Spatial distribution of cancer stem cells in head and neck squamous cell carcinomas.

BACKGROUND: CD44 and aldehyde dehydrogenase 1 (ALDH1) are considered putative markers of highly tumorigenic cells (i.e., cancer stem-like cells) in head and neck squamous cell carcinomas. This small subset of cells is believed to be the primary responsible for tumor initiation and progression. The objectives of this study were (i) to evaluate the patterns of CD44 and ALDH1 expression in the tumor center and in the invasive front, as well as in adjacent non-tumor epithelium, and (ii) to correlate these findings with clinical parameters. MATERIALS AND METHODS: The sample comprised 44 patients with primary head and neck squamous cell carcinomas. Hematoxylin and eosin (HE) staining was used for histopathological tumor grading and for morphological analysis of adjacent non-tumor epithelium. Semiquantitative analysis was performed in histological sections immunostained for CD44 and ALDH1. RESULTS: ALDH1 immunostaining in the invasive front showed positive association with tumor size, regional metastasis, tumor histopathological grading, and disease progression. Moreover, expression of this marker in both tumor invasive front and adjacent non-tumor epithelium was related with more aggressive tumors. CD44 immunostaining was heterogeneous in all areas evaluated and did not show association with clinical data. CONCLUSION: Collectively, these data suggest that ALDH1 immunostaining in the invasive front and in adjacent non-tumor epithelium may help identify tumors with a more aggressive behavior, potentially contributing to improving treatment customization and the monitoring of patients with head and neck cancer.

Analysis of the proliferative potential of odontogenic epithelial cells of pericoronal follicles.

AIM: To evaluate the proliferative potential and the cell proliferation rate of odontogenic epithelial cells. MATERIALS AND METHODS: Forty-two cases of pericoronal follicles of impacted third molars were submitted to silver impregnation technique for quantification of argyrophilic nucleolar organizer regions (AgNOR) and immunohistochemical staining for EGFR and Ki-67. For AgNOR quantification, the mean number of active nucleolar organizer regions per nucleus (mAgNOR) and the percentage of cells with 1, 2, 3 and 4 or more AgNORs per nucleus (pAgNOR) were quantified. Ki-67 immunolabeling was quantified, whereas for EGFR, a descriptive analysis of staining patterns (membrane, cytoplasm or membrane + cytoplasm positivity) was performed. We evaluated the reduced epithelium of the enamel organ and/or islands of odontogenic epithelium present in the entire connective tissue. RESULTS: mAgNOR were 1.43 (1.0-2.42) and were significantly different among pericoronary follicles from upper and lower teeth (p = 0.041). Immunostaining of Ki-67 was negative in all cases. EGFR immunolabeling was found mainly in the cytoplasm and was more intense in islands and cords when compared to reduced epithelium of the enamel organ. CONCLUSION: Odontogenic epithelial cells of some pericoronal follicles have proliferative potential, suggesting their association with the development of odontogenic lesions. CLINICAL SIGNIFICANCE: The authors suggest that nonerupted, especially of the lower teeth, should be monitored and if necessary removed.

Exploring the mechanisms of alcohol-related damage in oral mucosa - is oxidative stress associated with the increase in cell proliferation in rat tongue epithelium?

