RESEARCH ARTICLE Open Access
Immunohistochemical detection of laminin-1 and
Ki-67 in radicular cysts and keratocystic
odontogenic tumors
Mohamed S Ayoub
1*
, Houry M Baghdadi
2
, Moataz El-Kholy
3
Abstract
Background: Odontogenic cysts are those which arise from the epithelium associated with the development of
teeth. Some odontogenic cysts were found to have special biological features that make them distinct from other
lesions. This study was conducted to detect the immunoepxression of laminin-1 and Ki-67 in both radicular cysts
(RCs) and keratocystic odontogenic tumors (KCOTs) and to examine the possible predictive value of these markers.
Methods: Thirteen cases of RCs and twelve cases of KCOTs were included in this study. Antibodies against laminin-
1 and Ki-67 were used as primary antibodies.
Results: ten cases out of thirteen cases of RCs were immunopositive to laminin-1. The immunonegative cases of
RCs showed high degree of inflammation inside the connective tissue wall. One case out of twelve cases of KCOTs
was immunopositive to laminin-1 and the rest were immunonegative. Seven cases out of thirteen cases of RCs
showed immunopositivity for Ki-67 with increased numbers of immunopositive cells when the inflammation was
severe in the connective tissue wall. All KCOTS were immunopositive to Ki-67.
Conclusions: The benign nature of radicular cysts and the aggressive behavior of keratocystic odontogenic tumors
could be explained by the expression of laminin and Ki-67. Laminin-1 and Ki-67 could be valuable markers for the
prediction of the biologic behavior of cystic lesions.
Background
Radicular cysts are a direct sequel to chronic apical peri-
odontitis following the death of dental pulp [1]. The
epithelial rests of Malassez in periapical granuloma may
be stimulated to proliferate by inflamm atory stimuli [2].
The morphological aspects of the epithelium have been
considered to reflect the functional activity of the RCs
[3]. RCs depict a thin, regular and atrophic layer of stra-
tified squamous epithelium, usually with mild to moder-
ate inflammatory reaction [4]. The underlying
supportive connective tissue might be focally or diffusely
infiltrated with mixed inflammatory cells population [5].
Keratocystic odontogenic tumor (KCOT), previously
known as odontogenic keratocyst (OKC), is a rela tively
common developmental odontogenic cyst that arises
from the dental lamina remnants [6]. An important
aspect of the OKC that should be underlined is that it
can represent one component of the nevoid basal cell
carcinoma syndrome (NBCS ) [7]. Several studies have
shown that the OKC is well recognized by its invasive
potential [8], thus it tends to grow within the medullary
cavity of bon e an d becomes a large lesion without caus-
ing obvious expansion [9].
Expression of laminin-1 in normal oral mucosa, odon-
togenic cyst s and odont ogenic tumors was examined in
several studies. Sections of normal oral mucosa and
odontogenic cysts stained for laminin-1 showed a dis-
tinct linear deposit of strong intensity at the basement
membrane junct ion but not in the cytoplasm of the
epithelial cells [10]. Sections of odontogenic t umors
stained for laminin-1 showed strong reactivity at t he
basement membrane junction as well as in the cyto-
plas m of all tumor cells. The expression of laminin-1 in
the cytoplasm of the tumor cells, but not in the n ormal
mucosa may be a useful marker to distinguish these two
types of epithelium [11] and it may suggest that
* Correspondence: msayoub56@yahoo.com
1
Professor, Oral Pathology Department, Faculty of Dentistry, Ain Shams
University, Cairo, Egypt
Full list of author information is available at the end of the article
Ayoub et al. BMC Clinical Pathology 2011, 11:4
http://www.biomedcentral.com/1472-6890/11/4
© 2011 Ayoub et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons
Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unres tricted use, distribution, and reproduction in
any medium, provided the original work is properly cited.
laminin-1 infl uences the prol iferation activity t oward
tumor potential [12].
Ki-67 antigen is the pr ototypic cell cycle relat ed
nuclear protein, expressed by proliferating cells in all
phases of the a ctive cell cycle (G1, S, G2 and M phase)
and r eaches a peak in the G2 and M phases. It rapidly
degrades after mitosis with a half life of detectable anti-
gen being an hour or less. It is absent in resting (G0)
cells. Ki-67 antibodies are useful in establishing the cell
growing fraction in neoplasms [13].
The a ims of this study were t o detect immunohisto-
chemically the expression of laminin-1 and Ki-67 in
radicular cysts and keratocystic odontogenic tumors and
also to examine the possible predictive value of these
markers.
Method
Specimen selection
Twenty-five formalin-fixed, paraffin-embedded tissue
blocks of odontogenic cysts were ob tained from the
archives of the oral pathology departments, Ain Shams
University, A lexandria University, and National Cancer
Institute, Cairo University. Thirteen cases were diag-
nosed as ra dicular cysts (RCs) and twelve cases were
diagnosed as keratocysti c odontogenic tumors (KCOTs).
