Journal of
Oral Pathology & Medicine
doi: 10.1111/j.1600-0714.2009.00838.x
Expression of tenascin and nucleolar organizer region in
ameloblastoma and ameloblastic fibroma
Sunitha Carnelio, Hitesh Vij
Department of Oral and Maxillofacial Pathology, Manipal College of Dental Sciences, Manipal, Karnataka, India
BACKGROUND: The aim of this study was to assess
the expression, distribution and comparison of tenascin,
a glycoprotein of the extracellular matrix in amelo-
blastoma and ameloblastic fibroma, both odontogenic
neoplasms with diverse biological behavior and to
understand the proliferative activity by using the
morphometric analysis.
METHODS: Paraffin embedded tissue from 25 cases of
odontogenic tumors i.e., ameloblastoma (n = 15) and
ameloblastic fibroma (n = 10) were used. The expression
of tenascin was evaluated using immunohistochemistry.
Morphometric analysis of nucleolar organizer regions
(NORs) from ameloblastoma and ameloblastic fibroma
was carried out by silver staining.
RESULTS: A heterogeneous expression of tenascin was
found in ameloblastoma which was mainly localized at
the epithelial–mesenchymal interface and a patchy dis-
tribution was observed in the stroma (80%), while strong
positivity was observed in the stroma and at the base-
ment membrane zone of ameloblastic fibroma (100%).
argyrophilic nucleolar organizer regions (AgNORs)
revealed higher mean counts in ameloblastoma (3.093 ±
0.902) when compared with those of ameloblastic fi-
broma (1.553 ± 0.250). Ameloblastoma presented more
than two NORs (two to five) per nucleus in majority of
the cells, while ameloblastic fibroma exhibited only one
NORs per nucleus.
CONCLUSIONS: Expression of tenascin in these neo-
plasms suggest that it could play a role in epithelial-
mesenchymal interaction, while AgNORs reveal that
ameloblastomas are more aggressive when compared
with ameloblastic fibromas.
J Oral Pathol Med (2010) 39: 223–229
Keywords: ameloblastic fibroma; ameloblastoma; morphometric
analysis; tenascin; tumor marker
Correspondence: Dr. Sunitha Carnelio, MDS, Associate Professor,
Department of Oral and Maxillofacial Pathology, Manipal College of
Dental Sciences, Manipal, Karnataka, India. Tel: 00919449257614,
Fax: 00918202570061, E-mail: sunithacarnelio@yahoo.co.uk
Accepted for publication July 24, 2009
J Oral Pathol Med (2010) 39: 223–229
ª 2009 John Wiley & Sons A/S Æ All rights reserved
interscience.wiley.com/journal/jop
Introduction
The spectrum of odontogenic lesions range from devel-
opmental anomalies to benign neoplasms and also
include a minority of frankly malignant ones. Odonto-
genic tumors are rare and constitute 1.3% of all oral
lesions and 2.5–15% of all oral neoplasms, among which
ameloblastoma has been the most frequently encoun-
tered neoplasms. It comprises approximately 1% of all
the cysts and tumors of the jaws and 11% of odonto-
genic tumors (1). According to their putative origin,
odontogenic tumors can be subdivided into epithelial,
epithelial-ectomesenchymal and ectomesenchymal neo-
plasms.
Ameloblastoma which has an ectodermal origin,
although benign, is known for its locally invasive
behavior with a high risk of recurrence, while amelo-
blastic fibroma is relatively less common and comprises
approximately 2% of all odontogenic tumors (2, 3).
These tumors, like normal odontogenesis, demonstrate
varying inductive interactions between odontogenic
epithelium and odontogenic ectomesenchyme of which
extracellular matrix (ECM) plays an important role.
ECM proteins have been shown to play a significant role
in cellular growth and differentiation via complex cell
matrix interactions mediated by a major part by
transmembrane integrin receptors. It acts as a major
modulator of cellular gene expression and influences
morpho-differentiation and is said to regulate the cell
dynamic behavior (4).
Tenascin, a large glycoprotein component of ECM
with a molecular weight of 190–250 kDa is involved in
cell-to-cell and cell-ECM interactions and is spatially
and temporarily expressed in a site restricted manner at
the epithelial-mesenchymal interface in embryonic and
fetal development and wound healing. A diverse
expression of tenascin has been reported in various
lesions, where it may affect cellular proliferation,
differentiation and migration (5). Till to date, there
are only a few reports on the role played by tenascin in
odontogenic tumors (5–7). As ameloblastoma are
highly destructive, we studied the cellular proliferation
by using silver (Ag) stain.
 Tenascin and AgNOR in odontogenic tumors
Carnelio and Vij
224
Nucleolar organizer regions (NORs), considered
important in protein synthesis are DNA loops that
encode ribosomal RNA (8), which can be visualized by
Ag staining method and are termed argyrophilic nucle-
olar organizer regions (AgNORs). NORs, in the human
karyotype are located on the short arms of the
acrocentric chromosomes 13, 14, 15, 21, and 22 (9).
Quantitative and qualitative changes of NORs can
imply the degree of cell nucleolar activity in hyperplastic
and neoplastic conditions, and a rise in the number of
AgNORs indicate proliferative activity indicating a state
of activation of cells (10). This technique plays a major
role in estimating the proliferation potential of neo-
plasms in the surgical pathological practice (11–13).
