Ki-67 immunohistochemical expression in mandibular ameloblastoma: A prognostic indicator for local recurrence

doi:10.4236/ojst.2013.39086

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Open Journal of Stomatology, 2013, 3, 520-526 OJST

http://dx.doi.org/10.4236/ojst.2013.39086 Published Online December 2013 (http://www.scirp.org/journal/ojst/)

Ki-67 immunohistochemical expression in mandibular

ameloblastoma: A prognostic indicator for local

recurrence

Ayman Hegab1*#, Mohmmad Shuman2†, Mohammed Abd El-Akher1#, Delaram Arwlan3

1Oral and Maxillofacial Surgery, Faculty of Dental Medicine, Al-Azhar University, Cairo, Egypt

2Oral and Maxillofacial Surgery, Faculty of Dental Medicine, Al-Azhar University, Assuit, Egypt

3Medcal City Teaching Hospital, Teaching Laboratory, Baghdad, Iraq

Email:*[email protected]

Received 12 September 2013; revised 12 November 2013; accepted 29 November 2013

which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

ABSTRACT

Objective: This study used Ki-67 to evaluate the pro-

liferative activity of ameloblastoma in an attempt to

provide a scientific basis for any differences in the

biologic behaviour that exists between the different

types of ameloblastoma and correlate the results with

the recurrence rate. Study design: We assessed Ki-67

expression by immunohistochemistry in 32 cases of

mandibular ameloblastoma. Formalin-fixed, paraffin-

embedded blocks were sectioned and used for Hae-

matoxylin & Eosin (H&E) staining. H&E slides were

used to re-evaluate the histological types of the amelo-

blastoma. Immunohistochemical analysis for Ki-67

with a labelled streptavidin-biotin-peroxidase com-

plex technique was performed on tumour sections

using Monoclonal Mouse Anti-Human Ki-67 Antigen

(clones MIB1, code M7240, Dako Corporation). Lym-

ph nodes (lymphoma) were used as positive control

for Ki-67. Results: The specimens comprised 18 cases

of Follicular ameloblastoma, 7 cases of Cystic amelo-

blastoma, 4 cases of plexiform ameloblastoma, and 3

cases of Acanthotic ameloblastoma. The results show-

ed that cellular proliferative ac tivity varied within the

ameloblastoma types. There was no statistically sig-

nificant difference in the Ki-67 Acanthotic, cystic and

follicular types of ameloblastoma, while plexiform

type showed statistically significantly higher levels

than the other 3 types. The mean Ki-67 labelling in-

dex of plexiform ameloblastoma ranged from 13 to 30

with a mean of 19.75. The mean values of Ki-67 were

statistically significantly higher in recurrent than

non-recurrent cases. Conclusion: The significant ex-

pression of Ki-67 could provide useful prognostic

markers for proliferative activity and good prognostic

indicators for recurrence rate of mandibular amelo-

blastoma.

Keywords: Mandibular Ameloblastoma; Recurrence;

Immunohistochemistry; Ki-67

1. INTRODUCTION

Ameloblastoma is a Benign Tumour of Odontogenic

epithelium with mature fibrous stroma without odonto-

genic ectomesenchyme. In the updated World Health Or-

ganization (WHO) classification, it was classified into

solid/multicystic type, extra-osseous/peripheral type, des-

moplastic type and unicystic type [1].

Histologically, there are two main patterns, follicular

and plexiform, in which various cellular changes such as

squamous metaplasia and granular transformation are

commonly seen, and when these are extensive, the terms

acanthomatous type and granular cell type are applied,

respectively. Cyst formation within the epithelial islands

of the follicular type is common. Other rare histological

variants, including basal cell ameloblastoma, desmoplas-

tic ameloblastoma and kerato ameloblastoma, have also

been described. Despite numerous studies, correlation

between these histological patterns and tumour behav-

iour has not been consistently established [2]. The me-

chanisms of oncogenesis, cytodifferentiation and tumour

progression of ameloblastoma are still largely unknown

[3,4].

