Erik Dabelsteen

Oral epithelial dysplasia classification systems: predictive value, utility, weaknesses and scope for improvement

At a workshop coordinated by the WHO Collaborating Centre for Oral Cancer and Precancer in the United Kingdom issues related to potentially malignant disorders of the oral cavity were discussed by an expert group. The consensus views of the Working Group are presented in a series of papers. In this report, we review the oral epithelial dysplasia classification systems. The three classification schemes [oral epithelial dysplasia scoring system, squamous intraepithelial neoplasia and Ljubljana classification] were presented and the Working Group recommended epithelial dysplasia grading for routine use. Although most oral pathologists possibly recognize and accept the criteria for grading epithelial dysplasia, firstly based on architectural features and then of cytology, there is great variability in their interpretation of the presence, degree and significance of the individual criteria. Several studies have shown great interexaminer and intraexaminer variability in the assessment of the presence or absence and the grade of oral epithelial dysplasia. The Working Group considered the two class classification (no/questionable/ mild - low risk; moderate or severe - implying high risk) and was of the view that reducing the number of choices from 3 to 2 may increase the likelihood of agreement between pathologists. The utility of this need to be tested in future studies. The variables that are likely to affect oral epithelial dysplasia scoring were discussed and are outlined here; these need to be researched in longitudinal studies to explore the biological significance of a low-risk or high-risk dysplasia.

REVIEW ARTICLE. CELL SURFACE CARBOHYDRATES AS PROGNOSTIC MARKERS IN HUMAN CARCINOMAS

Tumour development is usually associated with changes in cell surface carbohydrates. These are often divided into changes related to terminal carbohydrate structures, which include incomplete synthesis and modification of normally existing carbohydrates, and changes in the carbohydrate core structure. The latter includes chain elongation of both glycolipids and proteins, increased branching of carbohydrates in N‐linked glycoproteins, and blocked synthesis of carbohydrates in O‐linked mucin‐like glycoproteins. In mature organisms, expression of distinct carbohydrates is restricted to specific cell types; within a given tissue, variation in expression may be related to cell maturation. Tumour‐associated carbohydrate structures often reflect a certain stage of cellular development; most of these moieties are structures normally found in other adult or embryonic tissues. There is no unique tumour carbohydrate structure, since certain structures which are tumour‐related in one organ may be normal constituents of other tissues. Tumour‐associated carbohydrate changes have been used in the diagnosis of human cancers. Recently, however, it has been demonstrated that the expression of some carbohydrate structures is associated with prognosis. Tn, sialyl‐Tn, and T are cell membrane‐bound mucin‐like carbohydrate structures that may be expressed in tumours due to blocked synthesis of the core carbohydrate chain of mucin‐like structures. Their expression is strongly associated with prognosis in certain tumours, but the biological relationship between their expression and tumour progression is at present unknown. The blood group‐related carbohydrate structures Lex, sialyl‐Lex, ABH, and Ley are examples of terminal carbohydrate structures which are related to tumour prognosis. These structures are of increasing interest since they may function as adhesion molecules; adhesion of tumour cells to endothelial cells of blood vessels may be mediated by an interaction between sialosyl‐Lex and E‐selectin and studies indicate that Ley is related to cell motility. These findings are now the basis for tumour therapeutic experiments.

Tissue distribution of histo‐blood group antigens.

The introduction of immunohistochemical techniques and monoclonal antibodies to specific carbohydrate epitopes has made it possible to study in detail the tissue distribution of histo‐blood group antigens and related carbohydrate structures. The present paper summarizes the available data concerning the histological distribution of histo‐blood group antigens and their precursor structures in normal human tissues. Studies performed have concentrated on carbohydrate antigens related to the ABO, Lewis, and TTn blood group systems, i.e. histo‐blood group antigens carried by type 1, 2, and 3 chain carrier carbohydrate chains. Histo‐blood group antigens are found in most epithelial tissues. Meanwhile, several factors influence the type, the amount, and the histological distribution of histo‐blood group antigens, i.e. the ABO, Lewis, and saliva‐secretor type of the individual, and the cell‐and tissue type. Oligosaccharides with blood‐group specificity are synthesized by the stepwise action of specific gene‐encoded glycosyltransferases. In general, this stepwise synthesis of histo‐blood group antigens correlates with cellular differentiation. The H and the Se genes both encode an α1–2fucosyltransferase, which is responsible for the synthesis of blood group antigen H from precursor disaccharides. A new model for the participation of the Se/H‐gene‐encoded glycosyl transferases in synthesis of terminal histo‐blood group antigens in human tissues is proposed; the type and degree of differentiation rather than the embryologic origin determines whether it is the H or the Se gene‐encoded transferases that influence expression of terminal histo‐blood group antigens in tissues.

