REFERENCE ID: 2019-02956

Comparative study of cytomorphometric parameters of exfoliative buccal epithelial cells in

smokeless tobacco users with and without premalignant lesions-An in-vivo study

INTRODUCTION:

Environmental factors have proven to be contributing to etiology of cancers of orofacial region.

This can be attributed from the fact that prevalence of orofacial malignancies is seen more in

specific areas when compared to other areas of the same country or other other countries of the

world. Tobacco which is used by means other than smoking is termed as smokeless tobacco

[SLT]. It can be used as a snuff, either moist or dry which is usually inhaled through nose or can

be chewed. Smokeless tobacco is known to have harmful chemicals like nicotine which are

suspected to have a carcinogenic potential. Various Studies have shown that In Asian population,

the consumption of tobacco in chewable form is far more than any other form whereas snuff is

more commonly used in US [1]. Tobacco usage form show variations depending on age, gender

and socioeconomic status. In Southeast Asian population, smokeless form of tobacco is used on a

large scale, especially bywomen tobacco users.

Smokeless tobacco induces certain oral mucosal lesions which can present as: oral squamous

cell carcinoma (SCC), leukoplakia, erythroplakia, tobacco lime lesions, lichen planus or may

show some other form.

Along with all other factors, consumption of gutkha, paan, khaini, zarda, nas have known to

cause oral carcinoma [2].Studies done in Mumbai population have shown that the consumption

of gutkha in chewable form is seen in 70% college and 40% school students [2]. Even after the

ban imposed on gutkha by certain Indian states, it is actively being marketed everywhere.
Women in Indian city of wardha consume approximately 20% of gutkha, whereas it is 46.4% in

male population. [1].

One of the predisposing factors of occurrence of oral potentially malignant disorders is

smokeless tobacco which may further tranform to oral squamous cell carcinoma(OSCC).[1,3]

Early diagnosis and prompt intervention may prevent this transformation. Oral exfoliative

cytology is simple diagnostic technique for early revelation of premalignant and malignant

lesions.[1] It has been demonstrated that exfoliative cytology can also be used for assessment of

clinically suspicious lesions and carcinomatous lesions after definitive treatment.[4] Awareness

regarding the clinical signs and symptoms of OPMDs is very important among general

population and physicians to prevent the development of OSCC.

Formulation of diagnosis of oral malignancies can be made by using morphometry, which is

widely used for determination of quantitative parameters like nuclear size, cellular size, , optical

density, nuclear shape, nuclear texture, nuclear cytoplasmic ratio etc. The significant parameters

amongst all above stated ones are nuclear size, cellular size and ratio of nuclear cellular diameter

which occur in actively proliferating cells.[3] Papanicoloaou staining technique is used in

exfoliative cytology as it confers a varied colour to the cytoplasm of epithelial cells which is

based on the degree of differentiation of epithelial cells[2].

The present study was done to evaluate the changes produced by smokeless tobacco

consumption on buccal mucosal cells and comparatively assess the cytomorphometric

parameters obtained from buccal mucosal cells of smokeless tobacco users and non-users.
AIM:

The aim of this research was to comparatively study the cytomorphometric parameters of

exfoliative epithelial cells obtained from buccal mucosa in smokeless tobacco users with and

without premalignant lesions.

OBJECTIVES:

The objectives of this study were:

1. To assess the cytomorphometric alterations in exfoliated cells obtained from buccal mucosa

in smokeless tobacco users having premalignant lesions.

2. To assess the cytomorphometric alterations in exfoliated cells obtained from buccal mucosa

in smokeless tobacco users without premalignant lesions.

3. To comparatively assess the cytomorphometric alterations in exfoliated cells obtained from

buccal mucosa in smokeless tobacco users with and without having premalignant lesions.

REVIEW OF LITERATURE:

1. ALKA HARISH HANDE, MINAL S. CHAUDHARY used cytomorphometry to evaluate the

changes produced by tobacco on buccal mucosa. Comparison of cellular, nuclear sizes
and nuclear cellular size ratio was made in a study population of 125 subjects , which was
divided into 5 groups between 21-75 years those including : Groups N consisting of
normal subjects with no habit history, A consisting of tobacco users with no oral lesion,
B including subjects with tobacco lime lesion ,C including subjects with leukoplakia and
D including subjects with OSCC. Scrapings were obtained from buccal mucosa, of which
the smear was prepared on slide after fixing it. Cytomorphometric parameters were
analysed. “Univariate analysis of variance (ANOVA) showed a significant variation in
nuclear diameter, cellular diameter and ratio of nuclear diameter to cellular diameter.
Multiple comparison tests by Tukey–HSD procedure revealed a fall in the mean cellular
diameter, rise in the nuclear diameter and ratio of nuclear diameter to cellular diameter”.
It was concluded that cytomorphometric parameters can be used as earliest indicators of
malignancy.
 2. VERMA R, SINGH A, CHANDRA A, et al. used exfoliative cytology for detection of
premalignant and malignant lesions. The study was done on 90 subjects which were
divided into 3 groups: Groups with 30 subjects each of oral leukoplakia, OSCC
and clinically normal mucosa. Were made. Papanicolaou staining and
cytomorphometric assessment of keratinocytes was done. The tools used for
statistics were ANOVA, chi square test. The mean nuclear diameter of group A
was 65.47 while for group B, it was 107.97 and for group C, it was 139.02. The
cellular diameter were: group A(1535.80), group B(1078.51) and group
C(769.70). The N:C ratio of group A was 0.043,group B was 0.1 and group C
was 0.18. It was concluded that, cytomorphometry could be used as an effective
tool in diagnosing and determination of the prognosis of the lesions with
dysplasia which which may further progress to OSCC in future.

