Argyrophilic nucleolar organizer regions(AgNORs) in odontogenic myxoma (OM)and ameloblastic fibroma (AF)

Cristiane Martins1

Yasmin Rodarte Carvalho2

Maria Auxiliadora Vieira do Carmo1

1Department of Oral Pathology, School ofDentistry, Federal University of Minas Gerais(UFMG) and2Department of Oral Pathology, Sao PauloState University (UNESP), Brazil

Correspondence to:Maria Auxiliadora Vieira do CarmoDepartment of Oral Pathology, School of DentistryFederal University of Minas Gerais (UFMG), AvenidaAntonio Carlos, 6627 – Pampulha, CEP: 31270-901 – Belo Horizonte – Minas Gerais, Brazile-mail: dorinhav/freemail.com.br

Accepted for publication January 11, 2001

Copyright C Munksgaard 2001J Oral Pathol Med . ISSN 0904-2512

Printed in Denmark . All rights reserved

489

AbstractTen cases of odontogenic myxoma (OM) and six cases ofameloblastic fibroma (AF) were subjected to comparative analy-sis by the AgNOR technique, in order to determine a possibledifference in cell proliferation index between these lesions. Themean AgNOR number of the mesenchymal component of AFwas compared with its epithelial component and the differencewas not found to be statistically significant. The mean AgNORindex of the AF group was significantly higher than that of theOM group. Moreover, the mesenchymal component of AF dem-onstrated increased AgNOR numbers compared with that of OM(P∞0.05). These results suggest that the epithelial and mes-enchymal components of AF may have similar cell proliferativeactivity. However, the cell proliferative index of this lesionseems to be higher than that of OM.

Key words: ameloblastic fibroma; argyrophilic nucleolar organ-izer regions (AgNORs); odontogenic myxoma

J Oral Pathol Med 2001: 30: 489–93

Odontogenic myxoma (OM) is an uncommon, locally invasive, be-

nign neoplasm, consisting of rounded and angular cells lying in an

abundant mucoid stroma (1). Its odontogenic origin has been justi-

fied by its association with missing or unerupted teeth, the sparse

presence of islands of odontogenic epithelium within the neoplastic,

myxomatous tissue, and its localization in the maxilla and mandible

(1, 2). Microscopically, the tumour is composed of a myxomatous

tissue made up of an acidic mucopolysaccharide ground substance,

containing a sparse population of stellate or spindle cells with long

anastomosing processes. The tumour cells commonly demonstrate

moderate pleomorphism, but rarely exhibit mitoses (1).

Ameloblastic fibroma (AF) is a rare benign odontogenic tumour

with a characteristic histologic pattern, including ameloblastic epi-

thelium embedded in fibromyxoid connective tissue without forma-

tion of enamel or dentin (3). This lesion is not aggressive and does

Martins et al.

not infiltrate (4, 5). Nonetheless, AF is a rare tumour that does recur

if not completely removed (4).

Nucleolar organizer regions (NORs) are loops of DNA with genes

of ribosomal RNA (rRNA). These genes are highly active during

metaphase and during the reorganization of the nucleolus at the

end of telophase (6). AgNORs are NOR-associated nonhistone argyr-

ophilic acidic proteins. Thus, some authors have proposed that inter-

phase AgNOR quantity is related to the cell duplication time and

rate of proliferation (7).

Some authors have utilized the AgNOR technique in odontogenic

lesions, and they have encountered controversial results (8, 9). The

aim of our study was to investigate probable differences in the cell

proliferation index between OM and AF by means of a comparative

AgNOR quantification.

Material and methods

Ten cases of OM and six cases of AF, from the files of the Oral

Pathology Laboratory, School Dentistry, Federal University of

Minas Gerais (UFMG) and from the Department of Oral Pathology

of Sao Paulo State University (UNESP), were analysed.

Two adjacent sections of 4 mm and 3 mm from each paraffin

block were obtained. The first one was stained routinely with hema-

toxylin and eosin (H-E), and the other by the AgNOR method (10).

In brief, hydrated sections were incubated in a freshly prepared

solution of one part made by dissolving 2 g of gelatin in 1% aque-

ous formic acid to two parts of 50% aqueous silver nitrate. Sections

were left in this solution for 30 min at 45 æC. The sections were then

washed with deionized water and mounted in Canada balsam.

Black-brown dots within nuclei were counted in 100 mesenchy-

mal cells of OM. Islands of odontogenic epithelium were not ob-

served in the OM lesions studied. In the cases of AF, the AgNORs

were evaluated in 100 cells of the epithelial component and in 100

cells of the ectomesenchymal component. The counting was done

twice by two different observers in characteristic areas of the

lesions. Cells where dots were not visualized were not considered.

Table 1. Mean AgNOR counts in odontogenic myxoma and ameloblasticfibroma

Lesion Means

Ameloblastic fibromaEpithelium 1.79a

Mesenchyme 1.60b

Odontogenic myxoma 1.37c

a,b P±0.05 n.s. b,c P∞0.05.

490 J Oral Pathol Med 30: 489–93

Aggregated dots without a ‘‘halo’’ of nucleoplasm around them were

considered as one (9).

Routine light microscopy under ¿1250 magnification and oil im-

mersion using an eyepiece graticule of 0.025 mm2 was performed.

After using Spearman’s correlation coefficient to establish both

inter- and intra-observer reliability (r50.91), the counts were per-

formed and the AgNOR means established. The non-parametric

Wilcoxon test was used for comparative statistical analysis of the

epithelial and the mesenchymal components of AF. The Wilcoxon-

Mann-Whitney test for independent samples was used between the

ectomesenchymal components of both lesions.

