Are CD68 and Factor VIII-RA Expression Different in Central and Peripheral Giant Cell Granuloma of Jaw: An Immunohistochemical Comparative Study
Soudabeh SARGOLZAEÝ, Nasim TAGHAVÝ , Farzaneh POURSAFAR
Department of Oral Pathology, Shahid Beheshti University of Medical Sciences, School of Dentistry, TEHRAN, IRAN
Keywords: Giant cell granuloma, CD68, Factor VIII-RA , Jaw
Central giant cell granuloma and peripheral giant cell granuloma of the jaw and oral cavity are identical in histopathologic features,
although they are different in pathogenesis and clinical behavior. The aim of present study was to compare CD 68 and factor VIII related antigen
(VIII-RA ) immunoreactivity in central giant cell granuloma and peripheral giant cell granuloma to determine the biologic nature and clinical
behavior of these lesions which may lead to a better or new treatment modality.
Material and Method: CD68 and factor VIII-RA expression were examined immunohistochemically in 22 cases of central giant cell granuloma
(10 aggressive and 12 non- aggressive ) and 19 cases of peripheral giant cell granuloma. The Kruskal-Wallis test followed by the Dunn test was
used for data analysis.
Results: CD68 expression was observed in approximately 100% of multinucleated giant cells and 50% of mononuclear cells. Overexpression of
factor VIII-RA in the endothelial cells of capillary like vessels in the periphery of the lesions was prominent. A statistical significant difference for
CD68 intensity score in mononuclear cells among three groups (P=0.016) was observed. Indeed, factor VIII-RA intensity score in the endothelial
cells of central giant cell granuloma and peripheral giant cell granuloma showed significant difference (P=0.004).
Conclusion: These findings support the histiocyte/macrophage nature of multinucleated giant cells and mononuclear cells. Overexpression and
high intensity score of CD68 in mononuclear cells and the high intensity score of factor VIII-RA in endothelial cells represent less aggressive
behavior in central giant cell granuloma.
Central giant cell granuloma (CGCG) is an intraosseous
lesion of unknown etiology. There is much debate regarding
whether this entity presents a reactive process or a benign
neoplasm. CGCG is classified as aggressive and nonaggressive
based upon radiographic criteria and clinical
features (1,2). The lesions that recur after treatment or are
greater than 5 cm in size are considered aggressive types.
Tooth displacement, cortical perforation or root resorption
are also radiologic criteria of aggressive lesions. Nonaggressive
lesions exhibit few or no symptoms with slow
growth and constitute most of the cases (3,4). Histologically,
CGCG is characterized by multinucleated giant cells
(MGCs) in a fibroblastic vascularized background (5,6).
Some evidence indicates the histiocyte/macrophage nature
of MGCs, while others suggest an osteoclastic phenotype
(7-11). Fibroblasts make up the proliferative component
of CGCG because they express proteins related to the
cell cycle. They are also responsible for recruitment and
retention of monocytes and subsequent transformation of MGCs (7-10). Interestingly, there are no reliable markers
to predict the prognosis and clinical behavior of CGCG.
Peripheral giant cell granuloma (PGCG) is a relatively
common extraosseouss reactive lesion of the oral cavity.
Inflammatory or developmental reactions in the periosteum
or periodontal ligament have been proposed as possible
etiologic factors of PGCG. PGGG bears microscopic
resemblance to CGCG despite different clinical features.
MGCs presence in PGCG remains unclear (12,13).
CD68 is a transmembrane glycoprotein with an unknown
function. At a very low dose, it is expressed in most cell
types but strongly expressed by tissue macrophages,
human monocytes, osteoclasts and histiocytes (7,8). CD 68
expression in MGCs and mononuclear cells (MCs) of giant
cell lesions has been reported previously (11).
