Immunohistochemical localization of vitamin B12 R-binder in salivary gland tumors. Implications for cell differentiation

Vitamin B12 R-binder, a specific binding protein for vitamin B12, was studied immunohistochemically in normal and 106 neoplastic salivary gland tissues with a monoclonal antibody against vitamin B12 R-binder (R-binder). In normal salivary glands, R-binder localization was restricted to the ductal systems and to mucous acinar cells; serous acinar cells, myoepithelial cells and stromal connective tissues were consistently negative. Among salivary gland tumors, R-binder was present in 87% of pleomorphic adenomas, 100% of monomorphic adenomas, and 40% of adenoid cystic carcinomas; positivity was observed only on luminal surfaces of small ductular elements, indicating that the components closely related to ductal differentiation were rather small in population. R-binder could be detected both in lacunar and non-lacunar cells within chondroid areas of pleomorphic adenomas, suggesting the possibility that chondroid regions arise from metaplastic changes in ductal epithelial cells. In mucoepidermoid tumors, mucous cells and focal squamous cells exhibited cytoplasmic staining. The staining pattern for R-binder in epithelial components of adenolymphomas showed close similarities to those found in normal large excretory ducts. Two acinic cell tumors and one case each of myoepithelioma and malignant myoepithelioma exhibited negative reactivity for R-binder, showing that these neoplasms are solely composed of tumor cells without the characteristics of ductular differentiation. The immunohistochemical examination of salivary gland tumors, employing a monoclonal anti-R-binder antibody, may have some implications for cellular heterogeneity and differentiation in various tumors.     

KIT protein expression and analysis of c-kit gene mutation in adenoid cystic carcinoma.

The c-kit proto-oncogene encodes a transmembrane receptor tyrosine kinase (KIT), which is expressed in several normal human tissues, especially mast cells and interstitial cells of Cajal. Expression of KIT has been noted in several types of neoplasms and gene mutation has been shown as a mechanism of c-kit oncogene activation in some tumors. Recently, a single adnexal adenoid cystic carcinoma (ACC) was reported to demonstrate KIT expression, however, examination of KIT expression or c-kit mutation in ACC of salivary glands has not been performed. We examined archival tissue samples from 30 ACC of major and minor salivary glands for KIT protein expression by immunohistochemistry with a polyclonal antibody and c-kit gene mutation by polymerase chain reaction amplification and DNA sequencing. KIT protein expression was noted in 90% of ACCs. An association between the presence of at least 50% KIT positive neoplastic cells and Grade 3 ACC or a solid growth pattern was observed (P < .05). KIT expression in normal or nonneoplastic salivary gland tissue was absent. No c-kit juxtamembrane domain (exon 11) or phosphotransferase domain (exon 17) mutations were found in any of the tumors examined. In conclusion, KIT protein expression is correlated with tumor grade of salivary ACC. However, gene mutation of exon 11 or exon 17 is not a mechanism of c-kit activation in these neoplasms.     

Expression of S-100 protein and glial fibrillary acidic protein in cultured submandibular gland epithelial cells and salivary gland tissues. Histogenetic implication for salivary gland tumors.

S-100 protein and glial fibrillary acidic protein (GFAP) were studied in human salivary gland tissues and human cultured submandibular gland epithelial cells. Immunohistochemically, ductal cells in normal salivary gland tissues were positive for S-100 protein and GFAP, but myoepithelial cells were uniformly negative. Immunocytochemically, cultured submandibular gland ductal cells were positive for S-100 protein and GFAP. By immunoblotting analysis of the cultured cell lysates, a 6.5-kd S-100 protein was detected. This band corresponded to S-100 protein purified from bovine brain. The cultured submandibular gland cells expressed 49- and 54-kd GFAP polypeptides. These results have important implications for the histogenesis of salivary gland tumors.     

Expression and amplification of neu oncogene in pleomorphic adenomas of salivary gland.

Amplification of the neu oncogene and overexpression of its product was found to be a potential marker for aggressive biological behavior in breast cancer. However, the expression of the neu oncoprotein in normal breast ducts and myoepithelial cells has also been demonstrated. Hence, we examined normal salivary gland tissue and 15 cases of pleomorphic adenoma for the expression of the neu oncoprotein by immunohistochemistry using two polyclonal antibodies and 12 cases for the amplification of the neu oncogene using slot blot hybridization. Immunohistochemistry in the normal salivary gland revealed positive staining of all ductal cells. In the pleomorphic adenomas all cellular elements stained to a variable degree. The positive staining was seen in the ductal cells, in the solid sheets, and in chondroid, myxoid, and metaplastic foci. The normal salivary gland and 11 of 12 cases of pleomorphic adenoma showed no increase in copy number of the neu oncogene, whereas one case showed threefold amplification. These results indicate that the neu oncoprotein is expressed but the neu copy number is not increased in normal salivary gland epithelium and in most pleomorphic adenomas. The threefold amplification of the gene in one case may indicate an aggressive biological behavior.     

