Evaluation of Midkine Expression in Dentigerous Cysts, Odontogenic Keratocysts and Different Types of Ameloblastoma
Naghmeh JEIROODI1, Mahshid BAGHERPOUR1, Razieh ZARE1, Shima TORABĘ ARDAKANI1, Azadeh ANDISHEH TADBIR2
1Department of Oral and Maxillofacial Pathology, School of Dentistry, Shiraz University of Medical Sciences, SHIRAZ, IRAN
2Department of Oral and Dental Disease, School of Dentistry, Shiraz University of Medical Sciences, SHIRAZ, IRAN
Keywords: Dentigerous Cysts, Odontogenic Keratocyst, Ameloblastoma, Unicystic, Multicystic
Midkine is a heparin-binding growth factor whose expression is increased in most tumors, namely ameloblastomas. This study aimed
to compare Midkine expression in different odontogenic lesions.
Material and Method: This analytical cross-sectional study was performed on 52 definitely diagnosed odontogenic lesions including 15
dentigerous cysts, 13 odontogenic keratocysts, and 17 unicystic and 5 multicystic ameloblastomas archived from 1997 to 2015. Midkine
expression was examined in tissue samples through immunohistochemistry. The nonparametric Kruskal-Wallis and Mann-Whitney tests were
run as appropriate (P < 0.05).
Results: The frequency of Midkine expression was < 20% in 7.7%, 20-50% in 25%, and > 50 % in 67.3% of the samples, indicating significant
differences among the groups (P = 0.002). Moreover, the expression intensity was strong in 63.5%, moderate in 23.1%, and weak in 13.5% of
odontogenic lesion samples (P = 0.071). The total staining score was weak in 3.8%, moderate in 48.1%, and strong in 48.1% of the cells, displaying
significant differences between the study groups in this regard (P = 0.043).
Conclusion: Midkine can be considered as both a differentiating factor and a molecular-targeted therapy in odontogenic lesions. Yet, further
studies are required to approve the role of this cytokine in different biological and pathological stages of the tumors.
Odontogenic cysts are important pathologic lesions of
the oral cavity. Among the most prevalent developmental
odontogenic jaw cysts are dentigerous cysts (follicular).
These occur as a result of fluid accumulation between the
tooth crown and reduced enamel epithelium. It is treated
through enucleation and extraction of the involved tooth.
The prognosis of a dentigerous cyst is excellent and rarely
does it recur.
An odontogenic keratocyst is a relatively common
developmental odontogenic jaw cyst originating from the
remnants of dental lamina. Due to the genetic features,
tendency to recur, and high aggressiveness according
to WHO classification, the parakeratinized variant
of odontogenic keratocysts is classified as a benign
odontogenic tumor (1-3).
Ameloblastoma is a generally benign odontogenic tumor of
the jaw bone. Yet, it is quite likely to relapse after extensive
surgeries. Ameloblastoma is more prevalent in the mandible than the maxilla; and rarely does it occur in an
extraosseous location. Malignant ameloblastoma is quite
uncommon. The etiology is still unknown. Its occurrence
rate is estimated to be 0.5 in one million in a year; however,
this number is estimated to be much higher in some
regions such as South Africa (4-6). Most patients are 30-60
years old when diagnosed. There is no difference between
the sexes concerning the distribution of the disease. Bony
septa may result in a honeycomb appearance (7). Clinically
and radiographically, ameloblastomas are divided into the
three clinical groups of unicystic, peripheral, and solid/
multicystic, the last of which is the most frequent (8).
The multifunctional cytokine, Midkine, is a heparinbinding
growth factor with low molecular weight (13
kilodalton). It contributes to formation of different organs
through mesenchymal-epithelial interactions (9-12).
Although the role and biological effective mechanisms
of Midkine are not crystal clear, its angiogenic role in
tumorigenesis has been well proven (12).
Various investigations on Midkine expression in tumors
and cancers have reported up-regulation of the gene of this
cytokine in different tumors, namely Wilms tumor (13),
breast cancer (14), ovarian cancer (15), gastrointestinal
cancer (16), and bladder cancer (17). The findings indicate
that histochemical evaluation of Midkine can be used
to assess the state of cancers. This cytokine is therefore
becoming one of the markers of cancer (18). So far, no
research has evaluated the expression level of this marker
in different odontogenic lesions with various biological
behavior. Hence, the current study was designed to compare
the expression of Midkine in different odontogenic lesions.