Results

AgNORs were visualized as black or brown, defined intranuclear

homogenous dots (Figs. 1 & 2). Table 1 demonstrates no statistically

Fig. 1. Visualization of AgNOR dots in epithelial (E) and mesenchymal (M)cells of ameloblastic fibroma (silver staining, ¿1250).

AgNORs in odontogenic tumours

Fig. 2. Visualization of AgNOR dots in cells of odontogenic myxoma (silverstaining, ¿1250).

significant difference in AgNOR means between epithelial and ecto-

mesenchymal components of AF, but a significant difference in

AgNOR means was observed between the ectomesenchymal cells

of both lesions.

Discussion

The AgNOR technique has been useful in differentiating benign

tumours from malignant tumours (6), although it has been of little

value in the diagnosis of some tumours. It is more useful in research

than in routine diagnosis. Many studies have associated the AgNOR

technique with other proliferative cellular indices, in lesions from

respiratory tract, skin tumours, prostate and bladder lesions and

491J Oral Pathol Med 30: 489–93

acute leukemias (11–13). Moreover, the AgNOR technique has been

useful both in diagnosis and in prognosis of salivary gland tumours

(14).

There are some controversial results in the literature about

studies using the AgNOR technique in oral mucosal carcinomas and

in odontogenic tumours. Some authors compared the mean AgNOR

index among odontogenic cysts and ameloblastomas and demon-

strated that AgNOR counts have neither a diagnostic nor a prognos-

tic value with respect to these lesions (8). However, other authors

compared odontogenic cysts and unicystic ameloblastoma, and ob-

served that there were statistically significant differences in AgNOR

counts among these lesions; according to these authors, this sug-

gests that these differences may or may not be an indication of

variations in metabolic, proliferative and transcriptional activities

(9).

The use of the AgNOR technique in comparative studies of

ameloblastomas and adenomatoid odontogenic tumours demon-

strated that both lesions have similar cell proliferation activity, im-

plying that the difference between their clinical behaviour is prob-

ably not correlated with their cell proliferation indices (15).

The use of the AgNOR technique with proliferating cell nuclear

antigen (PCNA) expression in ossifying fibroma (OF) and peripheral

ossifying fibroma (POF) has suggested an increased proliferative

activity in OF when compared with POF. This result is in accord-

ance with the more aggressive clinical behaviour of OF (16).

Some authors analysed ameloblastoma and basal cell carcinoma

(BBC) by AgNOR and observed that, although these lesions are

neoplasms with similar clinical behaviour, they have cell popula-

tions with statistically significant differences in AgNOR counts (17).

There was no statistically significant difference in AgNOR

counts between the epithelial and the mesenchymal components of

AF. This observation suggests that these components have similar

cell proliferation indices that could be related to the mixed nature

of AF where both the epithelial and the mesenchymal components

are considered to be neoplastic (18). In spite of its benign nature,

the rare cases of recurrences with possible malignant transform-

ation would be an important factor in the neoplastic characteristic

of AF (19).

According to the literature, the neoplastic characteristic of the

ectomesenchyme of AF was due to interactions of epithelium and

mesenchyme, which are related to tumour growth factor b2 (TGF-

b2) (19). It is present in AF, but absent in OM. It was suggested

that absence of TGF-b2 in OM could result in loss of inductive

capacity of the epithelium and its involution or disappearance (20).

It has also been suggested that a possible explanation for that dif-

ference in epithelial and mesenchymal interactions is due to tenascin

and cellular fibronectin in AF, regulating the growth and fetal pheno-

Martins et al.

type of neoplastic cells. These proteins are absent in OM. It was

further suggested that an early odontogenic induction may result in

a transformation of the fibroblasts into cells with enzymatic activity

similar to mature odontoblasts. These fibroblasts might lose their

ability to produce and secrete collagen, resulting in either a fibroblast

with low differentiation or a metabolically immature fibroblast (20,

21). The synthesis and degradation rate of the collagen is the basis of

the equilibrium of the connective tissue. In OM, the presence of cavi-

tation areas suggests that degradation is higher than synthesis, while

in AF the organization of the connective tissue rich in fibroblasts sug-

gests that synthesis is higher than degradation (22).

According to some authors, the mean AgNOR counts may be

correlated more to synthesis than to the rate of cellular proliferation

(6). Thus, our results may reflect either the higher proliferation

index or a greater capacity for synthesis of AF than of OM.

In the present study the mean AgNOR counts in AF was higher

than in OM. Thus, our results suggest that, although AF is com-

monly delimited by a fibrous capsule, the rate of growth of AF is

probably higher than that of OM. Furthermore, the proliferative

index of AF is probably not related to its inability to infiltrate ad-

jacent tissues. Therefore, these factors seem to indicate that the

infiltrative and recurrent features of OM are probably not related to

its cell proliferation index.

Some authors consider the infiltrative and recurrent features of

OM to be related to its gelatinous consistency and lack of an envel-

oping capsule. Likewise, mitosis is rarely seen, and the growth ratio

of the OM depends on the accumulation of myxoid substance (1, 2).

As a consequence, its low metabolic activity and the absence of

mitosis may result in a lower AgNOR index. Although it is not

compatible with its infiltrative and recurrent growth, it is compat-

ible with its slow growth. Moreover, the neoplastic cells of OM have

a high alkaline phosphatase and ATPase activity and a low acidic

phosphatase activity. These observations suggest a low metabolic

activity, which is compatible with its slow growth (19).

In conclusion, our results suggest that the cellular proliferation

ratio of AF is higher than that of OM. Thus, the different clinical

behaviours between these lesions with regard to their infiltrative

ability is probably not related to the proliferative index of their

tumour cells.

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