BSTRACT Odontogenic keratocyst

BSTRACT
Odontogenic keratocyst (OKC), and ameloblastoma (AB) aggressive epithelial odontogenic
tumors with a high recurrence rate though, their aggressive nature are not totally understood, Many
epithelial tumors are characterized by stromal reaction, Myofibroblast is one of stromal component
that could contribute to the biologic behavior of these lesions, Fifteen cases of AB and OKC were
operated on under general anesthesia, all cases were subjected to immunohistochemical staining
with alpha-smooth muscle actin and flow cytometry analysis, ameloblastoma showed expression
of ? muscle actin between the follicles as well as around the blood vessels, while in OKC the
expression was mostly in connective tissue wall of cyst lining, as well as flow cytometry analysis
showed tumors were diploid and showed a percentage of cells in S -phase higher than 26%. OKC
had a higher value than AB but the values were close and statistically insignificant, we concluded
that the immunohistochemical expression of MF in AB & OKC has insignificant difference may
contribute to the similar proliferative potential of both AB& OKC, as well The high proliferation
rate of OKC reinforces its classification as a benign odontogenic neoplasm rather than a cyst
Objective To detect the proliferative potential of odontogenic keratocyst, versus ameloblastoma
and their correlation to presence of stromal myofibroblasts INTRODUCTION
Odontogenic keratocyst (okc), and
ameloblastoma (ab) are considered as aggressive
epithelial odontogenic tumors with a high recurrence
rate though, the pathogeneses of these tumors and
their aggressive nature are not totally understood. (1).
Ameloblastoma (Ab) is a locally destructive
benign epithelial odontogenic neoplasm. It may
originate from dental lamina rests, the epithelial
lining of an odontogenic cyst, the rests of enamel
organ or from the basal cells of the oral mucosa. It
comprises about 10% to 45.2% of all odontogenic
tumors and nearly 1% of all neoplasms of the oral cavity. Clinically, Ab arises as a painless swelling
with an expansion of the jaw bones. It could be
classified into 3 main types: solid or multicystic
ameloblastoma, uni-cystic ameloblastoma, and
peripheral ameloblastoma. The solid type is the most
common type, which has various histopathological
patterns as follicular, plexiform, basal cell, granular,
acanthomatous and desmoplastic patterns. (2)
Odontogenic keratocyst (OKC) is a benign
cystic lesion that arises from rests of dental lamina.
It is a locally destructive lesion that grows within
the medullary spaces of the bone in an anteroposterior
direction without causing a notable bony
expansion. Due to its aggressive behavior, it was
grouped among the benign epithelial odontogenic
tumor in 2005. (3)
Locally aggressive lesions that have high
recurrance rate after conventional surgical
enucleation, it is well known this is due to the
absence of capsule in AB & highly friable cyst wall
with satellite cysts within okc capsule, the highly
aggressive potential of both lesions was attributed
to many biological, molecular & genetic factors
in many studies however few studies were done
on the correlation between presence of the stromal
myofibrblast in both lesions with their invasive
potential. (4)
Myofibroblasts found in the connective tissue
stroma and characterized by the presence of a
contractile apparatus. They are found in normal
tissues such as blood vessels, lymph nodes and bone
marrow as well as in various invasive and metastatic
malignant tumors. (5).
Many epithelial tumors are characterized by
the local accumulation of connective tissue cells
and extracellular material; this phenomenon
has been called the stromal reaction. One of the
cellular components of the stroma reaction is
the myofibroblast, a modulated fibroblast that
has acquired the capacity to neoexpress alphasmooth
muscle actin (actin isoform of vascular
smooth muscle cells), and to synthesize important
amounts of collagen and other extracellular
matrix components. It is now well accepted that
the myofibroblast is a key cell for the connective
tissue remodeling, which takes place during wound
healing and fibrosis development. Myofibroblasts
are capable of remodeling connective tissue but also
interact with epithelial cells and other connective
tissue cells and may thus control such phenomena as
tumor invasion and angiogenesis. Few studies have
investigated the role of non-epithelial factors such
as myofibroblasts (MFs) that could contribute to the
biologic behavior of the lesions. So, this study was
used to investigate the presence of myofibroblasts in
OKC and ameloblastoma and to compare between
the presence of stromal myofibroblasts and the
biologic aggressiveness of the lesions. (6)
A debate regarding the management of these
pathologic entities is present in the literature
particularly with the presence of several histologic
subdivisions and subsequently the pathologic
and clinical behavior for each. Treatment options
discussed in the literature range from simple
procedures like marsupialization or enucleation
with curettage to radical resection with discontinuity
defects. Radiotherapy was also a choice. Choosing
a treatment modality is guided by factors like tumor
potential, behavior, physical form plus histologic and
growth characteristics, location in the maxillofacial
skeleton, tumor size, primary or recurrent growth.
More over the patient age and medical condition. (7)
Our aim is to correlate between the presence
of stromal myofibroblasts in relation to the
proliferative potential of odontogenic keratocyst,
and ameloblastoma
Material and Methods
This study was conducted on patients who have
been treated for AB and OKC in the department of
Oral & Maxillofacial surgery – Faculty of Dentistry/
South Valley University from Jan 2014 to June 2017.Fifteen cases were operated on under GA.
