DENTOALVEOLAR SURGERY

Does Smoking Affect the Ki67 and p53 Expressions in Asymptomatic Fully Impacted Lower Third Molar Follicles? Orc¸un Toptas¸, PhD,* Timuc¸in Baykul, PhD,y and Kayhan Bas¸ak, PhD, MDz Purpose:

Ki67 and p53 protein expressions are the most widely used markers to show the pathologic proliferation and early-stage tumoral alterations in vital tissues. The aim of this study was to compare Ki67 and p53 protein expressions in smokers’ and nonsmokers’ pericoronal follicles of asymptomatic impacted lower third molars (ILTMs).

Materials and Methods:

A cross-sectional study was planned. The study sample was derived from a population of patients who presented for evaluation and operative treatment of asymptomatic ILTMs. The predictor variable was smoking status, defined as smoker or nonsmoker. Outcome variables were Ki67 and p53 protein expressions in ILTM follicles. Other study variables were age, gender, tooth position, cigarette pack-year, epithelial layer staining, and inflammation. Independent-samples t test analyses were conducted with SPSS 10.0 (SPSS, Inc, Chicago, IL), with statistical significance set at a P value equal to .05.

Results:

The study sample was composed of 70 patients (35 in the smoker group) who contributed 60 follicles. There were statistical differences between the 2 groups for variables Ki67 and p53. Mean expression levels of Ki67 were 3.93  2.17 and 2.48  2.09, respectively, for smokers and nonsmokers (P = .011). Mean expression levels of p53 were 5.32  1.98 and 3.06  2.34, respectively, for smokers and nonsmokers (P = .000). Conclusion:

The present study showed that dental follicles of smokers have higher Ki67 and p53 protein expressions than nonsmokers’ follicles. Ó 2015 American Association of Oral and Maxillofacial Surgeons J Oral Maxillofac Surg 73:819-826, 2015

One of the common surgical procedures in dentistry is the removal of impacted third molars.1 Most impacted teeth are third molars,2 and the impaction incidence of lower third molars varies from 9.5 to 39% among different populations.3 There is still no general agreement among surgeons on the treatment of asymptomatic fully impacted third molars. Prophylactic removal of asymptomatic third molars has generated much discussion in dentistry.1 Asymptomatic means that there is no discomfort or pain from the impacted third molar, but it does not mean free of risk.4-6 Studies that have investigated asymptomatic impacted third molars have

adopted a follicular space smaller than 2.5 mm as radiologically asymptomatic.7,8 Pericoronal follicles of fully impacted third molars can lead to the development of cystic and tumoral alterations.9-12 Pathologic alterations of impacted lower third molars (ILTMs) are more frequently seen than of upper third molars.13 Dentigerous cysts, ameloblastomas, sulfur granules, and foreign body granulomas are the most commonly seen pathologic changes in dental follicles.13 Cell proliferation can be the precursor of pathologic changes, such as cystic alterations or squamous metaplasia, in dental follicle epithelium.14 Ki67 is a protein

*Assistant Professor, Department of Oral and Maxillofacial

Address correspondence and reprint requests to Prof Toptas¸:

Surgery, Faculty of Dentistry, Abant Izzet Baysal University, Bolu,

Department of Oral and Maxillofacial Surgery, Faculty of Dentistry,

Turkey.

Abant Izzet Baysal University, Bolu, Turkey; e-mail: toptasorcun@

yProfessor, Department of Oral and Maxillofacial Surgery, Faculty of Dentistry, S€ uleyman Demirel University, Isparta, Turkey.

yahoo.com Received February 26 2014

zPathologist, Department of Pathology, L€ utfi Kırdar Kartal

Accepted October 4 2014

Training and Research Hospital, I_stanbul, Turkey.

Ó 2015 American Association of Oral and Maxillofacial Surgeons

This study is supported financially by the S€ uleyman Demirel

0278-2391/14/01614-0

University Coordination Unit of Scientific Research Projects (Project

http://dx.doi.org/10.1016/j.joms.2014.10.015

number: 1773-D-09).

