Key words:
Keratocystic odontogenic tumor - matrix metalloproteinase-2 - odontogenic keratocysts
- receptor-activated nuclear factor kappa B - receptor-activated nuclear factor kappa
B ligand - solid/multicystic ameloblastoma
Introduction
Keratocystic odontogenic tumors (KOTs) and ameloblastoma (AB) are characterized by
slow growth with locally invasive behavior and high risk of recurrence[1] Both show infiltration into cancellous bone and destructive growth. That is why
in 2005, odontogenic keratocyst is renamed as “KOTs” by the World Health Organization[2] The receptor-activated nuclear kappa B (RANK) is a central activator of necrosis
factor-kappa B, which controls the transcription of DNA, and is the signaling receptor
for RANK ligand (RANKL). RANKL binds to RANK on the surface of preosteoclasts and
stimulates the development and activation of osteoclasts.[3] Many authors suggested that odontogenic epithelial cells are an important source
of RANK.[4],[5] Sandra et al.
[6] suggested that AB could induce osteoclastogenesis by secreting RANKL and tumor necrosis
factor α (TNF-α). da Silva et al.
[5] in their study on AB and odontogenic keratocyst (OKC) found that >90% of odontogenic
epithelium of both lesions expressed RANK. They suggested that RANK originating from
odontogenic epithelium enhances bone resorption due to its effect on tumor growth,
cell proliferation, or delaying apoptosis. MMP2 is a gelatinase of the matrix metalloproteinase
(MMP) family. It degrades many types of collagens such as native type IV, V, and X
collagen and denatured fibrillar type I, II, and III collagens.[7] It has been found that many members of MMP family take part in cancer invasion and
metastasis.[8] Interest in AB and KOTs has risen due to their aggressive clinical behavior, variety
of morphological patterns, and high recurrence rates. Since these tumors generally
exhibit localized bone destruction and tooth resorption, the differential expressions
of MMP2, RANK, and RANKL may be expected to find in these lesions. The aim of the
present study is to examine and evaluate the expression of MMP2, RANK, and RANKL in
both lesions, in an attempt for better understanding of biological behavior associated
with these odontogenic tumors from the points of invasiveness and osteoclastogenesis.
Materials and Methods
Tissue samples
The study was approved by the Research and Ethics Committee of College of Dentistry
at University of Baghdad (REC. 25-2014). The paraffin blocks were retrieved from the
archives of the Oral Diagnosis Department, College of Dentistry, University of Baghdad.
The diagnosis of each case was confirmed by two oral pathologists through the examination
of hematoxylin and eosin-stained sections. Data, regarding name, age, sex, and site
of the lesion, were collected from the routine histopathology files of the same cases.
Recurrent AB and KOTs were excluded from the study.
The expression of MMP2, RANK, and RANKL was evaluated in the tissue specimens of 14
KOT and 13 solid/multicystic AB (SMA). The SMA was subdivided into eight follicular
and five plexiform patterns, while KOTs were categorized into those associated with
daughter epithelial nests (KOT with + dentigerous cyst [DC]) group (n = 5) and those without (KOT with – DC) group (n = 9). All tests were carried out on 4 μm sections from formalin-fixed paraffin-embedded
blocks of SMA and KOT. The expressions were calculated in the odontogenic epithelial
cells as well as the stromal cells.
Immunohistochemical staining
The paraffin blocks were cut serially into approximately 5 μm thick sections on charged
slides. Using Dako Autostainer, the slides were deparaffinized with xylene for 30
min, washed with absolute 100% alcohol for 15 min, and then washed with 95% alcohol
and distilled water. Dako REALTM EnVisionTM, peroxidase/diaminobenzidine (DAB), and Mouse IG detection system were used for MMP2,
RANK, and RANKL. For antigen retrieval, 20-min heat-induced antigen retrieving was
performed with RANK and RANKL, while in MMP2, enzyme block solution was used only
(chemical retrieving). The retrieving solution used for RANK and RANKL was citrate
buffered (pH = 9 under 80°C for RANKL and pH = 6 under 85°C for RANK) and then washed
in phosphate buffered saline for 5 min. To block endogenous peroxidase activity, slides
were incubated with 3% hydrogen peroxide, washed in distilled water and then soaked
in phosphate buffer saline for 5 min.