CONTEXT: Alcohol consumption has been related to a cell proliferation increase in oral epithelium but its mechanism remains unclear. OBJECTIVE: The aim of this study was to investigate whether oxidative stress parameters are implicated in the induction of cell proliferation in rat tongue epithelium after different times of chronic alcohol consumption. MATERIALS AND METHODS: Cell proliferation was assessed in tongue epithelium using AgNOR (argyrophilic proteins related to active nucleolar organizer regions) quantification. Oxidative stress parameters [lipid peroxidation, protein carbonyls, superoxide dismutase activity and catalase (CAT) activity and immunocontent] and Nrf2 immunocontent were quantified in tongue homogenates. RESULTS AND DISCUSSION: Mean AgNOR numbers (mAgNOR) per nucleus was 2.22 ± 0.30 in ventral tongue epithelium after 120 days of alcohol consumption (vs. 1.87 ± 0.18 for control animals and 1.91 ± 0.23 for animals treated with alcohol for 60 days) indicating cell proliferation increase (p < 0.05, ANOVA followed by Tukey post hoc). Interestingly, 60 days of alcohol consumption induced changes in oxidative stress parameters, but no alteration in cell proliferation. Vitamin E co-treatment was conduced in order to evaluate its possible protective effects. The 120 day Tween + vitamin E + alcohol treatment induced an increase in mAgNORs when compared to the Tween + vitamin E treated group (respectively 2.10 ± 0.30 vs. 1.77 ± 0.11, p < 0.05, ANOVA followed by Tukey post hoc), showing that vitamin E co-treatment had no protective effects. In addition, an inverse association was observed between CAT activity and AgNORs quantity (R = -0.32; p < 0.05, Person's correlation) as well as the possible involvement of Nrf2 in alcohol-related damage. CONCLUSIONS: Our findings suggest that the increase in cell proliferation associated with alcohol-related damage has no direct relation with an imbalance in oxidative parameters. In contrast, our results indicate that hydrogen peroxide may be implicated in cellular signaling during proliferation in the oral mucosa.

Evaluation of cell proliferation rate in non-dysplastic leukoplakias.

OBJECTIVE: Analyze whether the most frequent cases of non-dysplastic leukoplakias, hyperkeratosis (H), acanthosis (A), and hyperkeratosis with acanthosis (HA) have similar cell proliferation rates and to compare them with epithelial dysplastic (ED) leukoplakias and normal oral epithelium (NOE). STUDY DESIGN: The sample comprised 10 cases of normal oral epithelium, 10 cases of hyperkeratosis, 10 cases of acanthosis, 10 cases of hyperkeratosis with acanthosis and 10 cases of epithelial dysplasia. The mean number of AgNORs per nucleus (mAgNOR) and the mean percentage of cells with 1, 2, 3 and 4 or more AgNORs per nucleus (pAgNOR) were recorded. RESULTS: The results of mAgNOR showed differences between disorders in the evaluation of the basal layer, of the parabasal layer, and in the overall evaluation. mAgNOR and pAgNOR=2 increased progressively from normal oral epithelium to hyperkeratosis with acanthosis, hyperkeratosis, acanthosis and epithelial dysplasia (p<0.05). Cell proliferation rate was different between different subtypes of non-dysplastic leukoplakias and this group presented a higher proliferative behavior when compared to normal oral epithelium. CONCLUSION: It may be suggested that non-dysplastic leukoplakias had different characteristics regarding cell proliferation rates and sometimes showed a proliferative behavior similar to that found in epithelial dysplasia. More studies should be conduced to increase knowledge about the biological profile of non-dysplastic leukoplakias, especially as it pertains to acanthosis.

Immunohistochemical analysis of the patterns of p53 and PCNA expression in odontogenic cystic lesions.

OBJECTIVE: the role of p53 expression in odontogenic lesions has not been fully determined, but has been associated with cell proliferation. The purpose of this study was to analyze p53 and proliferating cell nuclear antigen (PCNA) expression in 4 different odontogenic lesions. DESIGN: expression of p53 and PCNA was analyzed in radicular and dentigerous cysts, odontogenic keratocysts, and calcifying odontogenic cysts (Gorlin cysts) using monoclonal antibodies for detection of p53 and PCNA. RESULTS: PCNA expression was significantly greater in the basal layer of radicular cysts and in the suprabasal layer of odontogenic keratocysts; the percentage of p53 positive cells was significantly greater in the suprabasal layer of odontogenic keratocysts. CONCLUSIONS: The patterns of p53 and PCNA expression in dentigerous and radicular cysts were similar although the two lesions are of different origin. In odontogenic keratocysts and Gorlin cysts, results indicate a different pattern of tumor growth.

Effects of aging on mouse tongue epithelium focusing on cell proliferation rate and morphological aspects.