Patients were scrubbed, painted and draped
according to the standard surgical protocol. 2%
lignocaine with epinephrine was given at the surgical
site. Access for tumors of the anterior segment of
the maxilla or the mandible was done via intraoral
transmucosal approach. For tumors of the posterior
maxilla, a Weber- Ferguson flap was used along
with vestibular incision. While for those involving
the mandibular body, angle and/or the ramus a
combined approach using a standard submandibular
and intraoral transmucosal incisions. A resection
margin of uninvolved bone of 1.5-2 cm in solid
and multicystic lesions and 0.5-1 cm for unicystic
and peripherl lesions was a standard. The resection
margin always lies beyond the tumor invaded tissue
plane to assure having a tumor free margin. (Fig.1)
Immunohistochemical study of surgically treated
cases of OKC and ameloblastoma, fifteen cases each
was selected to make the study. Five-micrometer
thick sections were histologically prepared and
stained with hematoxylin and eosin stain to confirm
the diagnosis under light microscopy.
Three-micrometer thick sections were then
obtained from each case for immunohistochemical
staining with alpha-smooth muscle actin. The
slides were deparaffinized by passing them through
two changes of xylene for 5 min each. They were
hydrated in two changes of 100% ethanol for 1 min
each. The slides were then transferred to citrate
buffer and autoclaved for antigen retrieval at 15
lbs pressure for 15 min. After cooling, they were
washed in phosphate buffer solution. The slides were
then treated with protein block reagent for 10 min.
Immunohistochemical staining was then performed
using ?- smooth muscle actin as the manufacturer’s
instructions. The slides were then mounted in dpx
and observed under light microscope for the results.
Flow cytometric analysis
All steps took place in vacsera (egyptian
company for vaccines, sera, and drugs).
Sample preparation: paraffin-embedded
specimens were processed for DNA flow cytometric
analysis by a technique modified from that of Hedley
and co-workers. Sections (thickness, 40 pm) were
deparaffinized in xylene, rehydrated, and subjected
to trypsin digestion. After overnight’s incubation
the nuclear suspension was stained with ethidium
bromide (50 pg/ml), digested with RNAase, and
analyzed with an EPICS C flow cytometer (Coulter
Electronics, Hialeah, FL) using 488 nm argon laser
excitation.
The criterion for DNA aneuploidy was the
presence of two distinct GO/Gl peaks. Mean
coefficient of variation of the diploid peak was 5.5
(range, 3.2-7.0) which usually allowed detection
of DNA peaks with a DNA index greater than 1.10. Cell cycle analysis was done according to
the Baisch rectangular histogram as described
previously.’o S-phase was analyzed in such cases
where the evaluation was thought to be reliable (89
of 102 tumors). Flow cytometric data analysis was
performed by the Coulter Statpak software program
(Coulter Electronics, Hialeah, FL).
Analysis of DNA in paraffin-embedded samples
was done by isolating cell nuclei from paraffinembedded
tissues and used it to retrieve, for flow
cytometric analysis. This methodology is useful for
studying tumor progression and for assessment of
the prognostic significance of DNA ploidy and cell
cycle distribution (mainly s-phase cells) (Hedley
1989).
All the results were tabulated and statistically
analyzed using computer software named the
Statistical Package for Social Science (SPSS version
16). Data in the present study were presented as
mean and standard deviation (SD). The comparisons
between the study groups were determined by using
an independent-samples t test and paired t- test. The
level of significance was established at the value of
P; 0.05.
Results
A total number of thirty cases were taken for the
study, fifteen from each group. All cases showed
positive immunoreaction in the stroma for ?
smooth muscle actin. The ameloblastoma showed
expression of ? muscle actin between the follicles
as well as around the blood vessels (Fig. 2), while in
OKC the expression was mostly in c.t wall of cyst
lining (Fig. 3)
Image Analysis Results
Mean values of area fraction of smooth muscle
actin immunopositivity in odontogenic keratocyst,
and ameloblastoma was (4.8825 ; 4.4819)
respectively Statistical results
The stastical results showed an insignificant
correlation between AB ; OKC expression of
smooth muscle actin (P= 0.541) table (1) Flow cytometric results
All the examined tumors were diploid and
showed a percentage of cells in S -phase higher than
26%. OKC had a higher value than AB but the values
were close and statistically insignificant. (fig 5)
Discussion
The neoplastic cells lead to abnormalities within
the epithelium due to which the stroma changed to
reactive one, such neoplastic cells secrete TGF-?1
cytokine which leads to the differentiation of
fibroblasts into MFs. (8). The myofibroblasts release
cytokines and matrix metalloproteases which
destruct the ECM leading to tumor growth, invasion
and metastasis. (9)
In the present study we investigate the presence,
and area fraction of MFs using immunohistochemical
marker, which is ?-SMA in two odontogenic
lesions, which are OKC and ameloblastoma, and to
correlate the results with the aggressiveness of the
lesions by flow cytometry.
