Non-invasive measurement of the microvascular properties of non-dysplastic and dysplastic oral leukoplakias by use of optical spectroscopy.

Differential Path-length Spectroscopy (DPS) was used to non-invasively determine the optical properties of oral leukoplakias in vivo. DPS yields information on microvascular parameters such as the mucosal blood content, the microvascular blood oxygenation and the average micro-vessel diameter as well as on tissue morphological parameters such as the scattering slope and scattering amplitude. DPS measurements were made on non-dysplastic and dysplastic oral leukoplakias using a novel fiber-optic probe, and were correlated to the histological outcome of biopsies taken from the same location. Our data show borderline significant increases in mucosal blood content in dysplastic lesions compared to non-dysplastic lesions, with no changes in microvascular oxygen saturation and light scattering signatures. These results suggest that dysplastic and non-dysplastic leukoplakias may be discriminated non-invasively in vivo through differences in their microvascular properties, if they can be reproducibly quantified in the presence of a variable thickness keratin layer that optically shields the mucosal layer.

Scanning electron microscopic study of surface epithelial cells in erosive and nonerosive oral lichen planus.

AIM: Aim of the study is to study the surface epithelial cells of erosive, nonerosive lichen planus by scanning electron microscope (SEM) and to correlate it with that of normal mucosa and oral leukoplakia. MATERIALS AND METHODS: Twenty biopsies of lichen planus and five biopsies of normal mucosa were taken. Half piece of each biopsy was processed for H&E stain and another half for SEM study. RESULT: Surface epithelial cells were showing different features. CONCLUSION: Definite different features were seen in lichen planus-leukoplakia group than in normal oral mucosa. These changes are due to abnormality in cell maturation and differentiation. CLINICAL SIGNIFICANCE: SEM study is helpful in early detection of dysplasia. It can lead to early treatment of the lesion and thus prevention of squamous cell carcinoma.

A qualitative and quantitative estimation of AgNORS in dysplastic and non-dysplastic leukoplakias.

Using a silver staining technique, nucleolar organizer region--associated proteins (AgNORS) were studied in formalin, fixed paraffin embedded tissue blocks of normal oral buccal mucosa epithelium, leukoplakia with dysplasias and leukoplakia without dysplasias. Fifty cases, that comprised of 10 cases of normal oral buccal mucosa epithelium and 40 cases, of leukoplakia without dysplasia and with mild, moderate and severe leukoplakia were examined with respect to the relationship between AgNOR counts and histologic grading. The mean AgNOR count per nucleus increased from normal oral buccal mucosa epithelium to leukoplakia without dysplasia to leukoplakia with dysplasia. Higher counts, wider scatter and smaller size of AgNOR in the nuclei were seen as the grading of dysplasia increased from mild to severe. It is suggested that this method has potential in distinguishing between dysplastic and non dysplastic leukoplakias and for early diagnosis, prognosis and treatment planning of dysplastic lesions.

Ultrastructural changes in malignant transformation of oral mucosa.

Transmission electron microscopy (EM) has been used to identify the ultrastructural details of normal and cancerous human oral mucosa. However, inconsistent reports of structural descriptions have rendered transmission EM valueless in the diagnosis of oral squamous cell carcinoma (SCC) or as a prognostic indicator. To identify features of dysplasia for diagnostic purposes, normal mucosa, severe dysplasia, oral SCC and normal margin adjacent to oral SCC were used to compare the ultrastructural features of normal and premalignant oral mucosa and oral SCC. The preparatory stages of dehydration, embedding, cutting and positive staining for transmission EM were modified and tested to improve ultrastructural definition. Thin and discontinuous basal laminas were found in mucosa with severe dysplasia and normal margin adjacent to oral SCC. No basal lamina was identified in oral SCC. This study has shown that there are some ultrastructural changes during malignant transformation of oral mucosa. Together with other laboratory investigative techniques, transmission EM may be helpful in detecting malignant changes in oral mucosa.

[Ultrastructural differences between leukoplakia with and without dysplasia]. / Différences ultrastructurelles entre la leucoplasie sans et avec dysplasie.

Leukoplakias with similar clinical appearances have presented with very different histopathological characteristics. In this study we investigate in depth the cellular ultrastructure of the tissues which determines the clinical behaviour of these lesions.

