Immunohistochemical demonstration of bone morphogenetic protein-2 and type II collagen in pleomorphic adenoma of salivary glands

Immunohistochemical investigation of bone morphogenetic protein-2 (BMP-2) and type II collagen, two cartilage-associated proteins, was undertaken using monoclonal antibodies in 20 cases of salivary pleomorphic adenoma (PA) in order to explore their possible roles in chondroid differentiation of this tumor. Other salivary gland tumors, including adenoid cystic carcinoma (17 cases), polymorphous low-grade adenocarcinoma (10 cases), basal cell adenoma (3 cases), basal cell adenocarcinoma (1 case), and epithelial-myoepithelial carcinoma (2 cases), were also examined for comparison. In PA, BMP-2 immunoreactivity was detected in the luminal and non-luminal cells of the tubulo-ductal structures, plasmacytoid cells, and other scattered tumor cells in solid areas. In addition, tumor cells in chondroid areas in most cases (14/15), and stellate cells in myxoid areas in many cases (7/19), were also intensely labeled for BMP-2. Furthermore, BMP-2 was also detected in the non-neoplastic ductal cells in salivary glands, whereas no other salivary gland tumors were positively stained for this protein. Type II collagen was localized in the intercellular matrix of chondroid areas and in a few chondroid differentiating cells in myxoid areas, confirming its cartilage-specificity. A proportional relationship was observed between BMP-2 expression and chondroid formation, although BMP-2 was also stained in occasional PAs without chondroid formation. It is speculated that BMP-2 might be secreted by tumor cells and play a role in chondroid formation in PA by inducing some tumor cells to produce type II collagen and other chondroid matrical substances, like glycosaminoglycans. The expression of BMP-2 is specific to PA and may possibly be used as a useful marker in differentiating PA from other salivary gland tumors.     

Immunolocalisation of cartilage-derived retinoic acid-sensitive protein in pleomorphic adenoma of the parotid salivary gland

Pleomorphic adenoma of the parotid salivary glands often contains chondroid elements and may exhibit cartilaginous and osseous differentiation, although the latter is extremely rare. Twenty-nine pleomorphic adenomas (PAs) of the parotid gland were examined immunohistochemically for the distribution of cartilage-derived retinoic acid-sensitive protein (CD-RAP), a recently described marker of chondrocytes, which may be important in the morphogenesis and development of the salivary gland. In the normal parotid gland, the ductal cells expressed CD-RAP, but not the myoepithelial cells. In the pleomorphic salivary adenomas, the duct-like cells, but not the myoepithelial cells, expressed CD-RAP. Since many authorities consider myoepithelial cells to be the source of the chondroid matrix, it is surprising that these cells do not express the chondrocytic marker, CD-RAP. Putative neoplastic myoepithelium in the pleomorphic adenoma and some cells in the myxochondroid areas expressed S-100 and calponin.     

Immunohistochemical study of four histologic types of parotid gland pleomorphic adenoma

The immunohistochemical detection of lysozyme, lactoferrin, a1-antichymotrypsin and a1-antitrypsin was used to investigate the marker expression and histogenesis of each one of four histologic types of 20 parotid gland pleomorphic adenomas. Moreover, 10 adult and 20 neonate parotid glands were studied. The immunohistochemical analysis revealed that tumor types 1 and 2 are nearly identical immunohistochemically while types 3 and 4 differ from one another, as well as from types 1 and 2. The markers used failed to suggest that the tumor arises from epithelial cells of any specific anatomic part of the parotid gland.     

Immunohistochemical localization of members of the transforming growth factor (TGF)-β superfamily in normal human salivary glands and pleomorphic adenomas

Although pleomorphic adenoma is the most common type of salivary gland epithelial tumor, it frequently contains "mesenchymal"-like components, including myxoid or chondroid tissues. We reported previously that chondroid tissue formation in pleomorphic adenoma was associated with overexpression of bone morphogenetic proteins (BMPs) by neoplastic myoepithelial cells. BMPs belong to the transforming growth factor (TGF)-beta superfamily, so we hypothesized that pleomorphic adenoma may express TGF-betas and that these molecules may regulate mesenchymal-like tissue formation. To evaluate this hypothesis, we immunohistochemically examined TGF-beta1, -beta2 and -beta3 expression and localization in normal salivary glands and in 43 cases of pleomorphic adenomas. There was no evidence of TGF-beta1 expression in normal salivary glands or pleomorphic adenomas. Signals for TGF-beta2 in the normal salivary glands were observed in the intercalated ducts, while in pleomorphic adenomas they were observed in the inner ductal cells of the tubulo-glandular structures. Signals for TGF-beta3 in the normal salivary glands were observed in mucous cells, whereas in pleomorphic adenomas they were observed in the solid nests of neoplastic myoepithelial cells, in the portion showing squamous metaplasia, and in the inner ductal cells of tubulo-glandular structures. TGF-betas induce ectopic cartilage formation in vivo, but chondroid tissues in pleomorphic adenomas showed only weak TGF-beta3 expression. TGF-beta may be related to differentiation of the inner ductal cells and the neoplastic myoepithelial cells. In conclusion, pleomorphic adenomas expressed TGF-beta2 and -beta3, which may be associated with differentiation of the inner ductal cells and neoplastic myoepithelial cells.     

