Correlation between SPARC (Osteonectin) expression with immunophenotypical and invasion characteristics of pituitary adenomas

Pituitary adenomas are the third most common intracranial tumors. Invasive adenomas account for only 0.1–0.2% of pituitary tumors. SPARC is a matrix glycoprotein that plays a role in progression and invasiveness of neoplasms. In this study, we examined the potential role of SPARC in invasive pituitary adenomas. Forty pituitary adenomas have been examined with histopathological and immunohistochemical techniques. The cohort has been classified into two groups as invasive (n = 25) and non‐invasive (n = 15) utilizing the Hardy classification. Formalin fixed tissues have been stained with hematoxylin eosin. Ki‐67, p53, and SPARC monoclonal antibodies have been used. We did not detect any significant difference on Ki‐67, SPARC, and p53 expression patterns correlating with the pathological subtype or invasiveness. Only 24% of invasive adenomas had Ki‐67 levels over 1%. A total of 67.7% non‐invasive adenomas had Ki‐67 levels below 1%. We did not detect any relation between SPARC levels and invasiveness of pituitary adenomas. Absence of significant SPARC expression in tumor progression, sellar dilatation, erosion and destruction suggest that SPARC scores are not related with invasiveness or progressiveness of pituitary adenomas.     

Chromogranin a processing in human pituitary adenomas and carcinomas: analysis with region-specific antibodies

The expression of various chromogranin A (CgA) peptide fragments was examined with region-specific antisera in benign and malignant pituitary tumors. Analysis of the proconvertases responsible for proteolytic processing of CgA, prohormone convertase 1/3 (PC1/3), and PC2 was also performed. Adenomas were studied using tissue microarrays, and a larger tissue section of a subset of the prolactin (PRL) adenomas was used to compare to the tissue microarray analysis. Carcinomas were analyzed using large tissue sections. There were differences in CgA proteolytic products detected between the functional (PRL, adrenocorticotropic hormone [ACTH], and growth hormone tumors and the nonfunctional (gonadotroph and null cell) tumors, with the former group, expressing lower levels of many peptides. These differences were most notable in the PRL adenomas and carcinomas in which the region-specific antisera against vasostatin I and vasostatin II detected these fragments in the lowest percentage of tumors and/or had the weakest immunoreactivity. The CgA peptide fragment detected by CgA 176–195 (chromacin) antiserum was expressed by the highest percentage of most functional and nonfunctional benign and malignant pituitary tumors. ACTH carcinomas (n+3) were more strongly immunoreactive compared to the ACTH adenomas. These results show that there is differential expression of CgA peptide fragments and PC1/3 among different types of pituitary tumors and that ACTH pituitary carcinomas have higher levels of immunoreactive CgA peptide fragments compared to ACTH adenomas. This study also shows the utility of tissue microarrays in the analysis of a large group of tumors with region-specific antisera.     

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.     

High nuclear expression of protein arginine methyltransferase‐5 is a potentially useful marker to estimate submucosal invasion in endoscopically resected early colorectal carcinoma

Protein arginine methyltransferase‐5 (PRMT5), a major type II arginine methyltransferase, is an important epigenetic modifier with oncogene‐like properties because of its ability to repress the expression of tumor suppressor genes. We determined the correlations between PRMT5 expression or its cellular localization and malignant progression, with special reference to invasiveness, in colorectal adenomas and early colorectal carcinomas (CRCs). We performed immunohistochemical detection of PRMT5 in 108 endoscopically resected tissue samples (6 adenomas with low‐grade dysplasia, 34 adenomas with high‐grade dysplasia, 30 intramucosal carcinomas, and 38 submucosal invasive carcinomas). Early CRC (55 of 68, 80.9%) showed more frequent nuclear expression of PRMT5 than adenoma (15 of 40, 37.5%) (P < 0.001). Furthermore, high (strong staining in ≥ 50% of nuclei) nuclear expression of PRMT5 was more common in submucosal invasive carcinoma (21 of 38, 55.3%) than in intramucosal carcinoma (9 of 30, 30.0%) (P = 0.037). In conclusion, our data suggests that high nuclear expression of PRMT5 is a potentially useful marker for estimating submucosal invasion of early CRC in endoscopically resected specimens.     

Inactivation of the p16 gene in human pituitary nonfunctioning tumors by hypermethylation is more common in null cell adenomas

Recent studies have shown that methylation of the CpG island within the p16/CDKN2A/MTS1 (p16) gene is associated with loss of expression of p16 protein in pituitary tumors. We analyzed a series of 21 pituitary adenomas and three normal pituitaries along with a human pituitary cell line (HP75) for methylation of exon 1 by methylation-specific PCR, immunohistochemistry, and Western blotting. PCR analysis showed that 5/7 (71%) of null cell adenomas, but only 2/7 (29%) gonadotroph tumors were hypermethylated. In addition, 1 of 2 ACTH tumors but no GH (n=4) or PRL (n=1) adenoma examined were hypermethylated. Immunostaining and Western blot analysis of protein expression supported the methylation-specific PCR analyses. These results show that p16 gene silencing by hypermethylation is more common in null cell adenomas compared to other nonfunctioning adenomas such as gonadotroph tumors and that the role of p16 in the pathogenesis of pituitary adenomas is restricted to specific tumor subtypes. Supported in part by NIH CA90249 and by a grant from the Jarislowsky Foundation.     