Factor VIII Related antigen (VIII-RA), the most commonly
recommended endothelial cell marker, is produced by
endothelial cells and megakaryocytes (14,15). The literature
evidenced a direct activity of factor VIII-RA complex on osteoclastogenesis and a role in bone remodeling or bone
damage (16). Therefore, the objective of this study was to
compare the CD68 and factor VIII-RA immunoreactivity
in aggressive CGCG, non-aggressive CGCG and PCGG of
the jaw and oral cavity to determine the biologic nature and
clinical behavior of these lesions which may lead to a better
or new treatment modality.
In this retrospective study, 22 paraffin-embedded tissue
samples of CGCG (10 aggressive, 12 non-aggressive) and
19 samples of PGCG were collected from the archive of the
department of Oral Pathology, Shahid Beheshti University
of Medical Sciences, Tehran, Iran.
The CGCG classification into aggressive and nonaggressive
was conducted based on Choung and Kanban’s
classification system (3).
Clinical information on each case including age, gender and
location were determined. Root resorption, rapid growth,
tooth displacement, cortical thinning, recurrence, size and
perforation were obtained from medical records to classify
CGCGs. Cases without complete data and inadequate
paraffin-embedded tissue were excluded from the study.
Immunohistochemistry: 3 μm sections of routinely
processed paraffin-embedded blocks were cut and mounted
on adherent glass slides. The sections were deparaffinized
in xylene and rehydrated in graded ethanol series and
then treated with 3% hydrogen peroxide. Microwave
pretreatment for 15 minutes in citrate buffer (PH 6.0, 10
mM) was performed to retrieve antigen. The sections were
then incubated for one hour at room temperature with
primary antibodies: 1) CD68- mouse monoclonal antibody
(KP1, Dako, Denmark), 2) factor VIII-mouse monoclonal
antibody (F8/86, Dako, Denmark). This was followed by incubation with secondary antibody (Dako Envision TM)
for 30 minutes and finally diamiobenzidine (DAB) for
3-5 minutes. The slides were counterstained with Mayer’s
Lymph node germinal center and blood vessels in the
periphery of the lesions were used as positive control for
CD68 and factor VIII-RA, respectively. In the negative
control, non-immune serum was used instead of primary
CD68 and factor VIII-RA expression were assessed
in multinucleated giant cells, mononuclear cells and
endothelial cells in eight high power (x400) fields. Each
field was evaluated for the proportion of stained cells and
the staining intensity.
Staining intensity was graded as: 0, negative; 1, light
staining; 2, moderate staining; 3, intense staining. The
proportion score of stained cells for CD68 and factor VIIIRA
was assessed as: 0, no stained cells; 1, <25% stained
cells; 2, 25-50% stained cells; 3, >50% stained cells (17,18).
The data were stored and analyzed with the SPSS 21 Software
Package (SPSS, Inc, Chicago, IL, USA). Data analysis was
performed using the Kruskal-Wallis test followed by the
Dunn test. Significance was established at P-value < 0.05.
In total, 22 cases of CGCG and 19 cases of PGCG were
studied. The average age in non-aggressive CGCG and
aggressive CGCG, and PGCG were 24.52 ± 11.72, 22.67±
14.42 and 40.11±17.36, respectively. All groups occurred
more often in the mandible than the maxilla (Table I
Eight (66.6%) cases of non-aggressive CGCG presented as
painless swelling and 4 (33.4%) cases were asymptomatic.
Clinical features of aggressive CGCG cases are given in
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|Table I: Demographic details of patients with central giant cell granuloma and peripheral giant cell granuloma
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|Table II: Clinical Features of aggressive central giant cell
Immunohistochemical evaluation of the three groups
examined showed a positive reaction for both CD68 and
factor VIII -RA. The MGCs expressed CD68 in all cases
as cytoplasmic reactivity, high proportion score (score
III) and intense staining (Figure 1). CD68 was detected in some of MCs and endothelial cells (Figure 2,3A,B) of
all groups (Table III). Factor VIII-RA immunoreactivity
was observed in MGCs, MCs and endothelial cells of all
categories. Notably, the expression was less prominent than
CD68. With respect to factor VIII-RA in MGCs, a score
of 2 was obtained in most PGCG cases (36.8%), whereas
a predominance of negative staining and score of 2 were
observed in both groups of CGCG (Table IV).
