Coexpression of cytokeratins, involucrin, and blood group antigens in oral squamous cell carcinomas.

The well and poorly differentiated oral squamous carcinomas preferentially express proteins, blood group antigens, and contain associated dendritic Langerhans' cells. Keratin pearls in well-differentiated carcinomas simulate the differentiation pathway of the normal oral squamous epithelium, whereas poorly differentiated carcinomas do not and appear more heterogeneous. Terminally keratinized cells correlate with involucrin and expression of blood group antigens in keratin pearls, a feature that differs from the nonkeratinizing normal epithelium in which such carcinomas arise. Dendritic Langerhans' cells are reduced in number in squamous carcinomas.

An immunohistochemical assessment of cellular proliferation markers in head and neck squamous cell cancers.

Prognostic information is essential for the evaluation, judgement and optimal treatment of patients with squamous cell cancers (SCCs) of the upper aerodigestive tract. Using immunohistochemical and flow cytometric techniques, we have studied the significance of cellular expression of the Ki-67 antigen, epidermal growth factor receptor (EGFR), the transferrin receptor (TFR) and DNA ploidy status in a prospective analysis of patients with SCCs of the head and neck region. All 42 fresh tumour samples (five well differentiated; 28 moderately differentiated; nine poorly differentiated) expressed both EGFR and TFR to varying degrees. Receptor expression was most marked on the peripheral invading margin of cancer cell islands although staining was also demonstrated in a random fashion within cellular islands and consistently along the basal cell layer of overlying stratified squamous epithelium. The percentage of cancer cells that reacted with the Ki-67 monoclonal antibody was assessed as low (less than 10%) in 15 samples (35.8%), intermediate (10-30%) in 19 samples (45.2%) and high (greater than 30%) in eight samples (19.0%). Eleven of 15 samples (73%) with a low percentage reactivity were DNA diploid, whereas seven of eight samples (87.5%) with a high percentage reactivity were DNA aneuploid. Poorly differentiated SCCs were significantly more often aneuploid than were either moderately or well differentiated tumours. Our results suggest that EGFR and TFR are widely distributed on SCCs, especially on proliferating cells at the invading tumour margin. In addition, there is a close spatial correlation between cells expressing EGFR, TFR and those expressing the Ki-67 antigen. Tumours in which the staining intensity for both EGFR and TFR was intense invariably expressed the Ki-67 antigen in a high proportion of cells. Further patient follow-up will be important in determining whether intense EGFR and TFR staining, combined with a high percentage reactivity with Ki-67 antibody and DNA aneuploidy, will ultimately define a subset of head and neck cancer patients with a poor clinical outcome.

The use of monoclonal anti-CEA antibody immunohistochemistry in detecting the origin of oral cavity metastasis.

A 75-year-old woman presented with a painless swelling in the right mandibular retromolar area and numbness of the left lower lip. Radiographic examination of the mandible demonstrated an osteolytic lesion of the ascending ramus. Biopsy revealed adenocarcinoma of obscure origin. Staining of the specimen with a monoclonal antibody specific to colon carcinoma revealed its origin. On subsequent examinations, a primary tumor in the rectosigmoid region with extensive lung, liver and skeletal metastases were diagnosed. This unusual case of colonorectal carcinoma, presenting as a metastatic lesion of the mandible, was readily diagnosed by a novel immunohistochemical technique that utilizes highly specific monoclonal antibodies.

Fatty acid composition of clinically healthy and malignant oral epithelium.

The fatty acid composition of epithelium from clinically healthy oral mucosa and or oral squamous cell carcinoma was analyzed by gas/liquid chromatography following extraction using a modified Folch technique and conversion of fatty acids to methyl esters. There were significant reductions in the relative proportions of palmitoleic and oleic acids and elevation of the relative proportions of palmitic, steric and arachidonic acids in samples from squamous cell carcinoma. These differences could reflect changes in cell membranes and/or fatty acid metabolism. Further studies are required to assess their functional, diagnostic and prognostic significance.

RAS oncogene product expression in normal and malignant oral mucosa.