*Corresponding author.

#Associate Professors of Oral and Maxillofacial Surgery, Faculty o

Dental Medicine, Al-Azhar University, Cairo, Egypt.

†Lecturer of Oral and Maxillofacial Surgery, Faculty of Dental Medi-

cine, Al-Azhar University, Assuit, Egypt.

Determination of epithelial proliferative activity is

apotentially useful means of investigating differences in-

biologic behaviour between tumours [5-7]. Assessment

OPEN ACCESS

A. Hegab et al. / Open Journal of Stomatology 3 (2013) 520-526 521

of cell proliferation in many types of tumours is an im-

portent adjunct to histologically based tumour classifica-

tion and has potential relevance as an indicator of tu-

mour-behaviour and treatment response and relapse.

Immunohistochemical assessment of cell proliferation

has advantages over the other techniques, such as tritiat-

edthymidine incorporation and flow cytometry; because

the tissue architecture remains intact and proliferating

cells can be visualized in relation to other histologic

characteristics [8]. The interpretation, merits, and clinical

value of the methods of proliferation assessment in tu-

mours are detailed by van Diest et al. [9].

The Ki-67 antigen, which is coded by a geneon chro-

mosome 10, is expressed in the G1, S, and G2 phases in

cycling cells [10,11]. The Ki-67 score partly correlates

with other proliferation markers like %S-phase cells, and

mitotic count [12-15]. Originally, the Ki-67 antigen

could only be studied in frozen sections. Recently, a se-

ries of Ki-67 antibodies has been developed by the Kiel

group that also works on paraffin embedded tumour ma-

terial after antigen retrieval [10,11]. The Ki-67 labelling

index was found to be prognostically relevant in several

studies [16,17].

The aim of the present study is to evaluate the Ki-67

labelled index in different histologic types of mandibular

ameloblastoma and their relation to proliferative activity

and local recurrence.

2. MATERIAL AND METHODS

2.1. Patients and Tissue Selection

A retrospective study of patients who underwent treat-

ment for mandibular ameloblastoma at Al-Azhar Univer-

sity Hospital, Cairo, Egypt from 2001 to 2009 was per-

formed. The study approved by the institutional ethics

committee and written informed consent was obtained.

Thirty two formalin-fixed, paraffin-embedded archival

biopsy specimens were randomly obtained from the De-

partment of Oral and Maxillo facial Surgery at Al-Azhar

University Hospital. Patient’s files were reviewed to re-

trieve clinical data, pathology reports, surgical treatment,

clinical follow-up and recurrence. All histologic slides

were re-evaluated, applying the criteria of the World

Health Organization for histological typing of ameloblas-

toma.

2.2. Histopathology and Immunohistochemistry

Formalin-fixed, paraffin-embedded blocks were sec-

tioned (3 - 4 µm in thickness) and used for Haematoxy-

lin & Eosin (H&E) staining. H&E slides were used to re-

evaluate the histological types and characteristics of the

ameloblastoma.

The paraffin-embedded tissue sections were also used

for Immunohistochemical staining for the antigens Ki-67.

Sectioning: 3 - 4 micrometer section is done on the par-

affin block on special glass slide and put in oven on 56

degree.

Deparaffinization with xyline and dehydrated with de-

creasing grades of ethanol alcohol. Demasking of the

antigen: Pre-treatment of formalin-fixed, paraffin-em-

bedded tissue sections with heat-induced epitope retrie-

val (HIER) is required for 20 minutes.

Immunohistochemical analysis for Ki-67 with a la-

belled streptavidin-biotin-peroxidase complex technique

was performed on tumour sections using Monoclonal

Mouse Anti-Human Ki-67 Antigen (clone MIB1, code

M7240, Dako Corporation) is used at a dilution range of

(1:75 - 1:150) for 20 minute at room temperature.

Visualization: The recommended visualization system

is Peroxidase/DAB+, Rabbit/Mouse using 20 minutes in-

cubation at room temperature.