MMP2 antibody at a dilution of 1:50 (Abcam No: Ab3158, Monoclonal Mouse Anti-Human
MMP2 Antigen, CA-4001/7CA19E3C, Abcam, USA), RANK antibody at a dilution of 1:50 (Abcam
No: Ab12008, Monoclonal Mouse Anti-Human MMP2 Antigen, 9A725, Abcam, USA), and RANKL
at a dilution of 1:75 (Abcam No: Ab 45039, Monoclonal Mouse Anti-Human RANKL Antigen,
12A668, Abcam, USA) were used as primary antibodies. Slides were incubated overnight
at 4°C with MMP2, RANK, and RANKL. Positive control slides were obtained according
to the antibodies manufacturer’s data sheet. For MMP2 monoclonal antibodies, tissue
blocks of breast carcinoma were used.[7] For RANK, the bone tissue located at the periphery of the lesions of SMA and KOT
was used, while that for RANKL, tissue blocks of peripheral giant cell granuloma were
used.[5] The negative control slides were prepared by substituting the primary specific antibodies
with nonimmune serum. All the above control samples were fixed and processed in a
way similar to that of test samples. The secondary antibody Dako REALTM EnVisionTM/HRP, Rabbit/Mouse IgG was reacted for 30 min, followed by DAB-chromogen solution
for 30 min being used to visualize the reaction. Finally, the sections were 0counterstained
with Mayer’s hematoxylin, dehydrated, coverslipped, and evaluated by light microscopy.
Signal specificity was demonstrated by the absence of immunostaining in the negative
control slides and its presence in the recommended positive controls. Odontogenic
epithelia and stromal cells in both SMA and KOT with clear brown membranous/ cytoplasmic
staining were considered to be positive for MMP2, RANK, and RANKL immunostaining within
a violet-blue tissue section background of hematoxylin staining. All the slides were
assessed blindly by two pathologists without prior knowledge of the corresponding
clinicopathological data, and the average of the two readings was obtained.
The specimens were examined and counted at x200 magnification using integration graticule
with an Olympus C x30 microscope. In the epithelia, positive cells were counted in
five contiguous and consecutive microscopic high-power fields. In the stroma, positive
endothelial and fusiform cells (fibroblasts) were quantified in five contiguous and
consecutive areas adjacent to neoplastic odontogenic epithelium;[5],[9] each field of the integration graticule had an area of 0.0061 mm2.[9] For accuracy, the counting rechecked at x 400 for each field. Results were obtained
and the mean of the percentage of positive cells was determined for each representative
field.
Statistical analyses were done using SPSS version 21 (Chicago., IL, USA). The statistical
significance of differences in RANK, RANKL, and MMP 2 reactivity was analyzed in both
lesions by the Mann-Whitney U-test and P < 0.05 was considered to indicate statistical significance.
Results
The demographic data of the studied sample are shown in [Table 1]. The mean age of patients from which the AB specimens were obtained ranged between
10 and 45 years (mean = 30.6 years). For KOT cases, the age range was between 15 and
45 years (mean = 31.9 years). All cases of AB occurred in the mandible, and for KOT,
the mandible was more frequently involved than maxilla (64.3% and 35.7%, respectively)
[Table 1].
Table 1:
Clinical parameters (age, sex, and site) for ameloblastoma and keratocystic odontogenic
tumors cases
|
n
|
sex
|
Age
|
Site
|
|
|
Male (%)
|
Female (%)
|
Range
|
Mean±SD
|
SE
|
Mandible (%)
|
Maxilla (%)
|
|
AB: Ameloblastoma, KOT: Keratocystic odontogenic tumor, SD: Standard deviation, SE:
Standard error
|
|
AB
|
13
|
7(53.8)
|
6(46.2)
|
10-45
|
30.6±9.16
|
2.64
|
13(100)
|
0
|
|
KOT
|
14
|
6(42.9)
|
8(57.1)
|
15-45
|
31.9±8.94
|
2.38
|
9(64.2)
|
5(35.7)
|
MMP2, RANK, and RANKL immunostaining was detected in the odontogenic epithelial cells
as well as in the stromal cells.
