Abstract

Background: The Odontogenic Keratocyst (OKC) is a clinicopathologically distinct form of

odontogenic cyst, known for its pathognomonic microscopic features, aggressiveness and

high recurrence rate. Transformation of the keratinized epithelial lining to non-keratinized

squamous epithelium is common and inflammation has been suggested to be responsible.

Apoptosis, also referred to as programmed cell death or physiologic cell death, plays diverse

roles in embryogenesis and normal homeostasis as well as in various pathologic conditions.

The bcl-2 is an anti-apoptotic protein, thought to regulate programmed cell death and to

facilitate cell survival. It is topographically restricted to cells in proliferating zones. It

prolongs the life span of epithelial cells with differentiation potential and allows proliferation,

differentiation and morphogenesis to proceed. Apoptotic cells have been detected in tooth

germ tissues and several epithelial odontogenic tumors; however odontogenic cysts have not

been sufficiently examined.

Methodology: A retrospective study was carried out comprising a total of 60 cases; (20

Ameloblastoma, 20 Keratocystic Odontogenic tumor and 20 Radicular cyst each). The

expression of bcl-2 was determined with respect to Localization, Area (percentage) and

Intensity of stained cells in the epithelium. The expression of bcl-2 was also determined in

the connective tissue stroma, by counting the endothelial, round and fusiform cells. Degree of

inflammation in the connective tissue stroma of all 3 lesions were evaluated and compared

with the area and intensity of bcl-2 stained cells in epithelium and connective tissue stroma.

Results: The area and intensity of bcl-2 expression in Keratocystic Odontogenic tumors was

higher followed by Ameloblastoma and lowest in Radicular cyst cases in the epithelium.

Whereas, in the connective tissue stroma area and intensity of bcl-2 expression was higher in

Keratocystic Odontogenic tumor and Radicular cyst than Ameloblastoma cases. Solid or

1
Multicystic variants showed statistically higher expression as compared to the Unicystic

variants of Ameloblastoma (p-value 0.009, 0.033, 0.011, 0.041). Keratocystic Odontogenic

tumor (44.170±22.258) and Radicular cyst (52.230±20.821) revealed high degree of

inflammation whereas Ameloblastoma cases (26.350±9.602) revealed far less amount of

inflammation. Inter-comparison of degree of inflammation with area and intensity of bcl-2

expression in Keratocystic Odontogenic tumor was significant in epithelium and connective

tissue both. In Radicular cyst and Ameloblastoma, Inter-comparison revealed significant

results in connective tissue and non-significant results in the epithelium.

Conclusion: High expression of bcl-2 in KCOT in the present study supports the general

agreement that some features of OKC are those of a neoplasia, notably the relatively high

proliferative rate of epithelial cells. The bcl-2 expression in the connective tissue cells

suggests that these cells may also be important as epithelial cells in the biological behaviour

of this odontogenic lesion.

Key words: Keratocystic Odontogenic tumor, Ameloblastoma, Radicular cyst, bcl-2,

apoptosis, inflammation.

2
Introduction

In multicellular organisms, cells are continuously shed and replaced. It is estimated that 1-

1011 cells die per day, equivalent to the turnover of an adult’s total body weight every 18–24

months. Apoptosis is the main mechanism by which cells are physiologically removed and

thus plays an important role in regulating tissues during embryogenesis and in normal

homeostasis. A dysfunctional apoptotic system can lead to either excessive removal or

prolonged survival of cells. Therefore, Dysregulation of apoptosis is involved in the

pathogenesis of a variety of diseases such as cancers, viral infections and immunological

disorders.1

The center-piece of the apoptotic program and the major effector arm of the cell death

program is the bcl-2 family of related proteins. The prototype member of this family is bcl-2,

an acronym for B-cell lymphoma/leukemia-2 gene, that was first discovered at the breakpoint

of the t(14;18) in a follicular non-Hodgkin's B-cell lymphoma. The bcl-2 gene family consists

of two opposing groups of proteins: Death antagonists (bcl-2, bcl-xL, bcl-w, Bfl-1, Brag- 1,

