Prashanta Shrestha

Proliferating cell nuclear antigen in malignant and pre-malignant lesions of epithelial origin in the oral cavity and the skin: an immunohistochemical study

Proliferating cell nuclear antigen (PCNA) is a nuclear protein synthesized in the late G1 and S phase of the cell cycle and immunohistochemical detection of the protein represents a useful marker for the proliferating fraction of cells in tissue specimens. A series of malignant and pre-malignant lesions of the oral cavity and skin were evaluated by the streptavidin biotin immunoperoxidase method for detection of this protein. Monoclonal anti-PCNA antibody (PC 10) labelled proliferating cells in all cases with varying intensity of nuclear staining. In squamous cell carcinoma (n=48), PCNA positivity correlated with the differentiation and atypia of the tumour cells; however, in poorly differentiated tumours, the relationship between PCNA expression and proliferation was lost. Basal cell carcinoma showed an increased growth fraction in tiny epithelial nests (mean 43.8, SD 6.0,n=20) than in neoplastic basal cells (mean 30.1, SD 6.9,n=8). The growth fractions were significantly higher in the pre-malignant lesions (leukoplakia, mean 22.3, SD 7.7,n=14; Bowens disease, mean 45.2, SD 11.7,n=12; senile keratosis, mean 41.2, SD 7.0,n=12) than in the normal mucosa (mean 9.8, SD 4.9,n=10), suggesting that cellular growth fractions correlate with the degree of dysplasia in pre-malignant lesions.

Localization of Ca2+-binding S100 proteins in epithelial tumours of the skin

Abstract The Ca2+-binding proteins S100A1, S100A2, S100A4, S100A6 and S100B were evaluated immunohistochemically in normal skin and skin appendage tumours. Epidermal basal cells, epithelial cells of sebaceous glands, hair follicle sheet epithelia and eccrine duct reacted strongly with an antiserum against human S100A2 but were nonreactive or weakly reactive to S100A1, S100A4, S100A6 and S100B. Varying types of skin appendage tumours and most peripheral cells in tumour nests of basal cell carcinoma and squamous cell carcinoma showed positive S100A2 immunoreactivity in neoplastic cells corresponding to basal cells but were nonreactive or faintly reactive for other S100 proteins. Langerhan’s cells and melanocytes were labelled by S100B. Basophilic cells of calcifying epithelioma were occasionally stained with S100A2 antiserum. Eccrine poroma did not react with any S100 antiserum. Mixed tumours of the skin containing neoplastic myoepithelial cells stained strongly for S100A2 and S100B but only faintly for S100A1, S100A4, S100A6. This is the first report on selective evaluation of different S100 proteins in normal skin. These antibodies are valuable tools for better characterization of skin appendage tumours.

Immunoreactivity of proliferating cell nuclear antigen in salivary gland tumours: An assessment of growth potential

Immunoreactivity of proliferating cell nuclear antigen (PCNA) was assessed to evaluate growth potential in surgically resected tissue specimens from 70 cases of benign and malignant salivary gland tumours. Three stage streptavidin-biotin immunoperoxidase immunostaining using monoclonal antibody to PCNA showed a heterogeneity of PCNA index and distribution. In normal salivary gland specimens, PCNA was demonstrated in the nuclei of few ductal and acinar cells. In pleomorphic adenoma a multiple nodular growth pattern was observed with positive immunoreactivity restricted to the nuclei of tubulo-ductal structures. Warthins tumour had positive nuclei in the outer cuboidal cells of epithelial component and germinal centres of lymphoid tissue. Myoepithelioma and acinic cell carcinoma showed slightly differing values and a statistically significant difference in the value of the index was observed in tumour cell aggregates of the cribiform type of adenoid cystic carcinoma and the solid undifferentiated type and between low/intermediate and high-grade mucoepidermoid tumours. PCNA is a useful marker of tumour cell proliferation; the index correlates with the grade of malignancy in salivary gland tumours.

Proliferating cell nuclear antigen in breast lesions: Correlation of c-erbB-2 oncoprotein and EGF receptor and its clinicopathological significance in breast cancer

Monoclonal anti-proliferating cell nuclear antigen (PCNA PC10), which is directed against a 36 kDa auxiliary protein for DNA polymerase delta specific for the S-phase of cell cycle, was used to measure tumour cell proliferation in 4 lactating breasts and 98 benign and malignant breast tumours. The percentage of PCNA-positive cells determined by point counting was significantly lower in the lactating breast [mean 3.6%, standard deviation (SD) 0.67,n=5] than in fibroadenoma and mastopathy (mean 23.7, SD 5.0,n=2). Primary breast carcinoma showed a PCNA index ranging from 2% to 36% (mean 12.3, SD 9.3,n=50), whereas in recurrent carcinoma the index was mean 28.5, SD 4.0. A high index was correlated with c-erbB-2 and epidermal growth factor (EGF) receptor membrane reactivity, worsening histological grade, poor survival and disease-free survival. The expression of c-erbB-2 and EGF receptor was associated with poor survival and disease-free survival in primary breast cancer patients.