Enhanced expression of cathepsin L in metastatic bone tumors.

Cathepsin L is a kind of cystein proteases which are known to facilitate the invasion and metastasis of tumor cells by degrading the components of basement membrane and extracellular matrix. This study was undertaken to investigate the expression of cathepsin L by Northern blot analysis with radiolabeled cDNA specific for cathepsin L in six normal tissues, two osteosarcoma cell lines, MG-63 and Saos-2, six primary bone tumors and six metastatic bone tumors. In six normal tissues, the highest level of cathepsin L was expressed in liver with the descending order of liver > lung > thymus > ovary > kidney > esophagus. One of the two osteosarcoma cell lines established from the primary sites expressed a high level of cathepsin L mRNA. Out of six primary bone tumors, three (50%) expressed cathepsin L mRNA, while all (100%) of six metastatic bone tumors expressed the mRNA. These results demonstrating the higher frequency of expression of cathepsin L in metastatic bone tumors suggest that cathepsin L may participate in tumor invasion and metastasis.     

Increased expression of cyclooxygenase-2 in human salivary gland tumors

We examined the immunohistochemical localization of cyclooxygenase (COX)-2 in human salivary gland tumors. Thirty salivary gland adenomas (SGA), 40 salivary gland carcinomas (SGC) and 15 normal salivary glands (NSG) were studied. NSG showed restricted COX-2 staining only in the epithelial cells of salivary ducts. In contrast, COX-2 protein was detected in 27 cases of SGA (90%), except for three myoepitheliomas, and in all cases of SGC (100%) at various intensities and in various fashions. Thirteen SGA (43%) and 36 SGC (90%) cases showed strong COX-2 staining predominantly in tumor cells containing ductal components, as did serous and mucous acinic components of acinic cell carcinomas, mucoepidermoid carcinomas and mucinous carcinomas. These findings may suggest that COX-2 in salivary gland tumors is expressed in tumor cells derived from pluripotential ductal epithelium that can histologically develop into either serous or mucinous acinar cells.     

Constitutive c-fos expression in osteoblastic MC3T3-E1 cells stimulates osteoclast maturation and osteoclastic bone resorption.

The effect of culture supernatants of c-fos-transfected MC3T3-E1 osteoblastic cells on osteoclastic bone resorption was studied. Human c-fos cDNA was integrated in the expression vector pH8, and the cells were transfected using the calcium phosphate precipitation technique. Osteoclastic bone resorption was quantified by the pit formation assay, and the osteoclast maturation from precursor was assessed by the generation of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells (MNC). The culture supernatants of MC3T3-E1 transfectants constitutively expressing c-fos gene enhanced osteoclast-like MNC formation from haematopoietic blast cells compared with those of control transfectants (P < 0.01). The culture supernatants also promoted osteoclastic bone resorption: the pit number, 118.7 +/- 38.5, was significantly higher than 19.0 +/- 10.1 of the control (P < 0.05). The absorption area, 12,394 +/- 3145 mm2, was significantly larger than 1646 +/- 314 mm2 of the control (P < 0.05). The culture supernatants also promoted bone resorption by purified chick osteoclasts (P < 0.05). The results show that constitutive expression of c-fos gene in osteoblastic MC3T3-E1 cells stimulates osteoclast maturation and osteoclastic bone resorption by releasing humoral mediator(s).     

Immunohistological analysis of tumour growth factor beta 1 expression in normal and inflamed salivary glands.

AIM: To determine whether transforming growth factor-beta 1 (TGF-beta 1) has a pathogenetic role in disease of the salivary glands. METHODS: An indirect immunohistochemical technique was used to analyse TGF-beta 1 expression in six specimens of normal salivary gland and 23 surgical specimens. RESULTS: TGF-beta 1 was strongly expressed in the ductal epithelial cells of normal salivary gland tissues (six of six cases) and in inflammatory conditions (eight of 11 cases). In contrast, TGF-beta 1 was not detectable in ductal epithelial cells expressing HLA-DR around infiltrating CD4+ CD45RO+ activated T cells, in the salivary gland tissue of patients with Sjögren's syndrome. CONCLUSION: Because TGF-beta 1 has an essential role in the mucosal immunity of salivary glands, abnormal expression of this cytokine must be regarded as a candidate in the pathogenesis of Sjögren's syndrome.     