In our study the area fraction percentage of MFs
were slightly higher in OKC than AB but without
any statistically significant difference. This was in
accordance with another study by Vered et al in 2005(10) states that OKC showed the lowest number
of MF positivity among the odontogenic cysts
studied, whereas OKC showed the highest MF
positivity.
Two other studies (11, 12) said that MF positivity
was higher in OKC than in ameloblastoma, which
was also in accordance with our results.
In contrary to our results, Deepa et al 2016
(13) said that ameloblastoma showed a marginally
increased count of MF when compared to OKC, but
this difference too was not significant.
Allison and Spencer (14) were shown that the
mean silver stained nucleolar-organizing regions
(AgNORs) count was highest in ameloblastoma,
followed closely by OKC. AgNOR, was a measure
of the clinical behavior of the lesions and those with
higher scores would be expected to exhibit a more
aggressive biologic behavior.
Lombardi and Morgan (15) confirm the presence
of the MFs in the wall of odontogenic cysts and
suggested that MFs may be part of a homeostatic
response to the distension of the cyst wall caused by
the cyst enlargement. So, increased number of MFs
in the stroma of OKC may be considered as directly
proportional to its reported aggressive behavior.
The presence of MFs in the stroma of SCC
especially at the invasive margins was related to
the role of these cells in the growth and progression
of the lesion, by their ability to alter the ECM.
The increased density of stromal MFs in poorly
differentiated oral SCCs has been related to the
involvement of MFs in the creation of a permissive
microenvironment in the stroma for tumor growth,
progression and invasion. (16)
The presence of MFs in the stroma just beneath
the lining epithelium in OKC and surrounding the
tumor islands in ameloblastoma, in our study, may
be an indicator to the role of these cells in the growth
and further progression of these lesions.
Our immunohistochemical results were in
accordance with flow cytometrical results which
revealed in the present study an increase in the
percentage of cells in S phase in all the studied cases
(all were above 26%).
Within the cell cycle, cell nuclei contain
different amounts of DNA according to the stage
of the cycle. Upon receiving proliferation signals,
diploid cells exit its resting state Gap 0 phase and
enter the Gap 1 phase. At Gap 1 phase, the diploid
cells maintain their ploidy (having two complete
sets of chromosomes) (2N). Replication of DNA
starts when the cells enter the synthesis phase. In
this phase, cells contain different amounts of DNA.
Replication of DNA persists until the DNA content
is twice that of the diploid state and be in a tetraploid
state (4N). These tetraploid cells in the Gap2 phase
prepare for the division and enter the mitosis phase
in which the cells divide into two identical diploid
(2N) cells. The daughter cells may enter another
division cycle or enter the resting stage. Based only
on DNA content, the M phase is identical to the G2
phase, and G0 is identical to G1.
So, the cell cycle is commonly described by the
G0/G1, S, and G2/M phases when based on DNA
content (17).
During apoptosis, extensive fragmentation DNA
occurs. The apoptotic cells are defined as “sub-G1”
cells. (18, 19)
The present study revealed an increase in the
percentage of cells in S phase in all the studied cases
(all were above 26%). The study of Ostovic et al,
2017(20) revealed an increase in the percentage of
cells of AB in the S-phase (25.9%) they consider
this percentage as an indication of an unregulated,
high proliferation.
Treatment of OKC and AB is a major challenge
for surgeons. Many surgical techniques have been
proposed aiming for complete removal and minimize
recurrent or residual disease. Unfortunately, there is no literature consensus on a standard management
protocol as each of these protocols have their
proponents and support to varying degrees in the
literature. Surgical techniques employed include:
enucleation and curettage, enucleation and chemical
fixation, enucleation and cryotherapy, enucleation
and peripheral osteotomy, marsupialization and
resection. Frequent results reported in the literature
after the usage of each technique or after comparing
techniques together guided its use.
Enucleation and curettage is considered the
most conservative treatment option for OKC
and AB. However, recurrence rates as high as
62.5% and 100% have been reported for OKCs
and AB correspondingly. Consequently it is not
recommended by most authors. (21, 22)
We used resection technique for managing
all cases enrolled in this study. Resection is the
only predictably curative procedure for patients
with OKC and AB that offers a recurrence rate
approaching zero.
We applied a resection margin of uninvolved
bone of 1.5-2 cm in solid and multicystic lesions
and 0.5-1 cm for unicystic and peripherl lesions to
assure having a tumor free margins. This was proved
by our histopathologic results. Tumor resection may
be completed in the form of composite, segmental,
or marginal. The drawn results of different research
work regarding high recurrence after enucleation
and curettage and better results after resection in
OKC and AB confirm the findings of this research
as the proliferating growth pattern of keratotic
odontogenic tumor and ameloblastoma could be
considered as the pathological base of their local
aggressive behavior.
Conclusion
The immunohistochemical expression of MF
in AB ;OKC has insignificant difference may
contribute to the similar proliferative potential of
both AB; OKC.
The high proliferation rate of OKC reinforces
its classification as a benign odontogenic neoplasm
rather than a cyst.

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