Enhanced tenascin immunoreactivity in leukoplakia and squamous cell carcinoma of the oral cavity: An immunohistochemical study

Tenascin is an extracellular matrix glycoprotein that shows a site restricted expression especially in areas of cell proliferation, cell motility, and tissue modeling at the epithelial-mesenchymal junction during embryogenesis. Tissue specimens obtained from surgery and/or biopsy for oral leukoplakia (n = 22) and squamous cells carcinoma (n = 36) were examined for the presence of tenascin by using monoclonal antibody. In normal tissue specimens (n = 5), tenascin immunoreaction appeared as a linear continuous lining at the immediate vicinity of basement membrane (n = 3). Hyperplastic epithelia in leuoplakia showed a distinct increase in tenascin immunoreactivity in the submucosa correlating with the degree of hyperplasis and/or dysplasia. In squamous cell carcinoma (SCC), the reactivity was most intense extending deeply into the underlying stroma with marked reaction around large tumour cell nests and the infiltrating tumour margin. The connective tissue stroma, however, in undifferentiated carcinoma showed traces of immunoreactivity. Positive immunoreactivity was seen around metastatic squamous cell carcinoma masses in regional lymph nodes. The stromal tissues infiltrated by inflammatory cells were usually unreactive while those with desmoplastic changes were positive for tenascin. The authors conclude that an enhanced expression of tenascin may play an important role during active phases of tumour cell proliferation and stromal changes in the premalignant and malignant lesions of the oral mucosa.

Tenascin: Growth and adhesion modulation—Extracellular matrix degrading function: an in Vitro study

Tenascin (TN), a recently characterised extracellular matrix protein, largely confined to the process with the development of embryo in areas of epithelial-mesenchymal interactions and in areas where there are morphogenetic movements and tissue patterning, has a highly restricted expression in adult tissues. The expression of TN is enhanced in a variety of human neoplastic lesions. However, function(s) and molecular mechanisms of enhanced expression in neoplastic lesions remain unclear. We employed human tongue carcinoma cells (SCCKN), human salivary gland adenocarcinoma cells (SGT-1), normal mouse embryonic fibroblasts (NIH3T3-3) and K-ras-2 transformed fibroblasts (Cle-H3) in an in vitro study to elucidate the biological roles of TN. In in vitro studies, all the cell lines examined had enhanced secretion of TN in the presence of transforming growth factor-beta in a dose-dependent manner and TN itself was found to possess a growth-enhancing activity. Moreover, studies on adhesion of the cell lines on coated substrates of fibronectin (FN), laminin (LN), tenascin (TN), TN/FN and TN/LN showed that all the cells adhere and spread well on FN and LN. However, on TN they attach poorly and remain rounded. The relative concentrations of TN and FN affected the cellular adhesion and morphology. In SCCKN and SGT-1, but not in NIH3T3 and Cle-He3 fibroblasts, a higher concentration of TN inhibited cellular adhesion on fibronectin, suggesting that cells attach poorly on TN, it may interfere with the action of fibronectin, and the relative concentrations of TN, FN or LN may affect cellular adhesion and morphology which may differ in different cell types. When TN was added in the growth medium of exponentially growing cells, the cells lost their cell to cell contact and were seen to be separating. The presence of these extracellular matrix proteins were further tested to determine whether they could modulate the secretion of proteolytic enzymes responsible for extracellular matrix degradation by tumour cells, when the neoplastic cells but not the non-neoplastic cells grown on FN/TN substrate showed positive immunofluorescence for collagenase. FN, LN or TN alone did not induce collagenase in the tumour cells. If the same is true in vivo, although a number of factors and interactions may implicate the ultimate outcome, the enhanced expression of TN in neoplastic lesions may have potential implications for tumour growth, differentiation, cellular adhesion, invasion and metastasis.

