Silencing of the imprinted DLK1-MEG3 locus in human clinically nonfunctioning pituitary adenomas

DLK1-MEG3 is an imprinted locus consisting of multiple maternally expressed noncoding RNA genes and paternally expressed protein-coding genes. The expression of maternally expressed gene 3 (MEG3) is selectively lost in clinically nonfunctioning adenomas (NFAs) of gonadotroph origin; however, expression status of other genes at this locus in human pituitary adenomas has not previously been reported. Using quantitative real-time RT-PCR, we evaluated expression of 24 genes from the DLK1-MEG3 locus in 44 human pituitary adenomas (25 NFAs, 7 ACTH-secreting, 7 GH-secreting, and 5 PRL-secreting adenomas) and 10 normal pituitaries. The effects on cell proliferation of five miRNAs whose expression was lost in NFAs were investigated by flow cytometry analysis. We found that 18 genes, including 13 miRNAs at the DLK1-MEG3 locus, were significantly down-regulated in human NFAs. In ACTH-secreting and PRL-secreting adenomas, 12 and 7 genes were significantly down-regulated, respectively; no genes were significantly down-regulated in GH-secreting tumors. One of the five miRNAs tested induced cell cycle arrest at the G2/M phase in PDFS cells derived from a human NFA. Our data indicate that the DLK1-MEG3 locus is silenced in NFAs. The growth suppression by miRNAs in PDFS cells is consistent with the hypothesis that the DLK1-MEG3 locus plays a tumor suppressor role in human NFAs.     

Immunohistochemical analysis of p27 (Kip1) in human pituitary glands and in various types of pituitary adenomas

p27 (Kip1) plays regulatory roles in the cell cycle by inhibiting the activity of cyclin dependent kinases (CDKs). This immunohistochemical study is aimed at elucidating the expression of p27 in human pituitary and in various types of pituitary adenomas in order to clarify its role in the regulation of proliferation. Sixteen normal pituitary glands and 179 human pituitary adenomas were used for immunohistochemical studies. The tissues were fixed in 10% formalin and embedded in paraffin. Indirect peroxidase method was performed after heat-induced antigen retrieval using a monoclonal antibody against p27 protein. p27 protein was expressed in the nuclei of all 16 normal human pituitary glands. p27 protein was also expressed in 128 of 179 cases of pituitary adenomas (71.5%). A marked decrease of p27 expression was noted in ACTH-secreting adenomas, 8/20 (40.0%), compared with other types of pituitary adenomas—GH-secreting adenomas, 35/46 (76.1%); PRL-secreting adenomas, 22/33 (66.7%); TSH-secreting adenomas, 8/11 (72.7%); and nonfunctioning adenomas, 55/69 (79.7%). These results suggest that p27 may play some role in the regulation of proliferation in all types of pituitary adenomas. The lower levels of p27 in ACTH-secreting adenoma is of particular interest with respect to the intermediate lobe-derived pituitary tumor developed in p27 knockout mice.     

神经内镜下经鼻蝶入路治疗垂体腺瘤25例疗效

目的:探讨神经内镜下经鼻蝶入路治疗垂体腺瘤的手术疗效。方法:2012年1月至2012年12月,应用神经内镜经鼻蝶入路治疗垂体腺瘤25例,其中巨大腺瘤7例,大腺瘤18例;无功能腺瘤14例,垂体泌乳素 (PRL) 腺瘤6例,生长激素(GH)腺瘤3例, PRL+GH腺瘤2例。术前均行头颅三维CT、MRI扫描及内分泌学检查。结果:本组25例,肿瘤全切除20例(80.0％),近全切除3例(12％),部分切除2例(8％)。术后尿崩3例,鼻出血1例。所有患者在术后第5 ～12天出院。术后随访1～10个月,患者临床症状和内分泌学指标均有所改善,未见肿瘤复发,无一例患者死亡。结论:神经内镜下经鼻蝶切除垂体腺瘤具有创伤小,术野清晰,手术操作简单、安全,术后并发症少,恢复快,住院时间短等优点,是治疗垂体腺瘤安全有效的手术方法。     

Expression of interleukin-6, interleukin-6 receptor (gp80), and the receptor's signal-transducing subunit (gp130) in human normal pituitary glands and pituitary adenomas.