However, there are only a few studies of AgNOR in
ameloblastoma (10, 14, 15).
Hence we have employed the use of AgNOR to study
the proliferative activity in these neoplasms as NOR-
related parameters assessed are biologically informative
and easy to monitor at a pathology laboratory (16). The
purpose of this study was to analyze the expression and
distribution of tenascin in ameloblastoma and amelob-
lastic fibroma with varied stromal back ground thus to
understand their phenotypical behavior. AgNOR stain-
ing was used to assess the cell proliferative activity.
Material and methods
This laboratory based study involved the use of buffered
formalin fixed paraffin embedded tissue, histopatholog-
ically diagnosed according to the WHO histological
typing of odontogenic tumors (17) of which 15 ame-
loblastomas and 10 ameloblastic fibromas were studied.
Eight specimens from human fetuses (13–20 week ges-
tation) were included in the study as control of which
four contained tooth germ in early bell stage and four in
the late bell stage of tooth development. A case series
analysis was carried out by immunohistochemistry
(IHC) for tenascin expression in ameloblastoma and
ameloblastic fibroma to know the nature and distribu-
tion of this glycoprotein and morphometric analysis was
assessed by the Ag staining as per Howat et al. (18).
Samples were fixed with 10% neutral buffered forma-
lin and embedded in paraffin. For IHC staining, 5 lm
sections were deparaffinized and immersed in 3%
methanol containing hydrogen peroxide for 30 min.
All sections were treated with 0.1% trypsin solution
at 37C for 1 h. Primary anti-sera, monoclonal anti-
human primary antibody for tenascin (clone BC-24,
code074K4819, IgG1 kappa isotope, Sigma Aldrich
Chemical Co., St. Louis, MO, USA) diluted 1:800 at
4C overnight (18 h) were incubated using streptavidin-
biotin immunostaining method. Visualization was per-
formed using freshly prepared 3,3¢ diaminobenzidine
tetrachloride (Dako Cytomation, Glostruop, Denmark)
chromogen for 10 min. The slides were then counter-
stained with Mayer’s hematoxylin and cover slipped.
For each batch of staining, a negative control was
performed, where the tissue sections were treated with
goat serum in phosphate buffered saline instead of the
primary antibody and also the specific primary antibody
J Oral Pathol Med
was replaced by a monoclonal IgG (Dako Cytomation)
with an irrelevant specificity (Aspergillus niger glucose-
oxidase) with the same subtype and concentration as the
specific primary antibody. The blood vessel basement
membrane was used as internal antigen positive control
for tenascin. Fetal tooth germ was also taken as positive
control.
The staining pattern of tenascin was observed using
light microscope at ·10 & ·40 magnification and
reviewed for its expression and location, i.e., (i) at
epithelial-connective tissue junction, (ii) in connective
tissue stroma, and (iii) cells within the follicle (peripheral
cells and stellate reticulum like cells). For the study
group, in each case thee fields were randomly selected
and evaluated. The intensity of expression of IHC
reactions was semiquantitatively assessed by two inde-
pendent observers to remove any possible bias using a
modification of criteria indicated by deOliveira et al.,
(19) as: 0 (absence of staining), + (moderate staining
greater than background staining), ++ (strong ⁄ intense
staining). Following this, the expression of tenascin in
each of the cases was evaluated for the above parameters
(location and intensity) considering its importance in the
disease process.
Method of staining with AgNOR
Sections were cut at 4 lm thickness from formalin fixed
paraffin wax-embedded blocks, were taken to water via
xylene and graded ethanol and were submitted to
AgNOR procedure at room temperature. The reaction
mixture comprised 2% gelatin in 1% aqueous formic
acid that was mixed in a proportion of 1:2 volume with
50% aqueous silver nitrate under dark room conditions.
These sections were then incubated in the dark with
AgNOR solution for 40–45 min. Further these sections
were dehydrated to xylene and resin mounted in dibutyl
phthalate xylene.
Controls
Sections of breast cancers known to have high AgNOR
counts were routinely used as controls. In addition, an
effective internal control proved to be the lymphocytic
infiltrates, normal melanocytes and vascular endothelial
cells that, on the average, had one AgNOR per cell.
Quantitation of AgNOR
Sections stained with AgNOR were examined indepen-
dently by two experienced histopathologists, the same
section being assessed for each lesion. Discrete dots in
100 cells were counted at ·1000 magnification under oil
immersion. The AgNORs were counted with the aid of a
graticule by using conventional light microscopy. The
standard number of regions assessed per slide was 10.
Statistical analysis for tenascin and Agnor
The intensity of expression of IHC reactions was
semiquantitatively assessed by two independent observ-
ers to remove any possible bias and to minimize the
interobserver variability, the scores of both observers
were subjected to Kendall’s tau-b test. Statistical anal-
ysis was performed using SPSS version 11.5 (Statistical
 Tenascin and AgNOR in odontogenic tumors
Carnelio and Vij