Tumor-specific downregulation and methylation of the CDH13 (H-cadherin) and CDH1 (E-cadherin) genes correlate with aggressiveness of human pituitary adenomas.

The gene products of CDH13 and CDH1, H-cadherin and E-cadherin, respectively, play a key role in cell-cell adhesion. Inactivation of the cadherin-mediated cell adhesion system caused by aberrant methylation is a common finding in human cancers, indicating that the CDH13 and CDH1 function as tumor suppressor and invasion suppressor genes. In this study, we analyzed the expression of H-cadherin mRNA and E-cadherin protein in 5 normal pituitary tissues and 69 primary pituitary adenomas including all major types by quantitative real-time RT-PCR (qRT-PCR) and immunohistochemistry, respectively. Reduced expression of H-cadherin was detected in 54% (28/52) of pituitary tumors and was significantly associated with tumor aggressiveness (P<0.05). E-cadherin expression was lost in 30% (21 of 69) and significantly reduced in 32% (22 of 69) of tumors. E-cadherin expression was significantly lower in grade II, III, and IV than in grade I adenomas (P=0.015, P=0.029, and P=0.01, respectively). Using methylation-specific PCR (MSP), promoter hypermethylation of CDH13 and CDH1 was detected in 30 and 36% of 69 adenomas, respectively, but not in 5 normal pituitary tissues. Methylation of CDH13 was observed more frequently in invasive adenomas (42%) than in non-invasive adenomas (19%) (P<0.05) and methylation of CDH1 was more frequent in grade IV adenomas compared with grade I adenomas (P<0.05). Methylation of either CDH13 or CDH1 was identified in 35 cases (51%) and was more frequent in grade IV invasive adenomas than in grade I non-invasive adenomas (P<0.05 and P<0.05, respectively). Downregulation of expression was correlated with promoter hypermethylation in CDH13 and CDH1. In conclusion, the tumor-specific downregulation of expression and methylation of CDH13 and CDH1, alone or in combination, may be involved in the development and invasive growth of pituitary adenomas.     

Pituitary Carcinomas

Pituitary carcinomas are defined by their metastatic growth. Most of them also invade into surrounding tissues. They should be classified by the site of their metastases (cerebrospinal, systemic, or combined) and by the presumable cell type of origin, respectively with the hormone being demonstrable by immunohistochemistry (adrenocorticotrophic hormone [ACTH], prolactin [PRL], growth hormone [GH], hormone-negative). Pituitary carcinomas develop from invasive adenomas. Nearly all tumors had been treated by surgery or X-ray before they metastasized. Since 1976, 37 cases demonstrated with modern methods were reported: 23 had metastasized into the brain or meninges, 10 showed extracerebral metastases, and 4 showed both types of metastases. In our collection of pituitary tumors, three carcinomas (0.13%) were identified: two with systemic metastases (one ACTH secreting and one PRL secreting) and one with meningeal dissemination and ACTH production. The diagnosis of pituitary carcinomas should be based on four criteria: a demonstrable metastasis, identification of the primary tumor as a pituitary tumor, similarity between the structure and immunohistological marker expression of metastsis and primary tumor, and exclusion of an alternative primary tumor.     

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.     

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.     

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.     

垂体腺瘤中E-cadherin和nm23蛋白表达与肿瘤生物学行为的关系

目的探讨垂体腺瘤中Ecadherin(Ecad)和nm23蛋白表达与肿瘤体积、激素分泌和侵袭性的关系。方法应用免疫组化SP法检测Ecad和nm23基因蛋白在23例侵袭性垂体腺瘤和24例非侵袭性垂体腺瘤组织中的表达。结果Ecad和nm23的表达在侵袭性垂体腺瘤组低于非侵袭性组(P<0.05),Ecad表达在非分泌型垂体腺瘤组低于分泌型组且与肿瘤体积呈负相关(P<0.05),Ecad与nm23表达间呈正相关(P<0.05)。结论Ecad与nm23表达降低可能与垂体腺瘤的侵袭性有关,Ecad表达降低可能影响垂体腺瘤细胞分化和促进细胞增殖,Ecad和nm23可作为评估垂体腺瘤侵袭能力的生物学指标之一。     