This retrospective cross-sectional study reviewed 52
paraffin-embedded tissue blocks of oral odontogenic
lesions collected from the archive of the Department of
Pathology, Shiraz School of Dentistry, in the time period
from 1997 to 2015. The samples included 15 dentigerous
cysts, 13 odontogenic keratocysts, and 17 unicystic and 5
Having checked the hematoxylin-eosin slides, those
samples with a definite diagnosis were selected. The severely
inflamed cases of odontogenic keratocysts and dentigerous
cysts were excluded from the study.
Immunohistochemical staining was carried out by using
the Envision Labeled Peroxides System (Dako; Carpentaria,
CA, USA). All samples were fixed in 10% buffered formalin.
They were then hydrated in graded alcohol and rinsed with
distilled water. Antigen retrieval was performed by using
the Dakocytomation target retrieval solution (pH=9) for 9
The endogenous peroxidase activity was inhibited by 3%
H2O2. Tissue sections were incubated with anti-Midkine
antibody (Abcam, ab66596) at 1/100 for 30 minutes.
Omission of the primary antibody and hepatocellular
carcinoma were respectively employed as negative and
An oral and maxillofacial pathologist examined the slides
randomly in the epithelium by using an optical microscope
at 100 and 400X. The cells were counted in a fixed single
blind fashion in 5 microscopic fields. The antibody reactive
cells were then counted in each field. Finally, the mean
percentage of stained cells as well as the mean staining
intensity was calculated.
The qualitative scores of immunohistochemical staining
were quantitatively analyzed by using the SID score
(staining-intensity-distribution); i.e., Distribution +
Staining Intensity of the stained cells. The staining of cells in all samples included the cell cytoplasm and membrane.
Only those cells with obvious staining were considered as
To determine the frequency of stained cells, the lack of
staining was scored as 0, staining < 20% scored as 1+, 25-
50% as 2+, and > 50% was scored as 3+. To determine
the staining intensity, lack of staining was scored as 0,
weak staining as 1+, moderate as 2+, and strong staining
was scored as 3+. To calculate the total lesion score, the
summation of staining and intensity < 3 was considered to
be weak, 3-5 was moderate, and = 6 was strong.
The distribution of data was evaluated through descriptive
statistics. Having assessed the abnormality of data, the
Kruskal-Wallis nonparametric test was used to compare
the staining ability, staining intensity, and total scores. If
needed, Mann-Whitney multiple comparison tests were
run to compare the marker expression among the four
groups. The obtained data was analyzed by using the SPSS
software, version 18 (P < 0.05).
A total of 52 cases of odontogenic lesions were taken from
the archive of the Department of Pathology of Shiraz
School of Dentistry. The suspicious and deficient cases
were excluded. The frequency and distribution of cases
with definite diagnosis was in four groups of dentigerous
cyst, odontogenic keratocyst, unicystic and multicystic
ameloblastoma. The patients demographic data is
displayed in Table I
Age was described using mean ± SD and sex was described
using frequency (%). There were no significant differences
for age and sex between the groups (Table II).
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|Table II: Comparison of age and sex variables between different tumor types.
The results of this study revealed that the Midkine factor is
highly expressed in odontogenic lesions. The frequency of
staining can be seen in Table III. Significant differences were
observed in the staining percentage (Midkine expression)
among the study groups (p=0.002). The Mann-Whitney
test showed higher expression of Midkine in multicystic
ameloblastoma than dentigerous cyst (p=0.009). Also the
Mann-Whitney test showed higher expression of Midkine
in OKC in comparison with DC (p=0.01). Midkine
expression in multicystic lesions was higher than unicystic
lesions (p=0.033). Our data showed higher expression of
OKC compared with unicystic lesions (p=0.005).
The pathologic evaluations revealed that the multicystic
ameloblastoma included follicular and plexiform samples.
All samples in this groups showed positive staining.