225
Package for Social Sciences, Chicago, Illinois, USA). band like immunoreactivity. The intensity of these
The P-value thus obtained was found to be non- patterns was weak (Fig. 1). The stroma exhibited patchy
significant in all. Thus tenascin expression graded by expression in most of the areas (Fig. 2; Table 3) with
one observer was considered for further statistical weak intensity in most of the areas. Most of the
analysis. With relation to AgNOR, data were recorded peripheral cells of follicles showed positive expression
for each pathologist to assess interobserver variation of tenascin in the cytoplasm.
using the sample correlation coefficient test. The mean
score of each lesion was calculated and further subjected Ameloblastic fibroma
to statistical analysis using Student’s t-test. In all cases of ameloblastic fibroma, the tenascin
expressivity at the epithelial-connective tissue junction
was found to be continuous and strong (Fig. 3; Tables 2
Results
In this study, the expression of tenascin in each of the 33
immunostained cases were reviewed in the region of
epithelial-connective tissue junction, connective stroma,
peripheral cells and stellate reticulum like cells which
comprised 15 cases of ameloblastoma, 10 cases of
ameloblastic fibroma and eight cases of tooth germ
tissue. The age of patients of ameloblastoma was in the
range of 31–74 years (mean age being 52.5 years,
Table 1) with a female majority, where as in cases of
ameloblastic fibroma the age ranged between 10 and
18 years (mean being 14 years, Table 1) with a male
preponderance. Mandibular posterior was found to be
the most common site for these lesions with two cases of
ameloblastoma occurring anteriorly.