Mcl- 1, and A- 1) and death agonists (Bax, Bak, BclxS, Bad, Bid, Bik, and Hrk). They differ

in their tissue and activation-dependent patterns as well as in their structure. The proteins

encoded by the bcl-2 family localize to the outer mitochondrial membrane, the nuclear

membrane, and the endoplasmic reticulum.2

Immunoreactivity for bcl-2 protein has been detected in enamel organs and dental lamina of

tooth germs as well as in odontogenic lesions like Ameloblastoma, Keratocystic Odontogenic

tumor, radicular cyst etc. to elucidate any relationship between histological features and

biological potential.3

3
Ameloblastoma is the most frequently encountered tumor arising from odontogenic

epithelium and is characterized by a benign but locally invasive behavior with a high risk of

recurrence4. Its incidence combined with its clinical behavior, makes it the most significant

odontogenic neoplasm of concern5. Several recent studies have detected genetic and

cytogenetic alterations in these epithelial odontogenic tumors6 however; the detailed

mechanisms of oncogenesis, cyto-differentiation, and tumor progression remain unknown4.

Cyst formation, like the development of neoplasms, involves a Dysregulation of the balance

between cell proliferation and cell death. Induced proliferation of epithelial cell rests (rests of

Serres or Malassez) in the jaw region plays an important role in the pathogenesis of

odontogenic cysts. Factors such as inflammation or trauma stimulate epithelial rests to

proliferate sealing off the inflamed or traumatized region from the surrounding healthy

tissues. Debris from dead cells in periapical lesions like radicular cyst contributes to the

formation and expansion of cysts by increasing osmotic pressure within the lumen of newly

formed cyst1. The role played by apoptosis in the pathogenesis of the most common jaw cyst

- periapical cysts occurring in tooth apices appears to involve the pro-apoptosis proteins of

Bax, Fas and FasL.1

Odontogenic Keratocyst, another common cyst arising from dental lamina or remnants of the

dental lamina7, has been deeply studied due to its aggressive clinical behavior with high

recurrence rates, distinct histopathologic features, singular growth mechanism, and genetic

alterations8. The potential for aggressive clinical behaviour and local recurrence resulted in its

recent classification as a benign odontogenic tumor with a new nomenclature: keratocystic

odontogenic tumor9. Several studies have demonstrated the higher proliferation activity of the

epithelial lining in keratocystic odontogenic tumors (KCOTs) in relation to odontogenic

cysts10. However, despite the fact that cell turnover is controlled by both cell proliferation and

4
apoptosis, few studies have evaluated apoptosis related proteins 11 and apoptotic index in the

epithelial lining of the odontogenic cysts12.

Generally, most of the Immunohistochemical studies on bcl-2 expression included epithelial

cell examination. There are a few studies in the literature examining bcl-2 expressions in

odontogenic epithelium and connective tissue cells of oral lesions. Hence, the present study

was aimed to evaluate bcl-2 expression and its distribution in the epithelial lining as well as

connective tissue cells of Ameloblastoma, Keratocystic Odontogenic tumor and Radicular

cyst.

Materials and Methods

Sample selection: The present study was carried out on 60 formalin-fixed paraffin-embedded

tissues 20 each of Ameloblastoma, Keratocystic Odontogenic tumor and Radicular cyst

samples, which were retrieved from the archives of Department of Oral and Maxillofacial

Pathology, Pacific Dental College and Hospital, Udaipur. Relevant clinical details were

obtained as per the case history Performa. All the other relevant patient clinical data was also

tabulated. None of the OKCs were associated with Nevoid basal cell carcinoma syndrome

(NBCS).

Immunohistochemistry: 4μm thick sections were cut and mounted on poly-L-lysine-coated

slides, air dried and heated at 45°C overnight. After deparaffinization and rehydration, the

sections were incubated in 0.01 M citrate buffer in a microwave oven for 15 min for antigen

retrieval. The slides were then washed in phosphate-buffered saline (PBS) for 30 min at room

temperature and incubated in 0.5% H2O2 in methanol for 10 min to block endogenous

peroxidase activity. Nonspecific antibody binding was blocked with 3% normal horse serum

in PBS. A mouse anti-human bcl-2 monoclonal antibody (Clone: Bcl-2-100, Dako, South San

Francisco, CA, USA) with a 1/100 dilution was applied as primary antibody and placed in a

5
humidified chamber at 4°C overnight. The sections were subsequently washed in PBS and

processed for detection of the positive immunohistochemical reaction using the strepavidin-

biotin complex immunoperoxidase technique. Rabbit-mouse antibody link solution (Dako

ABL008, South San Francisco, CA, USA) was used as secondary antibody to enhance the

sensitivity of the procedure and diaminobenzidine was applied as the chromogen. Sections

were finally counterstained with hematoxylin, cleared and mounted. Negative controls

consisted of phosphate buffered saline instead of primary antibody and a sample of

lymphoma and inflammatory cells within the cyst wall were used as the external and internal

positive controls.