Immunoreactivity was mostly observed in ameloblast-like
and stellate-reticulum like cells.
The staining intensity of Midkine factor is shown in Table
III. However, Based on Mann-Whitney test, the difference
was not significant among the groups (P=0.071)
The staining score can be seen in Table III. The Kruskal-
Wallis test revealed significant differences between the staining scores of the four types of odontogenic
lesions (P=0.043). Comparision between multicystic
ameloblastoma and dentigerous cyst showed higher
expression of Midkine in multicystic ameloblastoma
(p=0.010). Also higher Midkine expression was seen in
OKC than in dentigerous cyst (p=0.048).
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|Table III: Comparison of staining percentage, staining density and total scores of the studied odontogenic cysts
The unicystic form of ameloblastoma contained mural,
luminal, and intraluminal parts. The staining was positive
in the epithelial lining. The staining was detected to be
higher than 50% in all 13 cases of odontogenic keratocysts.
The cells showed staining in the cytoplasm of the basal
and parabasal cells. In dentigerous cysts, 6 out of 15 cases
showed >50% staining (Figure 1A-D).
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|Figure 1: Cytoplasmic Midkine Staining in Odontogenic Lesions Tissue Samples. A) Ameloblastoma (IHC; x100). B) Odontogenic
keratocyst (IHC; x200). C) Dentigerous cyst (IHC; x100). D) Unicystic ameloblastoma (IHC; x200)
Midkine is a heparin-binding cytokine that enhances
growth, survival, migration and other activities of the target
cells. The present research reviewed the expression level of
the Midkine gene in oral odontogenic lesions including
dentigerous cyst, odontogenic keratocyst, unicystic and
multicystic ameloblastomas. The obtained results revealed
that Midkine factor is highly expressed in odontogenic
Few studies have investigated the expression of the Midkine
cytokine in odontogenic lesions such as ameloblastoma.
Gramage et al. observed the regulation of expression of the
Midkine gene and protein through epithelial-mesenchymal
interaction in the tooth germ and suggested that Midkine
plays an imperative role in the molecular cascade that
controls the growth and development of the tooth (19).
Scheper et al. studied the Midkine expression in 34 patients
with ameloblastoma and 4 cases of ameloblastic carcinoma.
They found that the mentioned marker was expressed in
67% of cases, out of which 23.5% was weak, 14.7% was
moderate, and 29.4% was strong. A statistically significant
difference was observed between unicystic and multicystic
cases (18).The present study was different in assessing all
types of odontogenic lesions, and achieving total score of
strong staining in 48.1% of samples.
In the current study, expression >50% was observed
in 52.9% of unicystic ameloblastomas, 100% of both
multicystic ameloblastomas and odontogenic keratocyst
tumors, and 40% of dentigerous cysts. Overall, 67.3% of
odontogenic cysts showed above 50% Midkine expression.
The difference was significant among the study groups.
Sandra et al. investigated the expression and performance
of Midkine in 37 cases of tissue ameloblastoma, and
ameloblastoma cell lines (AM-1), and altered HPV-16DNA
cell lines. They found through the immunohistochemical
method that Midkine is expressed in the outer layers of
ameloblastoma-like and AM-1 cells in 70% of cases (20).
Likewise, Fujita et al. examined Midkine expression in
different odontogenic lesions including 50 ameloblastoma
samples. Midkine was located in the columnar cell
cytoplasm in 54.5% of cases (30 out of 55) (21). Our
results are therefore consistent with the findings of other
studies concerning the changes in Midkine expression in
An important unappreciated hint in recent studies is the
relationship between the Midkine cytokine expression and
the pathological and clinical parameters. We investigated
in the present study whether this protein contributes
to different pathologic behavior. It was revealed that
Midkine expression was stronger in multi-cystic lesions
and odontogenic keratocysts than in dentigerous cysts. The
finding suggests that this protein is expressed at a higher
rate in the presence of invasive behavior.
