Fumihiko Shinozaki

Relationship between labeling indices of KI-67 and brdurd in human malignant tumors

The relationship between labeling indices of Ki‐67 reactive antigen expressed by cycling cells and BrdUrd incorporated into S‐phase cells was investigated in 20 patients with malignant tumors. Both of the labeling indices varied greatly from patient to patient; the labeling index of Ki‐67 ranged from 37.5% to 1.9% with an average value of 16%, and the BrdUrd labeling index ranged from 23.4% to 1.6% with an average of 9.3%. The Ki‐67 labeling index was higher than the BrdUrd labeling index. In general, the values of the Ki‐67 labeling index were parallel to those of the BrdUrd labeling index, and the relation Y = 1.59X + 1.15 (r = 0.89) was obtained. In human solid tumors, therefore, the growth fraction can be easily estimated from the S‐phase fraction size, and vice versa.

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.

The significance of PCNA and p53 protein in some oral tumors

The expression of PCNA and p53 protein was evaluated in a total of 75 cases of benign and malignant lesions of the oral cavity, comprising 50 squamous cell carcinomas (SCCs), 14 leukoplakias, and 11 pleomorphic adenomas. The DNA histogram of 20 SCCs was measured by flow cytometry. p53-positive cells were frequently seen in SCCs, but were rare in leukoplakias and pleomorphic adenomas. The PCNA labeling index (LI) was higher in SCCs than in other benign lesions. The expression rate of p53 protein was markedly elevated in SCCs obtained from smoking patients, when compared to nonsmoking patients. DNA ploidy did not show a close relationship with PCNA and p53 expression. The mean value of PCNA LI for 22 cases carrying positive p53 protein was 52.3%, which was higher than that of p53 protein negative cases (35.7%). The Kaplan-Meier survival curve of the patients who were negative for p53 was significantly more favorable than for patients who were positive for p53 (P < 0.01, Cox-Mantel test). These results suggest that PCNA and p53 LI are markers for the malignant potential of the oral mucosa, and are a useful indicator suggesting a poor prognosis.

Detection of numeric abnormalities of chromosome 17 and p53 deletions by fluorescence in situ hybridization in pleomorphic adenomas and carcinomas in pleomorphic adenoma

A fluorescence in situ hybridization (FISH) technique using specific DNA probes allows for the detection of chromosomal aberrations and gene deletions and gains, even in interphase nuclei in human solid tumors. A high frequency of aberrations of chromosome 17 and mutation of the p53 gene have been reported in some human tumors. The correlation of p53 expression with abnormalities of chromosome 17 and p53 gene deletion in salivary gland tumors has not yet been investigated.

The nucleolar organizer regions associated protein (Ag-NORs) in salivary gland tumors

A silver colloid technique used to identify nucleolar organizer regions associated protein (Ag-NORs) has been applied to 20 salivary gland tumors. The method was readily applicable to the preparations of paraffin-embedded sections and the Ag-NORs were enumerated with ease. A significant difference was found between the numbers of Ag-NORs in the nuclei of malignant salivary gland tumors, such as adenoid cystic carcinoma, mucoepidermoid tumor and adenocarcinoma (with a mean of from 2.05 to 2.78 per nucleus) and those of benign salivary gland, such as pleomorphic adenoma, adenolymphoma (Wartin tumor) and clear cell adenoma (with a mean of from 1.47 to 1.72 per nucleus). It is proposed that the Ag-NORs technique, which is rapid, simple, and inexpensive, may be useful in the differential diagnosis of malignant and benign salivary gland tumors.

DETECTION OF EPSTEIN-BARR VIRUS IN ORAL PAPILLOMA

Fifty‐one cases of malignant and non‐malignant oral diseases were investigated for Epstein‐Barr virus (EBV). EBV DNA was detected by polymerase chain reaction analysis in 2 of 4 papillomas, but not in other tissues including 36 squamous cell carcinomas and 4 leukoplakias. The copy numbers of EBV DNA in the two positive samples were estimated to be 120 and 36 per cell, respectively. Intense EBV DNA signals were detected on papilloma cells by in situ hybridization. DNAs for the benign and malignant types of human papilloma virus were not detected in papilloma tissues. The present results suggest that EBV is a causative agent of oral papilloma.

An expression of squamous cell carcinoma-related antigen in oral cancers

The relationship between serum level and stainability of squamous cell carcinoma-related antigen (SCC-Ag) in carcinoma of the oral cavity was examined. The amount of serum SCC-Ag was measured in 60 of 97 patients by a radioimmunoassay system. 63.3% showed elevated serum SCC-Ag levels above 2.0 ng/ml. The specimens taken from 97 patients with oral squamous cell carcinoma (SCC) were stained with a monoclonal antibody against the SCC-Ag by using an immunoperoxidase method. Tumor cells with keratinization in well-differentiated SCC stained well with the antibody, whereas cells without keratinization in poorly-differentiated SCC stained weakly, or not at all. Serum levels of the antigen were significantly higher in cases of invasive cancers, but serum levels of antigen correlated with neither the degree of morphological differentiation of the tumor, nor with the intracellular content of the antigen. These observations suggest that the parenchymalstromal relationship is one of the decisive factors in determining the serum SCC-Ag level.

Immunohistochemical determination of growth fraction in human tumors.

The growth fraction in 93 cases of human tumors was estimated by an immunohistochemical staining using Ki-67. The tumors consisted of the following: 14 oral cancers, 14 breast cancers, 9 gastric cancers, 9 uterine cancers, 8 ovarian cancers, 6 colo-rectal cancers, 6 thyroid cancers, 5 esophagus cancers and 22 miscellaneous tumors. Regional labeled cells were predominantly found in the periphery of the tumor nests in squamous cell carcinomas. However, in adenocarcinomas the labeled cells were randomly distributed in tumor cell nests. The Ki-67 labeling index varied greatly from case to case (almost 0 to 50.9% with an average of 17.3%), even within the same organ group. The growth fraction was independent of the histological pattern, although thyroid cancers showed a lower labeling index than other malignant tumors. The labeling indices in benign tumors were lower than those in malignant tumors. The usefulness of this method for the estimation of biological behavior of human tumors is suggested.

Analysis of major histocompatibility complex (MHC) class I antigen in the rat salivary gland.

This antigen was examined in rats of different ages (new-born, 3, 5, 7, 10, 12 and 14 days after birth and adult) by immunofluorescence and immunoelectron microscopy. Changes in each kind of salivary gland when graft versus host disease was induced in recipient rats were also investigated. Monoclonal antibodies (HAM 2 or OX 18) specific to rat MHC class I antigen were used and these were detected by FITC-conjugated anti-mouse immunoglobulin. With HAM 2, MHC class I antigen in the submandibular gland was mostly located in the secretory duct cells; this expression was first found 10 days after birth. The antigen was found on the cell surfaces of the secretory duct cells by immunoelectron microscopy. With OX 18, MHC class I antigen was mainly found in the secretory duct cells, but weak expression was also found in the acinar cells. Localization of the antigen, by HAM 2 and OX 18 was less evident in the secretory duct cells of parotid and sublingual glands. When graft versus host disease was induced, MHC class I antigen (HAM 2) was observed in both acinar and secretory duct cells of the submandibular gland.