AgNORs in Hyperplasia, Papilloma and Oral Squamous Cell Carcinoma

Linaena Méricy da Silva FONSECA
Maria Auxiliadora Vieira do CARMO

Departamento de Clínica, Cirurgia e Patologia Odontológicas, Faculdade de Odontologia, Universidade Federal de Minas Gerais, Belo Horizonte, MG, Brasil

Braz Dent J (2000) 11(2): 105-110 ISSN 0103-6440

Introduction | Material and Methods | Results | Discussion | Resumo | References

Ten inflammatory fibrous hyperplasias, ten papillomas, and nineteen oral squamous cell carcinomas were analyzed by the AgNOR technique to determine if different disturbances of oral epithelia presented different AgNOR counts. The papilloma group showed higher mean AgNOR counts (3.15 ± 0.58) than the hyperplasia group (1.98 ± 0.24) and smaller than the well-differentiated oral squamous cell carcinoma group (6.56 ± 1.25) and poorly differentiated oral squamous cell carcinoma group (7.07 ± 1.60). The differences among the groups of lesions were statistically significant (P<0.05) except between the well differentiated oral squamous cell carcinoma group and the poorly differentiated oral squamous cell carcinoma group. Our findings suggest that the cellular proliferation ratio in papillomas is greater than hyperplasias and smaller than carcinomas.

Key Words: AgNORs, hyperplasia, papillomas, oral carcinoma.


Nucleolar organizer regions (NORs) are loops of DNA which contain ribosomal RNA genes. They are transcribed by RNA polymerase I and are of vital importance for the ultimate synthesis of protein (Egan and Crocker, 1992). AgNORs are acidic proteins associated to the NORs which are selectively stained by a silver colloid technique. A series of studies indicates that the quantity of protein AgNOR is related to the rapidity of cell proliferation and there is evidence of a relationship between AgNOR counts and the prognosis of malignant tumors (Derenzini and Trerè, 1994; Derenzini, 2000). Thus, the quantification of these proteins has been a useful method in diagnostic pathology especially in the differential diagnosis between benign and malignant tumors and helpful in recognizing limitrophic lesions (Ghazizadeh et al., 1997; Irazusta et al., 1998).

Inflammatory fibrous hyperplasia is a common lesion that results from chronic injury to the oral mucosa. It is frequently found under unstable dentures and is composed of flaps of hyperplastic connective tissue covered by a stratified squamous epithelium that usually shows acanthosis (Priddy, 1992).

Papilloma is a benign neoplasm with exophytic growth, stratified squamous epithelium arranged in papillary projections, giving it a clinical "cauliflower" appearance. The color is most often whitish and the most common location is the tongue and palate (Abbey et al., 1980). There is some evidence that HPV is implicated with oral papillomas (Ward et al., 1995).

Oral squamous cell carcinoma is the most common malignant neoplasm of the oral cavity. Its incidence in the world is increasing and the survival index continues to be small (50%), despite the progress of diagnosis and treatment of other malignant tumors. Thus, there is a need for improvement in early detection of oral carcinomas because treatment is more effective and the morbidity is minimal (Warnakulasuriya, 2000). Tobacco and alcohol are the two most important known risk factors for the development of oral cancer (Andre et al., 1995). However, the majority of initial alterations of oral cancer and precancerous lesions are not readily recognized in clinical or in histopathological examination (Warnakulasuriya, 2000). The basic biology of initiation and progression of various neoplasms is still obscure. Most invasive oral carcinomas are preceded by a preinvasive stage that may last for years (Altemani et al., 1999). Tumor progression in epithelia has been classified as normal, hyperplastic (non-dysplastic), dysplastic, carcinoma in situ and invasive carcinoma (Meyskens, 1991). However, in some instances, histopathological features are not defined sufficiently to determine the true nature of these disturbances. Thus, AgNOR staining is a helpful histochemistry technique to provide more information about the cellular status (Derenzini, 2000) .

Thus, the purpose of this study was to determine whether distinct disturbances of the oral covering epithelium had significant differences in their AgNOR counts and whether these counts were related to histological grade of oral squamous cell carcinoma.

Material and Methods

Ten inflammatory fibrous hyperplasia specimens, ten papilloma specimens, and nineteen oral squamous cell carcinoma specimens (seven well differentiated and twelve poorly differentiated) were selected from the Pathology Laboratory Archives of Minas Gerais Federal University School of Dentistry. Histological classification of the carcinomas was done by two experienced pathologists based on WHO criteria (Wahi et al., 1971).

Two adjacent 4-µm sections were obtained from each routinely processed paraffin block. One was stained with hematoxylin and eosin, the other section was stained using the argyrophilic technique described by Ploton et al. (1986). In brief, hydrated sections were put in a solution of ethanol and acetic acid in a proportion of 50% before the incubation with the solution formulated by dissolving 2 g gelatin in 1% aqueous formic acid to two parts of 50% aqueous silver nitrate solution. The sections were incubated in this solution for 30 min at 45°C and then washed in deionized water, and mounted in Canada balsam.

