 7 (100%); nonfunctioning adenomas, 29 of 44 (65.9%), including gonadotropin-positive, 16 of 22 (80.0%). The ISH signals for subunits α and βA were strongly expressed in gonadotropin-positive adenomas among the nonfunctioning adenomas. The mRNA signals were low and infrequent in the GH-producing adenomas. Inhibin and activin subunit α localization did not demonstrate cell-type specificity in pituitary adenomas. In contrast, subunit βA demonstrated predominant positivity in the functioning pituitary adenomas (ACTH- and TSH-secreting) and nonfunctioning adenomas (including gonadotropin-positive adenomas). The present results suggest that the functional role of inhibin and activin in the differentiation of cells in normal human pituitary glands and adenomas is present in subunit βA.     

Coexpression of galanin and adrenocorticotropic hormone in human pituitary and pituitary adenomas.

Galanin is a neuropeptide that regulates the secretion of several pituitary hormones, including prolactin (PRL) and growth hormone (GH). Galaninlike immunoreactivity (Gal-IR) and galanin mRNA in the rat anterior pituitary is cell lineage specific, with predominant expression in lactotrophs and somatotrophs. The authors examined the cellular distribution of human Gal-IR in seven normal postmortem pituitaries and 62 pituitary tumors by immunoperoxidase staining. In contrast to the rat, Gal-IR in human anterior pituitaries was present in corticotrophs scattered throughout the gland, but not in lactotrophs, somatotrophs, thyrotrophs, or gonadotrophs. Distinct Gal-IR also was present in hyperplastic and neoplastic corticotrophs in 19 of 22 patients with Cushing's disease. In noncorticotroph cell tumors, unequivocal Gal-IR was present in 5 of 11 GH-secreting tumors associated with clinical acromegaly, 9 of 18 nonfunctioning pituitary adenomas, and 2 of 14 prolactinomas. Of these galanin-positive tumors, four of the five GH-secreting adenomas, six of the nine nonfunctioning adenomas, and both of the prolactinomas also contained adrenocorticotropic hormone immunoreactivity (ACTH-IR). Immunostaining and in situ hybridization on adjacent sections using an 35S-labeled probe complementary to human galanin mRNA demonstrated predominant galanin expression in normal corticotrophs. Immunoelectron microscopy confirmed the presence of Gal-IR in pituitary cells characteristic of corticotrophs in both normal and neoplastic pituitaries. Thus, as in the rat, galanin gene expression in the human pituitary is cell-type specific. Unlike the rat, however, human galanin gene expression is restricted to the corticotroph lineage. Studies of tumors confirmed the observed coexpression of galanin and adrenocorticotropic hormone. The divergent cell type specificity of galanin production in human and rat pituitaries reflects different patterns of gene activation in these two species. In addition, these results suggest that galanin in the human pituitary may participate locally in the regulation of the hypothalamic-pituitary-adrenal axis.     

垂体生长激素细胞腺瘤的电镜及免疫电镜观察

24 cases of growth hormone(GH)-producing pituitary adenomas were studied with electron microscopy and immunoelectron microscopy by protein A-gold complex, 6 cases were identified as densely granulated GH adenoma and 15 cases as sparsely granulated GH adenoma, among which 4 cases were proved by immunoelectron microscopy to be containing granules with prolactin(PRL) activity simultaneously. Intracytoplasmic fibrous bodies were often seen in the sparsely granulated cells anyhow, not all those cells with fibrous bodies possess the secretory granules with GH activity, and fibrous bodies were also detected in some PRL cells of certain mixed type adenoma. This suggests that fibrous bodies might not be the specific morphological feature of pituitary growth hormone cell adenomas.     