An immunohistochemical study of XIAP expression in pleomorphic adenoma and carcinoma ex pleomorphic adenoma

Background: X‐linked inhibitor of apoptosis protein (XIAP) is a member of the inhibitor of apoptosis proteins family of caspase inhibitors. Expression of XIAP in various neoplasms has been associated with aggressive behavior. The biological progression from pleomorphic adenoma (PA) to carcinoma ex pleomorphic adenoma (CXPA) has been poorly understood. We studied XIAP expression by immunohistochemistry in PA and CXPA. Materials and methods: Formalin‐fixed, paraffin‐embedded representative sections of 14 cases of PA and seven cases of CXPA (four invasive and three intracapsular) were stained with anti‐XIAP (# 610763; BD Biosciences, San Jose, CA, USA) following citrate‐based antigen retrieval. Granular cytoplasmic staining was considered positive and intensity was assessed from weak (1+) to strong (3+). PAs were morphologically evaluated for cellularity, cytological atypia and mitotic activity. Results: Of the seven PAs composed mostly of myxohyaline stroma with scattered ductal elements, two tumors showed no staining and five showed rare (<1%) 1+ positive cells. Of seven more cellular PAs, five had sheets of tumor cells comprising more than 50% of the tumor and two had sheets comprising more than 80% of the tumor (cellular PA), focal to diffuse 2+ to 3+ staining was observed. Tumor cells with strong staining often exhibited cytological atypia in the form of nuclear enlargement and contour irregularity, prominent nucleoli and eosinophilic cytoplasm. Mitotic activity was occasionally seen in cellular areas expressing XIAP. All cases of CXPA demonstrated diffuse 3+ staining in the carcinomatous component and 1+ to focally 3+ staining in cellular areas of the underlying PA. Conclusion: Increased expression of XIAP from PA to cellular PA to CXPA and in atypical cells within cellular areas of PA adds to our growing understanding of defective apoptotic pathways in malignant transformation in this group of salivary gland tumors and suggests an adenoma to adenocarcinoma model of progression. Further correlation with other oncogene expression may provide insight into the multiple molecular pathways that are affected in these tumors. Targeted therapy of XIAP may play a future role in the management of CXPA.     

Immunohistochemical evaluation of the small and large proteoglycans in pleomorphic adenoma of salivary glands

This study investigated the immunolocalization of small and large proteoglycans (PGs), including decorin, biglycan, PG-M/versican and aggrecan, in salivary pleomorphic adenoma (PA) using monoclonal and polyclonal antibodies. In addition, a polyclonal antibody, A0082, recognizing blood vessels was also used to help identify truly mesenchymal tissues in PA. Decorin reactivity was detected only in tumor capsule and interstitial tissue of non-neoplastic salivary gland, but not in the tumor tissue. Biglycan was frequently revealed throughout the matrix of small chondroid regions and in the peripheral portion of larger chondroid regions. PG-M/versican was mainly localized to the truly mesenchymal tissues in PA and the innermost portion of tumor capsule. On the contrary, aggrecan was extensively expressed in the non-luminal epithelial areas as well as in the myxoid and chondroid areas, but not in the truly mesenchymal tissues. These findings suggest that aggrecan is the most widely distributed PG in PA and may be produced mainly by non-luminal tumor cells. The absence of aggrecan from the truly mesenchymal tissues argues against its origin from this source. Both aggrecan and biglycan may play important roles in the chondroid differentiation and morphogenesis of PA.     