Immunoreactive tenascin in tumours of salivary glands: evidence for enhanced expression in tumour stroma and production by tumour cells

Tenascin, a large molecular weight extracellular glycoprotein expressed at the epithelial-mesenchymal interface during morphogenesis in embryo, wound healing and in the stroma of various benign and malignant tumours was evaluated in a series of primary epithelial tumours of salivary glands using a monoclonal antibody. Normal salivary glands (n = 5) had linear delicate band-like immunoreactive tenascin in relatively large excretory or intralobular ducts. Pleomorphic adenomas (n = 40) had heterogeneity of expression in modified myoepithelial cell-associated myxoid, hyaline and chondroid areas. Warthins tumours (n = 10) had a linear immunoreactivity profile of tenascin just adjacent to the basal cells of the epithelial tumour component. A heterogeneity of expression with intense to low or negative stromal immunoreactivity was observed in adenoid cystic carcinomas (n = 8), mucoepidermoid carcinomas (n = 8), epithelial-myoepithelial carcinomas (n = 4), polymorphous low-grade carcinomas (n = 3), papillary cystadenocarcinomas (n = 15) and undifferentiated carcinomas (n = 3). In addition, small cystic spaces or lumens of epithelial-lined tubulo-ductal structures in numerous salivary tumours had positive immunoreactivity for tenascin, suggesting its production by the epithelial tumour component. An enhanced expression of tenascin in salivary tumours suggests a role of this protein in the stromal remodelling and tumour growth.

Expression of cytokeratins in Warthin's tumour (adenolymphoma) of parotid glands: Specific detection of individual cytokeratin types by monoclonal antibodies

This study evaluated the distribution of cytokeratins detected by monoclonal antibodies directed against individual keratin proteins in normal human salivary glands and epithelial tumour cells of Warthins tumour arising in parotid glands to determine a more precise mapping of their cellular distribution. The normal salivary ducts showed the presence of cytokeratin 7, 8, 18 and 19 in the intercalated, striated and excretory ducts, the primary keratins of stratified and simple epithelia with a profile very similar to the non-cornified epithelium of the oral mucosa. The basally located cells of salivary gland ducts other than myoepithelial cells were reactive for keratins 7 and 19 suggesting a close similarity in profile of keratin in the basal cells of the oral epithelium. In Warthins tumour, keratins 7, 8, 18 and 19 were consistently detected in the epithelial cells of the tumour, a profile with a tendency to mimic the same in normal ductal epithelium. The distribution, however, was diverse and a heterogeneity was observed in the basal and luminal cells of Warthins tumour which differed even in different areas of the same tumour specimen.

Expression of Tenascin in odontogenic tumours

We investigated the expression of tenascin in a series of odontogenic tumours (n = 63) of epithelial and epithelial-ectomesenchymal origin by using immunohistochemical methods. A heterogeneity of expression of tenascin was observed in odontogenic tumours. The heterogeneity was most prominent in odontogenic tumours not forming calcified tissues. In these ameloblastomas and adenomatoid odontogenic tumours, tenascin was mainly localised at the epithelial tumour cell-mesenchymal tissue interface. In the calcifying epithelial odontogenic tumour, ameloblastic fibroma and odontoma, a widespread stromal immunoreactivity was observed which was, however, unreactive in the calcified masses. The stellate reticulum-like cells and granular cells of ameloblastoma also showed a positive immunoreactivity for tenascin. The results of the present study suggest that expression of tenascin in the stromal tissue of odontogenic tumours differs according to the potential of forming calcified masses by the tumour cells irrespective of tumour cell morphology.

Expression of tenascin in hamster buccal pouch mucosa during experimental carcinogenesis

Experimental carcinogenesis by topical application of 7,12-dimethylbenz(a)anthracene (DMBA) in hamster buccal pouch mucosa was evaluated for expression of tenascin, an extracellular matrix glycoprotein expressed at the epithelial-mesenchymal interface during embryonic and fetal development, wound healing and in the stroma of various neoplastic lesions, by using immunohistochemical methods. The buccal pouch mucosa in normal hamsters showed immunoreactive tenascin either as a linear delicate band or without reactivity at the immediate vicinity of the basement membrane. During carcinogenesis, in the second to fourth week of application of DMBA, the hyalinous changes in the submucosal connective tissue had a weak but diffuse immunoreactivity for tenascin. The hyperkeratinised and hyperplastic mucosa following 5 weeks of application of DMBA showed focal areas of enhanced expression in the vicinity of the basement membrane. Subsequently, specimens showing hyperplasia, dysplasia, carcinoma in situ and invasive carcinomas had comparatively more widespread stromal immunoreactivity where the extent of enhanced reactivity positively correlated with the advancing lesion. These results compared with the results of expression in human normal mucosa, leukoplakia and squamous cell carcinoma of the oral cavity (Shrestha et al., Oral Oncol, Eur J Cancer 1994, 30, 132-137) suggest that the expression of tenascin in experimental carcinogenesis of hamster buccal pouch mucosa, as a model, faithfully mimics the same in human oral mucosa.