垂体腺瘤中MMP-9及TIMP-1表达与肿瘤生物学行为的关系

目的　探讨MMP 9及其抑制因子TIMP 1在垂体腺瘤中表达与肿瘤生物学行为的关系。方法　应用免疫组化S P法检测上述基因蛋白在 2 3例侵袭性和 2 4例非侵袭性垂体腺瘤组织中的表达。结果　侵袭性垂体腺瘤组中MMP 9的表达和MMP 9表达超过TIMP 1的比例高于非侵袭性腺瘤组 (P <0 0 5 ) ;TIMP 1在侵袭性垂体腺瘤组的表达有降低的趋势 ,但无统计学意义 (P >0 0 5 ) ;MMP 9与TIMP 1表达呈正相关 (P <0 0 5 )。结论　MMP 9表达上调导致其和TIMP 1的表达失衡与垂体腺瘤侵袭性有关 ,MMP 9可作为评估垂体腺瘤侵袭性的分子生物学指标     

Light bodies in human pituitary adenomas

Summary Light bodies are large cytoplasmic granules originally described in the gonadotrophic cells of the rat pituitary gland. In order to determine whether similar bodies occur in the human anterior pituitary gland, 89 pituitary adenomas and periadenomatous tissue from 20 cases were examined by transmission electron microscopy. Double membrane bound bodies with filamentous internal structure identical to rodent light bodies were identified in 10 hormone-producing adenomas: 5 PRL, 1 PRL-GH, 2 GH, and 2 ACTH-producing tumours. No light bodies were found in the remaining 79 tumours nor in the pituitary cells in periadenomatous tissue from 20 cases. These results show that some human pituitary adenomas may contain light bodies identical to those seen in gonadotrophs of rat pituitary.Abbreviations PRL prolactin - GH growth hormone - ACTH adenocorticotropic hormone - FSH follicle-stimulating hormone - LH luteinizing hormone     

神经生长因子受体在垂体腺瘤中的表达

目的：探讨两种神经生长因子（ＮＧＦ）受体（ＴｒＫＡ和ｇｐ７５）在各类垂体腺瘤中的表达。方法：采用免疫细胞化学ＳＡＢＣ法,检测了４４例经病理证实的垂体腺瘤中两种ＮＧＦ受体的表达情况。结果：ＴｒＫＡ在垂体ＰＲＬ分沁腺瘤中的过度表达率６９．２％,而在其它类型垂体腺瘤中的过度表达率为１９．４％,两者比较差异有非常显著性（Ｐ〈０．００５）。ｇｐ７５在垂体ＰＲＬ分泌腺瘤中的过度表达率在４６．２％,而在其它类型     

Immunocytochemical analysis of the synaptic proteins SNAP-25 and Rab3A in human pituitary adenomas. Overexpression of SNAP-25 in the mammosomatotroph lineages

SNAP-25 and Rab3A were originally identified as synaptic proteins involved in neuronal membrane traffic. Recently, both proteins have been detected in several mammalian endocrine cell types and have been proposed as essential components of the exocytotic pathway in neuroendocrine cells. In this study, the expression of SNAP-25 and Rab3A was analysed in biopsied human anterior pituitary tumours (21 cases) by immunocytochemical methods. No differences in Rab3A immunoreactivity were observed between tumour and normal pituitary cells. Strong SNAP-25 immunoreactivity was detected in tumour cells of prolactinomas (n=3). Several growth hormone (GH)/prolactin (PRL) tumours also displayed intense SNAP-25 immunolabelling (n=3), whereas the remaining GH-secreting adenomas (n=4) exhibited moderate to weak SNAP-25 immunoreactivity. In contrast, SNAP-25 near-background immunostaining was observed in tumour cells of adrenocorticotrophic hormone (ACTH)-secreting tumours (n=4) and non-secreting tumours (n=7), as well as in normal pituitary cells. Since SNAP-25 and Rab3A have been shown to be involved in exocytotic events in rodent endocrine cells, overexpression of SNAP-25 protein in PRL and GH/PRL tumour cells might be implicated in the mechanism of exocytosis of the neoplastic human mammosomatotroph lineages. © 1997 John Wiley & Sons, Ltd.     

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     

Folliculostellate cells in pituitary adenomas: Studies of hormonal profile and tumor vascularity

The hormonal immunoreactivity and vascularity of pituitary adenomas containing folliculostellate (FS) cells have been compared with those of tumors in which such cells were not identified. FS cells were present in variable numbers in 36 of 92 tumors. Adenomas immunoreactive for growth hormone (GH), adrenocorticotropic hormone (ACTH), or prolactin (PRL) contained FS cells in 40–50% of cases. Those immunoreactive for glycoprotein hormones and alphasubunit contained FS cells in 67–85% of cases, a statistically significant correlation. When alpha-subunit was also present in GH-, GH/PRL-, and ACTH-immunoreactive tumors, a higher proportion contained FS cells (57–91%). These data suggest a correlation between the presence of FS cells and glycoprotein immunoreactivity in pituitary adenomas. Vascular channels identified by the binding of the lectinUlex europaeus were quantified in the two types of tumors. Those containing FS cells were not more vascular than those without FS cells, which suggests that FS cells do not play a significant role in the regulation of intratumoraf vascularization in human pituitary adenomas.