Expression of alpha-methylacyl-CoA racemase (P504s) in various malignant neoplasms and normal tissues: astudy of 761 cases

Alpha-methylacyl CoA racemase (AMACR), also known as P504S, plays an important role in peroxisomal beta-oxidation of branched-chain fatty acids. It has recently been shown that AMACR is highly expressed in prostate cancer and that it may be an important diagnostic marker for prostate carcinoma. However, little is known about expression of AMACR in normal tissues and other malignant tumors. In this study, we investigated expression of AMACR in 539 malignant tumors and 222 normal human tissues of various types by immunohistochemical analysis. mRNA levels of AMACR in normal organs and in selected tumors were assessed by real time PCR. In normal tissue, high expression of AMACR mRNA was identified in liver, kidney and salivary gland, while AMACR protein was detected in liver (hepatocytes), kidney (tubular epithelial cells), lung (only bronchial epithelial cells), and gallbladder (only mucosal epithelial cells). High expression of AMACR mRNA was found in prostate, liver, and kidney cancers but rarely in stomach and bladder cancers. A high percent of adenocarcinomas arising from these organs express AMACR, including 17 of 21 (81%) of hepatocellular carcinomas and 18 of 24 (75%) of renal cell carcinomas. In addition, carcinomas arising from tissues normally not expressing AMACR were also positive for the antigen, including 17 of 18 (94%) prostate carcinomas, 9 of 29 (31%) of urothelial carcinomas, and 4 of 15 (27%) of gastric adenocarcinomas. Two hundred and fifty cases of adenocarcinomas from lung, breast, pancreas, bile duct, adrenal gland, salivary gland, ovary, thyroid and endometrium were negative or rarely positive for AMACR. Neuroendocrine carcinomas rarely expressed AMACR. Melanomas, squamous cell carcinomas, basal cell carcinomas, soft tissue tumors (including epithelioid sarcomas and synovial sarcoma), thymomas, and germ cell tumors were negative for AMACR. Our data provide important baseline information for using AMACR in clinical practice and also are valuable in furthering understanding of the pathogenic role of AMACR in malignant neoplasms.     

Association of hepatocyte growth factor expression with salivary gland tumor differentiation

To clarify the significance of hepatocyte growth factor (HGF) expression in salivary gland tumors, HGF distribution in tissue sections and HGF concentrations in saliva and serum were examined. Sixty salivary gland adenomas, 61 salivary gland carcinomas and three autopsy fetuses were studied. Hepatocyte growth factor expression was observed in the duct-type luminal cells by immunohistochemical staining and in situ hybridization. However, HGF failed to be expressed in acinar cells and myoepithelium of normal salivary gland tissue. Hepatocyte growth factor tended to be expressed more intensely in benign salivary gland tumors than in malignant salivary gland tumors (P < 0.0001). In highly malignant tumors, the expression was limited in some cases. Salivary and serological HGF concentrations of 18 patients, comprised of 12 benign cases and six malignant cases, were analyzed before and after operation by an ELISA system. The concentrations were distinctly elevated after operation, in both saliva and serum, compared to before operation (P < 0.0005). However, there were no significant relationships between HGF concentration and histology, age, gender, size or location. Our findings suggest that HGF may play an important role in the development of salivary ducts of normal salivary tissues and differentiation of ductal structures of their neoplasms, while HGF kinetics in saliva and serum would be less likely to reflect the neoplastic character, benign or malignant.     

胶质瘤细胞c—fos和c—myc基因表达与血小板源生长因子B链的？…

目的 探讨胶质瘤细胞ｃ－ｆｏｓ和ｃ－ｍｙｃ基因表达和血小板源生长因子Ｂ链的纯合二聚体自分泌环活性的改变及其相互关系。方法 用原位杂交和免疫组化方法观察了６７例人胶质瘤组织。结果 ６７例中,ｃ－ｆｏｓ ｍＲＮＡ,ｃ－ｆｏｓ蛋白,ｃ－ｍｙｃ ｍＲＮＡ及ｃ－ｍｙｃ蛋白阳性率分别为;１００％,１００％,８５．１％,８３．６％。     

Differential expression of epithelial cell adhesion molecule in salivary gland neoplasms