Interleukin-6 (IL-6) is an important cytokine in cell proliferation and differentiation in several organs. It has also been reported that IL-6 plays a role in secretion or release of pituitary hormones in pituitary hormone-secreting cells and pituitary adenomas, but convincing data in situ have not yet been reported. In this study, we examined the participation of IL-6 in the production of pituitary hormones and the differences between human normal pituitary glands and pituitary adenomas by determination of the localization or expression of IL-6, IL-6 receptor (IL-6R, gp80), and the signal-transducing subunit (gp130) of the receptor using immunohistochemical staining and RT-PCR. IL-6 was mainly expressed in ACTH- and FSH/LH-secreting cells in normal pituitary glands, as shown by double staining. gp 80 and gp130 were coexpressed in almost all GH- and PRL-secreting cells and in approximately 30% of FSH/LH-secreting cells. RT-PCR showed that IL-6 mRNA was expressed in only one of all the pituitary adenomas examined, whereas gp 80 and gp 130 mRNAs were detected in all these pituitary adenomas. In conclusion, IL-6 was mainly expressed in ACTH- and FSH/LH-secreting cells, and the receptors were expressed in GH-, PRL- and FSH/LH-secreting cells in human normal pituitary glands. Furthermore, our data emphasized that the mechanism of IL-6 function in human pituitary adenoma cells is distinct from that in normal pituitary cells.     

神经内镜下经鼻蝶入路治疗垂体腺瘤25例疗效观察

目的：探讨神经内镜下经鼻蝶入路治疗垂体腺瘤的手术疗效。方法：2012年1月至2012年12月,应用神经内镜经鼻蝶入路治疗垂体腺瘤25例,其中巨大腺瘤7例,大腺瘤18例;无功能腺瘤14例,垂体泌乳素（ PRL）腺瘤6例,生长激素（ GH）腺瘤3例, PRL＋GH腺瘤2例。术前均行头颅三维CT、MRI扫描及内分泌学检查。结果：本组25例,肿瘤全切除20例（80．0％）,近全切除3例（12％）,部分切除2例（8％）。术后尿崩3例,鼻出血1例。所有患者在术后第5～12天出院。术后随访1～10个月,患者临床症状和内分泌学指标均有所改善,未见肿瘤复发,无一例患者死亡。结论：神经内镜下经鼻蝶切除垂体腺瘤具有创伤小,术野清晰,手术操作简单、安全,术后并发症少,恢复快,住院时间短等优点,是治疗垂体腺瘤安全有效的手术方法。     

Galectin-3 expression in functioning and silent ACTH-Producing adenomas

Galectin-3 (Gal-3), a β galactoside-binding protein, has been implicated in a variety of biological functions including cell growth, differentiation, tumor cell adhesion, angiogenesis, tumor progression, and metastasis. We recently reported that Gal-3 was expressed in a subset of normal pituitary cells and tumors including PRL, ACTH, and in folliculostellate (FS) cells and tumors [1,2] and that Gal-3 had an important regulatory role in pituitary cell proliferation [1]. We further investigated the expression of Gal-3 protein in ACTH- and PRL-producing tumors and the expression of various galectin mRNAs by RT-PCR in pituitary adenomas and normal pituitary. Most silent ACTH subtypes 1 and 2 adenomas were negative or only focally positive for Gal-3 expression compared to functioning ACTH tumors from patients with Cushing’s disease and Nelson’s syndrome. In the normal pituitary, Gal-3 was expressed in less than 1% of the basophil-invading cells (ACTH cells present in the posterior pituitary) and in a subset of the anterior lobe ACTH-positive cells. RT-PCR analyses showed that many members of the galectin family including galectins 1, 2, 3, 4, 5, 6, 7, 8, and 9 were expressed in normal pituitary and in functioning ACTH- and PRL-producing tumors. These results indicate that Gal-3 is associated with functioning ACTH and PRL tumors and is expressed infrequently in silent ACTH adenomas, suggesting that Gal-3 protein and/or gene is altered in non-functioning ACTH tumors. The use of ACTH and Gal-3 immunostaining should help in the diagnosis of silent ACTH adenomas.     