Matrix metalloproteinase-2 immunostaining
Strong expression of MMP2 was seen in ABs and KOTs, with higher value in SMA tumor
cells than keratocyst lining epithelia (73.29% and 63.87%, respectively). However,
the difference was not statistically significant (P = 0.09) [Table 2]. MMP2 expression was higher in epithelial cells of plexiform AB (79.9%), followed
by KOT associated with daughter epithelia nests (76.37%) and then follicular AB (68.57%),
and the least was in KOT not associated with daughter epithelial nests (56.06%) as
shown in [Figure 1] and [Table 3]. MMP2 expression in stromal cells was almost the same in SMA and KOT (48.66% and
50%, respectively), with no significant differences between the histological subgroups
[Table 2].
Figure 1: (a-d) Matrix metalloproteinase-2 expression in odontogenic epithelium, follicular
ameloblastoma, plexiform ameloblastoma, keratocystic odontogenic tumor (−dentigerous
cysts), keratocystic odontogenic tumor (+dentigerous cysts)
Table 2:
The comparison between matrix metalloproteinase-2 expression of the odontogenic epithelia
and stromal cells of solid/multisystem ameloblastoma and keratocystic odontogenic
tumor
|
Tissue group
|
Cells
|
Total number
|
Positive MMP2
|
Negative MMP2
|
Mean±SD
|
SE
|
P
|
|
MMP2: Matrix metalloproteinase-2, SMA: Solid/multicystic ameloblastoma, KOT: Keratocystic
odontogenic tumor, SD: Standard deviation, SE: Standard error
|
|
SMA
|
Odontogenic epithelium
|
13
|
12
|
1
|
73.29±10.92
|
3.16
|
0.09
|
|
KOT 14
|
|
14
|
13
|
1
|
63.87±16.36
|
4.53
|
|
|
SMA
|
Stromal cells
|
13
|
12
|
1
|
48.66±12.03
|
3.47
|
0.7
|
|
KOT
|
|
14
|
13
|
1
|
50.00±14.69
|
4.07
|
|
Table 3:
Comparison between matrix metalloproteinase-2 area percentage in the odontogenic epithelia
and stromal cells of follicular ameloblastoma, plexiform ameloblastoma, keratocystic
odontogenic tumor (negative dentigerous cysts), and keratocystic odontogenic tumor
(positive dentigerous cysts)
|
Tissue group
|
Cells
|
Total number
|
Positive MMP2
|
Negative MMP2
|
Mean±SD
|
SE
|
P
|
|
✶Significant at P≤0.05. AB: Ameloblastoma, KOT: Keratocystic odontogenic tumor, SD: Standard deviation,
SE: Standard error, MMP2: Matrix metalloproteinase-2, DC: Dentigerous cysts
|
|
Follicular AB
|
Odontogenic epithelia
|
8
|
7
|
1
|
68.57±8.52
|
3.22
|
0.06
|
|
Plexiform AB
|
|
5
|
5
|
0
|
79.9±11.29
|
5.05
|
|
|
KOT (negative DC)
|
|
9
|
8
|
1
|
56.06±9.56
|
3.38
|
0.01*
|
|
KOT (positive DC)
|
|
5
|
5
|
0
|
76.37±18.65
|
8.07
|
|
|
Follicular AB
|
Stromal cells
|
8
|
7
|
1
|
52.57±4.98
|
1.88
|
0.16
|
|
Plexiform AB
|
|
5
|
5
|
0
|
43.2±17.23
|
7.70
|
|
|
KOT (negative DC)
|
|
9
|
8
|
1
|
46.87±13.15
|
4.65
|
0.3
|
|
KOT (positive DC)
|
|
5
|
5
|
0
|
55.0±17.16
|
7.67
|
|
Receptor-activated nuclear factor kappa B immunohistochemical staining
Odontogenic epithelial cells of AB strongly expressed RANK, which is significantly
higher than that of KOT (67.55 ± 16.83 versus 51.8 ° 17.15) as seen in [Table 4] and [Figure 2], whereas RANK expression in stromal cells did not show any significant differences
between lesions.