 Proper positive and negative controls were performed.

Lymphnodes (lymphoma) were used as positive con-

trol for Ki-67 and using 20 minutes heat-induced epi-

tope retrieval in Target Retrieval Solution, and 20

minutes incubation at room temperature with the Pri-

mary antibody.

 The recommended negative control is Dako Mouse

IgG1, diluted to the same mouse.

IgG concentration as the primary antibody.

2.2.1. Interpretation

Slides were scanned by ×40 magnification. Ten cellular

are as selected (i.e. the so-called hot spots) and evaluated

at ×400 magnification. Staining is seen as a dark brown

to black nuclear signal in cells expressing the Ki-67 an-

tigen. Ki-67 is expressed in all phases of the cell cycle

and is therefore considered a marker of proliferating

cells.

Ki-67 proliferation index is assessed by point counting

and reported as percent positive.

2.2.2. Assessmen t o f Ki-67

Ki-67 labelling index was done by calculating the ratio

of positive nuclei in relation to total number of neoplastic

nuclei in 10 HPFs. The labelling index (number of posi-

tive tumour cells/total number of tumour cells expressed

as a percentage) was calculated in every specimen.

2.3. Statistical Analysis

Microstat7 for windows statistical package (Microstat

Co.) was used for statistical analysis of this study. One-

Way ANOVA was used to evaluate the mean values of

the Ki-67 in different types of ameloblastoma while in-

dependent “t” test was used to compare Ki-67 levels in

recurrent and non-recurrent cases.

Difference between mean values was considered sta-

tistically significant when p ≤ 0.05.

A. Hegab et al. / Open Journal of Stomatology 3 (2013) 520-526

522

OPEN ACCESS

3. RESULTS

This study was carried out on retrospective 32 cases of

mandibular ameloblastoma treated in oral and maxillofa-

cial surgery department-Al-Azhar university hospital

from the period from 2001 to Jan. 2009 with follow up

period until 2011. The male: female ratio was 3:1, with

24 (75%) males and 8 (25%) females. Age distribution

ranged from 21 to 78 years, with a mean age of 35.7

years. Most of the cases occurred in the second and third

decades of life. The specimens comprised 18 cases of

Follicular ameloblastoma (56.2%), 7 cases of Cystic

ameloblastoma (21.8%), 4 cases of Plexiform ameloblas-

toma (12.5%), and 3 cases of Acanthotic ameloblastoma

(9.4%). Tumours were predominantly located in the mo-

lar-angle and angle-ramus regions of the mandible (Ta-

ble 1).

Recurrence of the ameloblastoma occurred in 8 cases

out of 32. The recurrence was higher in male more than

female.

4 cases of the recurrence were plexiform ameloblas-

toma (100%) and the other 4 cases occurred in the Fol-

licular ameloblastoma (22%).

The results showed that cellular proliferative activity

varied within the ameloblastoma types. There was no

statistically significant difference in the Ki-67 Acan-

thotic, cystic and follicular types of ameloblastoma,

Table 1. Summary of clinico-pathologic finding of the studied cases.