Evaluation methods: The specimens were examined in Olympus BX60 microscope attached

to a colour video camera (Olympus Analysis Five). All stained areas demonstrating positivity

for bcl-2 were determined with respect to Localization, Area (percentage) and Intensity of

stained cells at a magnification of ×40 and the number of positively stained cells was counted

on 10 representative areas of the epithelium using a ×40 objective in a minimum of 100 cells

in the full length of the epithelium. The expression of bcl-2 was also determined in the

connective tissue stroma, by counting the endothelial, round and fusiform cells at a

magnification of ×40 and the number of positively stained cells was counted on 10

representative areas as specified by Tenkkesin MS et al 20127

The Area of staining (Immunoreactivity) in the epithelium was defined by: (Vered M et al

200926): (0) no staining; (1) low staining 1% to 10% positive; (2) intermediate staining 11%

to 50% positive; (3) high staining more than 50% positive. Whereas, the intensity of bcl-2

positivity was estimated as follows (Jahanshahi GH et al 2006 17): (-) less than 5% stained

cells, (±) 5-9% cells stained positively, (+) between 10 and 24% cells stained positively, (++)

between 25 and 50% cells stained positively and (+++) Greater than 50% of tumor cells

stained positively. The degree of inflammation was assessed in the connective tissue stroma

6
adjacent to the epithelial lining in which the cell count had been performed (magnification,

×100) and also approximal to bcl-2 negative cases (Jahanshahi GH et al 2006 17). A lack of

inflammatory cells was scored as (-), less than 30 inflammatory cells as (+), 30 to 59

Inflammatory cells as (++) and 60 or more inflammatory cells as (+++).

The number of positive cells was divided into the total number of cells counted in the whole

area. The result was multiplied by 100 to find the percentage of positive cells. Collected data

was analysed using the SPSS software for Windows (Version 17). One way ANOVA (F-test)

was carried out for comparing the parameter for multiple groups. Comparison between

groups was carried out using the Student’s ‘t’ test. Correlations between bcl-2 positivity in

different lesions and degree of inflammation were derived using Karl Pearson’s, with the

level of significance set at p≤0.05.

Results

Positive bcl-2 expression was considered in the epithelium of the odontogenic lesions when

at least 10% of the cells were stained. All Keratocystic odontogenic tumors (Fig. 1a) and

Ameloblastoma (Fig. 1b) and 5 out of 20 radicular cysts (Fig. 1c) were positive for bcl-2.

In 13 of the 19 stained OKCs, positively stained cells were observed in the basal layer while

in the other bcl-2 positive OKCs (7 of 20), the stained cells were in the basal/supra basal

region. In radicular cyst positive cells were located in the basal/supra basal layers of only 2

cases. In Ameloblastomas bcl-2 was detected mainly in the peripheral layer whereas only a

few cells were positively stained in the central layer of epithelial tumor islands (4 cases).

Higher bcl-2 staining area and intensity of the connective tissue was found in keratocystic

odontogenic tumor as compared to radicular cysts and least in ameloblastomas. All

7
Keratocystic odontogenic tumors (Fig. 1d), 19 out of 20 Ameloblastoma (Fig. 1e) and 19 out

of 20 radicular cysts (Fig. 1f) were positive for bcl-2.

Mean and standard deviation values of bcl-2 staining area and intensity of the stained cells in

the epithelium and connective tissue of studied odontogenic lesions is shown in Table I.

Significant difference were observed between Ameloblastoma, KCOT and Radicular cyst,

(Anova, P = 0.00 Table 1 Graph 1). In multiple comparisons, bcl-2 staining area and intensity

revealed a statistically significant difference between KCOT and Ameloblastoma (Student ‘t’

test, P = 0.008, 0.000, 0.012 & 0.000, Table 2) and between Ameloblastoma and Radicular

cyst (Student ‘t’ test, P = 0.001, 0.001, 0.057, 0.000, Table 3). There was no significant

difference of bcl-2 staining area and intensity between Radicular cyst and KCOT in the

connective tissue (Student ‘t’ test, p = 0.46, 0.497 Table 4), whereas epithelial bcl-2 staining

area and intensity were significantly higher in Keratocystic Odontogenic tumors (Student ‘t’

test, P = 0.000 Table 4).