The aim of this study was to investigate cell proliferation rate and certain morphological features of mouse epithelium as aging progresses. Tongue biopsies were performed on female mice (Mus domesticus domesticus) at 2, 8, 14 and 20 months of age as indicative of adolescence, adulthood, early senescence and senescence, respectively. Histological sections of tongue were stained with hematoxylin-eosin and subjected to silver staining for active nucleolar organizer region counting. Cell proliferation rate and epithelial thickness analysis were carried out. Analysis of variance detected no differences between the groups in terms of numbers of silver-stained dots associated with nucleolar proteins. There was an increase in mean epithelial thickness in adult animals, followed by a gradual reduction until senescence. Mean keratin thickness presented an increase at 8 and 20 months of age. This difference is probably related to puberty, growth or dietary habits. Aging has no influence on oral epithelial proliferation rate in mice. A gradual reduction in epithelial thickness is a feature of aging in mammals. A conspicuous increase in the keratin layer was observed in senescence as an adaptative response to the reduction in epithelial thickness. These results suggest that aging affects the oral epithelium maturation process through a mechanism that is not related to cell proliferation.

Nuclear changes in tongue epithelial cells following panoramic radiography.

This study aimed to investigate the effect of radiation from panoramic radiographs on the cells of the lateral border of the tongue by evaluating nuclear changes. Forty-two patients were included: 22 had one radiograph (Group I), and 20 required a repeat radiograph due to error in the first exposure (Group II). Material for the cytopathologic evaluation was collected before radiographs and 10 days later. Smears were stained with the Feulgen reaction and micronuclei, buds, broken eggs, karyorrhexis and binucleate cells were scored. The comparison of nuclear changes before and after radiation exposure in both groups revealed a statistically higher number of broken eggs, buds, karyorrhexis and binucleate cells 10 days after exposure (P=0.01). The number of karyorrhexis and binucleate cells was greater in group II (P=0.01). There was no change in the frequency of micronuclei before and after the radiographs. Radiation emitted during panoramic radiographs increased the number of nuclear anomalies (except micronuclei) in exfoliated cells of the lateral border of the tongue. This effect was more pronounced when the patients were exposed to a repeat radiograph, without however implying increased risk of irreversible tissue damage.

Fibronectin Modulates Cell Adhesion and Signaling to Promote Single Cell Migration of Highly Invasive Oral Squamous Cell Carcinoma.

Cell migration is regulated by adhesion to the extracellular matrix (ECM) through integrins and activation of small RhoGTPases, such as RhoA and Rac1, resulting in changes to actomyosin organization. During invasion, epithelial-derived tumor cells switch from laminin-enriched basal membrane to collagen and fibronectin-enriched connective tissue. How this switch affects the tumor migration is still unclear. We tested the hypothesis that ECM dictates the invasiveness of Oral Squamous Cell Carcinoma (OSCC). We analyzed the migratory properties of two OSCC lines, a low invasive cell line with high e-cadherin levels (Linv/HE-cad) or a highly invasive cell line with low e-cadherin levels (Hinv/LE-cad), plated on different ECM components. Compared to laminin, fibronectin induced non-directional collective migration and decreased RhoA activity in Linv/HE-cad OSCC. For Hinv/LE-cad OSCC, fibronectin increased Rac1 activity and induced smaller adhesions, resulting in a fast single cell migration in both 2D and 3D environments. Consistent with these observations, human OSCC biopsies exhibited similar changes in cell-ECM adhesion distribution at the invasive front of the tumor, where cells encounter fibronectin. Our results indicate that ECM composition might induce a switch from collective to single cell migration according to tumor invasiveness due to changes in cell-ECM adhesion and the resulting signaling pathways that alter actomyosin organization.

Quantification of silver-staining nucleolar organizer region in epithelial cells of tongue of mice after exposure to, or intake of, alcohol.

The purpose of the current study was to evaluate the effect of alcohol on the proliferative activity of epithelial cells in the lingual mucosa of mice by means of silver-staining nucleolar organizer region (AgNOR) count and area measurements. Forty-eight CF1 mice were separated into three groups. The test groups were submitted to topical exposure to, or intake of, 40% (volume/volume) ethyl alcohol. Biopsy specimens were collected from the middle third of the dorsal tongue at 0, 6, and 12 months, and samples were stained according to the AgNOR technique. Mean number and mean area of AgNOR per nucleus were calculated for 50 basal layer cells and 50 intermediate layer cells. Increases in mean number and mean area of AgNOR per nucleus in intermediate cells were observed at 12 months in the alcohol intake group (P < .05). Results showed that intake of 40% alcohol increased epithelial cell proliferation in the dorsal surface of lingual mucosa.