Proto-oncogenes are important in both normal cellular differentiation and in carcinogenesis. The majority of transforming genes belong to the ras family and the ras gene product has been shown to be elevated in some oral carcinomas. RAP-5 monoclonal antibody was used to determine the expression of the p21ras protein in normal and neoplastic oral mucosa in an immunohistological study. The expression of p21ras protein was generally restricted to acanthous cells with strong staining in normal oral mucosa and well-differentiated carcinomas. In contrast, the p21ras protein was not detected in significant amounts in severely dysplastic lesions and poorly differentiated carcinomas. These results suggest that expression of p21ras is a normal feature of more fully differentiated tissues, both normal and neoplastic, and is not useful as an indicator of cell proliferation or 'malignant potential'.

The behaviour of human oral squamous cell carcinoma in cell culture.

This study examined the initial behaviour of 48 human oral squamous cell carcinomas (SCC) in cell culture. The early outcome of these cultures (contamination, absence of cell growth, epithelial cell senescence/fibroblast overgrowth, extended keratinocyte growth) did not reflect the clinical characteristics of the tumours of origin. Four new human oral SCC cell lines were characterized more extensively. Each cell line was immortal, 3T3-independent, and expressed low degrees of anchorage independence (CFE less than 4 per cent). Two of the four cell lines were tumorigenic in athymic mice. All of the cell lines expressed keratin intermediate filaments and two showed weak co-expression of vimentin. A wide range of keratins were expressed by the tumour xenografts; cornified keratins (K1, K10) were only expressed in the absence of K19 and vimentin, and vice versa. The nuclear:cytoplasmic ratio and the degree of serum independence correlated with each other and with the STNMP clinical grading of the tumours of origin.

Rapid in vitro bromodeoxyuridine labeling method for monitoring of therapy response in solid human tumors.

In vitro bromodeoxyuridine (BrdUrd) incubated single-cell suspensions obtained from solid tumors were fixed on slides for subsequent sample processing. As dispersal of nuclei largely was avoided, only small amounts of cells were needed for examination. The sensitivity of detecting even low BrdUrd incorporation rates could be improved by treatment with intense DNA denaturation conditions. This technique was applied to monitor cytokinetic response to chemotherapy and radiation in oral carcinomas by analysing biopsies taken consecutively in the course of treatment. By combining BrdUrd labeling and DNA flow cytometry, cells arrested in S phase easily could be distinguished from cells showing continuous proliferation.

Immunohistochemical localization of c-myc oncogene product and EGF receptor in oral squamous cell carcinoma.

Subcellular distribution of c-myc oncogene product and EGF receptor in oral squamous cell carcinomas was examined by using immunohistochemical method. Immunohistochemy for c-myc oncogene showed three staining patterns. Reactive products were demonstrable in the nucleus, in the perinuclear cytoplasm with granular appearance and in the entire cytoplasm. Concomitant stainings of each reactive pattern were observed. Occasionally, the reactive product was strongly deposited on the chromatin under mitotic phase. EGF receptor positive cases were found in only 4 of 28 cases. Though reactive products were usually found in the cytoplasm, the cell surface staining was detected in the only one case. In this case, cells under mitotic phase were strongly stained. The fact that the strong staining both of c-myc and EGF receptor in cells under mitotic phase suggested the close relation between the expression of these genes and the proliferation of carcinoma cells.

Establishment and characterization of four new squamous cell carcinoma cell lines derived from oral tumors.

Four cell lines were established from squamous cell carcinomas (SCC) of the oral cavity. Cell lines AW 13516 and AW 8507 were derived from poorly differentiated SCC and epidermoid carcinoma of the tongue respectively. Cell line AW 10498 was derived from moderately differentiated SCC of the lower alveolus, and AW 9803 grew from a well-differentiated SCC of a retromolar trigone. The cultures showed typical epithelial cell morphology, numerous mitotic figures, occasional multinucleated giant cells, individual cell diskeratosis and nuclear and nucleolar abnormalities. The cell lines AW 13516 and AW 8507 were fast growers with a doubling time of 35.5 h and 31.9 h, respectively, which was independent of the initial seeding density. Cell lines AW 10498 (doubling time 52.2 h) and AW 9803 (doubling time 66 h) showed slower growth and had shorter doubling times at higher seeding densities. The presence of cytokeratins was detected in all the four cell lines by using polyclonal antikeratin antisera in indirect immunofluorescence and in Western blotting. None of the cell lines expressed major histocompatibility complex (MHC) class II antigens. MHC class I antigens were expressed by three cell lines but not by AW 9803. Flow cytometric analysis of DNA content and chromosomal studies suggested the presence of polyploidy and aneuploidy in all the four cell lines. Ultrastructural studies revealed typical epithelial cell features, such as the presence of desmosomes, tonofilaments and keratin bundles.