Haematoxylin and e osin stained sections were used to
confirm the diagnosis.
Immunohistochemical procedures
For all specimens 4 μm sections were cut and mounted
on positivel y charged glas s slides. Sections were deparaf-
finized with xylene and rehydrated in graded ethyl al co-
hol, sections were immersed in citrate buffer solution of
pH 4.8 and were put in the microwave oven before
staining procedures.
For immunostaining a universal kit (R&D Systems;
USA) was used, peroxidase anti- peroxidase method of
immunostaining using the streptavidin-biotin system
was carried out, 3% hydrogen peroxide was applied to
the sections to block the endogenous peroxidase activity.
The sections were immunostained with anti-laminin1
primary antibody (clone AL-2, R&D Systems, USA) and
anti-Ki-67primaryantibody(cloneBGX,Biogenix
Corp., USA). The tissue sections were i ncubated over-
night at room temperature. Sections were then covered
by the link antibody followed by the streptavidin label-
ing antibody. After rinsing with PBS, DAB chromogen
was applied to the sections followed by counter stain,
and then sections w ere dehydrated in graded alcohol,
cleared in xylene and mounted.
Image analysis
For each positive section, four microscopic fields showing
immunopositivity were selected and photomicrographs
were captured at a magnification of 20×. Images were
then transferred to the computer system for analysis
using the image analysis software (Image J, 1.43r, NIH,
USA), to determine the following:
1. Area fraction of immunopositivity for both laminin-
1 and Ki-67. Area fraction was calculated as th e ratio of
immunopositive area to the total area of microscopic
field.
2. Number of immunopositive cells for Ki-67
Statistical Analysis
Statistical analysis was carried out on the tabulated data
using (SPSS 16.0) software. The performed statistical
tests included Student’s T-test to compare between the
expression of each marker in the t wo lesions and Pear-
son’s correlation to determine the correlation between
laminin-1 and Ki-67.
Results
I. Immunohistochemical Results
A) laminin-1
Lesions were considered positive when minimal brown
staining was dete cted in the field. The immunopositivity
at the basement membrane a rea of the blood vessels
and in relation to inflammatory cells was excluded dur-
ing analysis. The immunopositive reaction appeared as
brown linear staining at the basement membrane of the
epithelial cells.
Ten cases out of the thirteen RCs were immunoposi-
tive to laminin-1 representing 76.9%. The y showed a
discontinuous linear deposition at the basement mem-
braneoftheliningepithelium(Figure1).Thethree
immunonegative cases showed variable degrees of
inflammatory reaction in the connective tissue wall.
One case out of the twelve cases of KCOTs revealed
immunopositive reaction to laminin-1 representing
8.3%. The reaction a ppeared as a cont inuous linear
deposition at the basement membrane area (Figure 2).
Immunonegative reaction was observed in the remaining
eleven cases of the KCOTs.
B) Ki-67
Immunopositivity for Ki-67 appeared as brown reticular
reaction confined to the nucleus. Seven cases of the
thirteen RCs were immunopositive to Ki-67 representing
about 53.8% where there was severe inflammation in the
connective tissue. They showed a nuclear staining
mainly in the basal cells. The number of immunoposi-
tive cells increased with the in creased inflammatory
reaction in the connective tissue (Fi gure 3). Immunone-
gative reactions were observed in the remaining six
cases of the RCs.
All cases of KCOTs were positive for Ki-67. The reac-
tion was nuclear and confined to the basal and supraba-
sal cells of the epithelial lining (Figure 4).
Ayoub et al. BMC Clinical Pathology 2011, 11:4
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Figure 1 Photomicrograph of RC showing d iscontinuous linear deposition of laminin-1 at the basement mem brane area (laminin-1
×200).
Figure 2 Photomicrograph of KCOT showing continuous linear deposition of laminin-1 at the basement membr ane area. (laminin-1
×200).
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Figure 3 Photomicrograph of RC showing Ki-67 immunopositive basal cells. Note the severe inflammatory reaction in the connective
tissue (Ki-67 ×200).
Figure 4 Photomicrograph of KCOT showing Ki-67 immunopositvity mostly confined to the basal and suprabasal cells (Ki-67 ×200).
Ayoub et al. BMC Clinical Pathology 2011, 11:4
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II. Statistical Results
Statistical analysis for lamini-1 i mmunoexpression in
both RCs and KCOTs was not valid as laminin-1 was
expressed in only one case of KCOT.
For Ki-67, keratocystic odontogenic tumor showed a
statistically significant higher mean area fraction a nd
higher mean number of Ki-67 immunopositive cells
when compared with RCs (Tables 1, 2).