Epithelial cell adhesion molecule (EpCAM) is the epithelial-specific molecule expressed on various epithelial cell types. The function of EpCAM involves cellular adhesion, proliferation, and signaling in both normal tissues and cancers. The purposes of this study were to investigate the EpCAM expression in salivary gland neoplasms and examine its relationship with pathologic characteristics. Forty-two cases of salivary gland neoplasms, including 20 mucoepidermoid carcinomas (MECs), 11 adenoid cystic carcinomas (ACCs), 9 pleomorphic adenomas (PAs), and 2 polymorphous low-grade adenocarcinomas (PLGAs) were enrolled. Epithelial cell adhesion molecule expression was analyzed immunohistochemically using MOC-31 and BerEP4 antibodies. Results showed that the majority of MECs and all PLGAs showed EpCAM expression in more than 50% of neoplastic cells, whereas most PAs and ACCs did not express this protein. In MECs, most EpCAM-positive neoplastic cells were clear cells, glandular epithelial cells, and intermediate cells, whereas squamous cells and mucous cells were largely negative. The expression was limited to ductal epithelium in EpCAM-positive PAs and ACCs. The decreased EpCAM expression in MECs was significantly associated with microscopically diminished cystic components, the presence of small nest invasion at invasive front, cellular anaplasia, vascular invasion, and high pathologic grade. These data suggested that EpCAM showed different expression pattern among salivary gland neoplasms and in different grades of MECs.     

Significance of TLR4/MyD88 expression in breast cancer

Objective: To investigate the expression of TLR4/MyD88 in breast cancer, and explore the relationship between their expression and breast cancer tumor growth and invasion. Methods: We examined the protein expression of TLR4 and MyD88 in 60 cases of histologically confirmed breast cancer. The relationship of their protein expressions with clinical features including age at diagnosis, tumor size and stage, lymph node metastasis and distant metastasis were analyzed. Results: The IHC results showed that TLR4 and MyD88 were expressed in 63.3% (38/60) and 58.3% (35/60) of malignant breast tumors respectively. TLR4 expression in breast cancer were significantly higher than in fibroadenoma (n = 4, 20.0%) and adjacent normal tissues (n = 2, 10.0%) (P < 0.001). MyD88 expression in breast cancer were also significantly higher than in fibroadenoma (n = 4, 20.0%) and adjacent normal tissue (n = 3, 15.0%) (P < 0.001). The gene expressions of TLR4 and MyD88 were significantly higher in breast cancer than in fibroadenoma and adjacent normal tissues (P < 0.05). The protein expressions of TLR4 and MyD88 were also significantly associated with poor clinical features (P < 0.05). Conclusion: TLR4 and MyD88 expression might be associated with breast cancer growth and regional and distant metastases.     

Oligoclonality of T cells in salivary glands of autoimmune MRL/lpr mice.

The aim of this study was to obtain further information about exocrine glandular immunopathology and the potential of the MRL/lpr strain as a model of Sjögren's syndrome. Immunoenzyme staining (ABC technique) and monoclonal antibodies defining CD3 T-cell receptor (TcR) alpha beta, gamma delta and TcR V beta 2, V beta 4, V beta 6, V beta 7, V beta 8.1,2, V beta 10b and V beta 11 were used to identify the mononuclear cells (MNC) in salivary gland infiltrates and lymph nodes of 2- and 4-5-month-old female MRL/lpr mice. TcR alpha beta + cells dominated clearly over TcR gamma delta + cells in both salivary glands and lymph nodes. In addition, to be expressed on lymphocyte-like cells, TcR gamma delta + cells also had a dendritic appearance. The frequency pattern of TcR expression in early inflammation (2 months) was V beta 8.1, 2 > V beta 6 > V beta 4 > V beta 10b > V beta 2 > V beta 7 > V beta 11. Clear differences in frequencies could be found between salivary glands and lymph nodes in established sialadenitis (4-5 months). Particularly V beta 4, V beta 8.1,2 and V beta 10b showed expansion in salivary glands at > or = 4 months. In conclusion, this study shows a diverse repertoire of TcR at local sites of MNC infiltration in autoimmune MRL/lpr mice. However, with increasing age it also shows a preferential utilization of certain V beta gene products.     

Prognostic role of urokinase-type plasminogen activator in human gliomas.