819

820 that increases in epithelial cell proliferation15 and has a vital role in cell proliferation, but this role has not been explained.16 Cell cycle analysis has shown that the Ki67 marker is expressed in G1, G2, and S phases in mitosis and is not expressed in resting cells.17 The Ki67 antibody can be used as a proliferation marker in human tumors in situ.18 Staining in active proliferation phases and detection convenience are the major reasons of the popularity of Ki67 as a cell proliferation marker.15 P53 is a transcription regulator gene and it is activated by cell damage. After DNA damage, the p53 antibody binds DNA directly and recognizes the damage. In this situation, the cell cycle can be stopped in G1 phase or apoptosis can be started by the p53 protein. Therefore, the absence, malfunction, or mutation of the p53 gene causes the proliferation of damaged cells. This uncontrolled proliferation could be the origin of tumors and malignant alterations.19,20 P53 is a tumor suppressor gene that increases in the nucleus at early stages of tumoral alterations.21 It also can be detected at early stages of cancer and some benign pathologies.22 A positive correlation between Ki67 and p53 has been shown in vital tissues by many researchers.23-25 Ki67 and p53 incidences increase in cysts, lichenoid lesions, impacted tooth follicles, and situations of proliferative changes in the oral mucosa.14,26-29 A positive correlation also has been indicated between tobacco smoke and Ki67 staining.30 Tobacco smoke contains many carcinogenic agents, and these agents can stimulate the cell proliferation and tumoral alteration chain.30-32 The purpose of this study was to compare Ki67 and p53 expression intensity and distribution in smokers’ and nonsmokers’ pericoronal follicles of asymptomatic ILTMs and to determine the effect of smoking on the pathologic potential of the dental follicles of ILTMs. The authors hypothesized that smoking could cause or accelerate pathologic alterations in dental follicles. The specific aims of the study were to 1) compare Ki67 expressions in dental follicles of ILTMs in smokers and nonsmokers, 2) compare p53 expressions in dental follicles of ILTMs in smokers and nonsmokers, and 3) show the correlation between Ki67 and p53 expressions.

SMOKING AND KI67 AND P53 EXPRESSIONS

pared according to basic principles of patient-oriented manuscript writing.33 STUDY DESIGN

To address the research purpose, the authors designed and implemented a cross-sectional study. The study population was composed of all patients presenting for evaluation and management of asymptomatic fully ILTMs. To be included in the study sample, patients had to be free from any systemic diseases and have radiologically and clinically asymptomatic fully ILTMs. Patients were excluded as study subjects if they had any symptom history (pain, swelling, etc) with the ILTM and any systemic disease. Follicular spaces of teeth were measured from orthopantomographs by each author without regard to the magnification factor reported by the manufacturer. Follicular spaces smaller than 2.5 mm were included in the study. Third molar surgeries were carried out under local anesthesia. STUDY VARIABLES

Smoking status was the predictor variable of this study. Ki67 and p53 protein expressions in ILTM follicles were the outcome variables. Ki67 and p53 expression intensity scores of samples were determined by multiplying the percentage of positively stained cells (0, no staining; 1, #1% stained cells; 2, 1 to 10% stained cells; 3, 10 to 33% stained cells; 4, 33 to 67% stained cells; 5, $67% stained cells) by staining intensity (0, no staining; 1, weak staining; 2, moderate staining; 3, intense staining; Figs 1-5).34 The mean Ki67 and

Materials and Methods This study was performed at the Department of Oral and Maxillofacial Surgery, Faculty of Dentistry, S€ uleyman Demirel University (Isparta, Turkey). The ethics committee of the Faculty of Medicine, S€ uleyman Demirel University approved the study (number 0314; April 16, 2008), which was conducted in accord with the Declaration of Helsinki. This report was pre-

FIGURE 1. Suprabasal staining indicating a moderate increase in Ki67 expression (original magnification, 400). Toptas¸, Baykul, and Bas¸ak. Smoking and Ki67 and p53 Expressions. J Oral Maxillofac Surg 2015.

TOPTAS¸, BAYKUL, AND BAS¸AK

821

FIGURE 4. Moderate intensity of p53 expression at the basal and suprabasal layers of the epithelium (original magnification, 400). FIGURE 2. Moderate basal Ki67 expression (original magnification, 400). Toptas¸, Baykul, and Bas¸ak. Smoking and Ki67 and p53 Expressions. J Oral Maxillofac Surg 2015.

p53 scores of the smoking and nonsmoking groups were compared by independent-samples t test. Age was another variable in this study. Patients were divided by age as younger than 22 years and at least 22 years old. The 2 groups were compared with by the Mann-Whitney U test. Gender was another variable. The effect of smoking status on Ki67 and p53 expressions in ILTM follicles was evaluated in male and female groups by c2 test. Tooth positions were classified as distoangular, vertical, mesioangular, and horizontal. Ki67 and p53 expression scores in ILTM follicles were compared in tooth position groups by c2 test.