In both variants of ameloblastomas SAM lesions had a statistically significant high score of

bcl-2 staining area and intensity both in the epithelium and connective tissue (Student ‘t’ test,

p = 0.009, 0.33, 0.011, 0.041, Table 5, Graph 2, Fig 2 a&b)

Radicular cyst displayed highest degree of inflammation, Keratocystic odontogenic tumor

also displayed a considerable inflammatory component, whereas Ameloblastoma

demonstrated a mild to intermediate degree of chronic inflammation with mean values

(49.740±17.933, 48.710±23.511, 26.350±9.602 respectively, Table 6, Graph 3, Fig 3)

Keratocystic Odontogenic tumor revealed an increased epithelial and connective tissue bcl-2

area and intensity in the presence of inflammation (Karl Pearson’s coefficient, p = 0.006,

0.000, 0.005, 0.000) In Ameloblastoma and Radicular cyst, comparison of degree of

inflammation with bcl-2 expression showed an increased connective tissue expression and

8
intensity of bcl-2 in the presence of inflammation, (p = 0.000) whereas epithelial bcl-2 area

and intensity were not related to inflammation (p = 0.520, 0.110, 0.828, 0.657) (Table 7)

Discussion

Odontogenic cysts and tumors are unique to the jaws and originate from the tissue associated

with tooth development. They comprise an unusually diverse group because odontogenesis is

a complicated process in which cells in various stages of differentiation participate in a

complex, predetermined manner.13 Although, they share the same sources of odontogenic

epithelium, they exhibit different degrees of aggressiveness in their biological behaviour.

Odontogenic cysts are expansile lesions with favourable prognosis whereas tumors are

destructive lesions with variable prognosis.14 In addition to cellular pleomorphism and

atypism mitotic activity is another important factor in determining tumoral behaviour. The

bcl-2 is an anti-apoptotic protein and is now one of the most useful markers to determine the

aggressiveness of many tumour behaviours.15

In the present study, odontogenic epithelium of Keratocystic Odontogenic tumor revealed

strong positive staining for bcl-2 in the whole thickness of epithelium, in basal and supra

basal layers and low expression of bcl-2 in the basal and suprabasal layers in Radicular cysts

cases. These findings are in accordance with earlier studies done by Piatteli A et al 1998 16 and

Jahanshahi Gh. et al in 200617. However, Muzio L et al 199918 reported bcl-2 staining area in

Keratocystic Odontogenic tumor to be restricted to the basal layer only. Since, the basal layer

of normal mucosal epithelium is also positive for bcl-2 the lack of expression in the upper

layers may be due to a decrease in the dividing ability and termination of the cell’s life

span.13,19

Ameloblastoma in the present study revealed medium expression of bcl-2 in the peripheral

cell layer of the tumor islands and low expression in the central stellate reticulum like layer.

9
These findings are in accordance with earlier studies done by Mitsuyasu et al 1997 20 and

Sandra et al 2001.21 In a study by Florescu A et al in 2012 22, bcl-2 expression was present in

88.23% of the investigated ameloblastomas, predominantly in columnar cells from the

peripheral zone.22 Similar studies in the literature communicate that around 90% of

ameloblastomas are positive for bcl-2 similar to our study, which indicates that bcl-2

expression may be related to differentiation and proliferation of odontogenic epithelium, and

bcl-2 overexpression may be associated with the ameloblastoma development.23,24,25 These

results indicate that in ameloblastomas bcl-2 protein could function primarily as anti-

apoptotic factor, which reflects the proliferative activity of neoplasms. Also, the expression of

bcl-2 suggests the aggressive nature of odontogenic tumor and these results will be beneficial

in the differential diagnosis of odontogenic tumors and other tumors that occur in the mouth. 5

In addition, it is estimated that the bcl-2 protein may play a role in maintaining stem cell

population in peripheral layers of tumor islands of which are recruited proliferating cells.20