Fetal tooth germs

Tenascin yielded positive expression on the submucosal Figure 1 Showing weak staining in basement membrane in amelo-
blastoma (· 40).
connective tissue. Strong ⁄ intense accumulation was
observed in the dental papilla under basement mem-
brane and osteogenic tissue, whereas epithelial tissue
showed a negative reaction with tenascin in both early
and late bell stage.

Ameloblastoma
Tenascin expression varied from case to case and a
heterogeneous expression was also observed within the
same section. It was observed within the same section
although in majority of the cases it was discontinuous
(Table 2). The tumor islands had predominantly linear

Table 1 Intergroup comparison of age (in years)

Number of Mean
Group cases (n) Age (yrs) SD
Ameloblastoma 15 46.867 6.782
Figure 2 Stroma showing patchy tenascin expression in ameloblas-
Ameloblastic fibroma 10 13.100 4.408
toma (· 40).

Table 2 Intergroup comparison of pattern of staining (continuous ⁄ discontinuous) at the epithelial-connective tissue junction

F1 F2 F3
Pattern A AF A AF A AF
Continuous (%) 4 (26.7) 10 (100.0) 5 (33.3) 10 (100.0) 3 (20.0) 10 (100.0)
Discontinuous (%) 11 (73.3) 0 (0) 10 (66.7) 0 (0) 12 (80.0) 0 (0)

A, ameloblastoma; AF, ameloblastic fibroma; F1, field 1; F2, field 2; F3, field 3.
P-value for F1, F2, F3 <0.001 significant.

J Oral Pathol Med

 Tenascin and AgNOR in odontogenic tumors
Carnelio and Vij

226
Table 3 Intergroup comparison of pattern of staining (patchy ⁄ diffuse) in stroma

F1 F2 F3
Pattern A AF A AF A AF
Patchy (%) 12 (80.0) 0 (0) 12 (80.0) 0 (0) 12 (80.0) 0 (0)
Diffuse (%) 0 (0) 10 (100.0) 0 (0) 10 (100.0) 0 (0) 10 (100.0)
Absent (%) 3 (20.0) 0 (0) 3 (20.0) 0 (0) 3 (20.0) 0 (0)

A, ameloblastoma; AF, ameloblastic fibroma; F1, field 1; F2, field 2, F3, field 3.
P-value for F1, F2, F3 <0.001 significant; Chi-square value for F1, F2, F3 = 25.

ma are shown in Tables 6 and 7. The average number of

AgNOR dots per nucleus in ameloblastoma was signif-
icantly higher (ranging from two to five per nuclei;
Fig. 4), when compared with ameloblastic fibroma. The
average mean area occupied by AgNOR in ameloblastic
fibroma was larger (1.152 ± 0.391) when compared
with ameloblastoma.

Discussion
As morphogenesis and cell differentiation in the devel-
oping tooth are controlled by a series of reciprocal
interactions between the epithelial tissue and mesenchy-
mal tissue, it has been pointed out that development of

Table 6 Quantitative evaluation of NOR dots & area in the two types
Figure 3 The tenascin expression is strong and continuous in of odontogenic lesions
basement membrane & diffuse in the stroma (· 25).

and 4). The stromal expression was diffuse and intense Range of
in all areas (Fig. 3; Tables 3 and 5). A positive expres- NOR dots ⁄ AgNOR Coefficient
Lesion type nuclei area (mm2) of variation
sion of tenascin with weak intensity was observed in the
cytoplasm of peripheral and stellate reticulum like cells Ameloblastoma 2–5 0.233–1.84 29.16 (nucleus)
of the follicles. (n = 15) 85.19 (area)
Ameloblastic 1.2–2.1 0.62–1.87 16.12 (nucleus)
fibroma (n = 10) 34 (area)
Morphometric analysis
The results of the morphometric analysis of AgNOR NOR, nucleolar organizer region; AgNOR, argyrophilic nucleolar
dots from both ameloblastoma and ameloblastic fibro- organizer region.

Table 4 Intergroup comparison of intensity of staining at the epithelial-connective tissue junction

F1 F2 F3
Intensity A AF A AF A AF
Weak (%) 14 (93.3) 0 (0) 14 (93.3) 0 (0) 14 (93.3) 0 (0)
Strong (%) 1 (6.7) 10 (100.0) 1 (6.7) 10 (100.0) 1 (6.7) 10 (100.0)

A, ameloblastoma; AF, ameloblastic fibroma; F1, field 1; F2, field 2; F3, field 3.
P-value for F1, F2, F3 <0.001 significant.