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|Figure 1: CD68 stained multinucleated giant cells and mononuclear
cells in peripheral giant cell granuloma (CD68; x100).
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|Figure 2: CD68 stained endothelial cells in peripheral giant cell
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|Figure 3: CD68 stained A) mononuclear cells, B) multinucleated giant cells in central giant cell granuloma (CD68; x200).
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|Table III: Distribution of CD 68 proportion score and intensity score among groups
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|Table IV: Distribution of VIII-RA Proportion score and intensity score among groups
The analysis of factor VIII-RA expression in MCs revealed
no reactivity in most cases of the groups. On the other hand,
positivity of factor VIII-RA in endothelial cells presented a
discrete predominance of score 0 and score 2 in all groups
(Figure 4,5). Immunoreactivity was more remarkable at the
periphery of the lesions especially in PGCG. Data analysis demonstrated no significant difference among groups
regarding the CD68 and factor VIII-RA expression by
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|Figure 4: Factor VIII-RA stained endothelial cells in central giant
cell granuloma (Factor VIII-RA ; x200)
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|Figure 5: Factor VIII-RA stained endothelial cells in peripheral
giant cell granuloma (Factor VIII-RA ; x200)
The Kruskal-Wallis analysis showed a significant difference
in CD68 intensity score in MCs among groups (P=0.016),
with a higher intensity score observed in non-aggressive
CGCG (Table V). Pairwise comparison of CD68 intensity
score of MCs using the Dunn test showed significant
difference between aggressive and non-aggressive CGCG
(p=0.045). No significant difference was seen between
aggressive CGCG and PGCG. The percentage of CD68
positive MCs was less in aggressive CGCG compared with
the non-aggressive type. However, the difference was not
significant (p>0.05). CD68 positive endothelial cells were
statistically significantly higher in PGCG than CGCG (p=0.016) (Table VI), but no difference between aggressive
and non-aggressive CGCG was found (p=0.838). Regarding
the overall expression of factor VIII-RA, data analysis
showed a significantly higher intensity score in endothelial
cells of PGCG (p=0.004) (Table VII).
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|Table V: Comparison of CD 68 intensity score of mononuclear cells among groups
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|Table VI: Comparison of endothelial cells CD 68 Proportion score among groups
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|Table VII: Comparison of endothelial cells factor VIII-RA intensity score among groups
Despite histochemistry, immunohistochemistry and
ultrastructure studies focused on giant cell lesions of the
jaw and oral cavity, the pathogenesis and nature of these
lesions are still elusive. Neoplastic, reactive, inflammatory
and proliferative vascular processes have been proposed
(3, 19). Many believe that prominent MGCs are a reactive
component and have phagocyte, osteoclast or endothelial
cell origin (11,19). The stromal MCs are mostly spindle
cells with fibroblastic/myofibroblastic and endothelial
differentiation that is responsible for the proliferative
activity of the lesions (20-23).
To our knowledge, this is the first study to examine and
compare CD68, a specific macrophagic/histiocytic marker,
and factor VIII-RA, a specific endothelial marker, in
aggressive CGCG, non-aggressive CGCG and PGCG.
Our results showed CD68 expression in approximately
100% of MGCs as well as a fraction of MCs in the three
groups studied. This suggests a histiocyte/macrophage
origin for some of the cellular components and an important
role played by stromal MCs in MGC development through
fusion, which is consistent with previous studies (12,24-26).
Of note, the presence of functional characteristic osteoclast
like bone resorption, growth inhibition by calcitonin, and
expression of osteoclast markers (TRAP, MB1) in MGCs
confirm the osteoclastic phenotype of these cells (10,11,
Vk et al. reported less CD68 positive MGCs in CGCG
compared with PGCG but we did not find any significant
difference between the groups (17). This can be attributed to
the smaller sample size and evaluation of CGCG separately
in two subtypes.