HGH,PRL, and ACTH Gene Expression in Clinically Nonfunctioning Adenomas Detected with Nonisotopic In Situ Hybridization Method

In situ hybridization (ISH), which can manifest the specific gene expression of anterior pituitary hormones (mRNA), as well as immunohistochemistry (IHC), is needed to clarify the endocrine function of pituitary adenomas. With the aid of nonisotopic ISH, which has several advantages over isotopic ISH, we examined the expression of pituitary hormone mRNAs in 14 clinically nonfunctioning adenomas, which were considered to be a subtype of gonadotroph adenomas. Gene expression of growth hormone (GH; 4/14), prolactin (PRL; 5/14), adrenocorticotroph hormone (ACTH; 4/14), and gonadotropin were detected with our nonisotopic ISH studies. It is suggested from our ISH studies that some clinically nonfunctioning adenomas are composed of hormone (or subunit) producing cells and may be derived from plurihormonal primordial stem cells.     

Silent mixed growth hormone cell-prolactin cell pituitary adenoma

The case of a 51 -year-old man with recurrent nonfunctioning pituitary adenoma is presented. Despite clinically and endocrinologically normal pituitary function in regard to growth hormone and prolactin, many growth hormone- and prolactin-positive cells were immunohis-tochemically detected in adenoma tissue. Furthermore, a quite rare tumor of silent mixed growth hormone cell-prolactin cell pituitary adenoma was confirmed by the double-labeling immunoelectron-microscopical study.     

Pituitary tumors in the elderly

The present study was designed to investigate the incidence and immunohistochemical characteristics of pituitary tumors in the elderly. In our surgical collection of 1925 cases, we examined tumor tissue from 15 patients over 80 years of age. Pituitaries obtained at routine autopsies from 692 subjects over 80 years of age were also investigated. Of the 15 surgical cases studied, the majority of patients presented with chiasmatic syndromes, likely caused by macroadenomas. Gonadotroph adenomas were the most frequently diagnosed tumor type, followed by null-cell adenomas and oncocytomas. There is only one case with GH cell adenoma. Among 692 autopsy cases, 79 (11.4%) pituitaries were found to contain adenomas in the anterior lobe. In one pituitary, two separate adenomas were detected, hence the number of adenomas in our material was 80. All autopsy cases were microadenomas except one. The mean diameter of adenomas was 2.2 mm. ACTH cell adenomas were the most frequently diagnosed tumor type, followed by PRL cell adenomas and null cell adenomas. The occurrence of pituitary adenomas discovered after routine autopsy in the elderly was common, although these tumors were not found frequently in surgical cases over 80 years of age. Our immunohistochemical study revealed that many tumors contained one or more than one anterior pituitary hormone, although almost all pituitary adenomas were considered to be clinically inactive in surgical and autopsy cases.     

Presence of neurophysins in the human pituitary corticotrophs, Cushing''s adenomas, and growth hormone-producing adenomas detected by immunohistochemical study.

Neurophysins have been recognized as the carrier proteins of vasopressin and oxytocin. The distribution of neurophysins is immunohistochemically confirmed in the hypothalamus, median eminence, and posterior lobe of the pituitary gland. The authors detected neurophysins in the human corticotrophs and pituitary adenomas with the use of the immunohistochemical method with antiserum to human neurophysins, which did not cross-react with adrenocorticotropic hormone (ACTH), beta-endorphin, and corticotropin-releasing factor. All of ten pituitary glands obtained by autopsy revealed the presence of neurophysin-positive cells in the anterior, intermediate, and the posterior lobes. The neurophysin-positive cells were similar to the corticotrophs in shape and distribution. Simultaneous staining for ACTH and neurophysins in the serial sections revealed that neurophysin-positive cells were also ACTH-positive. One hundred twenty-four cases of pituitary adenoma operated upon were investigated. All of 7 Cushing's adenomas were composed of neurophysin-positive cells. Six tumors with giantism showed sparsely distributed neurophysin-positive cells. No neurophysin-positive cells were observed in any other cases. This study is the first reported evidence of the presence of neurophysins in the human corticotrophs and pituitary adenomas.     