Table 5 Intergroup comparison of intensity of staining in stroma

F1 F2 F3
Intensity A AF A AF A AF
Weak (%) 14 (93.3) 0 (0) 14 (93.3) 0 (0) 14 (93.3) 0 (0)
Strong (%) 1 (6.7) 10 (100.0) 1 (6.7) 10 (100.0) 1 (6.7) 10 (100.0)

A, ameloblastoma; AF, ameloblastic fibroma; F1, field 1; F2, field 2; F3, field 3.
P-value for F1, F2, F3 <0.001 significant.

J Oral Pathol Med


Table 7 Comparison of NOR dots & area in ameloblastoma & ameloblastic fibroma
Ameloblastoma
Parameter N Mean
AgNOR ⁄ Nucleus 15 3.093
AgNOR area (mm2) 15 0.457
NOR, nucleolar organizer region; AgNOR, argyrophilic nucleolar organizer region.
Figure 4 Morphometric analysis of AgNOR dots in ameloblastoma
(· 100).
odontogenic tumors and cysts arising from remains of
odontogenesis is also dependent on these interactions
(4).
We observed a heterogenous pattern of tenascin
expression in ameloblastoma ranging from reactive to
unreactive areas in the same section. In majority of the
follicles, a continuous linear band with frequent breaks
at the basement membrane-connective tissue interface
with variation in intensity was observed. These results
were in accordance with the available literature which
proposes that peripheral columnar cells of follicular
ameloblastomas are comparable with inner enamel
epithelial cells of early bell stage and that the extracel-
lular glycoproteins being an integral component of
stromal connective tissue plays an important role in
tissue morphological features and behavior of tumor (5)
and that the frequent breaks in the ameloblastoma
indicates the aggressive nature of the lesion. Joshi et al.,
(20) stressed the fact that variation in tenascin expres-
sivity depends on both the type of surface to which it is
attached and the length of time it is allowed to attach.
Heikinheimo et al. (7) in their study found that the
immunoreactivity to tenascin was observed throughout
the stromal tissue of ameloblastoma which was not in
accordance with our study.
In this study, it has been observed that in all cases of
ameloblastic fibroma, a strong linear band of immuno-
staining for tenascin was observed around the basement
Tenascin and AgNOR in odontogenic tumors
Carnelio and Vij
227
Ameloblastic fibroma
SD N Mean SD P-value
0.902 10 1.553 0.250 <0.001
0.389 10 1.152 0.391 <0.001
membrane of ameloblastic follicles. These findings were
synchronous with the studies by Heikinheimo et al., (7)
who stated that the distribution of tenascin in amelob-
lastic fibroma would play an important role in bringing
about cellular differentiation. Our results on eight cases
of tooth germ (four early bell and four late bell) were in
concurrence with the studies of IRMA Thesleff et al.,
(21) which relates that the presence of tenascin is
necessary for the differentiation of dental papillary cells
into odontoblasts and that the ECM glycoprotein
appears to regulate the cell morphology and has more
restricted tissue distribution than fibronectin. Further
studies carried out on the role played by tenascin in
embryonic teeth, mammary glands and hair follicles
have shown that tenascin is present in dense, organ
specific mesenchyme surrounding the invaginating epi-
thelial bud, but not in the more distant mesenchyme and
is topographically restricted. Besides, this may reflect the
fact that tenascin-C, a developmentally regulated matrix
protein modulates cellular response to other matrix
proteins such as fibronectin. In the late bell stage, in
areas of calcification and areas where dental papillary
cells were completely differentiated into odontoblasts,
there was loss of expressivity. Our observations on
stromal distribution of tenascin in ameloblastomas has
revealed expression to be patchy i.e., in few areas there
was expressivity of tenascin with decreased intensity,
while in some areas there was lack of expression. This
reflects the fact that ameloblastomas are characterized
by neoplastic epithelium in a mature connective tissue
stroma and that the expression of this glycoprotein in
adult tissue (mature connective tissue) is normally
restricted. Alternatively there could be increased degra-
dation of tenascin by specific enzymes like matrix
metallo proteases (MMP’s) or tenascin inhibitory fac-
tors (22, 23). The frequent breaks in tenascin expression
as well its paucity in the stroma might be related to
MMP’s. In our study, majority of cases of ameloblastic
fibroma revealed that the stroma showed a strong
staining for tenascin. This could be explained by the fact