Genetic and epigenetic alterations of the blood group ABO gene in oral squamous cell carcinoma

Loss of histo‐blood group A and B antigen expression is a frequent event in oral carcinomas and is associated with decreased activity of glycosyltransferases encoded by the ABO gene. We examined 30 oral squamous cell carcinomas for expression of A and B antigens and glycosyltransferases. We also examined DNA from these tumors for loss of heterozygosity (LOH) at markers surrounding the ABO locus at chromosome 9q34, for loss of specific ABO alleles, and for hypermethylation of the ABO promoters. Loss of A or B antigen expression was found in 21 of 25 tumors (84%) and was a consistent feature of tumors lacking expression of A/B glycosyltransferases. LOH at 9q34 was found in 7 of 27 cases (26%), and one case showed microsatellite instability. Among 20 AO/BO cases, 3 showed loss of the A/B allele and 3 showed loss of the O allele. Analysis of the proximal ABO promoter by methylation‐specific PCR and melting curve analysis showed hypermethylation in 10 of 30 tumors (33.3%), which was associated with loss of A/B antigen expression. ABO promoter hypermethylation was also found in hyperplastic or dysplastic tissues adjacent to the tumors, suggesting that it is an early event in tumorigenesis. Collectively, we have identified molecular events that may account for loss of A/B antigen expression in 67% of oral squamous cell carcinomas.

ABO Blood-group Antigens in Oral Cancer

Tumor progression is often associated with altered glycosylation of the cell-surface proteins and lipids. The peripheral part of these cell-surface glycoconjugates often carries carbohydrate structures related to the ABO and Lewis blood-group antigens. The expression of histo-blood-group antigens in normal human tissues is dependent on the type of differentiation of the epithelium. In most human carcinomas, including oral carcinoma, a significant event is decreased expression of histo-blood-group antigens A and B. The mechanisms of aberrant expression of blood-group antigens are not clear in all cases. A relative down-regulation of the glycosyltransferase that is involved in the biosynthesis of A and B antigens is seen in oral carcinomas in association with tumor development. The events leading to loss of A transferase activity are related, in some instances, to loss of heterozygosity (LOH) involving chromosome 9q34, which is the locus for the ABO gene, and in other cases, to a hypermethylation of the ABO gene promoter. The fact that hypermethylation targets the ABO locus, but not surrounding genes, suggests that the hypermethylation is a specific tumor-related event. However, since not all situations with lack of expression of A/B antigens can be explained by LOH or hypermethylation, other regulatory factors outside the ABO promoter may be functional in transcriptional regulation of the ABO gene. Altered blood group antigens in malignant oral tissues may indicate increased cell migration. This hypothesis is supported by studies showing that normal migrating oral epithelial cells like malignant cells show lack of expression of A/B antigens, and by studies that target ABH antigens to key receptors controlling adhesion and motility, such as integrins, cadherins, and CD-44.

T (Thomsen–Friedenreich) antigen and other simple mucin-type carbohydrate antigens in precursor lesions of gastric carcinoma

In a previous report we suggested that T antigen appeared to be associated with gastric carcinoma. To verify this hypothesis and characterize the pattern of expression of simple‐mucin type carbohydrate antigens (Tn. sialyl‐Tn and T before and after neuraminidase) in normal gastric mucosa and precursor lesions of gastric carcinoma, we studied the mucosa adjacent to 100 cases of gastric carcinoma, gastric biopsies of 60 dyspeptic patients, eight adenomatous polyps and eight hyperplastic polyps. The expression of the antigens was more related to the cell type and underlying lesions than to the coexistence of carcinoma. The most distinctive findings concerned intestinal metaplasia, dysplasia and hyperplastic lesions. In intestinal metaplasia, Tn was found mostly in columnar cells and sialyl‐Tn in goblet cells. T was more prevalent in incomplete intestinal metaplasia than in complete. A high prevalence of sialyl‐Tn expression and cell membrane immunoreactivity for T antigen, similar to those previously found in gastric carcinomas, were observed in three adenomatous polyps, one hyperplastic polyp, five cases of adenomatous dysplasia in the neighbourhood of intestinal carcinomas and four cases of marked foveolar hyperplasia, three of which were from the mucosa adjacent to diffuse carcinomas. We conclude that adenomatous and hyperplastic lesions share with gastric carcinomas features of aberrant glycosylation, namely the cell membrane expression of T antigen.