Microscopic analysis of the temporomandibular joint in rabbits (Oryctolagus cuniculus L.) using an occlusal interference.

The purpose of this study was to assess the tissue alterations in the temporomandibular joint (TMJ) of the New Zealand White rabbit (Oryctolagus cuniculus L.), after a unilateral occlusal interference insertion on the animal's right side back teeth. A total of 36 animals were used, thirty of which belonged to the experimental group and six to the control group. We established three experimental periods: 24 hours, three days and seven days. The control group animals were divided two by two; each pair followed the same experimental periods of the former one. The experimental group animals were submitted to the use of a 0.3 mm thick metallic cap with a visor. All animals were euthanized, and the TMJs were removed. Using a microscope for examination we observed, in all experimental periods, the presence of intra-articular hemorrhage in the supra- and infra-disk compartments as well as in the retro-disk zone. There were no inflammatory cells detected. The thickness of the condylar fibrocartilage presented significant alterations among the animals of the three experimental groups. In the left TMJs no inflammatory cells were detected. The results suggest that the insertion of a unilateral occlusal interference in rabbit back teeth does not cause any inflammatory intra-articular process within seven days; however, it does cause bilateral intra-articular hemorrhage and a larger compression of the condylar fibrocartilage in the joint opposite the side where the interference is placed. We also concluded that, in order to do research on the temporomandibular joint using animals, it is necessary have an independent (or separate) group of animals as controls.

Endothelial cell-secreted EGF induces epithelial to mesenchymal transition and endows head and neck cancer cells with stem-like phenotype.

Emerging evidence suggests that endothelial cell-secreted factors contribute to the pathobiology of squamous cell carcinoma (SCC) by enhancing invasive migration and resistance to anoikis. Here, we report that SCC cells within the perivascular niche have undergone epithelial to mesenchymal transition (EMT) in a primary human SCC of a patient that developed distant metastases. Endothelial cell-secreted EGF induced EMT of human SCC cells in vitro and also induced acquisition of a stem-like phenotype. In vivo, tumor xenografts vascularized with EGF-silenced endothelial cells exhibited a smaller fraction of cancer stem-like cells (ALDH(+)CD44(+)) and were less invasive than tumors vascularized with control endothelial cells. Collectively, these results demonstrated that endothelial cell-EGF induces EMT and acquisition of stem-like properties by head and neck tumor cells. On this basis, we suggest that vascular endothelial cells contribute to tumor dissemination by secreting factors that endow carcinoma cells with enhanced motility and stemness.

Effect of topical chamomile on immunohistochemical levels of IL-1ß and TNF-α in 5-fluorouracil-induced oral mucositis in hamsters.

PURPOSE: The aim of the present study was to evaluate the effect of topical chamomile and corticosteroid treatment on the profile of tissue cytokines (IL-1ß and TNF-α) in 5-fluorouracil-induced oral mucositis in hamsters. METHODS: Thirty-six hamsters were randomly separated into three groups (12 animals each): Group I--without treatment (control); Group II-treatment with chamomile (Ad-Muc(®)); and Group III--treatment with corticosteroid (betamethasone elixir- Celestone(®)). The animals received an intraperitoneal injection of 5--fluorouracil on Days 0 and 2. On Days 3 and 4, the buccal mucosa was scratched and therapy was initiated on Day 5. Three animals from each group were killed on Days 0, 5, 10, and 14 and the buccal mucosa was removed. The streptavidin-biotin complex method was used to delineate the in situ distribution, localization, and semiquantitative analysis of IL-1ß and TNF-α. Data from the semiquantitative analysis of immunohistochemical staining were comparatively analyzed using the Kruskal-Wallis test, followed by Dunn's multiple comparisons test. RESULTS: The distribution and localization of IL-1ß and TNF-α immunolabeling were similar. These proteins exhibited a diffuse pattern distributed throughout the connective tissue. The epithelium and adipose tissue were negative for both proteins. The semiquantitative analysis revealed that immunolabeling of IL-1ß and TNF-α increased in all groups with the development of mucositis. On Day 10 (period of peak mucositis), the group treated with chamomile had lower scores for both pro-inflammatory cytokines. CONCLUSIONS: Treatment with topical chamomile reduced the tissue levels of IL-1ß and TNF-α, thereby demonstrating anti-inflammatory action in oral mucositis in hamsters.