Inhibition of growth and squamous-cell differentiation markers in cultured human head and neck squamous carcinoma cells by beta-all-trans retinoic acid.

Vitamin A and some of its metabolites such as beta-all-trans retinoic acid (RA) have been implicated in the regulation of differentiation of normal and malignant epithelial cells in vivo and in vitro. In the present study the effects of RA on the growth and differentiation of 7 cell lines derived from human head and neck squamous-cell carcinomas (HNSCCs) were examined. RA (greater than 0.01 microM) inhibited the proliferation in monolayer culture of 6 of 7 HNSCC cell lines. One cell line (UMSCC-35) was very sensitive, 5 (UMSCC-10A, -19, -30, -22B and HNSCC 1483) were moderately sensitive, and 1 (HNSCC 183) was insensitive. Three of the cell lines (UMSCC-22B, -30, and HNSCC 1483) were capable of forming colonies in semisolid medium--a capability that was suppressed by RA. The HNSCC cell lines expressed various levels of the squamous-cell differentiation markers type I (particulate, epidermal) transglutaminase (TGase) and cholesterol sulfate (CS). RA treatment (I microM, 6 days) decreased TGase activity by more than 50% in 3 (UMSCC-10A, -22B and 1483) of the 7 cell lines, and the effect on UMSCC-22B was dose-dependent. Type II TGase (soluble, tissue type) activity was detected in 3 cell lines, and after RA treatment its activity increased in HNSCC 1483 and 183 cells and decreased in UMSCC-19. Following RA treatment, CS levels decreased by 20, 25, 70, 76, 89 and 91% in cell lines UMSCC-30, -10A, 183, UMSCC-35, -22B, and HNSCC 1483, respectively. The suppression by RA of CS accumulation in the 1483 cells was dose-dependent. Cholesterol sulfotransferase activity, which is responsible for CS synthesis, was suppressed by 40-97% after RA treatment of UMSCC-19, -22B, and HNSCC 1483. Our results demonstrate that RA inhibits the growth and decreases the level of 2 squamous differentiation markers in HNSCC cells.

Ha-ras oncogene product in human oral squamous cell carcinoma.

The expression of Ha-ras oncogene product (Ha-ras p21) in the biopsy or operation materials derived from 70 oral squamous cell carcinomas, 10 oral leukoplakias and 10 normal oral mucosae was examined immunohistochemically using anti-Ha-ras p21 antibody. Ha-ras p21 was detected in 43 (61%) of 70 carcinomas, 3 (30%) of 10 oral leukoplaskias and 3 (30%) of 10 normal oral mucosae. Patients with Ha-ras p21-positive carcinomas had a significantly worse prognosis than those with Ha-ras p21-negative carcinomas. These observations strongly suggest that the expression of Ha-ras p21 is a common event in oral squamous cell carcinomas, and that Ha-ras p21 serves as a tissue tumor marker for determining the prognosis of patients with oral squamous cell carcinomas.

Factor XIIIa-containing cells and fibrosis in oral and maxillofacial lesions: an immunohistochemical study.

The distribution of subunit A of blood coagulation factor XIII (FXIIIa) was investigated by the avidin-biotin-peroxidase complex (ABC) method in various oral and maxillofacial tissues. These tissues were from normal tongue, gingiva, lip, and submandibular gland, and from Dilantin gingival hyperplasia (one case), pyogenic granuloma (three cases), peripheral fibroma (four cases), squamous cell carcinoma (seven cases), chronic sclerosing submandibular adenitis (two cases), and fibrous dysplasia of the mandibular bone (one case). The distribution of collagenous components was examined in the same tissues by means of the Sirius red F3BA method. By means of the ABC method, FXIIIa was detected in the cytoplasm of certain connective tissue cells in each of the tissues examined. These FXIIIa-containing cells were sparse in the normal tissues but evidently abundant in the fibrous connective tissue of inflammatory and neoplastic lesions. In the present study, the close relationship between the distribution of FXIIIa-containing cells and that of collagenous components is demonstrated. The role that FXIIIa-containing cells play in the process of fibrosis is discussed.