Urokinase-type plasminogen activator (u-PA) is a 54-kd enzyme shown to participate in tissue degradation under certain normal and pathological conditions, including cancer invasion and metastasis. Increased u-PA expression has been found in cancers of the breast, lung, colon, and prostate, and correlated with worse outcome in patients with lung and breast cancer. We examined the correlation between u-PA expression in gliomas and patient survival. Seventy-seven gliomas from 41 men and 36 women (ages 2 to 73) were immunostained for u-PA using monoclonal antibody 394 directed against human urokinase. The tumors included 32 grade 4, 16 grade 3, and 20 grade 2 astrocytomas (Daumas-Duport scale), and 9 pilocytic astrocytomas. Strong cytoplasmic staining was found in tumor cells of all grade 4, most of the grade 3, and a few of the lower grade tumors. Adjacent normal brain tissue showed faint staining associated with subpial cell processes and white matter fibers. The fiber staining was stronger in brain tissue infiltrated by tumor cells. Cytoplasmic u-PA staining in tumor cells was scored from 0 (no staining) to 6 (strong and widespread staining). The mean u-PA scores were 5.08 +/- 0.19 (mean +/- SEM) for grade 4, 3.97 +/- 0.46 for grade 3, 1.65 +/- 0.39 for grade 2, and 1.22 +/- 0.60 for pilocytic astrocytomas. The statistical analysis was based on cytoplasmic staining only. Analysis of variance revealed significant differences between the mean u-PA scores of different grades (P < 0.02 between grades 4 and 3, and P = 0.0001 between grades 4 or 3 and 2, and between grades 4 or 3 and pilocytic), except between grade 2 and pilocytic astrocytomas. Univariate analysis indicated that u-PA score > or = 4 (P = 0.0001), tumor grade 4 (P = 0.01), and age > 50 (P < 0.001) were all significant predictors for shorter disease survival. A three-way interaction model by multivariate analysis indicated that u-PA score > or = 4, tumor grade 4, and age > 50, taken together, were significant factors for shorter patient survival (P < 0.02). We conclude that u-PA may be used as a prognostic tool in conjunction with tumor grade and patients' age in predicting survival for patients with gliomas.     

Role of antibody to S100 protein in diagnostic pathology

Normal tissues and various tumors were examined for S100 protein, using anti-S100 protein antiserum, in an immunoperoxidase reaction. Among normal tissues, in addition to the previously reported presence of S100 protein in some neurons, glial, and Schwann cells of the nervous system, melanocytes and Langerhans cells of the skin, interdigitating reticulum cells of lymph nodes, and chondrocytes, we demonstrated it in myoepithelial cells and ducts of sweat glands, salivary glands, and the breast, serous glands of the lung, fetal neuroblasts, and sustentacular cells of the adrenal medulla. Among neoplasms, S100 protein previously has been reported in neurogenic tumors, melanomas, and neuroblastomas; we have demonstrated it in mixed sweat gland tumors, histiocytosis X, pleomorphic adenomas of the salivary gland, medullary carcinomas of the breast, bronchioloalveolar carcinomas of the lung, sustentacular cells of pheochromocytomas, teratomas of the ovary, and tumors of cartilage (enchondromas, osteochondromas, and chondrosarcomas). With S100 protein producing tumors, a normal progenitor cell was identified, indicating that demonstration of S100 protein in tumors confirmed their origin.     

Expression and regulation of the ΔN and TAp63 isoforms in salivary gland tumorigenesis clinical and experimental findings

The TP63 gene, a TP53 homologue, encodes for two main isoforms by different promoters: one retains (TA) and the other lacks (ΔN) the transactivation domain. p63 plays a critical role in the maintenance of basal and myoepithelial cells in ectodermally derived tissues and is implicated in tumorigenesis of several neoplastic entities. However, the biological and regulatory roles of these isoforms in salivary gland tumorigenesis remain unknown. Our results show a reciprocal expression between TA and ΔN isoforms in both benign and malignant salivary tumors. The most dominantly expressed were the ΔN isoforms, whereas the TA isoforms showed generally low levels of expression, except in a few tumors. High ΔNp63 expression characterized tumors with aggressive behavior, whereas tumors with high TAp63 expression were significantly smaller and less aggressive. In salivary gland cells, high expression of ΔNp63 led to enhanced cell migration and invasion and suppression of cell senescence independent of TAp63 and/or TP53 gene status. We conclude the following: i) overexpression of ΔNp63 contributes to salivary tumorigenesis, ii) ΔNp63 plays a dominant negative effect on the TA isoform in the modulation of cell migration and invasion, and iii) the ΔN isoform plays an oncogenic role and may represent an attractive target for therapeutic intervention in patients with salivary carcinomas.