In the connective tissue stroma bcl-2 area and intensity in Keratocystic Odontogenic tumor

and Radicular cyst was higher than Ameloblastoma. Low positivity in Ameloblastoma may

be attributed to the mature fibrous connective tissue stroma as compared to the highly

inflamed stroma of the odontogenic cysts. These results are in contrast to study done by

Tekkesin MS et al in 20127 in which there was no significant difference in expression of bcl-2

between Keratocystic Odontogenic tumor and Ameloblastoma, as well as between Radicular

cyst and Ameloblastoma. So far in the literature no studies have been cited that have

compared the staining intensity of bcl-2 in these 3 lesions only the area of bcl-2 staining has

been compared.

In the present study, overall comparison for the expression of bcl-2 in all 3 odontogenic

lesions revealed statistically significant higher expression in the whole thickness of

epithelium of Keratocystic odontogenic tumor and predominantly in the peripheral cell layer

10
of the tumor islands of 8 ameloblastomas. On the other hand, Radicular cyst samples revealed

very low bcl-2 expression. These results are in accordance with that found by Mitsuyasu et al

in 199720 and Sandra et al in 200121. This could lead to aggressive growth pattern of

Keratocystic Odontogenic tumor and Ameloblastoma. Vered M et al in 2009 26, found that the

results of Keratocystic Odontogenic tumor were similar to Ameloblastoma but different from

the other Odontogenic cysts similar to our study. The author suggested that their results

supported the notion of Keratocystic Odontogenic tumor having a neoplastic nature . One

Radicular Cyst sample expressed bcl-2, which might reflect a case of clinically aggressive

lesion. The controversy in the literature between studies that found RC and DC lesions

negative (Piattelli A et al 199816) or positive for bcl-2 (Loyola AM et al 200527, Kolar Z et al

200628) could be in part a result of the different clinical behaviours displayed by the examined

lesions. Emerging from these observations is the assumption that the accumulation of

deranged cell-cycle factors that are induced by or act in parallel to the SHH pathway confers

a more aggressive phenotype to the lesions and it is known that activation of the antiapoptotic

factor bcl-2 is partly performed by the SHH pathway29.

In the present study inter-comparison between different variants of Ameloblastoma revealed a

significantly higher expression of bcl-2 area and intensity both in the epithelium and the

connective tissue of Solid or Multicystic variants as compared to the Unicystic variants of

Ameloblastoma. These results are in accordance with the study done by Vered M et al in

200926, where SAM demonstrated intermediate and low scores in basal and stellate reticulum

layers and only a few SAM samples had a high score similar to our study, on the other hand

UAM samples demonstrated less positivity for bcl-2 expression and one case of UAM was

negative for bcl-2.

According to a study done by Kumamoto H et al in 199730, reactions in granular cell

Ameloblastomas tended to be more evident than in plexiform and follicular Ameloblastomas,

11
and those in non-malignant Ameloblastomas tended to be more evident than in malignant

ones.30 In our study also granular cell variant showed more positivity as compared to other

variants. In contrast Kumammoto H et al in 199931, said that no expression of bcl-2 family

proteins was found in keratinizing areas or granular cell clusters in acanthomatous or granular

cell Ameloblastoma, suggesting terminal differentiation of tumor cells. Most tumor cells of

basal cell ameloblastomas showed intensely positive staining for bcl-2 and bcl-x proteins but

low or no reactivity for bax protein, and this type of Ameloblastoma is thought to possess

more proliferative activity than any other type of Ameloblastoma. Malignant ameloblastomas

were characterized by decreased expression of bcl-2 and enhanced reactivity for Bax proteins

compared with the enamel organs and benign ameloblastomas. These results suggest that

malignant transformation of odontogenic epithelium might be related to the p53 gene, which

can down-regulate bcl-2 and up-regulate bax. But, further investigation of malignant

ameloblastomas is required to establish any correlation between the bcl-2 family proteins and

malignant transformation of odontogenic epithelium. Again in 2001 Kumamoto H et al 32

suggested that cytoplasmic granularity might be caused by increased apoptotic cell death in

neoplastic cells and its following phagocytosis by neighbouring cells. According to a study

done by Wang J et al in 200624, bcl-2 expression increased in recurrent and Ameloblastoma

cancerization. A moderate negative correlation between bcl-2 and Bax protein was found. In

our case also recurrent Ameloblastoma samples showed more positivity for bcl-2 expression.

