Lucia Stefaneanu

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.

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.

Dopamine D2 Receptor Gene Expression in Human Adenohypophysial Adenomas

The inhibitory effects of dopamine on adenohypophysial cells are mediated via dopamine subtype 2 receptor (D2R). Dopamine agonists inhibit hormone release and induce tumor shrinkage in most prolactin-secreting adenomas, whereas in other adenoma types such effects are sporadic. We investigated D2R gene expression by in situ hybridization (ISH) and immunocytochemistry in different types of pituitary adenomas. By ISH, a variable D2R signal was detected in 79 of 89 cases: 4 of 6 densely granulated and 8 of 8 sparsely granulated somatotroph, 4 of 4 mammosomatotroph, 7 of 7 mixed somatotroph-lactotroph, 4 of 4 acidophil stem cell, 16 of 16 sparsely granulated lactotroph, 11 of 16 corticotroph (functioning and silent), 3 of 4 silent subtype 3, 5 of 5 thyrotroph, 5 of 6 gonadotroph, 5 of 6 null cell, and 7 of 7 oncocytic adenomas. By immunocytochemistry, D2R protein was localized in cytoplasm and nuclei of 60 of 62 adenomas. In lactotroph adenomas, long-acting bromocriptine (BEC-LAR) induced a major increase in D2R mRNA, which was not accompanied by increased D2R immunoreactivity, suggesting mRNA stabilization. In conclusion, D2R gene is expressed in the majority of pituitary adenomas representing all tumor types. The significance of nuclear localization of D2R protein remains to be clarified.

Pituitary lactotrophs and somatotrophs in pregnancy: a correlative in situ hybridization and immunocytochemical study

SummaryLactotroph hyperplasia is a prominent finding in the adenohypophyses of pregnant women. In order to elucidate the morphogenesis of this change, pituitaries from 16 women in various phases of pregnancy were collected at autopsy and studied by histology, immunocytochemistry and in situ hybridization. The results showed that the increase in the amount of prolactin (PRL) mRNA paralleled the progressive lactotroph hyperplasia. The presence of mitoses in PRL-immunoreactive cells provided evidence that proliferation of preexisting lactotrophs contribute to lactotroph accumulation. Growth hormone (GH) immunoreactive cells showed a marked reduction in GH mRNA indicating that GH synthesis was inhibited. In many GH-immunoreactive cells, PRL mRNA became apparent. These findings demonstrate that GH is stored following discontinuation of GH synthesis. It appears that, when PRL is secreted in excess during pregnancy, somatotrophs are recruited to produce PRL. These somatotrophs begin to express PRL mRNA, transform to bihormonal mammosomatotrophs and possibly later to lactotrophs, contributing to PRL production. Mature somatotrophs may be regarded as reserve cells in the adenohypophysis, having the potential to switch hormone synthesis and to convert to mammosomatotrophs and possibly lactotrophs.

Prolactinomas in male and female patients : A comparative clinicopathologic study

OBJECTIVEnTo explore the basis of the gender-based differences in endocrine and surgical findings in patients with prolactinoma (prolactin cell adenoma) as well as in their clinical outcome.nnnMATERIAL AND METHODSnIn young or reproductive-age female patients, older women (beyond 40 years of age), and male patients, we systematically studied the following factors: operative and endocrine features (tumor size, invasiveness, preoperative serum prolactin level, and biochemical outcome), specific biologic variables (mitotic index, MIB-1 labeling index, and p27 immunoreactivity), and hormone receptor status (estrogen and progesterone receptor proteins as well as dopamine D2 receptor messenger RNA).nnnRESULTSnOf the various factors assessed, the preoperative prolactin level and MIB-1 labeling index were lower in young female patients in comparison with older female and particularly male patients. Hormone levels were also positively associated with mitotic activity as well as the MIB-1 labeling index. Although invasion was infrequent in microadenomas of young female patients, no statistically significant differences in tumor size or invasiveness were noted among the three patient groups. Absence of differences in invasiveness may, in part, be explained by artifacts of case selection.nnnCONCLUSIONnThe basis for the observed differences in proliferative activities in tumors of the three study groups is not readily apparent but may reflect differences in the endocrine milieu or the effect of sex steroid hormone receptors, tumoral vascularity, or specific growth factors.