Expression of the cell cycle regulation proteins p53 and p21WAF1 in different types of non-dysplastic leukoplakias.

OBJECTIVES: The aim of this study was to analyze the immunolabeling of two cell cycle protein regulators, p53 and p21WAF1, in non-dysplastic leukoplakias with different epithelial alterations: acanthosis, hyperkeratosis and acanthosis combined with hyperkeratosis, and compare them with dysplastic leukoplakias. MATERIAL AND METHODS: This was a prospective cohort study involving 36 patients with oral homogeneous leukoplakias. excisional biopsies were performed and the patients remain under clinical follow-up. The leukoplakias were divided into four groups: 6 acanthosis, 9 hyperkeratosis, 10 acanthosis combined with hyperkeratosis, and 11 epithelial dysplasias. Paraffin-embebeded sections were immunostained for p53 and p21WAF1. Five hundred cells from the basal layer and 500 from the parabasal layer were counted to determine the percentage of positive cells. A qualitative analysis was also carried out to determine the presence or absence of immunohistochemical staining in the intermediate and superficial layers. Groups were compared with ANOVA (p<0.05). Pearson's correlation coefficient was used to test for associations between the two markers, p53 and p21WAF1. RESULTS: No leukoplakia recurred and no malignant transformation was observed whitin a follow-up period of 3-6 years. The mean percentage of p53 staining in the basal and parabasal layers was similar in all groups. p21WAF1 staining differed between layers was as follows: in the basal, only 3 to 4% of cells were stained, while in the parabasal, between 16 and 28% of the epithelial cells were stained in the four different studied groups with no statistically significant difference (p>0.05). CONCLUSIONS: Our findings failed to differentiate the non-dysplastic lesions by means of p53 and p21WAF1 immunostaining, notwithstanding similar profiles between non-dysplastic and dysplastic leukoplakias were observed.

A 10-year study of specimens submitted to oral pathology laboratory analysis: lesion occurrence and demographic features.

The purpose of the present paper was to describe the range of lesions histologically diagnosed in an oral pathology laboratory in southern Brazil. A retrospective study of 8,168 specimen analyses recorded between 1995 and 2004 was conducted. The records were retrieved from the Oral Pathology Laboratory, School of Dentistry, Federal University of Rio Grande do Sul, RS, Brazil. A total of 6,831 valid cases (83.63%) were examined. Of these, inflammatory lesions were the most common occurrences (n = 4,320; 63.24%). Benign and malignant tumors accounted for 7.66% (n = 523) and 1.9% (n = 130) of the occurrences, respectively. Significant associations were observed between nonneoplastic proliferative disorders and benign mesenchymal tumors in females, and between squamous cell carcinoma and leukoplakia in males. Most diagnoses were benign in nature and had an inflammatory etiology. The association of some demographic characteristics with the occurrence of lesions suggests that these characteristics should be considered in performing differential diagnoses.

Odontogenic epithelium: immunolabeling of Ki-67, EGFR and survivin in pericoronal follicles, dentigerous cysts and keratocystic odontogenic tumors.