Neural adhesion molecule (N-CAM) in pleomorphic adenoma and carcinoma ex-pleomorphic adenoma

BACKGROUND: The neural cell adhesion molecule (N-CAM) has been implicated in the behaviour of the adenoid cystic carcinoma. In vitro, it was demonstrated that N-CAM inhibits cell invasion. The aim of this study was to search for N-CAM in the most common salivary gland tumour that has a malignant counterpart. METHODS: We investigated the presence of N-CAM in pleomorphic adenoma and its malignant counterpart, the carcinoma ex-pleomorphic adenoma, using the immunohistochemistry technique. RESULTS: Neural cell adhesion molecule was expressed in all cases of pleomorphic adenoma, strongly labelling the luminal cells of the double-layered ductform structures. This expression was weaker in neoplastic myoepithelial cells and progressively diminished at a distance from the luminal cells. In carcinoma, ex-pleomorphic adenoma N-CAM was either totally absent or faintly present at the apical pole of the few luminal cells. CONCLUSIONS: As a result of the peculiar distribution of N-CAM in pleomorphic adenoma, we speculated that N-CAM behaves as a tumour-suppressor molecule, which is expressed in the benign neoplasm and which is down-regulated after malignancy, when the tumour assumes an invasive behaviour.     

Nucleolar organizing regions (NORs) in pleomorphic adenomas of the salivary glands

The number of nucleolar organizing regions (NORs) can be used to reveal the degree of cell activity or metabolism in histopathology specimens. Forty-one cases of pleomorphic adenomas of the salivary glands were studied with a silver nitrate colloid staining method to demonstrate the NORs in the cells of the solid and duct pattern areas and in chondroid areas. Acinic and intercalated ducts cells of the surrounding normal salivary glands were also examined. The data was analyzed statistically and it was found that the cells in the solid/ductal areas had a higher cellular activity than the chondroid cells. This difference is probably directly related with the possibility of malignant transformation and the incidence of the different types of malignant pleomorphic adenomas. Furthermore, it is concluded that the number of NORs in pleomorphic adenomas to define malignancy should be done discriminating between the different histologic patterns because of their different metabolic activities.     

涎腺多形性腺瘤恶变机制的研究进展

涎腺多形性腺瘤是最常见的涎腺上皮性肿瘤,多次复发或病程长者可恶变为恶性多形性腺瘤.涎腺多形性腺瘤的恶变是多因素、多基因变异的综合病变过程.研究发现,细胞遗传学改变、癌基因、抑癌基因、黏附分子及细胞外基质蛋白在涎腺多形性腺瘤的恶变过程中均有一定的作用.本文就多形性腺瘤恶变机制的研究进展作一综述.     

IGFBP-3在唾液腺多形性腺瘤中的表达及意义

目的:研究胰岛素样生长因子结合蛋白3(IGFBP-3)在唾液腺多形性腺瘤(SPA)中的表达.方法:采用Western blot检测IGFBP-3在40例SPA、40例正常腺体组织及10例恶性唾液腺肿瘤组织中的表达.采用实时荧光定量PCR(qRT-PCR)检测IGFBP-3 mRNA在50例SPA组织、50正常腺体组织及10例唾液腺恶性肿瘤中的表达.结果:IGFBP-3蛋白在唾液腺正常组织(N组:8.54 ±3.95)的表达(A值)显著高于多形性腺瘤组(PA组:4.78 ±2.07)及恶性肿瘤组(CA组:3.63 ±2.27)(P＜0.05).而PA组与CA组之间则不存在显著差异(P＞0.05).IGFBP-3 mRNA在SPA组织与恶性肿瘤组织中的表达均低于正常组织(PA/N组:0.654 ±0.387,CA/N组:0.452±0.229),PA组与CA组不存在显著差异(P＞0.05);不同包膜浸润程度的多形性腺瘤组织中相对含量的差异有统计学意义(P＜0.05),在年龄、性别及有无复发上无明显差异.结论:IGFBP-3在多形性腺瘤中的低表达可能减低了拮抗IGF-1R的作用,引起肿瘤细胞的增殖,促进肿瘤形成.     

Role of CD44 in tumor‐initiating cells of salivary gland pleomorphic adenoma: More than a surface biomarker

CD44, a cell‐surface glycoprotein, functions as a receptor for hyaluronic acid. Our research group has previously shown that CD44 is a biomarker for the CD44^hi cells (tumor‐initiating cells; TICs) in murine salivary gland tumors. However, little is known concerning the biological roles of CD44 in the tumorigenesis of pleomorphic adenoma. The present study is aimed to investigate the effects of CD44 on the proliferation, invasive capability, and apoptosis of TICs in vitro, as well as the tumorigenicity of TICs in vivo. The results demonstrated that knockdown of CD44 attenuated the malignant phenotype of TICs. Furthermore, in vivo xenograft studies indicated that CD44 knockdown inhibited tumorigenesis of pleomorphic adenoma. In addition, neither the CD44^low cells nor the CD44‐modified CD44^low cells developed neo‐tumors, which indicated that overexpression of CD44 did not enable the CD44^low cells to be transformed into TICs. Taken together, these data demonstrate that CD44 not only acts as a biomarker, but also functions as a key player in the tumor‐initiating capacity of TICs. These results shed light on the pathogenesis of salivary gland tumors and provide a potential therapeutic target for treating pleomorphic adenoma.     