Table 4:
Comparison between receptor-activated nuclear factor kappa B area percentage of the
odontogenic epithelia of solid/multisystem ameloblastoma and keratocystic odontogenic
tumor
|
Tissue group
|
Total number
|
Cells
|
Positive RANK
|
Negative RANK
|
Mean±SD
|
SE
|
P
|
|
✶Significant at P≤0.05. RANK: Receptor-activated nuclear factor kappa B, SMA: Solid/multicystic ameloblastoma,
KOT: Keratocystic odontogenic tumor, SD: Standard deviation, SE: Standard error
|
|
SMA
|
13
|
Odontogenic epithelia
|
12
|
2
|
67.55±16.83
|
5.07
|
0.02*
|
|
KOT
|
14
|
|
13
|
1
|
51.8±17.15
|
4.75
|
|
|
SMA
|
13
|
Stromal cells
|
10
|
3
|
45.22±18.74
|
5.92
|
0.59
|
|
KOT
|
14
|
|
13
|
1
|
41.34±18.49
|
5.13
|
|
Figure 2: (a-d) Receptor-activated nuclear factor kappa B expression in odontogenic epithelium,
follicular ameloblastoma, plexiform ameloblastoma, keratocystic odontogenic tumor
(−dentigerous cysts), keratocystic odontogenic tumor (+dentigerous cysts)
Plexiform AB highly expressed RANKL in their odontogenic epithelium in 81.08%, followed
by follicular type (62%), KOT associated with daughter epithelia nest (58.7%), and
then KOT with no daughter epithelia nest group (47.5%). A statistically significant
difference was seen only when plexiform pattern compared with both subtypes of KOT
(P = 0.03 and P = 0.009, respectively) [Table 5]. In the stroma, the expression was almost similar with a bit elevation of SMA (45.22%
and 41.34%) [Table 4].
Table 5:
The comparison between receptor-activated nuclear factor kappa B area percentage of
the odontogenic epithelia and stromal cells of follicular ameloblastoma, plexiform
ameloblastoma, keratocystic odontogenic tumor (positive dentigerous cysts), and keratocystic
odontogenic tumor (negative dentigerous cysts)
|
Tissue group
|
Total number
|
Cells
|
Positive RANK
|
Negative RANK
|
Mean±SD
|
SE
|
P
|
|
✶Significant at P≤0.05. AB: Ameloblastoma, KOT: Keratocystic odontogenic tumor, SD: Standard deviation,
SE: Standard error, RANK: Receptor.activated nuclear factor kappa B, DC: Dentigerous
cysts
|
|
Plexiform AB
|
5
|
Odontogenic epithelia
|
3
|
2
|
81.08±3.74
|
2.16
|
0.03*
|
|
Follicular AB
|
8
|
|
8
|
0
|
62.48±17.11
|
6.05
|
|
|
KOT (negative DC)
|
9
|
|
8
|
1
|
47.5±16.87
|
5.96
|
0.25
|
|
KOT (positive DC)
|
5
|
|
5
|
0
|
58.7±16.97
|
7.59
|
|
|
Plexiform AB
|
5
|
Stromal cells
|
3
|
2
|
38.33±2.88
|
1.66
|
0.009**
|
|
Follicular AB
|
8
|
|
8
|
0
|
48.17±2.14
|
8.36
|
|
|
KOT(negative DC)
|
9
|
|
8
|
1
|
48.2±14.53
|
6.49
|
0.25
|
|
KOT (positive DC)
|
5
|
|
5
|
0
|
37.06±20.28
|
7.17
|
|
Receptor-activated nuclear factor kappa B ligand immunostaining
In this study, RANKL was detected in the membrane and cytoplasm and in the extracellular
of AB and KOT cells as seen in [Figure 3].
Figure 3: (a-d) Receptor-activated nuclear factor kappa B ligand expression in odontogenic
epithelium, follicular ameloblastoma, plexiform ameloblastoma, keratocystic odontogenic
tumor (-dentigerous cysts), keratocystic odontogenic tumor (+dentigerous cysts)
Epithelial cells of AB expressed more RANKL than that of KOT without reaching statistical
significance (P = 0.06). However, stromal cells expressed RANKL more in AB than KOT with almost significant
differences [Table 6].