The aim of this study was to evaluate the biological profile of odontogenic epithelium by immunolabeling of epidermal growth factor receptor (EGFR), Ki-67 and survivin in keratocystic odontogenic tumors (KOT), dentigerous cysts (DC), and pericoronal follicles (PF). Immunohistochemical analysis was performed in 13 KOTs, 14 DCs and 9 PFs. Immunolabeling was analyzed in the basal and suprabasal layers of KOTs and DCs, and in the islands of odontogenic epithelium and/or reduced enamel epithelium of PFs. KOTs showed the highest proliferation rate among the three groups, mainly in suprabasal layers. EGFR immunolabeling was observed mainly in the cytoplasm in basal and suprabasal layers of KOTs and in the suprabasal layer of DCs. Immunolabeling in both membrane and cytoplasm was greater in PFs. In PFs, membrane-only staining was observed. Survivin immunolabeling showed a greater percentage of positive cells (scoring +++) in the suprabasal layer of KOTs. In DCs, both layers showed similar percentages of cells scoring +++; PFs showed the highest percentage of these cells. In KOTs, epithelial cells showed stimulus-independent neoplastic proliferative characteristics, suggesting the presence of a suprabasal proliferative compartment, maintained by inhibition of apoptosis. In DCs, the basal layer seemed to proliferate in response to stimulus. Although PFs showed low proliferative activity, the expression of EGFR indicates that some cells have a high capacity to respond to stimuli, which could probably explain the origin of odontogenic lesions.

Collagen and elastic fibers in odontogenic entities: analysis using light and confocal laser microscopic methods.

Dentigerous cyst (DC) and keratocystic odontogenic tumor (KOT) are odontogenic lesions arising from epithelial elements, such as those observed in dental follicles (DF), that have been part of the tooth forming apparatus. These lesions show different clinical and histological characteristics, as well as distinct biological behavior. This study aimed to qualify and quantify collagen and elastic fibers by means of histochemical techniques with light and confocal laser microscopic methods in three odontogenic entities. Eleven DF, 13 DC (n=10 with inflammation, n=3 without inflammation) and 13 KOT were processed to the following techniques: Hematoxylin and Eosin, Masson's Trichrome, Picrosirius, Direct Blue, and Orcein. DF and DC without inflammation exhibited collagen with similar characteristics: no parallel pattern of fiber orientation, thick fibers with dense arrangement, and absence of distinct layers. A comparison between DC with inflammation and KOT revealed similar collagen organization, showing distinct layers: thin collagen fibers with loose arrangement near the epithelium and thick fibers with dense arrangement in distant areas. The only difference found was that KOT exhibited a parallel collagen orientation in relation to the odontogenic epithelia. It may be suggested that the connective tissue of DC is a reactive tissue, inducing an expansive growth associated with fluid accumulation and inflammatory process, which in turn may be present as part of the lesion itself. In KOT, loosely arranged collagen may be associated with the behavior of the neoplastic epithelium.

Proliferative activity in clinically healthy oral mucosa exposed to tobacco smoking and alcohol: a longitudinal study using the agNOR staining technique.

OBJECTIVE: To evaluate cell proliferation in clinically healthy oral mucosa exposed to smoking and alcohol carcinogens over a period of 24 months using the AgNOR staining technique. STUDY DESIGN: Sixty patients were initially evaluated: 17 were control individuals, 25 were smokers and 18 were smokers and alcohol drinkers. Fifty-two of these patients were reevaluated. Specimens for cytology were obtained from swabs of lower lip mucosa, border of the tongue and floor of the mouth and underwent AgNOR staining for evaluation of mean number and mean area of AgNOR dots per nucleus and percentage of nuclei with > 3 and > 5 AgNOR dots. Student t and Kruskal-Wallis tests were used to compare values obtained. RESULTS: A statistically significant increase was found in mean number of AgNOR dots per nucleus in 2 groups. One group showed a tendency toward increase of these values. The results of the longitudinal evaluation (Kruskal-Wallis test) revealed a statistically significant difference in number and area of AgNOR dots in the cells of the lower lip. CONCLUSION: The increase of the variables suggests that the longitudinal evaluation of changes in cell proliferation in individuals exposed to smoking and alcohol carcinogens may be a useful monitoring tool.

Epidermal growth factor receptor distribution in pericoronal follicles: relationship with the origin of odontogenic cysts and tumors.