Morphometric study of nucleolar organiser regions (AgNOR) in HPV-associated precancerous lesions and microinvasive carcinoma of the oral cavity.

A morphometric study of silver-stained nucleolar organiser regions (NOR) was performed on histological sections from routinely paraffin-embedded blocks of 26 oral biopsy specimens (21 cases of leukoplakia consisting of 13 low, 4 moderate, 4 severe degree of dysplasia, and 5 cases of microinvasive carcinomas). In situ hybridisation (ISH) for HPV-DNA was performed on serial sections of the same samples. The following parameters were studied: V NOR (single AgNOR volume per nucleus), TV NOR (total AgNOR volume per nucleus), and R.I. (AgNOR's roundness index). The results highlight that not all the morphometric features of AgNORs allow the discrimination between lesions with dysplasia of low, moderate and severe degree and microinvasive carcinoma. TV NOR appeared useful, while the other morphometric parameters appeared statistically not significant i differentiating between the different lesions. These findings suggest that high values of TV NOR in oral dysplasia could represent a risk marker, identifying a subgroup of lesions with a worse prognosis, constituting then a possible indication for rigorous clinical management and/or for complex treatment of these HPV-associated preneoplastic lesions.

[A clinical and microscopic evaluation of the leukoplakia of oral mucosa]. / Ocena kliniczna i mikroskopowa leukoplakii blony jamy ustnej.

The authors present methods of treatment of 90 patients with leukoplakia of oral mucosa. In 60 cases tissue material taken from focus of leukoplakia, has been tested in microscope. In 8 cases the cancer has been identified. Using method of Carr-Price, the level of vit. A and beta-karoten has been appointed.

Time-related changes induced by zinc-deficient diet in the concentration of rat cheek epithelial membrane-coating granules.

Weanling male Sprague-Dawley rats were fed a diet containing 0.4 parts/10(6) zinc and controls were fed an identical diet supplemented with 40 parts/10(6) zinc. After 9, 18 and 27 days of zinc deficiency, specimens were excised from cheek epithelium and processed for transmission electron microscopy to study the concentration of membrane-coating granules (MCG). Their concentration was increased in the granular-cell layers of the zinc-deficient epithelium and became significantly greater after 18 and 27 days than 9 days of deficiency. MCGs appeared in the parakeratinized layers of zinc-deficient epithelium and their concentration became significantly greater after 27 days in comparison with 9 and 18 days of deficiency. Thus the intracellular retention of MCGs was increased in the granular and parakeratinized layers with the increase in time of zinc deficiency.

Micronuclei, a biomarker for chemoprevention trials: results of a randomized study in oral pre-malignancy.

Biomarkers are being sought that could serve as surrogate end points for chemoprevention trials. Micronuclei, cytoplasmic fragments of DNA, have been proposed as a biomarker and studied in oral pre-malignancy. This study evaluated micronuclei frequency in a randomized chemoprevention trial of oral pre-malignancy. A recent clinical trial evaluated the responses of pre-malignant oral lesions to 3 months of therapy with isotretinoin followed by 9 months of either low-dose isotretinoin or beta-carotene. For 57 study participants, micronuclei were counted in mucosal scrapings of the lesion and in normal-appearing mucosa at baseline and following 3 months and 12 months of therapy. Micronuclei counts were higher in scrapings from the lesion than in the normal-appearing mucosa. Following 3 months of isotretinoin, the micronuclei counts in scrapings of the lesion were significantly reduced. With treatment, the mean micronuclei count declined at 3 months. In a randomized comparison, both isotretinoin and beta-carotene maintained the suppression of micronuclei. The change in micronuclei count was not associated with the clinical or histological response to treatment. Chemoprevention treatment with isotretinoin led to a reduction in frequency of micronuclei, a marker of recent DNA injury, which was then maintained by both isotretinoin and beta-carotene.

Oral hairy leukoplakia in a child: confirmation of the clinical diagnosis by ultrastructural examination of exfoliative cytologic specimens.