Contribution of immunohistochemistry, electron microscopy, and cell culture to the characterization of nonfunctioning pituitary adenomas: A study of 40 cases

We studied 40 endocrinologically inactive pituitary adenomas by immunohistochemistry, electron microscopy, and cell culture in order to determine the incidence of gonadotropic adenomas and to classify nonfunctioning adenomas. Immunohistochemical studies using a large panel of monoclonal and polyclonal antibodies identified the following nonfunctioning adenomas: 20 gonadotropic adenomas, four silent corticotropic adenomas, one plurihormonal adenoma, and 15 nonsecreting adenomas. Among nonsecreting adenomas, ultrastructural study of 13 cases identified seven null cell adenomas and six oncocytomas. Silent corticotropic adenomas were classified into subtypes I, II, and III according to Kovacs and Horvath. Most often, gonadotropic adenomas displayed a varying number of oncocytic cells, characteristic secretory granules, and a prominent Golgi apparatus. Postembedding immunoelectron microscopy was performed on eight gonadotropic or nonsecreting adenomas, but this technique did not provide any additional information. Six gonadotropic adenomas and 10 so-called nonsecreting adenomas were studied in primary cell cultures. The six gonadotropic adenomas and seven of the 10 nonsecreting adenomas released gonadotropins in the culture medium. The use of in vitro results as a supplementary diagnostic criterion allowed classification of the 40 nonfunctioning adenomas as follows: 27 gonadotropic adenomas, four silent corticotropic adenomas, one plurihormonal adenoma, and eight nonsecreting adenomas. These results demonstrate a high proportion of gonadotropic adenomas among nonfunctioning adenomas (67.5%) and the usefulness of several techniques in characterizing this type of pituitary adenoma.     

GH-, PRL-, POMC-, beta-TSH-, beta-LH-, beta-FSH-mRNA in gonadotroph adenomas of the pituitary by in situ hybridization in comparison with immunostaining and clinical data

In situ hybridization (ISH) enables the visualization of specific mRNA for pituitary hormones. Our collection consists of 40 surgically removed pituitary adenomas that were classified as follicle stimulating hormone/luteinizing hormone (FSH/LH) cell adenomas by structure and by immunostaining (IH) for all pituitary hormones. All forty adenomas were regarded as clinically inactive. The aim of our study was to examine nonfunctioning adenomas by ISH for demonostration of mRNAs for all pituitary hormones. The results were compared with proliferation markers, invasiveness and clinical data. ISH detected signals for all pituitary hormones at a range of 30% for prolactin (PRL) to 85% for proopiomelanocortin (POMC). mRNA for β-FSH was detected in 70% and β-LH mRNA in 43% of adenomas. Thirty-three percent of adenomas revealed negative mRNA detection for β-LH but positive hormone content. The majority of adenomas (75%) expressed more than two mRNAs simultaneously, mostly the combination of POMC mRNA together with β-FSH mRNA and one to four others. Comparison with clinical data showed no significant differences except for one adenoma with a high Ki-67 index (>2.1% positive nuclei). This adenoma showed very high signals for PRL and β-TSH mRNA.     

Pituitary adenomas. An immunohistochemical study of hormone production and chromogranin localization.

Tumors from 42 surgically resected pituitaries and from 13 autopsy cases were studied immunohistochemically with polyclonal antisera to 7 anterior pituitary hormones and with a newly developed monoclonal antibody directed against human chromogranin for evaluation of the distribution of chromogranin in normal and neoplastic pituitaries. In addition, a prospective study was done for assessment of the prevalence, morphology, and endocrine cell types of pituitary tumors in 100 autopsy subjects. When these 55 pituitary adenomas were examined with monoclonal antibody (LK2H10) directed against human chromogranin, selective staining of normal adenohypophyseal cell types and pituitary tumors was observed. Most null-cell adenomas (12/14) were positive for chromogranin, whereas all prolactin (PRL)-producing adenomas (19/19) were negative. Growth hormone (GH) adenomas were focally positive (9/9). All oncocytomas (2/2), 1 thyrotropin (TSH) adenoma, and a follicle-stimulating hormone/luteinizing hormone adenoma were positive for chromogranin. One or more adenomas were present in 14% of the autopsy cases. The tumors occurred most frequently in patients in the fifth through the seventh decades of life. Immunohistochemical staining of 13 adenomas revealed 1 TSH, 1 ACTH, and 4 PRL-producing tumors, whereas 7 other tumors, which were null-cell or undifferentiated adenomas, failed to stain for any of the seven principle pituitary hormones. These results indicate that antibody LK2H10 to human chromogranin is useful in the immunohistochemical characterization of pituitary adenomas. Incidental pituitary microadenomas from autopsy-derived pituitaries most commonly produce PRL, or they belong to the null-cell or undifferentiated tumor group.     

