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.     

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.     

HGH,PRL and βHCG/βLH gene expression in clinically inactive pituitary adenomas detected by in situ hybridization

Summary Within our surgical collection clinically inactive pituitary adenomas represent 30.7% of all pituitary tumours. To characterize their endocrine activity we studied 40 clinically inactive pituitary adenomas with in situ hybridization (ISH) using cRNA probes labelled with³⁵S encoding growth hormone (GH), prolactin (PRL) and chorionic gonadotrophin (HCG). No tumour was associated with clinical evidence of elevated hormone secretion. A mild hyperprolactinaemia not correlated with hormone or the mRNA content of the cells was interpreted to be incidental in 11 patients. By histological analysis, immunohistochemistry (IH) and electron microscopy the adenomas were diagnosed as small cell chromophobic (n=16) and large cell chromophobic (n=8) adenomas, and oncocytomas (n=16). Gene expression of one or more hormones was identified by ISH in 18 of 40 adenomas in few cells. GH and PRL gene expression was rare (GH mRNA in 3 of 40 tumours and PRL mRNA in 8 of 40 tumours) whereas in 14 of 40 adenomasHCG/LH gene expression was identified in scattered cells. Five of 40 adenomas lacking hybridization signals revealed hormones by IH. The detection of mRNA was accompanied by positive immunostaining for the respective hormones in 72%. The combination of ISH and IH reveals good evidence that the hormones are synthesized in the tumours and not taken up from the serum and stored in the cells. The two methods used together permit a more precise analysis of tumour biology than each alone.Presented in part at the meeting on Non-secreting pituitary adenomas, Uppsala, Sweden, May 1990     

Pituitary adenomas and granular cell tumors. Incidence, cell type, and location of tumor in 100 pituitary glands at autopsy.

Incidentally detected pituitary adenomas were investigated in 100 pituitary glands at autopsy to determine the number, cell type, and location of tumors, and the presence of coexisting granular cell tumors in the neurohypophysis. Pituitary glands were sagittally sectioned at 1.5-mm intervals in toto and embedded in 1 cassette to orient location of each tumor. Twenty-four pituitary glands harbored adenomas, most smaller than 3 mm and the largest 6 x 5 x 4 mm. Two pituitary glands contained double adenomas of immunocytochemically different cell types. Of the 26 adenomas, 10 had lactotrophs, 2 had mixed lactotrophs-somatotrophs, 1 had mixed lactotrophs-luteinizing hormone cells, and 12 were nonfunctioning. One adenoma with adenocorticotropic hormone cells was also detected. Thus 25 of 26 (96%) adenomas were either lactotrophic or nonfunctioning; this percentage is much higher than that of surgically resected tumors. Twenty-two tumors were contiguous with or adjacent to the capsule from which the adenomas originated. Nine granular cell tumors were noted in the neurohypophysis; 3 coexisted with pituitary adenomas. Fourteen additional cases revealed small granular cell nests. Thus the incidental finding of nonfunctioning pituitary adenomas is relatively common in adults (24% of cases in this study), and the coexistence of pituitary adenomas and granular cell tumors may suggest a possible histogenic connection between anterior and posterior pituitary tumorigenesis.     

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.     

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.     

Immunohistological studies on clinically silent pituitary adenomas

Clinically inactive adenomas represent 30.7% of all pituitary tumors in our surgical collection of 616 cases. Ninety-six tumors were studied immunohistologically with many antibodies for their hormone content. Morphological classification of these adenomas reveals oncocytic adenomas in 42 cases (44%), small cell chromophobe adenomas in 33 (34%), large cell chromophobe adenomas in 14 cases (15%), undifferentiated mucoid cell adenomas in 4 cases (4%) and undifferentiated acidophil adenomas in 3 cases (3%). Immunohistological studies performed with the six pituitary hormones GH, prolactin, ACTH, TSH, LH and FSH and additionally with α-subunit demonstrated nearly all possible combinations of hormones in adenoma cells. The most frequently occurring (29%) was LH (in 3% of adenomas alone); α-subunit followed in frequency (24%), with FSH present in 21%. Combinations of 2 hormones were detected in 16%, of 3 in 13% and of more than 3 hormones in 2%. All 6 hormones and α-subunit were negative in 48% of adenomas. It must be concluded 1) that many clinically silent adenomas are LH- or FSH- or α:-subunit-positive and therefore probably originate from gonadotropic cells, and 2) that clinically silent adenomas of acidophil cell type or mucoid cell type are rare. Although many of these adenomas apparently do not secrete the hormone which they immunohistologically contain, determining the plasma levels of the gonadotropins, and especially the α-subunit in clinical studies, may obtain a suitable and helpful clinical marker in the diagnosis of “endocrine inactive” adenomas, and especially of their recurrences.     

