Kazuto Yamada

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.

Measurement of proliferating cell nuclear antigen (PCNA) and its clinical application in oral cancers.

The PCNA score was measured in oral squamous cell carcinoma (SCC), and its relationship to other cell proliferation markers, Ki-67 score, S-phase fraction (SPF), and AgNORs counts was investigated. The PCNA score ranged from 0.4% to 43.5% with an average value of 22.8%, the Ki-67 score ranged from 4.9% to 40% with an average of 24.1%, and the SPF ranged from 0.4% to 32.5% with an average of 12.4%, while AgNORs counts ranged from 2.53/nucleus to 7.03/nucleus with an average of 4.74/nucleus. These four parameters were closely interrelated. There was a significant difference in PCNA score between malignant and nonmalignant lesions, suggesting a difference in growth activity. The mean PCNA score decreased significantly from 20.0% to 8.0% after cancer chemotherapy. The response of cancer cells to anticancer agents may be estimated by consecutive measurement of PCNA, since the PCNA score dropped after treatment in cases showing a favorable prognosis.

Immunohistochemical expression of amelogenins in odontogenic epithelial tumours and cysts

Amelogenins, enamel proteins in odontogenic tumours, were detected immunohistochemically using a monoclonal antibody. They were strongly expressed in amyloid-like material, ghost cells, and the cells surrounding ghost cells of calcifying epithelial odontogenic tumours and cysts, whereas calcified bodies within the tumours and cysts showed negative staining. The expression of amelogenins was also positive in tumour cells of ameloblastoma, adenomatoid odontogenic tumour, squamous odontogenic tumour and ameloblastic fibroma. Peripheral tumour cells of the follicular ameloblastoma were positive with relatively intense staining. Undifferentiated or flattened tumour cells of adenomatoid odontogenic tumour and non-keratinized tumour cells of the squamous odontogenic tumour showed marked staining. Reduced ameloblasts in the odontoma displayed the strongest staining for amelogenins. The study suggests that biosynthesis of amelogenins may occur in the homogeneous materials of calcifying epithelial odontogenic tumours and cysts.

Immunohistochemical expression of epidermal growth factor receptor in salivary gland tumours

Immunohistochemical localization of epidermal growth factor receptor (EGFR) in normal salivary glands and tumours (108 cases) was studied using a monoclonal antibody. In the normal salivary glands, EGFR was occasionally detected in ductal segments of intercalated, striated, and excretory ducts, but not in acinar cells. The frequency of positive EGFR staining in salivary gland tumours was not high: pleomorphic adenoma, 33.8%; mucoepidermoid tumour, 25.0%; adenolymphoma, 44.4%; and sialoadenocarcinoma, 66.6%. Pleomorphic adenomas showed positive staining for EGFR on the luminal side of luminal cells and in squamous metaplastic cells of tumour tissue. Some modified myoepithelial cells were also reactive whereas outer spindle tumour cells were unstained. Adenolymphomas regularly exhibited positive EGFR staining in the cell membrane; mucoepidermoid carcinoma displayed positive staining in cell membranes in epidermoid tumour cells and cytoplasmic staining in mucous-secreting tumour cells. Sialocarcinomas revealed cell membrane staining and whole cytoplasmic staining for EGFR. The immunohistochemical localization of EGFR could be classified into two types, one the cell membrane-positive type found in epithelial tumour cells, and the second the cytoplasmic positive type seen in normal ductal cells, the luminal tumour cells of pleomorphic adenomas and mucous-secreting tumour cells.

Prenatal development of human major salivary glands and immunohistochemical detection of keratins using monoclonal antibodies

The major salivary glands were examined from 69 human fetuses ranging from 10 to 40 weeks of gestation. Prenatal growth curves of developing salivary glands could be established by histological scoring, and development was divided into the early developmental stage (EDS) from 10 to 18 weeks, early intermediate developmental stage (EIDS) from 19 to 24 weeks, late intermediate developmental stage (LIDS) from 15 to 32 weeks, late developmental stage (LDS) from 33 to 40 weeks. Characteristic morphogenesis and cytodifferentiation occurred in glandular duct cells during the period of EIDS and LIDS. In the LDS, acini and ducts of the salivary glands histologically developed into a mature state similar to adult glands. Immunohistochemical staining with monoclonal antibodies (MoAbs) PKK1, KL1, K8.12, K8.13, K4.62, RPN 1160, 1162, 1163, 1164, and 1165 was performed. During the fetal period, keratin expression as revealed by MoAbs PKK1, KL1, K8.12 was well established, and the staining pattern for each of these antibodies was comparable. Other antibodies showed rare or negative staining except K8.13 which had a diffuse, non-specific staining pattern. Accordingly, the proliferation and cytodifferentiation of fetal stage keratin staining in ductal cells as revealed by MoAbs PKK1, KL1, and K8.12 showed a heterogenic distribution in both luminal and basal cells. It is a characteristic finding that the cytodifferentiation of ductal luminal cells precedes ductal basal cells. Ductal basal cells stained with MoAb K8.12 and show heterogeneity of keratin distribution continuously until the full term of gestation. The keratin staining of oral epithelium was also examined to compare with distribution of salivary gland ductal cells and oral epithelial cells. In the present study, the developmental sequence of salivary gland cells and the immunohistochemical properties of keratin proteins in these cells were described in relation to the histogenesis of 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.

