Transforming growth factor-α and oral fibroma: immunohistochemical and in situ hybridization study

PURPOSE: Transforming growth factor-alpha (TGF-alpha) is usually expressed in cell lines derived from sarcomas. It is known as a mitogen for fibroblasts. The aim of this study was to determine whether there were any differences in the expression pattern of TGF-alpha between normal oral mucosa and oral fibroma. PATIENTS AND METHODS: Fourteen pathologic specimens (6 males and 8 females; 37.2 +/- 23.2 years) and 10 normal oral mucosal specimens (5 females and 5 males; 43.8 +/- 17.7 years) were used for this study. Identification of TGF-alpha was sought by using immunohistochemistry and in situ hybridization. RESULTS: The samples from normal oral mucosa did not express TGF-alpha. One sample from oral fibroma did not express TGF-alpha (7.1%). Five samples from oral fibroma expressed TGF-alpha sparsely (35.7%). Eight samples showed diffuse expression of TGF-alpha (57.1%). The immunopositive reaction to TGF-alpha in oral fibroma was localized in the basal layer and the fibroblasts that resided beneath the epithelium. This pattern was also shown in the in situ hybridization study as well. CONCLUSION: TGF-alpha is expressed in oral fibromas. It suggested that TGF-alpha might play a role in fibroblast proliferation in oral fibromas.     

Immunohistochemical expression of EGFR in oral leukoplakia: Association with clinicopathological features and cellular proliferation

Objectives: to investigate the immunoexpression of epidermal growth factor receptor (EGFR) in a sample of oral leukoplakias (OL) and to determine the receptor' s association with dysplasia, tobacco consumption, lesion site, and proliferation rate. Although EGFR should be overexpressed in some oral leukoplakias, the factors that may interfere with this expression and the influence of this receptor on epithelial proliferation have yet to be investigated. Study Design: Samples of oral leukoplakias (48) and of normal oral epithelium (10) were immunohistologically examined for expression of EGFR. Immunohistochemistry for Ki-67, and p27 were also performed in leukoplakias. EGFR expression was associated with clinical and pathological features. Results: EGFR was positive in 62.5% of the leukoplakias and 50% of normal oral epithelium. The number of EGFR positive OL located in high-risk sites was significantly higher than EGFR positive OL located in low-risk sites. Most of the p27 negative leukoplakias were EGFR positive, and the p27 index in the parabasal layer was diminished in the presence of dysplasia. Positivity for EGFR was not associated with dysplasia, tobacco exposure, or Ki-67. Conclusion: EGFR is expressed in leukoplakia regardless of dysplasia, but EGFR positivity should be more frequent in lesions sited in areas of high cancer risk. The association between EGFR and p27 may represent an important mechanism in the control of cellular proliferation and malignant progression of oral epithelium and therefore warrants further investigation.     

Immunohistochemical expression of EGFR in oral leukoplakia: Association with clinicopathological features and cellular proliferation

Objectives: to investigate the immunoexpression of epidermal growth factor receptor (EGFR) in a sample of oral leukoplakias (OL) and to determine the receptor’s association with dysplasia, tobacco consumption, lesion site, and proliferation rate. Although EGFR should be overexpressed in some oral leukoplakias, the factors that may interfere with this expression and the influence of this receptor on epithelial proliferation have yet to be investigated. Study Design: Samples of oral leukoplakias (48) and of normal oral epithelium (10) were immunohistologically examined for expression of EGFR. Immunohistochemistry for Ki-67, and p27 were also performed in leukoplakias. EGFR expression was associated with clinical and pathological features. Results: EGFR was positive in 62.5% of the leukoplakias and 50% of normal oral epithelium. The number of EGFR positive OL located in high-risk sites was significantly higher than EGFR positive OL located in low-risk sites. Most of the p27 negative leukoplakias were EGFR positive, and the p27 index in the parabasal layer was diminished in the presence of dysplasia. Positivity for EGFR was not associated with dysplasia, tobacco exposure, or Ki-67. Conclusion: EGFR is expressed in leukoplakia regardless of dysplasia, but EGFR positivity should be more frequent in lesions sited in areas of high cancer risk. The association between EGFR and p27 may represent an important mechanism in the control of cellular proliferation and malignant progression of oral epithelium and therefore warrants further investigation. Key words:Oral leukoplakia, EGFR, p27, Ki-67, epithelial dysplasia.     