Localization of glycosaminoglycans (GAGs) in pleomorphic adenoma (PA) of salivary glands: an immunohistochemical and histochemical evaluation

The tumor matrix of salivary pleomorphic adenoma (PA) is characteristically rich in glycosaminoglycans (GAGs), which contribute to its complex histoarchitecture. This study evaluated the microscopic localization of various GAGs in 17 PAs, using a panel of anti-GAG monoclonal antibodies and biotinylated hyaluronic acid (HA)-binding protein. Both epithelial and mesenchymal-like tissues were confirmed to contain GAGs. Luminal epithelial cells mostly lacked GAGs, whereas GAGs were seen both in the cytoplasm and cell membrane of non-luminal epithelial cells. In addition, small intercellular accumulations of GAGs were often present in solid epithelial areas, implying the epithelial origin of GAGs. GAGs did not appear to be a main component of the hyaline matrix. The myxoid region was consistently stained for both chondroitin 6-sulfate (CS-6) and HA but variably for chondroitin 4-sulfate (CS-4), dermatan sulfate (DS) and keratan sulfate (KS); heparan sulfate (HS) was not detected. The chondroid region showed increased staining for CS-6 but reduced staining for HA when compared with the myxoid region. In addition, CS-4, DS and KS were seen both in chondroid cells and the territorial matrix, whereas HS was present only in the cells. It is suggested that GAGs in PA are mainly produced by non-luminal cells and influence the proliferation, differentiation, secretory activity and shape of tumor cells, thus contributing to the morphological diversity of this tumor.     

Precancerous foci in pleomorphic adenoma of the salivary gland: recognition of focal carcinoma and atypical tumor cells by P53 immunohistochemistry

BACKGROUND: It is still controversial if atypical tumor cells scattered in salivary pleomorphic adenomas are precancerous and how carcinoma arises in pleomorphic adenomas. METHODS: We studied clinicopathologically the frequency and variation of cellular atypia among tumor cells and examined the expression status of p53 gene products as well as proliferating cell nuclear antigen (PCNA) in 101 surgical materials of pleomorphic adenomas. RESULTS: Histopathologically, atypical tumor cells were found in 51% of the cases examined. Their mode of distribution was classified into three groups: focal (six cases, 6%) which could be identified as focal carcinoma, measuring less than 1 mm in diameter; sporadic (15 cases, 15%) and singular (30 cases, 30%). These atypical cells were located mainly within sheet-like nests of tumor cells but not in chondroid or fibro-hyaline foci. Immunohistochemically, most of the atypical cells were positive for p53 gene products and PCNA. CONCLUSION: The results indicated that atypical cells with p53 protein accumulation in their nuclei could be regarded as cells in a precancerous state not yet forming an apparent carcinomatous nest. Some cell population with these atypical cells are likely to form focal carcinomas and then to an apparent form of carcinoma in pleomorphic adenoma.     

Biosynthesis of glycosaminoglycans and aggrecan by tumor cells in salivary pleomorphic adenoma: ultrastructural evidence

The present study attempted to discover the sites of synthesis of various glycosaminoglycans (GAGs) and aggrecan in salivary pleomorphic adenoma (PA) with the use of a highly sensitive and specific post-embedding immunogold-silver staining technique at the ultrastructural level. Silver particles representing various GAGs and aggrecan were found to accumulate frequently in the intercellular spaces of non-luminal cells in the epithelial clusters and were dispersed in the myxoid matrix of the mesenchyme-like areas. Furthermore, the non-luminal epithelial cells were demonstrated to contain immunopositive intracytoplasmic vesicles and vacuoles, some of which were of Golgi complex origin. In contrast, intracellular silver particles for hyaluronic acid were mostly attached to the inner surface of the cell membrane. These observations agree well with the current theories of the biosynthesis of GAGs and proteoglycans and provide direct evidence for the production of various GAGs and aggrecan by tumor epithelial cells of PA. Such findings support the ideas that in PA a loss of epithelium occurs by stromalization following epithelial secretion of extracellular matrix substances and transformation of epithelium to mesenchyme represents the basic principle of the tissue heterogeneity in this tumor.     