No Age Sex Duration & Site Type Recurrence Ki-67

1 21 Y F 2Y/angle-ramus region Cystic ameloblastoma − 2

2 33 Y M 1Y/angle-ramus region Follicular ameloblastoma − 5

3 36 Y F 6M/molar-ramus region Follicular ameloblastoma − 4

4 30 Y M 18M/body-angle region Plexiform ameloblastoma + 13

5 23 Y M 6M/molar-angle region Cystic ameloblastoma − 2

6 42 Y F 1Y/body-angle region Follicular ameloblastoma + 6

7 36 Y M 1Y/molar region Cystic ameloblastoma − 4

8 28 Y F 6M/body-angle region Cystic ameloblastoma − 6

9 35 Y M 3Y/angle-ramus region Follicular ameloblastoma − 8

10 37 Y F 2Y/canine-premolar region Cystic ameloblastoma − 3

11 31 Y M 18M/molar-ramus region Cystic ameloblastoma − 2

12 78 Y F 10Y/symphysis-angle region Acanthotic ameloblastoma − 2

13 42 Y M 1Y/body molar region Follicular ameloblastoma + 10

14 38 Y F 6M/body molar region Follicular ameloblastoma − 9

15 29 Y F 2Y/molar angle region Follicular ameloblastoma − 3

16 37 Y M 1Y/Molar angle region Cystic ameloblastoma − 4

17 33 Y M 2Y/body region Follicular ameloblastoma − 6

18 24 Y M 1Y/body molar region Follicular ameloblastoma − 3

19 35 Y M 2Y/molar angle region Acanthotic ameloblastoma − 2

20 33 Y M 18M/molar angle region Follicular ameloblastoma − 2

21 42 Y M 6M/molar angle region Plexiform ameloblastoma + 20

22 29 Y M 1Y/body molar region Follicular ameloblastoma − 2

23 65 Y M 3Y/symphysis-molar region Follicular ameloblastoma − 6

24 31 Y M 1Y/molar angle region Follicular ameloblastoma − 4

25 37 Y M 2Y/molar angle region Follicular ameloblastoma − 5

26 29 Y M 1Y/molar angle region Follicular ameloblastoma + 15

27 41 Y M 3Y/body molar region Follicular ameloblastoma + 15

28 35 Y M 1Y/molar angle region Plexiform ameloblastoma + 30

29 36 Y M 2Y/molar angle region Acanthotic ameloblastoma − 6

30 28 Y M 6M/molar angle region Follicular ameloblastoma − 3

31 36 Y M 1Y/molar angle region Plexiform ameloblastoma + 16

32 32 Y M 2Y/molar angle region Follicular ameloblastoma − 4

A. Hegab et al. / Open Journal of Stomatology 3 (2013) 520-526 523

while plexiform type showed statistically significantly

higher levels than the other 3 types (Figure 1). The mean

Ki-67 labelling index (LI) of plexiform ameloblastoma

ranged from 13 to 30 with mean of 19.75 (Table 2 and

Figure 2).

Statistical analysis of the Ki-67 between the recurrent

and non-recurrent cases showed that, the mean values of

Ki-67 was statistically significantly higher in recurrent

than non-recurrent cases (Table 3 and Figure 3).

4. DISCUSSION

Ameloblastoma have been categorized broadly into 3

biologic variants: cystic (unicystic), solid, and peripheral.

(a) (b)

(e) (f)

(g)

Figure 1. (a) Lymphnode germinal center (Lymphoma ×400)

Ki-67 as a positive control; (b) Acanthotic ameloblastoma (H&

E); (c) Acanthotic ameloblastoma (×400), Ki-67 labeling index

reaching 6%; (d) Follicular ameloblastoma (H&E); (e) Follicu-

lar ameloblastoma Ki-67 labeling index reaching 8%; (f) Plexi-

form ameloblastoma (H&E); (g) Plexiform ameloblastoma

(×400) Ki-67 labeling index reaching 20%.

Table 2. Ki-67 means levels in ameloblastoma different types.

Mean St Dev

Acanthotic 3.33 2.31

Cystic 3.29 1.50

Follicular 6.11 3.94

Plexiform 19.75 7.41

Figure 2. Plexiform type showed statistically significantly

higher levels than the other 3 types.

Table 3. Independent “t” test results comparing Ki-67 levels in

recurrent and non-recurrent cases.

Mean ± St Dev

Non-recurrent Recurrent “t” value Probability

4.042 ± 1.99 15.63 ± 7.15 7.336 3.6E−08

Figure 3. The mean values of Ki-67 was statistically signifi-

cantly higher in recurrent than non-recurrent cases.

The literature indicates that the cystic variant is biologi-

cally less aggressive and has a better response to enucle-

ation or curettage than does the solid ameloblastoma [18,

19].