So far, a number of Immunohistochemical studies have examined KCOT employing various

markers of apoptosis.3,18,16,33 However, the question remains open as to why OKCs form cysts

but do not form tumor masses in spite of their high potential to proliferate. Kichi E et al in

200512, stated that the reasons why OKCs are observed as cystic lesions but not as tumor

masses in spite of their high potential to proliferate seem to be that:

12
(i) Cells constituting the lining epithelium have prominent proliferative activity in the
basal and suprabasal layers;
(ii) Apoptosis is inhibited by the expression of bcl-2 protein in the basal layer;
(iii) Apoptosis occurs in the surface layer to regulate the thickness of the lining
epithelium;
(iv) p53 contributes probably as an apoptosis-related protein as well as a marker of
cellular proliferation;
(v) Abundant keratin produced by apoptosis and other exudates from cyst wall may
facilitate an expansion of cyst cavity.

Study by Vered M et al 200926, provide further support to the assumption that an OKC can be

of a neoplastic rather than a cystic nature. This is based not only on the expression of the

PTCH and its downstream factors, SMO and GLI-1, but also on the analysis of the

immunohistochemical profile of OKC, which is comprised of the SHH-related proteins and

the SHH-induced bcl-2 oncoprotein. The quality and quantity of the interactions between the

SHH and other cell cycle regulatory pathways most probably work synergistically to define

the individual phenotype and the corresponding biological behaviour of OKCs. The

activation of atleast two cell cycle regulatory systems is ‘switched on’ in OKCs as this lesion

evolves. According to the literature, other molecules with functions that cause these lesions to

have built-in’ aggressive biological behaviour have been identified, such as IPO38 and cyclin

D1.9 This may also serve as an explanation for the observation that not all OKCs demonstrate

a similar pattern of clinical behaviour, and that there is a large spectrum of variations among

them. Therefore, the type and number of disturbed cell cycle molecules would more

appropriately define the profile of the lesion and ultimately dictate its phenotype.

Investigations on the Immunoreactivity of bcl-2 protein have been demonstrated in KCOTs

and recent studies report that bcl-2 positive cells are predominantly located basally, thus

supporting the concept that apoptosis does not occur in the basal cells of the lining

epithelium. However, TUNEL-positive cells have been detected exclusively in the surface

layer of KCOTs, indicating marked levels of apoptosis. Thus, bcl-2 inhibits apoptosis to

13
facilitate cellular proliferation in the basal and suprabasal layers, whereas apoptosis maintains

the homeostasis of the thickness of the lining epithelium and allows the synthesis of large

amounts of keratin in the surface layer of KCOTs. Considering that there is a regulated

balance between cell proliferation, cell differentiation and cell death in this type of lesion,

this may explain why KCOTs, though portraying a neoplastic behaviour, with an increase

potential to proliferate, do not tend to form tumor masses.34

Inflammation is a protective response intended to eliminate the initial cause of cell injury as

well as the neurotic cells and tissues resulting from the original insult. 35 In several pathologic

conditions, inflammation results in epithelial hyperplasia and metaplasia, such as radicular

cysts. Transformation of the keratinized epithelial lining to non-keratinized squamous

epithelium is common in Keratocystic Odontogenic epithelium and inflammation may be

responsible. It is possible that inflammation may alter not only the morphology but also the

proliferative potential of the epithelial lining.36

In our study, inflammation was assessed in all the lesions revealing Radicular cyst with

highest degree of inflammation followed by Keratocystic Odontogenic tumor whereas

Ameloblastoma samples showed far less amount of inflammation due to its mature fibrous

connective tissue stroma. These findings are in contrast to some studies performed earlier,

Browne RM et al in 197537 stated that there was no evidence that Keratocystic Odontogenic

tumor arise in a foci of inflammation, their walls are characteristically free of inflammation

apart from presence of scattered foci.37 M Shear et al in 2002 supported this saying that

inflammatory exudate played a negligible role in the Keratocystic Odontogenic tumor

enlargement which was pointed out in a number of studies.38,39,40

However Forssell K et al 198041 stated that although Keratocystic Odontogenic tumor is

classified as a developmental lesion, inflammation is found in the connective tissue wall in