OBJECTIVE: Investigate the distribution of epidermal growth factor receptor (EGFR) in pericoronal follicles as a predictor of progression to odontogenic cysts and tumors. STUDY DESIGN: Immunohistochemical EGFR staining patterns (membrane-only, cytoplasm-only, or combined membrane and cytoplasmic staining) in the reduced enamel epithelium and nests of odontogenic epithelium associated with follicles of impacted molar teeth were evaluated. The staining pattern of 20 specimens of pericoronal follicle was compared with that of 16 normal oral mucosa samples and to squamous cell carcinoma samples. RESULTS: Combined membrane and cytoplasmic staining was observed for normal oral mucosa mostly in proliferating layers (basal and suprabasal), decreasing in intensity toward the surface. Seven epithelial nests presented membrane-only staining, and the majority presented either a cytoplasm-only or a combined staining pattern. The staining patterns observed in reduced enamel epithelium were cytoplasm-only and combined. CONCLUSION: EGFR membrane-only staining may be an indicator of increased potential for epithelial nests to become odontogenic cysts or tumors.

Expression of the cell cycle regulation proteins p53 and p21WAF1 in different types of non-dysplastic leukoplakias

OBJECTIVES: The aim of this study was to analyze the immunolabeling of two cell cycle protein regulators, p53 and p21WAF1, in non-dysplastic leukoplakias with different epithelial alterations: acanthosis, hyperkeratosis and acanthosis combined with hyperkeratosis, and compare them with dysplastic leukoplakias. MATERIAL AND METHODS: This was a prospective cohort study involving 36 patients with oral homogeneous leukoplakias. excisional biopsies were performed and the patients remain under clinical follow-up. The leukoplakias were divided into four groups: 6 acanthosis, 9 hyperkeratosis, 10 acanthosis combined with hyperkeratosis, and 11 epithelial dysplasias. Paraffin-embebeded sections were immunostained for p53 and p21WAF1. Five hundred cells from the basal layer and 500 from the parabasal layer were counted to determine the percentage of positive cells. A qualitative analysis was also carried out to determine the presence or absence of immunohistochemical staining in the intermediate and superficial layers. Groups were compared with ANOVA (p<0.05). Pearson's correlation coefficient was used to test for associations between the two markers, p53 and p21WAF1. RESULTS: No leukoplakia recurred and no malignant transformation was observed whitin a follow-up period of 3-6 years. The mean percentage of p53 staining in the basal and parabasal layers was similar in all groups. p21WAF1 staining differed between layers was as follows: in the basal, only 3 to 4% of cells were stained, while in the parabasal, between 16 and 28% of the epithelial cells were stained in the four different studied groups with no statistically significant difference (p>0.05). CONCLUSIONS: Our findings failed to differentiate the non-dysplastic lesions by means of p53 and p21WAF1 immunostaining, notwithstanding similar profiles between non-dysplastic and dysplastic leukoplakias were observed.

Head and neck squamous cell carcinoma: decline in clinical stage at the time of diagnosis in a reference hospital in southern Brazil

Objective: Developing countries have a high incidence of head and neck squamous cell carcinoma (HNSCC). Risk factors are smoking and alcohol consumption; socioeconomic status and oral health may be associated with etiology. The aim of this study was to evaluate the sociodemographicprofile and oral health of patients with primary HNSCC, as well as the clinical and histopathological characteristics of the tumor. Material and Methods: We evaluated 78 patients; data about sex, age, skin color, schooling, oral hygiene, smoking, alcohol consumption and socioeconomic status were collected using a structured questionnaire. An intraoral examination provided dataabout caries, missing teeth and dental prosthesis. Hospital records were reviewed to collect clinical tumor information. Results: Mean age was 57.6 years; most participants were male,white, former or current smokers and moderate or high consumption of alcoholic beverages with low socioeconomic and educational levels. The majority of patients were disease-free at 2 year-follow up. Classification showed 60.2% of the tumors as T1 and T2 and 59% had no regional involvement. Most tumors were found in the mouth, and the tongue was the most frequent site. Histopathological examination revealed that 57.7% of the tumors were classified as moderateand poor prognosis. Conclusion: The profile of patients with HNSCC was similar to that found in other populations, but there is a decline in clinical stage at the time of diagnosis, and detecting this tumor at an early stage can be an effective mean to determine a better prognosis for patients