D. M. Chisholm

The association between tumour progression and vascularity in the oral mucosa.

Tumourigenesis in experimental models is associated with the formation of new blood vessels (angiogenesis). Recent studies have suggested that tumour angiogenic activity may be inferred in histological sections by measuring the density of the vasculature. The purpose of this study was to determine whether the transition from normal to dysplastic and neoplastic tissue in the oral mucosa is accompanied by quantitative or qualitative changes in the vascularity of the tissue, and how the estimate of vascularity is influenced by the vessel marker and method of assessment. A total of 100 specimens of normal oral mucosa, dysplastic lesions, and squamous cell carcinomas were examined. Sections were immunostained with the pan‐endothelial antibodies to von Willebrand Factor (vWF) and CD31, or with an antibody to the αvβ3 integrin, previously reported to be a marker of angiogenic vessels. Vascularity was quantitated by two different methods: highest microvascular density (h‐MVD) and microvascular volume, as determined by point counting (MVV). The results showed that vascularity, measured by the MVV method using antibodies to either vWF or CD31, increased significantly (P<0·0001) with disease progression from normal oral mucosa, through mild, moderate, and severe dysplasia to early and late carcinoma (76 paraffin‐embedded tissues examined). In contrast, h‐MVD did not discriminate between dysplastic lesions and carcinoma. A similar percentage of the total vessel volume (MVV) and density (h‐MVD) were positive for αvβ3 in 24 frozen tissues examined, including normal oral mucosa. It is concluded that there is a close association between vascularity and tumour progression in the oral mucosa. Morphometric analysis reflecting microvascular volume is more informative than the currently popular analysis of microvascular density. The expression of αvβ3 in the vasculature of oral tissues does not necessarily reflect the presence of angiogenic vessels.

An analysis of mandibular fractures in Dundee, Scotland (1977 to 1985)

A retrospective study was undertaken to assess mandibular fractures presenting over the period 1977-1985 in Dundee, Scotland. The data collected included age, sex, aetiology, month in which injury occurred, anatomical site of fracture, associated maxillofacial trauma and treatment modalities. The majority of fractures were sustained by males in the age group 20 to 29 years. Assault was the major cause of trauma followed by falls and road traffic accidents. The posterior body region was found to be the most common fracture site in the mandible. The level of such trauma has more than doubled, since a similar study was undertaken between the years 1961 to 1970.

Apoptosis, proliferation, and angiogenesis in oral tissues. Possible relevance to tumour progression

Experimental animal models have demonstrated that angiogenesis is essential for tumour progression, whilst sustained tumour growth requires a positive balance between tumour cell proliferation and cell death (apoptosis). The aim of this study was to determine the relative contribution of apoptosis, proliferation, and angiogenesis to disease progression in the oral mucosa. Histological sections of 47 archival specimens were examined; these included four groups of oral tissues: normal mucosa (n=12), moderate dysplasia (n=11) severe dysplasia (n=6), and squamous cell carcinoma (n=18). Apoptotic cells were visualized by in‐situ end‐labelling of DNA, proliferative cells by staining with Ki‐67 antibody, and blood vessels with von Willebrand factor (vWF) antibody. One‐way analysis of variance showed that indices of apoptosis (AI), proliferation (PI), and angiogenesis (vascularity) increased significantly with disease progression from normal oral mucosa, through dysplasia, to carcinoma (p<0.0001 for every index). The increase from normal mucosa to moderate dysplasia was significant for PI and vascularity, but not for AI. In contrast, the increase from dysplasia to carcinoma was significant for AI and vascularity, but not for PI. These data suggest that disease progression in the oral mucosa is accompanied by angiogenesis and increases in both epithelial proliferation and apoptosis. Net epithelial growth results from proliferation starting earlier and proceeding at a higher rate than apoptosis. Copyright

Assessment of vascularity in histological sections: effects of methodology and value as an index of angiogenesis in breast tumours.