Molecular markers in the surgical margin of oral carcinomas

BACKGROUND Local or regional lymph node recurrence is the most common pattern of treatment failure in oral squamous cell carcinoma (SCC). The local recurrence rate is 30% even when the surgical resection margin is diagnosed as tumour free. Accumulation of genetic changes in histologically normal epithelium in the surgical resection margin may explain the local recurrence rate. The purpose of this study is to investigate the presence of senescence markers, which may represent early malignant changes in the margin that in routine pathological evaluations are classified as histologically normal. METHODS Formalin-fixed, paraffin-embedded surgical specimens from 16 consecutive patients with oral SCC and a clear surgical margin were obtained. The margin was analysed by immunohistochemistry for p53, p16, Chk2, Laminin-5 and glycosylated oncofetal fibronectin. RESULTS Two patterns of p53 expression were found in the histologically normal epithelium in the surgical resection margin. One was characterized by no protein expression in the majority of cells, except for small clusters of basal and parabasal cells with nuclear staining. The other was characterized by p53 expression in the nuclei of most basal cells. The expression of p16 was confined to small groups of cells in the basal cell layer whereas Chk2 was only seen in one case. Upregulation of the stromal proteins, Laminin-5 or glycosylated oncofetal fibronectin, was only seen at regions of invasion. CONCLUSION Small groups of cells expressing p53 and p16 were found in the surgical resection margin that appeared to be histologically normal and may represent early malignant changes.

Gliadin uptake in human enterocytes. Differences between coeliac patients in remission and control individuals.

The pepsin trypsin digest of the wheat prolamin gliadin (PT-gliadin) is deleterious to the small intestinal mucosa of coeliac patients. The handling of PT-gliadin by the intestinal epithelium in coeliac patients in remission and control individuals was investigated by in vivo instillation of PT-gliadin. The uptake of PT-gliadin was monitored by immunofluorescence microscopy of intestinal biopsy specimens, using affinity purified PT-gliadin antibodies. Control individuals show weak staining in the apical region of the enterocytes thereby showing an uptake of PT-gliadin. Coeliac patients have a conspicuous fluorescence in relation to the lateral membrane/intercellular space of enterocytes and intense staining intracellularly in the apical region. There is only weak staining in the enterocytes after the instillation was terminated, indicating an intracellular clearance. The study shows that normal enterocytes are able to take up PT-gliadin. The increased uptake in coeliac patients might be of importance for the pathogenesis either by direct toxicity or by presentation to immunocompetent cells. Furthermore, the results are in agreement with the suggestion of a functional alteration in the zonula occludens in the intestinal epithelium of coeliac patients.

A multivariate study of the prognosis of oral squamous cell carcinomas. Are blood group and hemoglobin new prognostic factors

Because blood groups and hemoglobin concentration have been associated with the risk of the development of some cancers, this study evaluated the significance of ABO and Rhesus blood groups and hemoglobin concentration as prognostic factors in oral squamous cell carcinoma (SCC). The authors examined all registered primary SCC of buccal and maxillary alveolar mucosa in the Norwegian population between 1963 and 1972. The biopsy specimens from these patients were reevaluated and borderline cases excluded. The remaining 111 cases were included in the study, and features recorded on first admission were included in the survival analyses. ABO and Rhesus blood groups were found in 99 of these patients. Multivariate survival analysis showed that tumor size, hemoglobin concentration, stage, and Rhesus blood groups were significant prognostic factors, but sex, age, treatment, duration of symptoms, ABO blood groups, and clinical appearance of the tumors were not. The prognostic value of Rhesus blood groups and hemoglobin concentration has not been previously reported for oral SCC.

Oral fibroblasts produce more HGF and KGF than skin fibroblasts in response to co-culture with keratinocytes.

The production of hepatocyte growth factor (HGF) and keratinocyte growth factor (KGF) in subepithelial fibroblasts from buccal mucosa, periodontal ligament, and skin was determined after co‐culture with keratinocytes. The purpose was to detect differences between the fibroblast subpopulations that could explain regional variation in epithelial growth and wound healing. Normal human fibroblasts were cultured on polystyrene or maintained in collagen matrix and stimulated with keratinocytes cultured on membranes. The amount of HGF and KGF protein in the culture medium was determined every 24 h for 5 days by ELISA. When cultured on polystyrene, the constitutive level of KGF and HGF in periodontal fibroblasts was higher than the level in buccal and skin fibroblasts. In the presence of keratinocytes, all three types of fibroblasts in general increased their HGF and KGF production 2–3 times. When cells were maintained in collagen, the level of HGF and KGF was decreased mainly in skin cultures. However, in oral fibroblasts, induction after stimulation was at a similar level in collagen compared to on polystyrene. Skin fibroblasts maintained in collagen produced almost no HGF whether with or without stimulation. The results demonstrate that the secretion of KGF and HGF in both unstimulated fibroblasts and in fibroblasts co‐cultured with keratinocytes is dependent on the type of fibroblasts. In general, the periodontal fibroblasts had the highest level of cytokine production. This high level of growth factor production may influence the proliferation and the migration of junctional epithelium and thereby influence the development of periodontal disease.