14
the majority of cases which may be attributed to communications with the oral mucosa either

by perforations of the cortical bone documented in up to 39% of KCOT or by the periodontal

ligament in cases where the cyst is close to adjacent teeth and in cases following

decompression treatment where inflammation is always present. In his study Kaplan I in

2004,36 said that inflammation was shown in 75% of the cases and the inflammation score

was moderate to high in 45% of the cases. 36 Similarly, in a study done by Jahanashi GH et al

200617 60% of KCOT showed moderate to high inflammation whereas 25% showed low

inflammation and only 15% showed no signs of inflammation in KCOT, whereas Radicular

cysts samples showed, 80% moderate to high inflammation and 20% cases with low

inflammation.17

In the present study, comparison of degree of inflammation with bcl-2 expression using Karl

Pearson’s Coefficient of Correlation in Keratocystic Odontogenic tumor revealed an

increased epithelial and connective tissue area and intensity of bcl-2 staining in the presence

of inflammation suggesting significant relation between the two.

It has been shown that growth factors and cytokines (interleukin-1,6 and TNF) are released

during inflammatory events. Lie et al in 199342 observed that epidermal growth factor

expression by odontogenic rests and cysts (Keratocystic Odontogenic tumor and Dentigerous

cyst) is related to the presence of inflammation within the adjacent connective tissue. 42 Also it

has been demonstrated that growth factors and cytokines may positively or negatively

modulate cell proliferation, growth and differentiation. Interestingly, a study in which

Keratocystic Odontogenic tumor were transplanted into a Thymic mice demonstrated that the

features of the epithelial lining are only maintained in the presence of its cystic wall. Thus it

is reasonable to suggest that growth factors and cytokines released by the inflammatory

infiltrate present in the fibrous tissue capsule of Keratocystic Odontogenic tumors may be

responsible for greater proliferative activity in inflamed lesions compared to non-inflamed

15
lesions.43 However, these results are in complete contrast to earlier studies done in the

literature. Piatteli’s et al 199816 showed bcl-2 Immunoreactivity decreased significantly in the

presence of inflammation. He said that, inflammation in OKC may change the morphologic

appearance of the epithelial lining or it may in some way alter the proliferative potential of

the cells, although Jahanashi GH et al 2006 17, was unable to demonstrate a significant

relationship between the degree of inflammation and bcl-2 Immunoreactivity, which may be

due to an inadequate sample size or an improper measuring method.

In Radicular cyst and Ameloblastoma-Unicystic variant (as the solid or multicystic variant

has a mature fibrous stroma and thus less inflammation), comparison of degree of

inflammation with bcl-2 expression revealed an increased connective tissue area and intensity

of bcl-2 revealing significant relation between the 2, which might be due to increase in the

number of inflammatory cells such as lymphocytes. Whereas, bcl-2 staining area and

intensity in the epithelium were not related significantly to inflammation.

These findings are in contrast to a study done by Tosios KI et al in 2000 44, where RCs with

intense inflammatory changes showed higher Bax expression and lower bcl-2 expression than

RCs with less inflammatory changes. These results suggest that these bcl-2 family members

regulate some balance that contributes to proliferation and cell death of lining epithelium of

inflammatory periapical lesions.44 However on reviewing the literature no previous studies

were found where inter-comparison between bcl-2 expression and inflammation was done in

Ameloblastoma.

Conclusion

In conclusion, the results of the present study suggest that Odontogenic Keratocyst has a high

proliferative and survival activity and that might be one of the reasons why Odontogenic

Keratocyst has a high recurrence rate. The proliferation potential of the epithelium and the

16
overexpression of various anti-apoptotic proteins in odontogenic epithelial tumors are quite

significant for their clinical behaviour providing further support to the assumption that an

OKC can be of a neoplastic rather than a cystic nature. We have also demonstrated that

connective tissue cells may also be important as epithelial cells in the biological behaviour of

these odontogenic lesions. Although these results support that Keratocyst Odontogenic tumor

is a neoplasm, with expression of bcl-2 similar to Ameloblastoma rather than Radicular cyst,

there are not enough genetic studies and only two studies so far have directly compared the

expression of bcl-2 in these three lesions all together. Further studies on genomic changes

may help better understanding of the pathogenesis of odontogenic keratocysts.