Immunohistochemical analysis of RCAS1 in human pituitary adenomas.

It has been reported that RCAS1 (receptor-binding cancer antigen expressed on SiSO cells) acts as a ligand for a receptor present on normal peripheral lymphocytes and induces apoptotic cell death. It is expressed in uterine and ovarian carcinomas, especially in invasive cancers. This immunohistochemical study is aimed to elucidate the expression of RCAS1 in human pituitary adenomas in order to clarify its role in their proliferative regulation and invasiveness. Five normal pituitary glands, 50 human pituitary adenomas, and one malignant glioma were subjected to immunohistochemical studies. In normal pituitary glands, immunostaining of RCAS1 and MIB-1 was not found. In malignant glioma, large numbers of cell nuclei were positive for MIB-1 (MIB-1 index: 28%), and RCAS1 was detected both in the cytoplasm and on the membrane of the tumor cells. Expression of RCAS1 was noted in 48% of pituitary adenomas immunohistochemically (60.0% of growth hormone-secreting adenomas, 60.0% of prolactin-secreting adenomas, 42.9% of adrenocorticotrophin-secreting adenomas, 40.0% of thyroid-stimulating hormone-secreting adenomas, 33.3% of nonfunctioning adenomas, and 44.4% of gonadotropin-subunit-positive adenomas). It showed no correlation with tumor type, size, and invasiveness. The statistically significant relationship between RCAS1 and MIB-1 positivity was identified in our study. These results suggest that expression of RCAS1 as well as MIB-1 positivity predict the growth potential of individual pituitary adenomas.     

Growth hormone-releasing hormone receptor mRNA in acromegalic pituitary tumors.

The growth hormone (GH)-releasing hormone receptor (GHRH-R) has been recently cloned and found to be a member of a new family of seven transmembrane receptors that includes secretin, vasoactive intestinal peptide, calcitonin, and corticotropin-releasing factor. GHRH-R mRNA has been demonstrated by Northern blot analyses to be present specifically in the anterior pituitary gland. To determine the precise cellular localization of this receptor in normal anterior pituitary and pituitary adenomas, GHRH-R mRNA was analyzed in 2 normal human pituitary glands and 16 human pituitary adenomas using in situ hybridization. GHRH-R was specifically localized in somatotroph cells in the normal pituitary. In the adenomas, all GH-producing adenomas originating from acromegalic patients demonstrated up-regulation of GHRH-R mRNA when compared with levels in the normal pituitary. Only one of five clinically nonfunctioning adenomas, a gonadotroph luteinizing hormone/follicle-stimulating hormone-positive adenoma, exhibited up-regulation of this receptor message. Adrenocorticotrophic hormone-secreting and prolactin-secreting adenomas did not express GHRH-R message. In summary, GHRH-R is specifically expressed in somatotrophs and GH-producing adenomas, suggesting that GHRH-R may influence GH release in adenomas similar to this receptor's actions in the normal somatotrophs and may be involved in the growth of GH-secreting adenomas.     

Acidophil stem cell adenoma of the human pituitary.

Among 87 pituitary adenomas, four neoplasms had a superficial resemblance to undifferentiated cell adenomas and some fine structural features of both sparsely granulated adenomatous growth hormone and prolactin cells. Misplaced exocytosis, fibrous bodies, and multiple centrioles were sometimes revealed within the same cell and usually were accompanied by oncocytic transformation, mitochondrial alterations, and abnormal centriologenesis. The patients had normal or low blood growth hormone levels and elevated or normal prolactin values. All the tumors that were tested contained immunoreactive growth hormone and prolactin, irrespective of the blood hormone levels. The four tumors could represent a hitherto unclassified adenoma type and derive from the common, committed precursor of the two acidophils. The term acidophil stem cell adenoma is proposed to designate this entity.     