Elevated expression of p21 (WAF1/Cip1) in hormonally active pituitary adenomas

Although most pituitary adenomas arise sporadically, molecular studies show alterations of known oncogenes and/or tumor suppressor genes in a small percentage of adenomas, and the molecular pathology of most is unknown. The p21 gene is a universal inhibitor of cyclin-dependent kinases and serves as a cell-cycle blocker and cell-growth inhibitor. Pituitary adenomas (n = 54) were immunophenotyped for hormone production (prolactin, growth hormone, adrenocorticotropin, thyrotropin, follicle-stimulating hormone, and luteinizing hormone), and expression of p21 was determined by immunohistochemistry. The percentage of cells expressing p21 for each tumor was evaluated blindly with regard to hormone status, and expression of p21 was then correlated with the results of hormone immunotyping. Results show a striking difference in the expression of p21 between immunonegative adenomas and hormone-producing tumors. Whereas 71% (10/14) of nonfunctional adenomas exhibit p21 expression in fewer than 5% of cells, 77% (31/40) of hormone-producing adenomas show expression in more than 25% of cells, and of these, 68% (21/31) show expression in more than 75% of cells. Overexpression of p21 is particularly striking for growth hormone-producing tumors, of which 92% (11/12) show expression in more than 75% of cells. Hormone-producing pituitary adenomas express much more p21 than do immunonegative adenomas. These high levels of p21 expression represent the most widespread molecular genetic alteration demonstrated to date in pituitary adenomas.     

Growth hormone-releasing hormone expression in pituitary somatotroph adenomas, studied by immunohistochemistry and in situ hybridization using catalyzed signal amplification system

Growth hormone-releasing hormone (GHRH) is a well-known hypothalamic hormone that stimulates the synthesis and release of growth hormone (GH) as well as the proliferation of GH-producing cells in the anterior pituitary gland. Recent reports have shown GHRH synthesis in pituitary somatotroph adenomas, but GHRH immunoreactivity has not been shown in previous studies. To confirm the role of locally generated GHRH for the progression of somatotroph adenomas, we investigated the expression of GHRH in 25 pituitary somatotroph adenomas immunohistochemically, through the use of both conventional avidin-biotin-complex (ABC) method and novel catalyzed signal amplified (CSA) system. In addition, we investigated the expression of GHRH mRNA and GHRH receptor mRNA with in situ hybridization (ISH) using the CSA system. The weak immunopositivity of GHRH was observed in only 2 adenomas (8.0%) of 25 somatotroph adenomas using the ABC method. In contrast, 15 adenomas (60.0%) of 25 somatotroph adenomas were immunopositive for GHRH, as shown by CSA system. Very few of nonsomatotroph adenomas were immunopositive for GHRH using the CSA system. The expression of GHRH mRNA was confirmed, using the CSA-ISH system in 13 adenomas (72.2%) of 18 somatotroph adenomas. In 11 adenomas (61.1%) of 18 somatotrophic adenomas, the expression of GHRH receptor mRNA was demonstrated using the CSA-ISH system. This is a first report that clarified histopathologically GHRH production in pituitary somatotrophic adenomas. The demonstration of GHRH and its receptor expression is meaningful in clarifying the autocrine or paracrine regulation of GHRH in GH production and progression of pituitary somatotroph adenomas.     

Expression of Rab3, a Ras-related GTP-binding protein, in human nontumorous pituitaries and pituitary adenomas.

Rab proteins are low molecular weight GTP-binding proteins. Among these proteins, the Rab3 isoforms are considered to be involved in the exocytosis of synaptic vesicles and secretory granules in the central nervous system and anterior pituitary gland. In recent reports, the expression of Rab3 isoforms in anterior pituitary glands of mammalian species was extensively investigated. In the present study, we investigated the localization of Rab3 protein in 5 human nontumorous pituitaries and 114 human pituitary adenomas using immunohistochemical methods. In five human nontumorous pituitaries, Rab3 protein was expressed in the cytoplasm of anterior pituitary cells. Double staining for anterior pituitary hormones revealed the expression of Rab3 in growth hormone-secreting cells, but rare expression was observed in the other anterior pituitary hormone-secreting cells. Among the pituitary adenomas, 71 (62.3%) of 114 pituitary adenomas were positive for Rab3. Among the different pituitary adenoma types, the incidence of Rab3 immunopositivity was highest in growth hormone-secreting adenomas (100%), followed by adrenocorticotropic hormone-secreting adenomas (71.4%), thyroid-stimulating hormone-secreting adenomas (57.1%), nonfunctioning adenomas (56.0%), and prolactin-secreting adenomas (33.3%). After an embedding immunoelectron microscopic study, Rab3 was localized along the limiting membrane of secretory granules in the Rab3-positive pituitary adenomas. Western blotting showed the molecular weight of Rab3 to be 25 kDa in the pituitary adenomas, which were immunohistochemically positive for Rab3 protein. These results suggested that Rab3 might be involved in regulating the exocytosis of secretory granules of the anterior pituitary cells, especially growth hormone-secreting ones, which are particularly characterized by densely granulated cytologic features.     