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Table 1: Overall mean and standard deviation values

Area Intensity
Odontogenic lesion Connective Connective
Epithelium Epithelium
Tissue Tissue

28.640± 16.575± 23.405± 13.420±

Ameloblastoma
22.381 11.135 24.359 12.094

Keratocystic 49.605± 45.045± 44.510± 43.815±

Odontogenic tumor 24.786 27.176 26.126 28.709

9.680± 38.600± 11.065± 38.060±

Radicular cyst
6.951 23.385 14.124 24.158

“p-value” 0.00 (S) 0.00 (S) 0.00 (S) 0.00 (S)

Table 2: Inter-comparison between Keratocystic Odontogenic tumor and Ameloblastoma

Area Intensity
Odontogenic lesion Connective Connective
Epithelium Epithelium
Tissue Tissue

Ameloblastoma 28.640± 22.381 16.575± 11.135 23.405± 24.359 13.420± 12.094

Keratocystic
49.605± 24.786 45.045± 27.176 44.510± 26.126 43.815± 28.709
Odontogenic tumor
“p-value” .008 (S) .000 (S) .012 (S) .000 (S)

Table 3: Inter-comparison between Ameloblastoma and Radicular Cyst

Area Intensity
Odontogenic lesion
Epithelium Connective Tissue Epithelium Connective Tissue

Ameloblastoma 28.640± 22.381 16.575± 11.135 23.405± 24.359 13.420± 12.094

11.065±
Radicular Cyst 9.680± 6.951 38.600± 23.385 38.060± 24.158
14.124
“p-value” .001 (S) .001 (S) .057 (S) .000 (S)

Table 4: Inter-comparison between Keratocystic Odontogenic tumor and Radicular cyst

Area Intensity
Odontogenic lesion
Epithelium Connective Tissue Epithelium Connective Tissue
Keratocystic
49.605± 24.786 45.045± 27.176 44.510± 26.126 43.815± 28.709
Odontogenic tumor

Radicular Cyst 9.680± 6.951 38.600± 23.385 11.065± 14.124 38.060± 24.158

“p-value” .000 (S) .426 (NS) .000 (S) .497 (NS)

Table 5: Inter-comparison between different variants of Ameloblastoma

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 Area Intensity
Variant N Connective Connective
Epithelium Epithelium
Tissue Tissue
20.506± 13.587± 15.820± 10.287±
Unicystic 15
15.512 0.675 17.815 5.032
50.180± 25.540± 46.140± 22.820±
Multicystic 5
27.638 19.454 29.089 21.421
“p-value” .009 (S) .033 (S) .011 (S) .041 (S)

Table 6: Mean and Standard deviation values of inflammation in all the lesions

Lesion Inflammation
Ameloblastoma 26.350±9.602
Keratocystic Odontogenic tumor 44.170±22.258
Radicular cyst 52.230±20.821

Table 7: Inter-comparison of bcl-2 expression with degree of inflammation in different

odontogenic lesions:

Odontogenic
Inflammation Ameloblastoma Radicular cyst
keratocyst
r .592 .440 .052
Epithelial Count
“P-value” .006 (S) .052 (NS) .828 (NS)
r .793 .899 .859**
Connective Tissue Count
“P-value” .000 (S) .000 (S) .000 (S)
r .606 .368 .106
Epithelial Intensity
“P-value” .005 (S) .110 (NS) .657 (NS)
r .781 .905 .862**
Connective Tissue Intensity
“P-value” .000 (S) .000 (S) .000 (S)
Graph 1: Overall comparison of bcl-2 expression in Ameloblastoma, KCOT and Radicular
cyst

Graph 2: Comparison of bcl-2 expression in the two variants of Ameloblastoma;

Solid/Multicystic and Unicystic variants

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Graph 3: Assessment of Inflammation in all 3 lesions.
Figure
1: bcl-
2

expression in the epithelium of a) Ameloblastoma, b) KCOT, c) Radicular cyst and bcl-2

expression in the connective tissue of e) Ameloblastoma, f) KCOT, g) Radicular cyst
respectively.

Figure 2: bcl-2 expression in the variants of Ameloblastoma a) solid or multicystic, b)

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Unicystic respectively

Figure 3:
Inflammation in a)
Ameloblastoma
Mullticysticvariant,
b) Ameloblastoma
Unicystic variant,
c) KCOT, d)
Radicular cyst

23