Overexpression of c-Src in areas of hyperproliferation in head and neck cancer, premalignant lesions and benign mucosal disorders

To examine which proteins are responsible for the elevated protein tyrosine kinase (PTK) activity in human head and neck squamous cell carcinoma (HNSCC) and adjacent histologically normal epithelium, paraffin embedded sections of these tissues were stained for PTK c-Src. Using double labeling techniques and antibodies against both the proliferation marker Ki-67 and PTK c-Src, we have shown that c-Src is overexpressed in areas of hyperproliferation in HNSCC, dysplastic epithelium, benign papillomas and inflamed normal tissue. Our data indicate that c-Src is (one of) the protein(s) responsible for the increased PTK activity in HNSCC. We could not demonstrate that c-Src expression is responsible for the increased PTK activity in normal epithelium adjacent to tumour tissue. We assume that c-Src plays a role in the increased proliferation seen in (pre)malignant and benign epithelial lesions as well as in reactive inflammatory epithelial hyperplasia.     

口腔粘膜上皮癌变过程表皮生长因子受体的表达及意义

目的 研究口腔粘膜上皮癌变过程中表皮生长因子受体的表达及意义。方法 采用免疫组化Ｓ－Ｐ法对正常口腔粘膜,轻中重度粘膜上皮异常增生和高分发化鳞癌共８５例标本进行ＥＧＦＲ检测。结果 正常口腔粘膜全部为阴性。上皮异常增时,随病变程度加重,ＥＧＦＲ阳性表达也相应提高,至重度异常增生时,阳性表达率达最高峰。而高分化鳞癌时,表达率有所下降。结论ＥＧＦＲ的表达与口腔鳞癌的发生有关,可作为评估口腔上皮恶谱潜能的较     

口腔鳞癌癌旁上皮中细胞凋亡与细胞增殖的关系

目的 :探讨口腔鳞癌癌旁上皮中细胞凋亡与细胞增殖的病理意义及其相互关系。方法 :利用脱氧核糖核酸末端转移酶介导的dUTP缺口末端标记 (TUNEL)技术、增殖细胞核抗原 (PCNA)免疫组织化学染色对 30例口腔鳞癌标本癌旁上皮等4个不同区域中凋亡细胞及增殖细胞的分布和密度进行观察和比较。结果 :与正常区域相比较 ,癌旁上皮中凋亡细胞及增殖细胞分布异常且细胞密度增高 ;肿瘤组织中 ,凋亡细胞明显减少 ,增殖细胞明显增多。结论 :口腔鳞癌癌旁上皮中存在活跃的细胞凋亡及细胞增殖 ,它们在其癌变中具有重要意义。     

Epstein-Barr virus receptors but not viral DMA are present in normal and malignant oral epithelium

The presence and distribution of Epstein-Barr Virus receptors (EBVR's) on a range of normal (n = 18), dysplastic (n = 10) and malignant (n = 20) oral mucosa were studied by immunocytochemical methods using the monoclonal antibodies (MAb's) HB5 and B2. EBVR's were demonstrated as membrane staining of the spinous layers of normal non- and parakeratinized epithelium, indicating that EBVR's are differentiation-linked. This distribution was retained in dysplastic epithelium. Tissue from oral squamous cell carcinomas (SCC's) showed variable reactivity of only a few cells scattered randomly within the samples. Furthermore, a sensitive in situ hybridization (ISH) technique was used to determine if Epstein-Barr virus (EBV) was present in normal (n = 15) and oral squamous cell carcinoma tissue (n = 20). No EBV DNA was demonstrated within either normal or malignant epithelium, suggesting that the virus does not persist in normal oral stratified squamous epithelium nor is there any evidence for a role in oral carcinogenesis.     