that the extracellular microenvironment could represent
an immature papilla.
Tenascin is primarily described as a mesenchymal cell
product but recent studies have demonstrated that
epithelial cells also play an important role in its
production (24, 25). We observed that tenascin expres-
sion was weak in the peripheral and central cells of these
odontogenic tumors but a strong reaction in areas of
squamous metaplasia while few of the follicles stained
negatively. Further this could be confirmed by mRNA
and cDNA analysis for tenascin. As we were not able to
J Oral Pathol Med
 Tenascin and AgNOR in odontogenic tumors
Carnelio and Vij
228
correlate the local aggressive nature of ameloblastoma
with tenascin expressivity, we employed the Ag staining
method to identify the nature of epithelial proliferation,
wherein NOR counting has been used as a method to
assess the rate of cellular proliferation. However, during
mitosis nuclear fragmentation usually occurs resulting in
the dispersion of NOR’s throughout the nucleus (26).
NOR has been used as one of the parameters to assess
the proliferation of the cellular activity and was further
confirmed by studies showing a correlation between the
number of NOR’s and other cellular proliferation
parameters such as ki-67 antigen and DNA flow
cytometer (8). Our observation showed that the average
number of AgNOR dots per nucleus in ameloblastic
fibroma (1.55 ± 0.250) was significantly smaller when
compared with ameloblastoma (3.093 ± 0.902); con-
versely tumors with a low rate of cellular proliferation
are more likely to display one or two NORs (26) and
these results where in accordance with the work of
Martins et al., (27). In our series, we also observed that
the mean number of AgNOR dots were greater in
ameloblastoma when compared with studies of Rosa
et al., (28) and with that of Pinheiro et al., (29).
However, this difference is probably caused by our
image processing and analysis system. It has also been
reported that the increase in the number of AgNOR in
interphase is a reflection of their proliferation rather
than degree of malignancy (12). From the literature, we
can infer that the number of dots per nucleus is more
reliable and reproducible predictor of cell proliferation.
In our study, we have also found a significant increase in
the number of AgNOR dots in ameloblastoma.
It can also be speculated that rise in AgNOR material
denotes a rise in protein synthesis and this activity in
ameloblastoma would contribute in part to the growth
pattern of this neoplasm. Further, the secretion of
various proteolytic enzymes such as metalloproteinases
and an increase in production of metalloproteinases
have been proved in ameloblastoma and the release of
various mitogens as a result of the activity of MMP’s on
the matrix could enhance the proliferative activity of the
tumor (29). The mean area in ameloblastic fibroma was
larger when compared with ameloblastoma (Table 6).
However, there are no previous reports to compare the
same. Alternatively the increase in number and decrease
in size of NORS in ameloblastoma can be explained as
result of altered proliferation and differentiation mech-
anism in conjunction with the synthesis of new onco-
proteins. Follow-up information available from records
of 5 years duration in our patients have shown that in
one case of ameloblastoma which was completely absent
for tenascin and 10 cases of ameloblastoma which had a
heterogenous expression for tenascin had no recurrence.
Moreover, eight cases of ameloblastic fibroma are
keeping fine till to date.
Thus the findings of this study on the expression of
tenascin in benign odontogenic tumors has particular
significance in elucidating and distinguishing the epithe-
lial and epithelial-ectomesenchymal tumors of odonto-
genic origin in terms of outcome related to epithelial
mesenchymal interactions but could not correlate with
J Oral Pathol Med
prediction of the tumor behavior (aggressiveness). Fur-
ther studies on tenascin along with other glycoproteins
such as fibronectin, laminin etc. might give an insight
into molecular events critical for epithelial-mesenchymal
interactions and tumorigenesis that would emphasize its
role as a tumor marker. However, morphometric
analysis by Ag stain was informative in distinguishing
these lesions on the grounds of their biological behavior.
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Conflict of interest
None
Source of funding
Routine patient diagnostics, no extra funds.
J Oral Pathol Med