Pituitary adenomas in Cushing's disease. A histologic, ultrastructural, and immunocytochemical study.

Twenty-two pituitary adenomas in Cushing's disease were removed by transsphenoidal surgery. In six patients the pituitary tumor had become manifest following adrenalectomy (Nelson's syndrome). Sixteen tumors were microadenomas measuring from 2 to 9 mm, while two were diffuse invasive adenomas verified at postmortem examination. Light microscopy showed that the tumors were made of basophillic cells containing PAS-positive granules that stained blue with Herlant tetrachrome and lead hematoxylin. Immunocytochemical studies showed that the granules stained positively with antiserum to adrenocorticotrophic hormone (ACTH) or to beta-lipotropic hormone (beta-LPH) and the peroxidase-antiperoxidase complex. Electron microscopic study of the tumor cells showed ACTH and beta-LPH containing granules varying in size, shape, and amount. Perinuclear bundles of 70 A microfilaments constituted a specific ultrastructural finding.     

Detection of chromosomal imbalances in growth hormone-secreting pituitary tumors by comparative genomic hybridization.

Although recent molecular investigations have identified a number of genetic alterations that are associated with the development of pituitary adenomas, the exact pathogenesis mechanism of these tumors remains largely unknown. In this study, we used a genome-wide survey to detect specific genetic changes within the genome of pituitary adenomas. A series of 10 growth hormone-secreting adenomas were analyzed for their genetic imbalances on all 22 autosomes by comparative genomic hybridization (CGH). Chromosomal imbalances were detected in 8 GH-secreting adenomas, whereas 2 tumors had no detectable genetic abnormalities. Chromosome gains were more frequent than losses. Overrepresentation of whole or parts of chromosomes were detected in 5/10 (50%) in 19, 3/10 (30%) in each of 5, 9, and 22q, 2/10 (20%) in 17p12-q21, whereas DNA loss were 3/10 (30%) in 13q and 2/10 (20%) in 18. No detectable gain or loss of genetic material was observed in chromosomes 7, 8, 10, 12, 15, and 20. The findings of overrepresentation of chromosomes 5q, 9p, 17q and DNA loss of chromosome 18 were consistent with those detected in nonfunctioning adenomas (Daniely M, Aviram A, Adams EF, et al:J Clin Endocrinol Metab 83:1801-1805, 1998) suggesting that the development of pituitary tumors, at least in somatotroph and nonfunctioning adenomas, may share common pathway. Frequent amplifications in chromosomes 19 and 22q imply that candidate genes residing in these chromosomal regions may be involved in the pathogenesis of GH-secreting adenomas.     

Cytogenetic alterations in pituitary adenomas detected by comparative genomic hybridization.

Pituitary adenomas are benign monoclonal tumors that are either hormonally functional or nonfunctional. Although their histologic and immunocytologic characteristics have been studied extensively, cytogenetic studies are scarce. We have investigated the cytogenetic alterations and DNA ploidy patterns of 12 sporadic pituitary adenomas, including 2 growth-hormone-secreting tumors, 1 prolactinoma, and 9 nonfunctional adenomas, by comparative genomic hybridization (CGH) and laser scanning cytometry (LSC). CGH revealed that the mean number of sites of copy gain was significantly higher in functioning adenomas than in nonfunctioning tumors (P < 0.01). The most frequent change detected was loss of 13q (5 cases), with a minimal common overlapping region at 13q14. These findings suggest that a putative tumor suppressor gene on 13q14 may play an important role in the development of pituitary adenomas. DNA aneuploidy was detected by LSC in 3 of the 12 cases. The DNA aneuploid adenomas showed cytogenetic changes more frequently than did the DNA diploid tumors (P < 0.02).