Immunohistochemical study of syndecan-1 down-regulation and the expression of p53 protein or Ki-67 antigen in oral leukoplakia with or without epithelial dysplasia

BACKGROUND: Leukoplakia is an oral pre-cancerous lesion that sometimes develops into squamous cell carcinoma. Therefore, leukoplakia with epithelial dysplasia is useful for studying carcinogenesis at the cellular level. The purpose of this study was to evaluate a potential association between the loss of syndecan-1 expression and the expression of p53 protein and Ki-67 antigen, and to identify reliable markers for predicting malignant changes in oral leukoplakia with epithelial dysplasia. METHODS: Changes in the expression of syndecan-1, p53, and Ki-67 were examined immunohistochemically in 43 cases of oral leukoplakia with or without epithelial dysplasia. The subjects were categorized as: none, 13 cases; mild dysplasia, 5 cases; moderate dysplasia, 17 cases; and severe dysplasia, 8 cases. The expression of these molecules in normal oral epithelia (22 cases) was also investigated. RESULTS: Strong syndecan-1 expression was observed on the surface of keratinocytes in normal epithelium. Immunopositivity was lost gradually as the extent of epithelial dysplasia increased. In normal epithelium, p53 and Ki-67 appeared mainly in the basal cell layer, while they were more widely distributed in leukoplakia. Specifically, significant changes were observed in the labeling index of p53 and Ki-67 in leukoplakia as epithelial dysplasia progressed from mild to moderate or severe. CONCLUSION: Our results reveal that overexpression of p53 protein and Ki-67 antigen, and down-regulation of syndecan-1 expression in the lower part of the epithelium, are associated with dysplastic changes. Therefore, the down-regulation of syndecan-1 expression may be the most important reliable marker for dysplastic changes.     

Eosinophil-derived transforming growth factors (TGF-alpha and TGF-beta 1) in human periradicular lesions

Inflammatory mediators of periradicular lesions are poorly understood. Transforming growth factors-alpha and -beta 1 (TGF-alpha and TGF-beta 1) have been linked with the cellular processes for both soft and hard tissue wound healing. The purpose of this study is to demonstrate the cellular sources of TGF-alpha and TGF-beta 1 mRNA and protein in periapical lesions by in situ hybridization and immunohistochemistry. Nine periapical granulomas and nine periapical cysts were examined. TGF-alpha mRNA and protein were not detectable in the granulomas examined. However, eosinophils surrounding the periapical cysts demonstrated both TGF-alpha mRNA and protein. The vast majority of eosinophils present in the periapical granulomas and cysts also demonstrated TGF-beta 1 mRNA and protein. Other cells producing TGF-beta 1 were lymphocytes, fibroblasts, and monocytes. The presence of wound repair cytokines, such as TGF-alpha and TGF-beta 1, suggests a mechanism by which the host inflammatory response may participate in the repair and remodeling of periapical tissues.     

Osteopontin (OPN) distribution in premalignant and malignant lesions of oral epithelium and expression in cell lines derived from squamous cell carcinoma of the oral cavity

The objectives of this study were to assess the immunolocalization of human osteopontin (OPN) in oral lesions and to identify human cell lines of oral squamous cell carcinoma (OSCC) origin that express OPN mRNA. OPN was localized using immunohistochemistry in the following oral specimens: normal epithelium (n=6), epithelial hyperplasia (n=4), epithelial dysplasia (n=28), carcinoma in situ (n=11) and squamous cell carcinoma (n=43). Cell lines UMSCC-1, MDA TU 138, MDA 686LN, SCC4, SCC9, SCC25, CAL 27 and MDA 1483 were characterized for OPN mRNA expression using Northern blotting. OPN was not detected in normal oral epithelium. Intracellular and intercellular immunore-activity was seen in 75% of hyperplasias, 57% of dysplasias, 54% of carcinoma in situ and 67% of squamous cell carcinomas. UMSCC-1 expressed high levels of OPN mRNA. We conclude that OPN protein is detectable in premalignant and malignant lesions arising from oral epithelium. UMSCC-1 may be a useful cell line in which to conduct in vitro studies designed to clarify the role of OPN in OSCC.     