Table 6:
Comparison between receptor activator of nuclear factor kappa-B ligand area percentage
in the odontogenic epithelia of solid/multicystic ameloblastoma and keratocystic odontogenic
tumor
|
Tissue group
|
Total number
|
Positive RANKL
|
Negative RANKL
|
Mean±SD
|
SE
|
P
|
|
OKC: Odontogenic keratocyst, SMA: Solid/multicystic ameloblastoma, RANKL: Receptor
activator of nuclear factor kappa.B ligand, SD: Standard deviation, SE: Standard error
|
|
SMA
|
13
|
12
|
1
|
57.93±20.6
|
5.94
|
0.06
|
|
OKC
|
14
|
13
|
1
|
43.78±17.43
|
4.83
|
|
|
SMA
|
13
|
11
|
2
|
34.68±20.42
|
6.15
|
0.09
|
|
OKC
|
14
|
12
|
2
|
23.96±10.03
|
2.89
|
|
Regarding histological subtypes, epithelia of plexiform type expressed more RANKL
(65.1%) than follicular type (53.53%). Epithelial and stromal cells of KOT, on the
other hand, showed almost same expression in both subtypes without observable statistical
significant value [Table 7].
Table 7:
The comparison between receptor activator of nuclear factor kappa-B ligand area percentage
in the odontogenic epithelia and stromal cells of follicular ameloblastoma, plexiform
ameloblastoma, keratocystic odontogenic tumor (positive dentigerous cysts), and keratocystic
odontogenic tumor (negative dentigerous cysts)
|
Tissue group
|
Total number
|
Cells
|
Positive RANKL
|
Negative RANKL
|
Mean±SD
|
SE
|
P
|
|
AB: Ameloblastoma, KOT: Keratocystic odontogenic tumor, SD: Standard deviation, SE:
Standard error, RANKL: Receptor activator of nuclear factor kappa.B ligand, DC: Dentigerous
cysts
|
|
Plexiform AB
|
5
|
Odontogenic epithelia
|
5
|
-
|
65.1±23.75
|
10.62
|
0.34
|
|
Follicular AB
|
8
|
|
7
|
1
|
53.53±18.65
|
7.05
|
|
|
KOT (with negative DC)
|
9
|
|
8
|
1
|
43.16±19.57
|
6.92
|
0.87
|
|
KOT (with positive DC)
|
5
|
|
5
|
0
|
44.78±15.47
|
6.92
|
|
|
Plexiform AB
|
5
|
Stromal cells
|
5
|
0
|
38.44±25.72
|
11.5
|
0.56
|
|
Follicular AB
|
8
|
|
6
|
2
|
31.55±16.70
|
6.82
|
|
|
KOT negative DC)
|
9
|
|
7
|
2
|
26.9±11.0
|
4.15
|
0.23
|
|
KOT (with positive DC)
|
5
|
|
5
|
2
|
19.86±7.68
|
3.43
|
|
Discussion
Assessment of matrix metalloproteinase-2 expression in ameloblastoma and keratocystic
odontogenic tumor
MMP2 plays a major role in extracellular matrix remodeling because of its ability
to initiate and continue degradation of fibrillar collagen, especially type IV collagen
which is the major component of basement membrane.[10] Although numerous researches have verified that MMP2 plays an important role in
tumor invasion,[11] yet there are limited researches about its behavior in AB and KOT. The present study
as well as the others showed that the high expression and activity of MMP2 is related
to the aggressive behavior in both AB [12]-[15] and KOT,[16],[17] with an overall higher level in the first than in the latter.[12],[14],[16] There was a strong expression of MMP2 in the stroma of SMA and KOT, which is in
accordance with other studies,[18],[19] although one study observed the opposite.[15] The presence of MMPs in the tumor stroma could be attributed to tumor induction
since neoplastic cells express the protein extracellular inducers (EMMPRIN/ CD147)
on their surface, and this acts as a potent inducer for the production of MMPs by
stromal fibroblasts and endothelial cells.[20]
Furthermore, the high expression of MMP2 in the odontogenic epithelia of plexiform
type and KOT with daughter cysts (KOT with + DC) could suggest more aggressive behavior
of plexiform type of AB. KOTs have a tendency to form satellite cysts through detachment
of epithelial cell lining from the connective tissue wall. This suggests the important
role of MMP2 in this detachment. Wahlgren et al.