We report on a 12-year-old girl infected with human immunodeficiency virus (HIV) who developed oral hairy leukoplakia (OHL). This is the fifth child with OHL whose case has been described so far. The clinical diagnosis was confirmed by ultrastructural documentation of a herpes-type virus in the lesional scraping specimen. Exfoliative material was obtained by a painless and noninvasive procedure. We conclude that ultrastructural examination of lingual exfoliative cytological specimens from clinically suspected OHL is a noninvasive and painless method for confirming the clinical diagnosis of OHL.

Argyrophilic nucleolar organizer regions in the evaluation of tumour progression in the oral mucosa: correlation with tissue pathology.

The present study has analysed the numbers of argyrophilic nucleolar organizer regions (AgNOR) in normal tissues and in premalignant and malignant lesions of the oral mucosa in order to assess their potential as a biological marker for tumour progression. On comparison of AgNOR numbers in different lesions, carcinomas showed the highest number (4.65 +/- 0.98) compared to leukoplakias (2.38 +/- 0.47) and normal tissues (1.53 +/- 0.39). Spindle cell carcinomas and poorly differentiated squamous cell carcinomas had higher AgNOR counts than well-differentiated carcinomas. In various clinically different types of oral leukoplakia, the lowest AgNOR counts were observed in homogenous leukoplakia and the highest in speckled leukoplakia. No significant difference in AgNOR number was observed between non-dysplastic and dysplastic leukoplakia, although a significant difference was evident between dysplastic leukoplakia and normal oral mucosa. Correlating the AgNOR count and tumour progression, a significantly high positive correlation coefficient (r = 0.7969, P = 0.0000) was observed.

Ultrastructural variations and assessment of malignant transformation risk in oral leukoplakia.

Oral cancer is the most common malignancy in India, having a well defined precancerous state, leukoplakia. Various follow-up studies have shown that only 4 to 18% of oral leukoplakia lesions subsequently progress into invasive malignancy over a period of time. This study evaluates the potential of electron microscopy in the early detection of malignant changes in leukoplakia. The study revealed that of the 25 leukoplakia lesions examined, 7 showed changes akin to malignancy. Predominant ultrastructural alterations observed were reduplication and discontinuity of the basal lamina, basal cells with pleomorphic and bizarre nuclei containing perichromatin and interchromatin granules, prominent intercellular spaces and poorly formed desmosomes. Since none of these changes were evident under light microscopy the study demonstrates the value of electron microscopy in evaluating oral carcinogenesis. A long term follow-up of such patients is needed to understand the prognostic implications of these ultrastructural variation and how they can be used as a base line for better light microscopic evaluation.

Ultrastructural findings in mucocutaneous infections of patients seropositive to HIV.

Tissue samples from 19 HIV-seropositive immunocompromised patients suffering from oral hairy leukoplakia, chronic vesicular or ulcerative herpes simplex, chronic nonmetameric herpes zoster, secondary syphilis, condylomata acuminata, molluscum contagiosum, or disseminated cutaneous mycobacteriosis were examined ultrastructurally in order to better define the fine structure of the causative organisms in parasitic conditions and to clarify the host-parasite relationships. Taking into account the few data in the literature regarding the same disorders in immunocompetent subjects, no striking differences in the morphology of the infectious agents or in the types of parasitism were found. Nevertheless, isolated herpesvirus and papillomavirus virions were found outside the infected cells, and this observation, if confirmed in a larger series of cases, could suggest a persistent infectivity of the lesions in immunocompromised patients. Moreover, electron microscopy proved to be useful for diagnostic purposes; in one case of disseminated cutaneous mycobacteriosis, repeated cultures failed to grow the organism.

Lingual hyperkeratosis in dyskeratosis congenita: ultrastructural findings.

The first ultrastructural description of the mucosal lesions of lingual hyperkeratosis in dyskeratosis congenita is reported. The histology described may help explain the high incidence, later in life, of cancerous change in these leukoplakic patches.

Electron microscopic changes on the DMBA induced oral precancerous and cancerous lesion in hamster cheek pouch.

Experiments were performed to study the early and late ultrastructural changes during hamster cheek pouch carcinogenesis using a regimen of topical application of 9,10 dimethyl-1-1-2 benzanthracene (DMBA) twice a week in liquid paraffin oil. The DMBA was administered for a period of 2 and 4 1/2 months. Hamsters exposed to DMBA for 2 months developed moderate precancerous changes, whereas the hamsters treated with DMBA for 4 1/2 months developed frank and multiple oral tumors with a cauliflower appearance. The ultrastructural pathological changes seen were considerably increased at 4 1/2 months compared with a 2 month period of DMBA treatment. Untreated and solvent control hamsters cheek pouch treated for 2 and 4 1/2 months with liquid paraffin oil alone did not show any premalignant or malignant changes during this period.