The aims of this study were to (a) determine how the quantification of blood vessels in histological sections (vascularity) is affected by the methodology used and (b) assess the value of vascularity as an index of angiogenesis by comparing tumour and normal breast tissue. Archival specimens of breast, lung and oral carcinoma, oral dysplasia and normal breast tissue were used to test the effects of the following experimental variables on vascularity: pretreatment of the sections (enzymatic digestion, heating), endothelial markers (von Willebrand factor and CD31 antibodies), method of quantification (highest microvascular density, average microvascular density and microvascular volume) and interobserver variations. All the variables examined significantly affected the estimated vascularity; this depended on the type of tissue and method used. The pretreatment of the sections before staining was the most important variable, altering the vascularity ranking of the tumours. Vascularity in breast tumours was similar to that of the normal breast intralobular stroma, suggesting that an area of high microvascular density in the tumour does not necessarily represent tumour-induced angiogenesis. Contradictory results have been published regarding the value of vascularity as a tumour prognostic factor. Our results suggest that statistically significant differences in vascularity values are most likely to arise from failure to optimize the staining protocol and from the method used to assess vascularity.

Relationship between vascularity, age and survival in non-small-cell lung cancer

Lung tumours in the elderly show reduced growth potential; impaired angiogenesis may contribute to this phenomenon. Recent studies have suggested that the angiogenic potential of a tumour may be inferred by the vascularity measured in histological sections. The purpose of this study has been to determine whether vascularity is related to age, survival or other clinical parameters in resected non-small-cell lung cancer (NSCLC). A group of 88 consecutive patients with a follow-up period of at least 5 years was selected. The group exhibited a wide age range (37-78 years) and similar survival characteristics to those of the general NSCLC population. Tumour sections were stained with a pan-endothelial antibody (vWF) and vascularity was quantitated, without knowledge of the clinical details, by three methods: highest microvascular density; average microvascular density; and average microvascular volume. The results were analysed by non-parametric statistical tests. A correlation was found between all three methods of quantitation. Vascularity was not associated with age, sex, tumour type, stage, volume, size (TNM-T) nodal status (TNM-N) or survival. However, survival time was generally longer for patients with higher vascularity, reaching borderline significance (P = 0.06) for the average microvascular density values. Higher tumour volume (P = 0.02) and stage (P = 0.05) were associated with lower survival times. Using multivariate survival analysis, tumour volume was the only factor related to survival. We conclude that vascularity is not associated with age and has no significant prognostic value in NSCLC.

OVEREXPRESSION OF p53 IN NORMAL ORAL MUCOSA OF ORAL CANCER PATIENTS DOES NOT NECESSARILY PREDICT FURTHER MALIGNANT DISEASE

Recent reports of p53 positivity in the normal mucosa of some head and neck cancer patients have been taken as evidence for field cancerization and hence a likelihood of the development of further tumours, yet few papers report the clinical significance of this finding through long‐term follow‐up. The immunohistochemical detection of p53 expression in clinically and histopathologically normal oral mucosa taken from the wound margin following excision of oral cancer was assessed using the polyclonal antibody CM1. Fresh frozen biopsies of normal oral mucosa and the corresponding tumour from 21 oral cancer patients and of normal mucosa from 25 non‐cancer patients were assessed for p53 overexpression. The ‘normal’ mucosa was positive in 12 of the oral cancer patients and one of the non‐cancer patients. Second malignant tumours were seen in patients from whom p53‐positive ‘normals’ and p53‐negative ‘normals’ were recorded. In five of the p53‐positive ‘normals’, the corresponding cancer was p53‐negative. In one case, where ‘normal’ mucosa was available from more than one site, one region was positive, whilst the other was negative. No obvious difference in age, tobacco use, or recurrence rate was seen between positive and negative cases. All patients who were still alive were reviewed for a minimum of 5 years. Using Fishers exact test, no statistically significant difference was found for the rate of second malignant tumours occurring in patients with p53‐positive compared with p53‐negative normal mucosa. Thus, the detection of p53 in normal mucosa did not necessarily predict a further tumour.