Apoptosis in nontumorous and neoplastic human pituitaries: expression of the Bcl-2 family of proteins.

Analyses of apoptosis and of the apoptosis regulatory proteins Bcl-2, Bax, Bcl-X, and Bad were done in 95 nontumorous and neoplastic pituitary tissues by terminal deoxynucleotide transferase-mediated dUTP nick-end labeling (TUNEL), immunohistochemistry, and Western blotting. The apoptotic index was relatively low in all groups but was at least fourfold higher in pituitary carcinomas compared with any other groups. Pituitaries from pregnant and postpartum women had a fivefold higher apoptotic index compared with matched controls from nonpregnant females. Preoperative treatment of adenomas with octreotide or dopamine agonists did not change the apoptotic index significantly. The lowest levels of Bcl-2, Bax, and Bcl-X expression were in pituitary carcinomas as detected by immunostaining. An immortalized human pituitary adenoma cell line, HP75, developed in our laboratory using a replication-defective recombinant human adenovirus with an early large T-antigen, had a much higher level of apoptosis than nontumorous and neoplastic pituitaries. Treatment with transforming growth factor (TGF)-beta1 and protein kinase C (PKC) inhibitors increased apoptosis in this cell line. Analysis of the Bcl-2 family of proteins after treatment with TGF-beta1 and PKC inhibitors showed a 20% to 30% decrease in Bcl-X in the treated groups compared with controls. These results, which represent the first study of apoptosis in pituitaries from pregnant and postpartum cases and in pituitary carcinomas, indicate that 1) the apoptotic rate is low in nontumorous and neoplastic pituitary tissues but is relatively higher in pituitary carcinomas, 2) there are alterations in the expression of the Bcl-2 family of proteins in pituitary neoplasms with a decrease in Bcl-2 expression in pituitary carcinomas that may contribute to pituitary tumor pathogenesis and/or proliferation, and 3) cultured pituitary tumor cells respond to TGF-beta1 and PKC inhibitors by undergoing apoptotic cell death.

Pituitary Mammosomatotroph Adenomas Develop in Old Mice Transgenic for Growth Hormone-Releasing Hormone

It has been shown that mice transgenic for human growth hormone-releasing hormone (GRH) develop hyperplasia of pituitary somatotrophs and mammosomatotrophs, cells capable of producing both growth hormone and prolactin, by 8 months of age. We now report for the first time that old GRH-transgenic mice, 16 to 24 months of age, develop pituitary mammosomatotroph adenomas. These findings provide conclusive evidence that protracted stimulation of secretory activity can cause proliferation, hyperplasia and adenoma of adenohypophysial cells.

In situ hybridization study of pro-opiomelanocortin (POMC) gene expression in human pituitary corticotrophs and their adenomas

Pro-opiomelanocortin (POMC) mRNA was detected on paraffin sections by in situ hybridization (ISH) in corticotrophs of 12 nontumorous pituitaries, 11 functioning corticotroph, and 11 silent pituitary adenomas. ISH combined with immunocytochemistry for adrenocorticotrophic hormone (ACTH), a POMC-derived peptide, was also performed. ACTH immunoreactive cells of the anterior lobes and those invading the posterior lobe showed a high or moderate level of POMC mRNA that was not correlated with the intensity of ACTH immunoreactivity. Variable levels of POMC gene expression were present in Crookes cells, corticotrophs suppressed by glucocorticoid excess. Most functioning corticotroph adenomas and silent subtype 1 adenomas had an intense hybridization signal and ACTH immunoreactivity. In silent subtype 2 and 3 adenomas, POMC mRNA had a diffuse low level or was absent; in these adenomas ACTH immunoreactivity was diffuse, restricted to some cells, or negative. The results indicate that POMC gene is expressed in both normal and suppressed nontumorous corticotrophs. Intense signals for POMC mRNA are found in most functioning corticotroph adenomas. The difference between POMC gene expression in silent 1 and silent 2 and 3 adenomas suggests that different mechanisms are responsible for the lack of endocrine activity.

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.

Pituitary cells producing more than one hormone human pituitary adenomas

The existence of cells capable of producing more than one hormone in nontumorous human adenohypophyses and pituitary adenomas has been conclusively proved. In light of the evidence, current concepts on pituitary structure, function, and regulation as well as adenoma cytogenesis and classification have to be reconsidered.