AgNOR count as objective marker for dysplastic features in oral leukoplakia

BACKGROUND: Dysplasia is an important feature of leukoplakia. Because agreement among oral pathologists is poor regarding lesional diagnosis, silver stainable nucleolar organizer regions (AgNORs) as replicatory markers may have a place in objectively characterizing dysplasia in tissue specimens. METHODS: We studied 41 normal oral epithelia, 51 oral leukoplakia (26 dysplastic, 25 non-dysplastic), and 51 cases of squamous cell carcinoma specimens for their mean AgNOR counts. RESULTS: Mean AgNOR counts increased gradually from normal epithelium to non-dysplastic to dysplastic leukoplakia to squamous cell carcinoma. Using ROC analysis, we determined a mean AgNOR count cut-point (2.37) that can be used to distinguish between dysplastic and non-dysplastic leukoplakia. The test had a sensitivity of 75% and specificity of 83% with area under the curve being 88%. CONCLUSIONS: Mean AgNOR count could be a valuable criterion for defining objective parameters for diagnosis/determination of dysplasia distinguishing between dysplastic and non-dysplastic leukoplakia.     

The use of thrombomodulin to study epithelial cell differentiation in neoplastic and non-neoplastic oral lesions

Thrombomodulin (TM) is a glycoprotein originally isolated from rabbit lung vasculature and characterized as a natural endothelial anticoagulant. Thrombin binds to TM noncovalently with high affinity. Thrombin - TM complexes can activate protein C efficiently. Activated protein C inactivates factors Va and VIIIa and regulates the blood coagulation cascade. Thus TM converts thrombin from a procoagulant protease to an anticoagulant. TM is found on endothelial cells in veins, arteries and capillaries. Our previous study has shown that TM is also expressed on the cell surface of squamous epithelium. In the present study, we aimed to disclose differences in TM expression among normal, dysplastic, and malignant squamous epithelium in human oral mucosa by counting TM-positive cells in each lesion. TM was uniformly expressed in the spinous layer of normal human oral squamous epithelium. The number of TM-positive cells was not significantly different between normal epithelium, lichen planus and mild dysplasia. In contrast, in moderate and severe dysplasia and well-differentiated squamous cell carcinoma (SCC), there were significantly fewer positive cells compared with normal epithelium. In SCCs, the periphery and the central keratinized area of tumor islands were often negative. The proportion of TM-positive cells in poorly differentiated SCC was significantly lower than in well-differentiated SCC. These results indicate that TM may have diagnostic value in the histological examination of oral premalignant and malignant lesions.     

Immunohistochemical expression of p53, p16 and hTERT in oral squamous cell carcinoma and potentially malignant disorders

Oral carcinogenesis is a multi-step process. One possible step is the development of potentially malignant disorders known as leukoplakia and erytroplakia. The objective of this study was to use immunohistochemistry to analyze the patterns of expression of the cell-cycle regulatory proteins p53 and p16(INK4a) in potentially malignant disorders (PMD) of the oral mucosa (with varying degrees of dysplasia) and in oral squamous cell carcinomas (OSCC) to correlate them with the expression of telomerase (hTERT). Fifteen PMD and 30 OSCC tissue samples were analyzed. Additionally, 5 cases of oral epithelial hyperplasia (OEH) were added to analyze clinically altered mucosa presenting as histological hyperplasia without dysplasia. p53 positivity was observed in 93.3% of PMD, in 63.3% of OSCC and in 80% of OEH. Although there was no correlation between p53 expression and the grade of dysplasia, all cases with severe dysplasia presented p53 suprabasal immunoexpression. p16(INK4a) expression was observed in 26.7% of PMD, in 43.3% of OSCC and in 2 cases of OEH. The p16(INK4a) expression in OEH, PMD and OSCC was unable to differentiate non-dysplastic from dysplastic oral epithelium. hTERT positivity was observed in all samples of OEH and PMD and in 90% of OSCC. The high hTERT immunoexpression in all three lesions indicates that telomerase is present in clinically altered oral mucosa but does not differentiate hyperplastic from dysplastic oral epithelium. In PMD of the oral mucosa, the p53 immunoexpression changes according to the degree of dysplasia by mechanisms independent of p16(INK4a) and hTERT.     