Comparison of cytokeratin, filaggrin and involucrin profiles in oral leukoplakias and squamous carcinomas.

As the distribution pattern of cytokeratin (CK), filaggrin and involucrin has recently been suggested to discriminate between benign and malignant epithelial growths, biopsies of healthy oral mucosa, leukoplakias without and with dysplasia and squamous cell carcinomas were examined immunohistochemically using a panel of 4 monoclonal antibodies (AB) against different cytokeratin polypeptides (34 beta E12, KL1 and Pkk1) and filaggrin as well as a polyclonal AB to involucrin. Major and statistically significant differences were observed in the profiles of CKs (except Pkk1), filaggrin and involucrin between leukoplakias without and with epithelial dysplasia. However, the alteration in the expression of CKs, filaggrin and involucrin proved to be not a constant feature in leukoplakias with dysplasia as a considerable portion (20-25%) of them revealed the profiles of CKs, filaggrin and involucrin similar to those of benign leukoplakias, and vice versa. Immunostaining of these antigens did not define the diagnosis of dysplasia in leukoplakias more precisely than grading in conventional histology can do so far. However, immunohistochemical sensitivity in detecting a broad range of variation in the abnormal maturation patterns of keratinocytes in leukoplakias with dysplasia can be used to divide these lesions into subgroups to elucidate their prognosis in follow-up studies.

Spindle-cell carcinoma of the oral cavity and larynx. Immunohistochemical aspects.

Five cases of monophasic and 7 cases of biphasic spindle-cell carcinomas were analyzed immunohistochemically for the presence of vimentin and keratin type intermediate filaments in the pleomorphic spindle cells. Vimentin reactivity proved to be a consistent feature but keratin reactivity was more variable, this latter filament being lost in two cases initially presenting as pure squamous cell carcinomas showing dedifferentiation towards a pure monophasic spindle-cell tumour when recurring. The converse was also noted: acquisition of keratin in a monophasic spindle-cell tumour that recurred as squamous cell carcinoma. These results were considered to support the concept that spindle-cell tumours of the upper aerodigestive tract are a peculiar type of carcinoma and not a product of a pluripotent stem cell exhibiting bidirectional differentiation. Diagnostic implications are as follows: keratin positivity in a spindle-cell tumour substantiates its carcinomatous nature but its absence does not rule out a diagnosis of spindle-cell carcinoma.

[Immunohistochemical localization of laminin and its relation to the grade of histological malignancy in oral cancer].

The immunohistochemical localization of Laminin has been investigated with a monoclonal antibody in biopsy specimens from 27 patients with an invasive squamous cell carcinoma of the oral cavity. In normal mucosa, laminin was found distributed in a linear pattern in the epithelial-stromal junction, around the blood vessels, the nerve fibres, and the skeletal muscle bundles. In the cancerous tissues a variety in the linear staining around the cancer cell nests was seen. Extreme attenuation or complete loss of the linear staining of laminin was demonstrated in 8/27 specimens, and these tumors histologically showed a high grade of malignancy with H.E. staining.

Significance of DNA content in oral carcinoma.

Standard cytophotometric measurement of DNA in normal, leukoplakic and cancerous oral and oropharyngeal tissues with a Leitz Weitzler Aristophot Cytophotometer showed both 1% and 5% significance in different grades of malignancy and 5% as regards sites of malignancy. The differences were marked in different grades of malignancy and specially with progression of the lesion. Cytophotometry can be useful to diagnose the stages of carcinoma.

[An histologic and histochemical study of arteries in the stroma of oral squamous cell carcinoma]. / Istologike kai istochemike melete ton arterion tou syndetikou ypostromatos sto endostomatiko akanthokyttariko karkinoma.

A microscopical study of 175 oral squamous cell carcinomas demonstrated that 12% of the tumors showed small arteries in their stroma. The arteries occurred most frequently around the infiltrative border of the tumors. Focal intimal thickening was a common feature, while duplication of the internal elastic lamina or obliteration of the lumen appeared more rarely. The intimal thickenings consisted mainly of glycosaminoglycans, a fact supporting the hypothesis that they represented early atherosclerotic lesions. On the contrary, the duplication of the elastic lamina could be attributed to age-related changes. The complete closure of the lumen was brought about by a tissue rich in sulphated glycosaminoglycans; the uncertain pathogenetic mechanisms of endarteritis obliterans probably accounted for the development of this tissue.