Toptas¸, Baykul, and Bas¸ak. Smoking and Ki67 and p53 Expressions. J Oral Maxillofac Surg 2015.

Cigarette pack-years were calculated by multiplying smoking duration by the number of cigarettes per day. The effect of cigarette pack-year on Ki67 and p53 expressions in ILTM follicles was evaluated by correlation analysis. Epithelial layer staining levels of Ki67 and p53 were evaluated. Basal and suprabasal layer staining levels of the smoking group were compared with those of the nonsmoker group.

FIGURE 3. Weak Ki67 expression (original magnification, 400).

FIGURE 5. Weak intensity of p53 expression at the basal layer of the epithelium (original magnification, 400).

Toptas¸, Baykul, and Bas¸ak. Smoking and Ki67 and p53 Expressions. J Oral Maxillofac Surg 2015.

Toptas¸, Baykul, and Bas¸ak. Smoking and Ki67 and p53 Expressions. J Oral Maxillofac Surg 2015.

822 Inflammation in ILTM follicles was evaluated and scored (0, no inflammation; 1, mild inflammation; 2, moderate inflammation; 3, severe inflammation). Inflammation scores were compared with Ki67 and p53 expression scores by correlation analysis. Inflammation of ILTM follicles in the smoking and nonsmoking groups also was compared by c2 test. DATA COLLECTION METHODS

Specimens were fixed in 10% neutral buffered formalin and sent to the pathology laboratory for histopathologic and immunohistochemical analyses. Tissue samples were processed routinely and stained by hematoxylin and eosin and monoclonal antibodies to Ki67 and p53 (NCL-L-Ki67-MM1 and NCL-L-p53-DO7, respectively; Novocastra, Leica Biosystems Nussloch GmbH, Wetzlar, Germany). Smoking history was quantified by pack-years.35 DATA ANALYSES

Statistical analyses were conducted with SPSS 10.0 (SPSS, Inc, Chicago, IL), with statistical significance set at a P value equal to .05.

Results Seventy follicles were obtained from 35 smoking and 35 nonsmoking patients. After microscopic analyses, 10 follicles were excluded from the study because of a lack of epithelium. Sixty clinically and radiologically asymptomatic ILTM follicles were enrolled in the study (29 from the nonsmoker group and 31 from the smoker group). Patients’ ages ranged from 17 to 39 years (mean, 23.40  4.97 yr). Of the patients included, 56.7% were women and 43.3% were men (Table 1). In this study, the predictor variable was smoking status and the outcome variables were Ki67 and p53 staining intensities and distributions. Variables that could be related to the outcome and described the sample were age, gender, tooth position, cigarette pack-year, epithelial layer staining, and inflammation. The relation among pack-years (calculated by multiplying smoking duration by number of cigarettes per day), Ki67 scores, and p53 scores of the smoker group were analyzed and found to be statistically unimportant according to the correlation analyses (Table 2). The mean Ki67 and p53 scores of the 2 groups were compared with a c2 test to determine whether gender or position of the ILTM had an effect; the results were not statistically different (Table 2). The patients had been divided by age into 2 groups ( .05; Table 1).

Discussion The purpose of this study was to compare Ki67 and p53 expressions in smokers’ and nonsmokers’ pericoronal follicles of asymptomatic ILTMs and to determine the effect of smoking on the pathologic potential of ILTM dental follicles. The authors hypothesized that smoking could increase the pathologic potential of dental follicles. The specific aims of the study were to compare Ki67 and p53 expressions in dental follicles of ILTMs in smokers and nonsmokers and to show the correlation between Ki67 and p53 expressions. The results of this study showed that Ki67 and p53 expressions in the follicular epithelium of asymptomatic ILTMs was statistically higher in the smoker group than in the nonsmoker group and that there was a positive correlation between Ki67 and p53 expressions. The 2 age groups (

Does smoking affect the Ki67 and p53 expressions in asymptomatic fully impacted lower third molar follicles?

Ki67 and p53 protein expressions are the most widely used markers to show the pathologic proliferation and early-stage tumoral alterations in vital ti...
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