Reduction of syndecan-1 expression is associated with dysplastic oral epithelium

Syndecans are a family of integral membrane proteoglycans that participate in cell-matrix interactions and growth factor binding. Syndecan-1 expression is induced during keratinocyte differentiation and reduced in squamous cell carcinomas. The purpose of this study was to examine the alteration in syndecan-1 expression in dysplastic oral epithelium. Sixty-six oral biopsy specimens (43 epithelial dysplasias, 3 carcinoma in situ and 20 squamous cell carcinomas) were studied using immunohistochemical methods. The normal epithelium specimens were highly positive for syndecan-1. Fifteen of 46 dysplasias or carcinoma in situ specimens showed negative or weak staining for syndecan-1, two of which were totally negative. Intermediate and strong staining were observed in 17 and 14 dysplasias or carcinoma in situ specimens, respectively. Thirteen (65%) squamous cell carcinomas showed negative or weak staining for syndecan-1, seven of which were totally negative. Only three carcinomas had a strong syndecan-1 expression. Four of the 34 patients with dysplasia who were followed up developed squamous cell carcinoma. All these dysplasias had weak or totally negative syndecan-1 expression. The results suggest that the loss of syndecan-1 is associated with dysplastic changes in oral epithelium.     

Deranged expression of the E-cadherin/β-catenin complex and the epidermal growth factor receptor in the clinical evolution and progression of oral squamous cell carcinomas

BACKGROUND: Deranged expression and function of the E-cadherin/beta-catenin (E-cad/beta-cat) complex and the epidermal growth factor receptor (EGFR) have been implicated in the development and progression of carcinomas. METHODS: To estimate the role of these molecules in oral cancer, we investigated 75 primary oral squamous cell carcinomas (OSCCs) with adjacent normal and/or dysplastic mucosa, 30 paired metastases and 12 recurrences by immunohistochemistry. RESULTS: All three molecules were constitutionally expressed in the basal/parabasal layers of tumour adjacent 'normal' epithelium, in contrast to a significant increase of EGFR and heterogeneous expression of E-cad/beta-cat in dysplasia. In OSCCs, over-expression of EGFR correlated significantly with lower tumour grade and poor prognosis, loss of E-cad was a significant marker for shortened survival, reduced beta-cat staining was a predictive marker for lymph node metastasis. CONCLUSIONS: There is a perturbance in intercellular adhesion molecules and EGFR expression/function in oral cancer with major clinical impact. E-cad and beta-cat seem to inhibit EGFR to enhance the progression of OSCCs.     

Altered H-antigen reactivity as an early indicator of malignant transformation in oral epithelium

This study was undertaken to determine whether or not the blood group H-antigen reactivity of oral epithelium has value in predicting malignant transformation. Tissue from 3 groups of patients was studied retrospectively, using an immunoperoxidase technique. Two biopsy specimens from each patient, obtained at different times but from the same site, were examined for the presence of H-antigen. Group 1 consisted of 16 patients in which the initial biopsy was histologically benign, but the subsequent biopsy revealed epidermoid carcinoma. The initial biopsies in Groups II (17 patients) and III (17 patients) revealed epithelial dysplasia. Whereas the subsequent biopsy in Group II revealed carcinoma, the subsequent biopsy in Group III remained non-invasive. Normal epithelium from 64 patients was also studied.
The results showed that in normal epithelium, H-antigen-negative cells are rarely seen, but 81% of the initial benign specimens of Group I showed antigen-negative areas. Therefore, it was concluded that altered H-antigen reactivity in histologically benign epithelium may serve to predict eventual malignant transformation, and that immunologic de-differentiation precedes histologic dedifferentiation. A comparison between the initial biopsy specimens of Groups II and III yielded nearly identical results and showed that it was not possible, on the basis of the H-antigen reactivity, to predict which dysplastic lesions would progress to epidermoid carcinoma.