The clinical course in any given patient with an amelo-

blastoma is difficult to predict because there are few fea-

tures to aid the evaluation of the biological behavior of

the tumor. There is no clear evidence that histologic fea-

tures of ameloblastoma, such as follicular or plexiform

A. Hegab et al. / Open Journal of Stomatology 3 (2013) 520-526

524

patterns, determine the degree of invasiveness of indi-

vidual cases or allow prediction of likelihood of recur-

rence or malignant transformation [20]. The only prog-

nostically significant histologic factor is that unicystic

types are less aggressive especially if associated with

impacted molar teeth. Therefore, there is a need for a

predictive marker of clinical behavior inameloblastoma.

The proliferative activity of tumor cells has been

found to be an indicator of aggressiveness and to provide

useful prognostic in formation. Immunohistochemical

detection of the Ki-67antigen has provided a means of

evaluating turnout cell proliferation in a range of ma-

lignancies including carcinomas of the breast and the

head and neck [21-24].

Many investigations of tumor cell proliferative activ-

ity have used PCNA and Ki-67 in oral and other systemic

tumors. However, despite the years of intense research

on these relatively simple immunostaining techniques

and the routine use of such investigations in certain ma-

lignancies such as lymphomas, the routine application of

PCNA and Ki-67 in diagnostic histopathology of oral

tumors is as yet of little value [25].

Ki-67antigen is a more specific marker of proliferating

cells, maximally expressed during S phase, which is rap-

idly degraded after mitosis. The original antibody raised

against this 395 KD Ki-67 protein (pKi-67) is referred to

as Ki-67 antibody, a prototype for other antibodies that

also identify epitopes of pKi67, e.g., MIB-1 [26]. MIB-1

is an antibody that detects Ki-67 antigenin formalin-fixed,

paraffin-embedded tissue after antigen retrieval [27]. Ki-

67 is a nonhistone protein initially expressed in mid G1,

increasing in level through S and G2 and peaking in M.

It has been suggested that Ki-67 staining is more ac-

curate than the counting of mitoses or PCNA staining. Its

estimated half-life is 60 to 90 minutes [26]. However, not

all cells containing the Ki-67 antigen are actively prolif-

erating cells, and the use of Ki-67 to assess proliferative

activity is not advised in tissues over expressing p53 or

p21 [28]. It should further be noted that correlation be-

tween mitotic and Ki-67 labeling indices is variable [29-

34].

The Ki-67 labelling index was found to be prognosti-

cally relevant in several studies [16,17]. More recent stu-

dies using MIB1 paraffin antibody have confirmed its

prognostic value [35,36].

The results of the current study showed that; cellular-

proliferative activity as assessed by Ki-67 labeling indi-

ces varied within recurrent and non-recurrent cases of

ameloblastoma. There was a significant relation between

labeling index of nuclear proliferation marker Ki-67 and

recurrence of ameloblastoma. Also; recurrence rate was

highly significant in the plexiform ameloblastoma (100%)

which associated with the highest Ki-67 labelling index

followed by Follicular ameloblastoma (22%).

The result of Immunohistochemical data in the current

study indicated that the immuno expression of Ki-67 la-

beling index might be a good prognostic indicator for

proliferative activity and the local recurrence of man-

dibular ameloblastoma. The use of Ki-67 before surgical

excision of the ameloblastoma can be considered as an

important step in the treatment plan for evaluation of the

proliferative activity and subsequent the possibility of the

recurrence.

Even though, our results showed that both plexiform

and follicular types expressed the higher proliferative

activity and recurrence rate, but this cannot be consid-

ered as a role because of small sample size of our study.

Histologic types with high Ki-67 label index should be

treated with large safety margin to decrease the incidence

of recurrence.

5. CONCLUSION

The significant expression of Ki-67 could provide useful

prognostic markers for proliferative activity and good

prognostic indicators for recurrence rate of mandibular

ameloblastoma. Preoperative immunohistochemistry with

Ki-67 could be considered as an important step for the

treatment plan. Further studies on large sample size

should be considered.

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