Proliferative activity of salivary gland pleomorphic adenomas and myoepitheliomas as evaluated by the proliferating cell nuclear antigen (PCNA) labeling index (LI)

Thirteen examples of pleomorphic adenoma (PA) and 4 of myoepithelioma (Me, 2 plasmacytoid cell type, 2 mixed cell type) were examined with respect to their proliferative activity on the basis of proliferating cell nuclear antigen (PCNA) immunohistochemistry. In PA, PCNA labeling index (LI) in tubular/trabecular/solid areas was significantly higher than that in myxomatous or chondroid areas. Although the mean value of LI in PA and Me was not statistically different (PA; 3.02±1.03%, Me; 3.19±1.76%), the Me of mixed cell type composed of epithelial, spindle or clear neoplastic myoepithelial cells had significantly higher LI, indicating the possibility of more rapid growth than PA. The small difference in the mean value of PCNA LI between PA and the mixed cell type of Me, however, suggests that enucleation with a margin of normal uninvolved tissue remains the recommended treatment for Me, as well as for PA.     

Immunohistochemical localization of bone morphogenetic protein (BMP) in calcifying fibrous epulis

Immunohistochemical detection of bone morphogenetic protein (BMP) in calcifying fibrous epulis was performed to elucidate the biological process of ossification and cemento-ossification. In a total 25 cases, 15 (60%) showed positive BMP staining in bone forming areas. Histopathological features of developing hard tissues were varied, consisting of structures such as woven bone and cemento-osseous formations. BMP immunostaining was limited to osteoblasts and fibrous connective tissue surrounding the bone matrix. BMP was concentrated in the periodontal fibres and in dense fibrous structures in the cemento-osseous masses. On the basis of histopathological and immunohistochemical features, the histogenesis of ossifying and cemento-ossifying processes appear to be of two possible origins; the excessive proliferation of periodontal ligament and a meta-plastic process occurring in the connective tissue fibres (non-periodontal in origin), with the former being more common.     

MMP-9和VEGF在腮腺多形性腺瘤中的表达研究

目的 研究MMP-9和VEGF在腮腺多形性腺瘤中的表达情况及其与肿瘤良恶性的关系。方法 应用荧光实时定量PCR和Western blotting方法检测32对多形性腺瘤及其瘤旁组织中MMP-9和VEGF的表达。结果 32例多形性腺瘤中26例为良性,6例为恶性。MMP-9和VEGF的mRNA和蛋白表达量在多形性腺瘤组织中高于瘤旁组织（P＜0.05）;在恶性多形性腺瘤组织中高于良性多形性腺瘤组织（P＜0.05）。结论 MMP-9和VEGF在腮腺多形性腺瘤中高表达,并且可能与腮腺多形性腺瘤的恶变相关;腮腺多形性腺瘤肿瘤外1 cm为安全切除界限。     

Immunolocalization of basement membrane molecules in the stroma of salivary gland pleomorphic adenoma

In order to determine the participation of basement membrane molecules in formation of its characteristic stroma, 30 cases of salivary gland pleomorphic adenoma were examined by immunohistochemical staining for type IV collagen, laminin, heparan sulfate proteoglycan, and entactin. The stroma was histopathologically classified into four types: hyaline, fibrous, myxoid, and chondroid. Immunohistochemically, type IV collagen and laminin were more intensively localized in hyaline, fibrous and chondroid types of stroma, whereas heparan sulfate proteoglycan was more prominent in myxoid areas. The results suggest that the stroma contains these basement membrane components, which are possibly biosynthesized by epithelial tumor cells, and that histological variety of the stroma depends on proportion of local contents of each basement membrane molecule.     

腮腺多形性腺瘤中BMP-2基因的表达及意义

目的：检测腮腺多形性腺瘤中BMP-2基因的表达并探讨其病理意义。方法：分别抽提7例人腮腺多形性腺瘤组织标本的总RNA,应用人BMP-2 mRNA特异性引物和RT-PCR方法检测7例腮腺多形性腺瘤中BMP-2 mRNA的表达情况。结果：7例标本中有6例检测到人BMP-2 cDNA的特异性扩增条带。结论：BMP-2基因的表达可能参与腮腺多形性腺瘤中软骨样组织形成的病理过程。