Suppression of serum 1,25-dihydroxyvitamin D in humoral hypercalcemia of malignancy is caused by elaboration of a factor that inhibits renal 1,25-dihydroxyvitamin D3 production.

Although many patients with humoral hypercalcemia of malignancy exhibit reduced serum 1,25-dihydroxyvitamin D [1,25-(OH)2D] levels, N-terminal fragments of recently identified PTH-related protein as well as PTH itself elevate serum 1,25-(OH)2D concentrations. In the present study, the effect of tumor extracts from human tumor-implanted hypercalcemic nude rat models with high and low serum 1,25-(OH)2D on renal 1,25-(OH)2D3 production was examined using rat kidney cells in culture. Whereas tumors from rats with high serum 1,25-(OH)2D levels (OCC rats) contained only a single peak of cAMP production-stimulating activity (CPSA) in osteogenic sarcoma cells on reverse phase HPLC, tumor extracts from rats with low serum 1,25-(OH)2D levels (UCC rats) contained at least two peaks of CPSA. The main peak (peak A) was estimated to be approximately 17K by gel permeation chromatography, which was the same as the molecular size of the hitherto identified PTH-related protein, and a minor peak of CPSA (peak B) was estimated to be about 25K. When peak A or crude extracts of OCC tumors as well as human PTH-(1-34) were added to primary cultures of rat kidney cells, the production of 1,25-(OH)2D3 was significantly stimulated. In contrast, although peak B or crude UCC tumor extracts had no effect on 1,25-(OH)2D3 production in themselves, when they were added together with peak A or human PTH-(1-34) the stimulation of 1,25-(OH)2D3 production was almost completely inhibited. Both peak A and peak B enhanced cAMP production in cultured kidney cells, and the cAMP production by peak A was not affected by peak B. These results are consistent with the possibility that elaboration of an additional factor from tumor cells may be the mechanism by which serum 1,25-(OH)2D levels are suppressed in patients with humoral hypercalcemia of malignancy. The nature as well as the mechanism of action of this factor remain to be elucidated.

Altered keratin expression in buccal mucosal squamous cell carcinoma.

Cytokeratin pattern was analyzed in 14 moderately differentiated and 12 well-differentiated squamous cell carcinomas of buccal mucosa by SDS-PAGE, immunoblotting and two dimensional electrophoresis. These were compared with patterns of normal buccal mucosa and surrounding areas whenever possible. Normal buccal mucosa expresses keratin No. 4 (59Kd), 5 (58Kd), 13 (54Kd) and 14 (50Kd). Keratin No. 4 (59Kd) and 14 (50Kd) were expressed by 20 of 26 tumors studied, while many of the tumors did not express keratins No. 5 (58Kd) and 13 (54Kd). Keratin No. 1 (67Kd) and 16 (48Kd) were aberrantly expressed by 9 well-differentiated tumors. Keratin No. 17 (46Kd) and 18 (45Kd) were expressed by 10 and 8 tumors of 14 moderately differentiated tumors. Six tumors which showed involvement of alveolar mucosa, expressed some keratins expressed by its normal counterpart. Their altered expression was consistent with the differentiation pattern as stated earlier. Non-expression of keratins 5 and 13 seems to be the result of malignant transformation and is seen in the majority of tumors, while appearance of aberrant keratins seems to be related more to the degree of differentiation of the tumor.

Cytokeratins in hamster cheek pouch epithelium during DMBA-induced carcinogenesis.

The pattern of keratin expression in hamster cheek pouch epithelium during 15-wk of DMBA-induced carcinogenesis was studied. The sequential changes in cytokeratins of premalignant and malignant tissues and comparative investigation of normal epithelial tissues were examined during a weekly sequential DMBA-induced chemical carcinogenesis. Keratin polypeptides of normal pouch epithelium appear in a molecular weight range of 43-67 kd and 5-6 proteins can be identified. The disappearance of high molecular weight keratin (61-67 kd) was observed from the 6-wk DMBA-treated premalignant group to the 15-wk DMBA-treated malignant group. An additional keratin polypeptide was noted initially on the 11th-wk-DMBA-treated group and remained to the 15th-wk-DMBA treated group.