AgNORs were counted in 100 nuclei from significant areas of each specimen under 1000X magnification and oil immersion using a 0.025 mm2 eyepiece graticule. The significant areas were considered as those showing histopathologic morphologic criteria of each lesion. In the cases of hyperplasia, the areas with thickened epithelium and acanthosis were considered significant, whereas in the papilloma lesions, the significant areas were characterized by squamous epithelium arrayed in finger-like projections and basilar hyperplasia. In the oral squamous cell carcinomas, the invasive islands and cords of malignant epithelial cells were considered significant.

Only individual AgNORs were counted. Aggregated dots were counted as one when it was not possible to observe a halo of nucleoplasm among them. Where the closely aggregated dots were separated by a halo of nucleoplasm, the dots were counted separately (Coleman et al., 1996).

The Pearson's correlation test was used to calculate inter- and intra-observer differences. The means were compared statistically by the Student t-test.


AgNORs were visible as black or brown dots within the nuclei of epithelial cells. They were more regular in hyperplasias and papillomas (Figures 1, 2). Differences in size and shape were observed in oral squamous cell
carcinomas (Figure 3). The quantitative results are summarized in Table 1.

The differences observed among the groups of lesions were statistically significant (P<0.05) except between the group of well differentiated oral squamous cell carcinoma and the group of poorly differentiated oral squamous cell carcinoma.


The significant differences observed in this study among hyperplasias, papillomas and carcinomas are in accordance with studies which reported that AgNOR technique has been a useful method in diagnostic pathology, especially for the differentiation of benign and malignant tumors (Irazusta et al., 1998).

Several studies have shown variations in the number, shape and volume of AgNORs of normal mucosa cells and malignant cells (Coleman et al., 1996). In our study the AgNORs were more regular in the hyperplasias and papillomas. Significant differences in size and shape were seen in the oral squamous cell carcinomas.

Neoplastic cells generally exhibit a rise in the synthesis of normal and abnormal products and thus frequently feature a significant rise in AgNOR material (Yue et al., 1999). However, the exact significance of changes in AgNOR counts from one lesion to another is not fully understood. AgNOR counts rise with increased cell ploidy, with increased transcriptional activity and in stages of active cell proliferation (Derenzini, 2000). Moreover, AgNOR counts may not be a good indicator of cell proliferation but rather represent variations in metabolic or transcriptional activity (Coleman et al., 1996).

It is interesting to observe that although hyperplasia and papilloma are both benign lesions with similar clinical behaviors, we could verify significant differences in AgNOR counts between these groups with higher values for the papilloma group. Thus, we can predict that it was probably due to the neoplastic nature of the papilloma. However, some authors did not observe significant differences between normal mucosa and papillomas (Cabrini et al., 1992).

In the present study, the AgNOR means of the well differentiated and poorly differentiated oral squamous cell carcinomas were not statistically different. This finding is in accordance with other studies that also observed no relationship between AgNOR counts and the histologic pattern of the lesion (Piffko et al., 1999). These results suggest that AgNOR is related to malignant transformation, but not prognosis (Miyaguchi et al., 1994). However, other reports established a significant correlation between the mean AgNOR numbers and the prognosis of the lesion (Xie et al., 1997). It is interesting to note that the establishment of the histological differentiation grade in the present report was based on only one section of the specimen. Thus, this would not be representative of the entire lesion.

Our findings strongly suggest that the cellular proliferation ratio in papillomas is higher than hyperplasias and smaller than carcinomas.

The differences encountered among the groups could be due to the different etiological factors related to each lesion. However, these factors are not totally known and further studies are required for better understanding of the differences in the index of the cellular proliferation among different disturbances of the epithelium covering the oral mucosa and their relationship to these etiological factors.


Fonseca LMS, do Carmo MAV: AgNOR em hiperplasia, papiloma e carcinoma epidermóide de boca. Braz Dent J 11(2): 105-110, 2000.

Dez casos de hiperplasia fibrosa inflamatória, 10 papilomas e 19 carcinomas epidermóides de boca foram analisados pela técnica da AgNOR para se determinar se esses distúrbios do epitélio bucal possuiam números de AgNOR diferentes. O grupo dos papilomas apresentou contagens médias maiores de AgNOR (3,15 ± 0,58) quando comparadas às do grupo de hiperplasias (1,98 ± 0,24) e menores médias de AgNOR quando comparadas aos casos de carcinoma epidermóide de boca bem diferenciado (6,56 ± 1,25) e aos carcinomas epidermóides pobremente diferenciados (7,07 ± 1,60). As diferenças entre os grupos de lesões foram estatisticamente significantes (P<0,05) exceto entre o grupo de carcinomas epidermóides de boca bem diferenciados e o grupo do carcinomas pobremente diferenciados. Nossos achados sugerem que o índice de proliferação celular nos papilomas é maior que nas hiperplasias e menor que nos carcinomas epidemóides de boca.

Unitermos: AgNOR, hiperplasia, papiloma, carcinoma epidermóide bucal.


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Correspondence: Dr. Maria Auxiliadora Vieira do Carmo, Departamento de Clínica, Cirurgia e Patologia Odontológicas, Faculdade de Odontologia, Universidade Federal de Minas Gerais, Rua Conde Linhares, 141, Cidade Jardim, 30380-030 Belo Horizonte, MG, Brasil. Tel: +55-31-291-1199. Fax: +55-31-291-5600 or +55-31-291-7282. E-mail:

Accepted May 26, 2000
Eletronic Publication october, 2000