Immunohistochemical localization of epidermal growth factor (EGF) and EGF receptor in human oral mucosa and its malignancy.

The immunohistochemical localizations of human epidermal growth factor (hEGF) and EGF receptor (EGFr) in oral tissues, including normal mucosa, leukoplakia and squamous cell carcinoma were examined by the use of monoclonal antibodies to hEGF and EGFr. In normal mucosa and leukoplakia, immunostaining of hEGF was limited to an underlying layer of connective tissue near the epithelium. The intensity of extracellular staining appeared to increase with the degree of epithelial malignancy and was eventually most striking in the stroma of invasive carcinoma. The epithelial cells in normal mucosa, leukoplakia, and squamous cell carcinoma showed negligible immunoreactivity for hEGF. Expression of EGFr appeared to be associated with the proliferative activity of cells and/or epithelial malignancy. In normal mucosa, anti-EGFr monoclonal antibody reacted only with the basal cell layer. In all sections of leukoplakia, the positive cells for EGFr were found in the prickle cell layer in addition to the basal cell layer. Most tumour cells in squamous cell carcinoma were strongly positive for EGFr. These findings indicate increased expression of hEGF and EGFr with malignancy. The characteristic localization of extracellular hEGF in the underlying connective tissue and in stroma of oral mucosal tumours suggests a possible epithelial-mesenchymal interaction in hEGF secretion.

Oral hairy leukoplakia in AIDS patients: an ultrastructural study.

Hairy leukoplakia is a specific oral lesion associated with the opportunistic development of Epstein-Barr virus in the oral epithelium. It is now considered to be an early sign of HIV-induced immunosuppression. Four cases of oral hairy leukoplakia (OHL) from the lateral borders of the tongue of male AIDS patients were investigated by transmission electron microscopy. At the ultrastructural level, herpes-like viral particles were detected in the oral lesions of all cases. Indirect immunofluorescence performed on two cases showed the presence of EBV antigens in the nuclei and the cytoplasm of the infected epithelial cells. None of the specimens contained ultrastructural evidence of human papillomaviruses.

Diagnosis of oral hairy leukoplakia by ultrastructural examination of exfoliative cytologic specimens.

Lingual exfoliative cytologic specimens (scrapings) were obtained from 18 patients positive for human immunodeficiency virus with clinical oral hairy leukoplakia. Buccal mucosal scrapings were obtained from 12 of these patients. The specimens were processed for examination by transmission electron microscopy (TEM). Sixteen (89%) of the lingual specimens revealed infection of keratinocytes by herpes-type virus. There was no evidence of virus infection in the 12 buccal mucosal scrapings. Fungal hyphae were seen by TEM in 14 (78%) of the lingual scrapings and two (17%) of the buccal scrapings. One exfoliative specimen and two biopsy specimens were stained for Epstein-Barr virus DNA with a DNA probe. The demonstration of herpes-type virions by TEM in keratinocytes from a lesion clinically suspected to be hairy leukoplakia provides direct, objective diagnosis. Furthermore, use of exfoliative cytologic specimens provides a clinically simple, noninvasive technique.

Further ultrastructural findings in epithelial cells of hairy leukoplakia.

Epstein-Barr virus and multivesicular structures appearing in lesions of hairy leukoplakia have been examined ultrastructurally in serial sections. It was found that both structures probably represent various levels of sectioning of transversely cut cell processes. The so-called viral nucleocapsid having a single membrane results when the plane of section is at right angles to one of the membranes but tangential to the other. While the so-called viral particle having two membranes would result if the plane of section is perpendicular to both the membranes of adjacent cells. The internal electron-dense core probably represents filaments enclosed within these processes and therefore does not represent DNA nucleotides. In addition, these nuclei contained bundles of filaments which probably represented tonofilaments. This was found to be due to tangential sectioning of the nuclear membrane surrounding deep cytoplasmic invaginations which led to the appearance of these filaments as if free within the nucleus.