A Silent Follicle-Stimulating Hormone-Producing Pituitary Adenoma in a Teenage Male

An 18-year-old male was referred to Toranomon Hospital seeking reoperation for recurrent clinically nonfunctioning pituitary adenoma. A pituitary macroadenoma was first suspected at age 15 due to intractable headaches. Endocrine data were unremarkable except slightly elevated serum follicle-stimulating hormone (FSH). Transsphenoidal surgery done at another hospital achieved partial tumor removal but the remaining tumor regrew 2 years after surgery. The recurrent tumor was completely and selectively removed on repeat surgery at Toranomon Hospital. Pathological examination confirmed a silent FSH-producing pituitary adenoma. Forty-five patients less than 20 years old underwent transsphenoidal surgery for pituitary adenoma at Toranomon Hospital between 1993 and 2010. Of the 45 patients, 36 (80.0%) had clinically functioning adenomas and the other 9 (20.0%) had clinically non-functioning adenomas. No patients, other than the present case, had a silent gonadotroph adenoma. In contrast, among 579 patients over 20 years old undergoing surgery for nonfunctioning pituitary adenomas between 2006 and 2010 at Toranomon Hospital, 304 (52.3%) had silent gonadotroph adenomas. Gonadotroph adenomas are more common with aging: for example, 37 (61.7%) of 60 patients more than 70 years old at the time of operation had gonadotroph adenomas. In conclusion, gonadotroph adenomas, especially silent gonadotroph adenomas, are extremely rare in childhood and adolescence.     

Detection of gonadotropin-releasing hormone receptor in normal human pituitary cells and pituitary adenomas using immunohistochemistry

Gonadotropin-releasing hormone (GnRH), which is a well-known regulator of gonadotroph function, has recently been considered to be a paracrine factor involved in the control of somatotroph, lactotroph, and corticotroph cells. GnRH action is initiated by binding to a specific cell surface receptor, the gonadotropin-releasing hormone receptor (GnRHR), which is expressed by follicle-stimulating hormone/luteinizing hormone (FSH/LH) cells. Using in situ hybridization techniques, GnRHR messenger ribonucleic acid (mRNA) has recently been detected in normal human anterior pituitary gland and in various pituitary adenomas, including FSH/LH-cell, growth hormone (GH)-cell, adrenocorticotropic hormone (ACTH)-cell, and null-cell adenomas. However, immunohistochemical studies indicating the specific cell distribution of GnRHR in normal pituitary cells have never been reported. The aim of the present investigation was to evaluate the immunohistochemical expression of GnRHR in different types of normal pituitary cells and related tumors. Using double-label immunohistochemical techniques on formalin-fixed and paraffin-embedded tissues and specific antibodies directed against pituitary hormones and GnRHR, we found GnRHR immunoreactivity not only in FSH/LH cells, but also in GH- and thyroid-stimulating hormone (TSH) cells. GnRHR was detected in FSH/LH-cell, GH-cell, mixed GH- and prolactin (PRL)-cell, and α-subunit (α-SU)/null-cell adenomas. The findings of this study suggest that the interaction between GnRH and GnRHR may play a role in paracrine/autocrine regulation of different types of normal pituitary cells and pituitary adenomas. Received: 24 January 2000 / Accepted: 12 April 2000     

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.     

Immunohistochemical analysis of tumor angiogenic factors in human pituitary adenomas

Microvessel density (MVD) has been studied in a number of neoplasias, and apparently, there is a relationship between angiogenesis and tumor progression, response to treatment, and outcome. In pituitary adenoma, the association between MVD and vascular endothelial growth factor (VEGF) with tumor behavior has been described, but correlation with other angiogenic factors such as fetal liver kinase 1 (Flk-1) or proliferative markers is unknown. We investigated MVD, VEGF, and its receptor Flk-1 expression in 60 human pituitary adenomas: 13 growth hormone cell adenomas, 7 prolactin cell adenomas, 5 corticotroph cell adenomas, 2 thyrotroph cell adenomas, and 33 nonfunctioning adenomas (30 gonadotroph cell adenomas and 3 null cell adenomas). We performed immunohistochemistry for CD34, Ki-67, VEGF, and Flk-1. To evaluate MVD, we used 2 methods: the number of vessels per square millimeter and the Chalkley method. Immunohistochemistry results were correlated, as well as with clinicopathologic factors. Adenomas with higher MVD were thyrotroph cell adenomas (299.9 +/- 87.5), and those with lower MVD were prolactin cell adenomas (168.6 +/- 63.3; P = .45, analysis of variance). We found a trend toward higher MVD in the adenomas of older patients (P = .142), but no difference was found regarding sex, extrasellar extension, or Ki-67 (P > .05). However, extrasellar extension was nearly significant when the Chalkley method score was high (P = .056). Low expression of VEGF was seen predominantly in prolactin cell adenomas, and high in nonfunctioning adenomas, or in cases of older patients (P < or = .032). Flk-1 score correlated with VEGF (P = .006). High expression was observed in nonfunctioning adenomas, cases presenting at older ages, and with extrasellar extension (P < or = .022). Our study shows that VEGF and Flk-1 are widely expressed in pituitary adenomas, predominantly in nonfunctioning adenomas and those presenting at older ages. Moreover, Flk-1 is associated with a more aggressive phenotype, and it may have potential therapeutic interest.