[17] mentioned that MMP2 can induce epithelial migration by fragmenting the basement
membrane material laminin-5 gamma-2 chain, which induced migration, tendency to detach
from the connective tissue capsule. This molecular behavior has been thought to be
responsible for the frequent recurrence of KOT after surgical enucleation.
Assessment of receptor-activated nuclear factor kappa B expression in ameloblastoma
and keratocystic odontogenic tumor
RANK, a member of the tumor necrosis factor receptor superfamily, is the signaling
receptor for RANKL. RANKL binds to RANK on the surface of preosteoclasts and stimulates
the development and activation of osteoclasts.
RANK originating from the epithelium has an effect on bone resorption, cell proliferation,
or delaying apoptosis and hence the tumor growth. The present study could indicate
greater bone resorption activity in SMA than KOT. The RANK expression in odontogenic
epithelia of SMA is significantly higher than in KOT. Tekkesin et al.
[9] observed the opposite findings. However, da Silva et al.
[5] observed almost the same findings of the present study.
RANK in stromal cells other than osteoclasts and their precursors may have influential
effects in several functions, such as immune cell regulation and inhibition of apoptosis
of osteoclasts.[6],[21] da Silva et al.
[5] and Tekkesin et al.
[9] showed different levels of positive stroma cells in AB and OKC; the present study
observed similar stromal RANK expression in both SMA and KOT with heavy staining at
the periphery of AB and KOT.
Expression of RANK, in both the epithelial and connective tissue cells of KOTs and
AB, indicates that RANK plays a role in local bone resorption of both lesions.
Assessment of receptor-activated nuclear factor kappa B ligand expression in ameloblastoma
and keratocystic odontogenic tumor
RANKL is a membrane-bound protein found on osteoblastic and activated T-cells. RANKL
binds to RANK on the surface of preosteoclasts and stimulates the development and
activation of osteoclasts. Osteoprotegerin (OPG) is a soluble decoy receptor for RANKL
that inhibits the pro-osteoclastogenic interaction between RANK and RANKL, thereby
inhibiting bone resorption.[22] The RANKL, RANK, and OPG system have been shown to be abnormally regulated in several
malignant osteolytic pathologies, including neoplastic and nonneoplastic odontogenic
lesions. The enhanced RANKL expression or decreased OPG levels play an important role
in tumor-associated bone destruction.[23] OPG and RANKL have also been detected in periodontal ligament cells and their expression
are considered to play a role in osteoclastogenesis and bone resorption in periodontal
diseases.[24]
RANKL expression in the present study confirmed the findings of previous studies where
tumor cells and stroma could act as a source of this osteoclastogenic factor. This
indicates that RANKL-producing tumor cells of AB and cystic cells of KOT are crucial
for osteoclastogenic behavior. The results of the present study also showed that both
factors RANK and RANKL had higher ratio when comparing SMA to KOT. This would account
to more aggressive nature and higher rate of recurrence in AB than KOT.[5],[6],[9],[25] This fact could be also true when comparing plexiform pattern with other histological
subtypes of SMA and KOT.[26] There is no known link in the pathway of synthesis, mechanism of action, overexpression
of MMP2, and the RANK and RANKL in both SMA and KOT.[27] MMP2 role of action is mainly degradation of extracellular matrix, while RANKL and
RANK are involved in mechanism of how the tumor expands in the bone, through stimulation
proliferation and activation of osteoclast and hence bone resorption.[28] Bone resorption mediated by activation of RANK and RANKL signalling pathway and
matrix degradation by acivation of MMP2 pathway in AB and KOT will identify the aggressive
bilogical behavior of both lesions. The fact that RANKL is required for osteoclast
development suggests that agents that inhibit its activity may be therapeutic. This
has provided the rationale for the development of targeted molecular therapy with
the ability to modulate RANK-induced osteoclastogenesis.
Conclusion
The high expression of MMP2, RANK, and RANKL is related to the biological behavior
of AB and KOT and may indicate close behavior of KOT to AB, which reinforce the possibility
of its recent classification as an odontogenic tumor. In addition, tumor epithelial
cells of plexiform AB and epithelial nests or satellite microcyst-associated KOT exhibited
distinct stronger expression of these markers compared with follicular AB and KOT
without microcyst or epithelial nests, respectively. This could reveal a more aggressive
behavior of these types of lesions.
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