Immunohistochemical examination of the paraadenomatous “normal” pituitary

Summary Prolactin cell hyperplasia has been described to occur in the paraadenomatous normal pituitary gland surrounding prolactinomas. However, compression of the glandular lobules and secretory cells alters profoundly the histological configuration of this tissue. No changes resembling those that occur during pregnancy are found. Immunohistochemical staining and counting of prolactin-(PRL-)secreting and growth hormone-(GH-)secreting cells in the normal, paraadenomatous pituitary gland obtained during extirpation of 24 prolactinomas and 5 adenomas causing acromegaly demonstrated that GH-secreting cells predominated in all biopsies obtained from acromegalic patients. PRL-secreting cells were more frequent than GH-secreting cells in 14 of 24 biopsies of the normal tissue surrounding prolactinomas. A particular predominance of PRL-secreting cells was found in patients with postoperative residual hyperprolactinemia. Direct comparison of adjacent sections demonstrates three cell types: One reacts with both antibodies and the other two react only with one or the other. We suggest that these groups are not stable but that cells belonging to one group can be transformed into cells belonging to the two other groups. Such a process, induced by extrahypophysary factors, may explain the shift of relative cell frequency observed in the normal pituitary gland surrounding prolactinomas.     

Immunohistochemical studies of chromogranins (A and C) in pituitary adenomas.

Expression of Chromogranin A and C was examined immunohistochemically on 45 surgically obtained pituitary adenomas. Positive rate of chromogranin A was 40.0% and chromogranin C was positive in 8.0% of 45 pituitary adenomas. Chromogranin A was expressed frequently in adenomas in which at least one of the FSH alpha, FSH beta, LH beta and TSH beta subunits was positive. It also expressed frequently in non-functioning adenomas. The positive rate of chromogranin A was low in GH or PRL positive adenomas. These findings suggest that chromogranin could be a parameter of the functional differentiation of the pituitary adenomas.     

Expression and clinical significance of Wnt players and survivin in pituitary tumours

Deregulation of the Wnt pathway has been implicated in oncogenesis of numerous tissues including the pituitary gland. Immunohistochemical localization and quantification of β-catenin, Cyclin D1, c-MYC and Survivin expression in 47 pituitary adenomas (35 non-functioning, seven GH-secreting, three prolactinomas, two ACTH-secreting tumour) and six normal controls was undertaken in this study and correlation of protein expression to patient and tumour characteristics analysed. β-catenin was strictly membrane-bound with no difference observed between normal and tumour tissue. In contrast, Cyclin D1 and c-MYC localization was nuclear and significantly higher in tumour versus normal tissue (p < 0.05). c-MYC expression correlated negatively with age at diagnosis (p = 0.006, R = −0.395) while Cyclin D1 expression correlated positively with age (p = 0.036, R = 0.306) and was higher in males than in females (p = 0.036). c-MYC expression was significantly lower in patients with functional tumours requiring octreotide treatment and in patients with non-functioning tumours suffering from hypopituitarism. Survivin expression was extremely low in tumours and absent in normal controls. Involvement of the canonical Wnt pathway appears to be minimal, given the segregation of β-catenin to the membrane. Our data suggest that c-MYC may have an important role in early pituitary tumorigenesis while Cyclin D1 is likely to promote tumour growth at a later stage. We also report a novel gender difference in Cyclin D1 expression, the biological significance of which merits further analysis. The reported reduction of c-MYC in functional tumours subsequently treated with octreotide further supports a role of c-MYC in early tumorigenesis and not in recurrence. The decrease in c-MYC in patients with hypopituitarism provides the first in vivo evidence for hormonal regulation of c-MYC expression.     

Ki-67 labelling index and invasiveness among anterior pituitary adenomas: analysis of 103 cases using the MIB-1 monoclonal antibody.