There are studies on the exact route of signal transmission
such as Notch that might play a role in creation of different
phenotypes of odontogenic lesions. However, among the
limitations of the current study was the inaccessibility of
further data; hence, we could not elaborate on the relation
between Midkine expression and the severity of tumor
Fujita et al. reported that Midkine was expressed in
keratinized cells of acanthomatous ameloblastoma and
keratocyst odontogenic tumors. In mixed odontogenic
tumors, except for odontoma, the immunoreactions
to Midkine were noted in the epithelial follicles
surrounding the ectomesenchymal odontogenic tissue
(21). Accordingly, Midkine can contribute to the growth
and activities of odontogenic tumors, as well as to cell
differentiation through molecular mechanisms similar to
The differentiation value of Midkine in odontogenic
lesions is among the disputable issues. In the present study, Midkine was expressed in both the cytoplasm and
membrane. No difference was mentioned between the two
different Midkine expression patterns (cytoplasmic and
membranous). Hence, further studies are required to clarify
the significance of various Midkine expression patterns.
The presence of similar inflammatory and epithelial cells
might interfere with the differential diagnosis of these
lesions. However, pairwise comparison of the staining
percentage of Midkine cytokine in these lesions revealed
significant differences between unicystic and multicystic
ameloblastoma (P=0.033) and odontogenic keratocyst
Significant differences were also observed in the expression
level of Midkine between odontogenic keratocyst and
dentigerous cyst (P=0.001), as well as multi-cystic
ameloblastoma and dentigerous cyst (P=0.009). Such
significant differences can imply that Midkine can be used
as a differential factor in these lesions.
Other studies reported that this cytokine was expressed in
diseased tissues by inflammatory cells like macrophages,
lymphocytes, and endothelial cells whereas such an
expression did not occur in normal tissues (22). Increase
and changes in Midkine expression were also reported in
other diseases and cancers, namely esophageal squamous
cancer (14,17,18,23), squamous cell carcinoma (24), and
meningioma (25). This proves its role as a candidate gene
in targeted therapy.
The results of studies suggest that immunohistochemical
evaluation of Midkine can be used as a way to examine the
cancer status; this cytokine is therefore becoming a marker
of cancer. Accordingly, Midkine can be used as a potential
target therapy in odontogenic lesions where therapeutic
measures can be taken according to its increase or decrease.
Scheper et al. found that Midkine expression increased
in human ameloblastoma (18). Meanwhile, Hatori et al.
reported that the expression of this cytokine increased
in inflammatory dental granuloma (22). They also added
that propagation of this cytokine was required to prevent
apoptosis. Hence, reduced expression of this cytokine by
genotoxic drugs increased the number of cancerous cells
that were entering the apoptosis stage. Seemingly, the
synergism effect of some medications can help treat the
cancer through targeting the DNA and Midkine gene in
Previous studies have revealed that genetic changes in the
path of the AKT protein resulted in hyperactivity of this
protein, as already reported in a number of cancers. In the laboratory environment, Midkine can phosphorylase and
activate the AKT kinase protein and increase the cell growth
through the SRC and extracellular path. Accordingly,
the cellular growth and proliferation continues and
apoptosis is reduced. As declared in a previous study,
stimulation of Midkine in cultured meningioma cells led to
phosphorylation of AKT.
Midkine can also protect the meningioma cells against
apoptotic death by decreasing active caspase-3. Therefore,
targeting the Midkine in the signal transmission route
and blocking it can be a useful therapeutic approach in
odontogenic lesions, particularly those with significant
In conclusion, the results of this study showed that
Midkine is expressed in several odontogenic lesions;
particularly in more invasive ones, this protein can indicate
the progress and survival of tumor behavior. Midkine can
be used as a differential method between odontogenic
lesions when hematoxylin-eosin staining does not work
for differentiating the lesions. Moreover, Midkine can be
a targeted-molecular therapy for odontogenic lesions. Yet,
further studies are required to approve the role of this gene
in different biological and pathological stages of tumor.
The authors thank the Vice-Chancellery of Shiraz
University of Medical Science for supporting this research
(Grant# 8895166). This manuscript is based on the thesis of
Mahshid Bagherpour for partial fulfillment of DDS degree.
The authors are grateful to Dr. M. Vossoughi from the
Dental Research Development Center of the Dental School
for the statistical analysis and Ms. Farzaneh Rasouli for her
help with the English editing of the manuscript.
Conflict of Interest
The authors declare no conflict of interest.
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