Multiple expression of keratins, vimentin, and S-100 protein in pleomorphic salivary adenomas

SummaryImmunohistochemical staining for S-100 protein and the intermediate filaments keratin and vimentin, was made in 41 salivary adenomas. In pleomorphic adenomas, great heterogeneity in the staining, as well as multiple and co-expressions of these proteins were found in the outer tumor cells of tubulo-ductal structures and modified myoepithelial cells, but not in the luminal tumor cells. All the outer tumor cells stained for S-100 protein, 97% for K8.12 keratin and 85% for vimentin. Of these cells, 29% showed multiple expression of K8.12 keratin, vimentin, and S-100 protein, and 17% showed co-expression of K8.12 and S-100 protein. Modified and neoplastic myoepithelial cells showed similar expressions of these proteins to those of outer tumor cells; myoepithelioma cells displayed the most complicated pattern, being positive for KL1, PKK1, and K8.12 keratins, vimentin and S-100 protein. In luminal tumor cells there was a heterogeneous expression of KL1 and PKK1 in 82%, and of KL1, PKK1, and K8.12 in only 14.7%. Based on the immunohistochemical findings obtained with different monoclonal antibodies in pleomorphic salivary adenomas, outer tumor cells may be derived from ductal basal cells and luminal tumor cells from intercalated duct cells.

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.

Prenatal development of myoepithelial cell of human submandibular gland observed by immunohistochemistry of smooth muscle actin and rhodamine-phalloidin fluorescence.

Immunostaining of monoclonal antibody (MoAb) of smooth muscle actin in paraffin sections and fluorescence of actin-specific phalloidin in cryostat sections were utilized to demonstrate the myoepithelial cells in prenatal and adult salivary glands of humans. In the early developmental stage (10-18 weeks) MoAb actin was weakly positive in the basal cells of the gland epithelium, and the positivity gradually accentuated at the basal portions of the terminal ducts and acini as the gestational period advanced. In the early intermediate developmental stage (19-24 weeks) the polyhedral myoepithelial cells were arranged in the basal portions of the acini and intercalated ducts. At this stage the myoepithelial cells produced phalloidin-positive spindle cytoplasmic processes. In the late intermediate developmental stage (25-32 weeks) the myoepithelial cells became flattened and formed dendritic processes to surround the acini and intercalated ducts. In the late developmental stage (33-40 weeks) numerous myoepithelial cells with well developed dendritic processes were demonstrable in the acini and intercalated ducts. In conclusion, it was found that the myoepithelial cells began to develop at 15-16 weeks of gestation when the acinar cells were still immature. The primitive myoepithelial cells were polyhedral in shape to form compact basal layer beneath the developing acinar cells during 19-24 weeks of gestation. In late gestational period the myoepithelial cells almost matured like the dendritic ones of adult salivary glands. However, the myoepithelial cells were never demonstrated in the striated and excretory ducts of the fetal salivary glands as opposed to its normal presence in the adult salivary glands. A possible aging process of myoepithelial cells was discussed in accordance with the histogenesis of transformed myoepithelial cells of salivary gland tumors.

Immunostaining of involucrin in odontogenic epithelial tumors and cysts

An immunoperoxidase method was used to detect involucrin in 47 odontogenic tumors and 35 radicular cysts. Of a total of 40 ameloblastomas, 9 cases were positive for involucrin expression and those positive cases exhibited acanthomatous or follicular patterns. Squamous odontogenic tumors were strongly positive for involucrin, whereas adenomatoid odontogenic tumors gave a negative staining reaction. Involucrin expression in odontogenic tumors was divided into three categories: single cell positive, focally positive, and squamous metaplastic cell positive. Radicular cysts showed a very irregular distribution of involucrin; nonstratified epithelium was generally negative or showed only trace staining for involucrin, whereas suprabasilar stratified squamous epithelial cells were strongly positive. Cells positive for involucrin in odontogenic tumors and in cystic epithelium are probably direct signs of epithelial differentiation; such cells were squamoid in appearance.