Evidence for field change in oral cancer based on cytokeratin expression.

It was hypothesised that one may be able to visualise field changes, which are proposed to exist around tumours, as alterations in keratin intermediate filament protein expression. Standard immunohistochemical analysis using a panel of monoclonal anti-keratin antibodies was applied to fresh tissue sections to look for subtle changes in epithelial differentiation not visible in H&E sections. Such changes were observed in clinically normal epithelium from oral cancer patients, involving primarily substantial expression of keratins K8/K7 (using CAM 5.2) in the basal cells of 12 out of 34 biopsies, and also a trend towards a reduction in the complexity of keratin differentiation. Monitoring such changes may prove to be a valuable adjunct to conventional H&E staining if found to have prognostic and diagnostic significance.

The Presence of Pericytes and Transitional Cells in the Vasculature of the Human Dental Pulp: An Ultrastructural Study

The aim of this study was to determine the ultrastructural characteristics of the microvasculature of healthy human dental pulp, with particular reference to pericytes. Pulp tissue was taken from healthy impacted third molars following extraction. Eight teeth were obtained from 17- to 25-year-old patients and pulp tissue was processed for examination using standard techniques for transmission electron microscopy. The pulp was rich in capillaries composed of endothelial and peri-endothelial cells in a 4 : 1 ratio. Endothelial cells contained typical and abundant Weibel–Palade bodies. Three types of peri-endothelial cells were identified: pericytes, transitional cells and fibroblasts. Pericytes were embedded within the capillary basement membrane. Transitional cells were partly surrounded by basement membrane, but separated from the endothelium by collagen fibrils; fibroblasts were outside, but adjacent to the basement membrane and closely associated with collagen fibrils. Pericytes and transitional cells, but not peri-endothelial fibroblasts, contained low numbers of dense bodies similar to the endothelial Weibel–Palade bodies. Our observations are consistent with the hypothesis that, during normal tissue turnover, some pericytes may originate from endothelium and migrate away from the vessel wall to undergo transition to a fibroblastic phenotype.

The association between epithelial proliferation and disease progression in the oral mucosa

The purpose of this study was to examine the possible association between epithelial proliferation and disease progression in the oral mucosa. Archival specimens of normal oral mucosa (n = 12), dysplasia (n = 17) and squamous cell carcinoma (n = 18) were sectioned and proliferating cells visualised by staining with Ki-67 antibody. The proliferative index of the epithelium (PI) was determined by total cell counts and point counting. Similar results were obtained using either method. Comparison of the three groups of tissues by one-way analysis of variance showed a significant increase in PI with increasing lesion severity (p < 0.001). The PI of both dysplasia and carcinoma groups was significantly higher than that of normal oral mucosa (p < 0.001). However, the difference between dysplasia and carcinoma groups was not significant. PI was not associated with tobacco or alcohol consumption. We therefore conclude that Ki-67 expression is an early marker of disease progression in the oral mucosa but, on its own, is not a good indicator of neoplastic transformation.

The relationship between localization of Na+, K+-ATPase and cellular fine structure in the rat parotid gland

SummaryA method for the ultrastructural localization of the Na+-pump enzyme, Na+, K+-ATPase has been applied to the rat parotid gland. A ouabain-sensitive final reaction product, dependent on the presence of K+ and Mg2+, was found to be evenly distributed along the basal and basolateral plasma membranes of acinar and striated duct cells. In both cases, it was localized predominantly in the cytoplasm of the extensive foldings of these membranes.It is concluded that the reaction product meets the criteria for valid localization of this enzyme and that potential Na+-pump sites have been demonstrated. This study supports previous findings in salivary glands and contributes to an increasing body of evidence that contraluminal Na+-pumps are common to both reabsorbing and secreting epithelial cells.