On the other hand, statistical analysis with Pearson’s
correlation coefficient revealed a positive linear correla-
tion between laminin-1 and Ki-67 immunopositive area
fraction. However, in RCs, this correlation was not pro-
ven to be statistically significant. (Table 3 and Figure 5).
Discussion
The results of the present study revealed t hat ten cases
outofthirteenofRCs(76.9%)showedimmunopositive
expression of laminin-1 at the basement membrane
zone of the epithelial lining. This is in ac cordance with
the results of Poomsawat et al. [14]. Interestingly, the
neg ative immunostaining for laminin-1 was observe d in
three RCs (23.1%) that showed severe inflammatory
reaction in the connective tissue wall of the cysts. This
result is in accordance to that reported by Furuyama
et al., [15] who stated that the ability of epithelial cells
to form continuous basement membrane was lost in the
presence of infl ammator y cytokines which enhances the
secretion of matrix metalloproteinase (MMP-9) and
(MMP-2).
On the other hand, negative i mmunoexpression of
laminin-1 was observed in eleven cases out of twelve
cases of OKCs included in this study. This finding is in
agreement with the results of Amorim et al., [16]. In
contrast Poomsawat et al., [14] concluded that laminin-
1 was expressed in RCs, dentigerous cysts and odonto-
gen ic keratocyst with different distribution patterns and
intensity. Also, Gurgel et al., [17] investigated the
expression of laminin-1 in twenty cases of odontogenic
keratocysts and found that laminin-1 was expressed in
all cases.
Seven cases of the RCs included in the present study
were immunopositive for Ki-67, which represent 53.8%
of the total cases. The expression was confined mainly
to the basal ce lls. Interestingly the surface area and the
number of immunoposi tive cells increased with t he
severity of inflammation in the con nective tissue. This
could be explained on the assumption that chronic
inflammatory reaction could act as stimulators causing
epithelial proliferation. An explanation similar to that
reported by Willoughby et al. [18] who concluded that
mild inflammatory injury stimulates epithelial prolifera-
tion, whereas more severe inflammation depresses it,
perhaps due to more extensive progenitor-cell damage.
Immunopositivity for Ki-67 was detected i n a ll cases
of KCOTs included in the study. The expression was
mainly in the supra-basal cells, a finding similar to that
reported by Kichi et al [19].
However, further studies utilizing a larger sample size
and more advanced methodological tools a re recom-
mended due to limited number of cases included in this
study.
Conclusions
Based upon the results of the present study, it could be
concluded that:
• The benign nature of radicular cysts and the
aggressive behavior of keratocystic odontogenic
tumors co uld be explained by the expression of
laminin and Ki-67.
Table 1 The means, standard deviation (SD) values and
results of Student’s t-test for the comparison between
Ki-67 area fraction in RC and KCOT
RC KCOT
Ki-67 Mean SD Mean SD P-value
0.72 0.29 2.68 0.55 <0.001*
*: Significant at P ≤ 0.05.
Table 2 The means, standard deviation (SD) values and
results of Student’s t-test for the comparison between
number of Ki-67 +ve cells in RC and KCOT
RC KCOT
Number of +ve cells Mean SD Mean SD P-value
152 65.8 682.8 257.5 <0.001*
*: Significant at P ≤ 0.05.
Table 3 Results of Pearson’s correlation coefficient for
the correlation between Lamini-1 and Ki-67 area
fractions in radicular cysts
Correlation coefficient (r) P-value
0.381 0.399
Figure 5 Scatter p lot showing positive correlati on between
Laminin-1 and Ki-67.
Ayoub et al. BMC Clinical Pathology 2011, 11:4
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• Laminin-1 and Ki-67 could be valuable markers for
the prediction of the biolog ic behavior of cystic
lesions.
Author details
1
Professor, Oral Pathology Department, Faculty of Dentistry, Ain Shams
University, Cairo, Egypt.
2
Associate Professor, Oral Pathology Department,
Faculty of Dentistry, Ain Shams University, Cairo, Egypt.
3
Assistant Lecturer,
Oral Pathology Department, Faculty of Dental Surgery, Modern Science and
Arts University, Cairo, Egypt.
Authors’ contributions
MSA participated in the study design, photomicrography of the
immunohistochemical results, interpreting and displaying the results of the
study, carried out the sequence alignment and drafted the manuscr ipt. H.M.B
participated in displaying the results of the study, writing the discussion of
the results and alignment of the references. ME carried out the
immunohistochemical technique, collection of the background references
and participated in writing the discussion of the results.
Competing interests
The authors declare that they have no competing interests.
Received: 9 December 2010 Accepted: 2 March 2011
Published: 2 March 2011
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Pre-publication history
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Cite this article as: Ayoub et al.: Immunohistochemical detection of
laminin-1 and Ki-67 in radicular cysts and keratocystic odontogenic
tumors. BMC Clinical Pathology 2011 11:4.
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