Despite no significant difference of CD68 expression in
MCs among groups, positive CD68 MCs in aggressive
CGCG was less than in non-aggressive CGCG and PGCG,
whih was in agreement with the Syrio et al. study (12).
Moreover, the intensity score of CD68 in non-aggressive
CGCG was significantly higher than aggressive CGCG.
Taken together, these findings suggest that there may be a
correlation between CD68 proportion and intensity score
of MCs and the behavior or aggressiveness of the lesions.
Most of the researchers evaluated CD31, CD34 and VEGF
expression as angiogenic markers in giant cell lesions
A few studies analyzed factor VIII-RA as a vascularization
marker in giant cell lesions (13, 18, 24). The current study
demonstrated factor VIII-RA immunoreactivity in MGCs,
MCs and endothelial cells in both PGCG and CGCG,
implying MGCs and MCs as the sources of factor VIII-RA in addition to endothelial cells. In contrast to previous
study (13), we found no significant difference regarding
percentage of factor VIII-RA positive endothelial cells
among the groups. This discrepancy may be due to the
evaluation method of factor VIII-RA expression. Matos
et al. only focused on microvessel density and showed
larger number of factor VIII-RA reactive vessels in PGCG.
Also in that study, a negative correlation between VEGF
expression and microvessel density in CGCG was observed,
supporting the function of VEGF in osteoclastogenesis. On
the other hand, possible interaction of factor VIII-RA in
osteoclastogenesis through regulating OPG, RANK and
RANK-L may be present. Baud’huin et al. (16) examined
the role of factor VIII-RA on osteoclastogenesis using a
cellular model. They concluded that the interaction between
factor VIII-RA and OPG can cause inhibition of RANK-L
induced osteoclastogenesis. Considering the significance of
OPG, RANK and RANK-L as essential osteoclast formation
components (30,31), further attempts to clarify the role and
correlation of factor VIII-RA with osteoclastogenesis may
lead to a new treatment modality in giant cell lesions.
One of the most important findings in the present study
was prominent factor VIII-RA immunoexpression in
capillary like blood vessels in the periphery of the lesions. Furthermore, the morphology of the reactive blood vessels
was completely different and slit-like in the center.
In line with present study, ultrastructural analysis of
the microvasculature of giant cell granuloma showed a
remarkable difference between the blood vessels of periphery
and those deeper within the lesion around MGCs. Blood
vessels of the periphery were lined with endothelial cells
and basal lamina, thus presenting mature and normal blood
vessels despite the central blood vessels (18). In accordance
with these finding, Quindere et al. indicated a lack of
collagen IV expression in blood vessels deeper within the
lesion confirming the existence of a poorly formed vascular
structure (13). However, Dewsnup et al. evaluated CD34
staining in giant cell lesions and no apparent difference
was observed between the central and peripheral portion
(28). Interestingly, we found that endothelial cells in PGCG
stained intensely with factor VIII-RA. This may represent
the reactive process of PGCG, targeting higher production of
pro-angiogenic factors and greater inflammatory reaction.
In conclusion, the results of present study supported
the histiocyte / macrophage nature of MGCs and MCs.
Furthermore, overexpression and high intensity score of
CD68 in MCs and high intensity score of factor VIII-RA
in endothelial cells represent less aggressive behavior in
This research is extracted from undergraduate thesis by
Dr.Poursafar, which was successfully completed under the
supervision of Dr. Sargolzaei and Dr. Taghavi, Department
of Oral Pathology, Dental School, Shahid Beheshti
University of Medical Sciences, Tehran, Iran , which was
supported by research deputy of Dental School, Shahid
Beheshti University of Medical Sciences, Tehran, Iran
(Code number 12846).
CONFLICT of INTEREST
The authors declare no conflict of interest.
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