AIMS: To investigate the relation between proliferative activity of anterior pituitary adenomas, quantified by the Ki-67 labelling index, and their invasive behaviour. METHODS: Expression of Ki-67 was evaluated in 103 anterior pituitary adenomas consecutively operated on in a 36 month period and correlated with surgical evidence of invasiveness. RESULTS: Non-invasive (n = 65) and invasive (n = 38) adenomas were identified from surgically verified infiltration of sellar floor dura and bone. The wall of the cavernous sinus was infiltrated in 16 cases. Forty one adenomas were non-functioning and 62 functioning (24 prolactin, 21 growth hormone, 10 ACTH, seven mixed). The overall mean (SD) Ki-67 labelling index was 2.64 (3.69) per cent (median 1.5). The mean index was 3.08 (4.59) per cent in functioning and 1.97 (1.78) per cent in non-functioning tumours; 5.47 (9.52) per cent in ACTH adenomas and 2.33 (2.42) per cent in others (p = 0.01); 3.71 (5.17) per cent in invasive and 2.01 (2.45) per cent in non-invasive adenomas (p = 0.027); and 5.58 (7.24) per cent in cavernous sinus infiltrating v 2.10 (2.39) per cent in cavernous sinus non-infiltrating adenomas (p = 0.0005). To identify a value of labelling index beyond which adenomas should be considered invasive and another beyond which cavernous sinus infiltration should be suspected, normality Q-Q plots were obtained: a threshold labelling index of 3.5% for invasive adenomas and of 5% for cavernous sinus infiltrating adenomas was defined, with statistically significant differences (p = 0.02 and p = 0.004, respectively). CONCLUSIONS: The Ki-67 labelling index can be considered a useful marker in determining the invasive behaviour of anterior pituitary adenomas.     

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.     

Analysis of epidermal growth factor receptor and activated epidermal growth factor receptor expression in pituitary adenomas and carcinomas.

Epidermal growth factor receptor plays an important role in the pathogenesis of many malignancies. Various growth factors, including epidermal growth factor receptor, have been shown to influence pituitary tumor growth and differentiation. To analyze the role of epidermal growth factor receptor in pituitary tumor development, we examined normal pituitaries (n=8), pituitary adenomas (n=158), and pituitary carcinomas (n=7) for expression of epidermal growth factor receptor protein and messenger RNA using tissue microarrays and RT-PCR. We also examined (a) the expression of phospho-epidermal growth factor receptor, the activated form of epidermal growth factor receptor, in pituitary tumors and normal pituitaries by immunohistochemistry and (b) the effects on epidermal growth factor receptor expression of treating pituitary cells (HP75 cell line) with epidermal growth factor. Epidermal growth factor receptor and the phosphorylated variant expression were present in normal pituitary cells. Epidermal growth factor receptor messenger RNA was also detected in normal pituitaries, pituitary adenomas, and carcinomas by in situ hybridization and RT-PCR. Most pituitary adenomas showed expression of epidermal growth factor receptor and the phosphorylated variant. Nonfunctional adenomas showed higher levels of expression of epidermal growth factor receptor (76 vs 34%) and of phospho-epidermal growth factor receptor (26 vs 8%) as compared to functional adenomas. Five of seven pituitary carcinomas showed strong expression of both epidermal growth factor receptor and phospho-epidermal growth factor receptor. When a human pituitary cell line (HP75) was cultured in the presence of epidermal growth factor receptor, there was an increase in the levels of both epidermal growth factor receptor and phospho-epidermal growth factor receptor after 5 h of treatment, thus confirming that epidermal growth factor receptor signaling was active in pituitary tumors. These results indicate that activated epidermal growth factor receptor is expressed in pituitary adenomas and carcinomas. Higher levels in pituitary carcinomas suggest a role in pituitary tumor progression.     

Oncogene Activation in Pituitary Tumors

Pituitary tumors constitute 10% of intracranial neoplasms and are mostly benign, monoclonal adenomas derived from single mutant cells. Pituitary oncogenes have been intensively studied and three of them, gsp, ccnd1, and PTTG are abundant in significant numbers of cases. gsp is present in approximately 40% of Caucasian patients with GH-secreting tumors and results from a mutated, constitutively active alpha subunit of Gs protein. Persistent activation of the cAMP-PKA-CREB pathway may lead to uncontrolled cell proliferation and GH secretion. ccnd1 is overexpressed cyclin D1, and cyclin D1 gene is amplified in some pituitary tumors. PTTG is expressed in most pituitary tumors. PTTG is localized to both the nucleus and cytoplasm and interacts with several protein partners. At least three tumorigenesis mechanisms are proposed for human PTTG. 1) PTTG and FGF form a positive feedback loop and stimulate tumor vascularity. 2) PTTG transactivates c-myc or other pro-proliferation genes. 3) PTTG overexpression causes aneuploidy. PTTG expression activates p53 and causes p53-dependent and -independent apoptosis. Due to lack of functional human pituitary cell cultures and appropriate animal models for pituitary tumors, many of the results reviewed here are obtained from heterologous systems.     

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.