METHODOLOGY:

The study population was 75 individuals which were further categorized into 3 groups:

 GROUP A included 25 cases without tobacco chewing habit and with no oral mucosal

lesion.

 GROUP B included 25 cases having tobacco chewing habit but without any oral mucosal

lesion.

 GROUP C included 25 cases having tobacco chewing habit and oral mucosal

premalignant lesion.

Every subject’s detailed tobacco consumption history was taken. Selection criteria of subjects

for the study was : tobacco usage 4-5 times daily for a minimum period of 8 years. Group A

consisted of 25 subjects with no tobacco consumption history and no oral mucosal

premalignant lesion which was taken as the control group. Group B included individuals

having tobacco chewing habit but no oral mucosal lesion . Group C included individuals

having tobacco chewing habit and oral mucosal premalignant lesion. The premalignant

lesions included in the study were leukoplakia, erythr oplakia, leukoerythroplakia, lichen
 planus, tobacco pouch keratosis. An age group of 25-75 years was included and individuals

were chosen from both the genders. After taking the informed written consent, blood sample

and cytological smear was taken from the subjects. Venepuncture of all the subjects was

done for determination of hemoglobin levels. The exclusion criteria was as follows: for

female population: 11 gm/dl and for males population: 12 gm/dl.

Moistened cytobrush with normal saline was used for scraping of buccal mucosa. Scrapings were

taken from clinically normal appearing buccal mucosa in groups A and B. In group C, a

representative area of the lesion was scraped. A smear of area 2.5x2.5 cm was prepared on a

glass side from scrapings.spray fixative was used to fix the smear. All cytological smears were

stained using Papinicolaou stain. Cellular Diameter, nuclear diameter, and Nuclear-cellular

diameter ratio were calculated using an automated image analysis from 100 cells from each

smear. “One way analysis of variance (ANOVA) was performed for the three groups to compare

the mean of the parameters. Comparison of the mean values between the groups was made using

multiple comparison test by Tukey-HSD procedure, using the statistics package SPSS 10.0 for

Windows”

RESULTS:

Cellular Mean Std Deviation F- Value P Value

Diameter
A 1017.97 177.96 77.29 <0.001
B 784.65 79.06
C 560.35 52.13

Table 1: Comparison among all 3 groups with respect to Cellular Diameter by ANOVA
 Cellular Diameter Comparison P value
1200
1000 A vs B <0.001
800
A vs C <0.001
600
400
200 B vs C <0.001
0
A B C

TABLE 2: Comparison among all three groups with respect to Cellular Diameter by Post hoc

tukey Test.

“ANOVA as used and comparison between groups A, B and C with respect to various

cytomorphometric parameters was made using multiple comparison test by Tukey-HSD

procedure, using the statistics package SPSS 10.0 for Windows”. Tables 1 and 2 show the

comparison between groups A, B and C with respect to cellular diameter. When the mean

differences were compared between all the three groups, cellular diameter of group A was more

than group B and group C. The mean differences amongst all three groups were considered to be

statistically significant. (p<0.01) .

Nuclear Diameter Nuclear Mean Std F- Value P Value

120
diameter Deviation
A 66.26 9.14 38.99 <0.001
100
80
B 90.94 9.06
60
40
20 C 113.06 26.06
0
A B C

TABLE 3 : Comparison between groups with respect to nuclear diameter followed by Post hoc
tukey Test.
Table 3 shows the comparison made between the nuclear diameters of groups A, B and C. Group
C showed the highest nuclear diameter and the lowest was shown by group A. The comparisons
of mean differences with respect to nuclear diameter between all three groups was found to be
statistically significant (p<0.01).

Nuclear-Cellular diameter ratio

0.2

0.15

0.1

0.05

0
A B C

TABLE 5 : Comparison between groups with respect to nuclear-cellular diameter ratio by

ANOVA.

Comparison P value

A vs B <0.001
A vs C <0.001
B vs C <0.001

TABLE 6: Comparison between groups with respect to nuclear-cellular diameter ratio by Post
hoc tukey test.

Tables 5 and 6 show comparison of ratios of the nuclear cellular diameter between all three
groups. It was concluded that nuclear cellular diameter ratio of Group C was more than groups
A and B. The differences amongst all three groups were found to be statistically significant
(0.01).
DISCUSSION:

Tobacco consumption in any form can cause cancers of orofacial region. Oral cancer in

smokeless tobacco users is often seen in alveolar or buccal surfaces, the area where the quid is

kept. In Central Maharashtra rural population, tobacco mixed with lime, commonly referred to as

khaini is placed commonly in the mandibular canine premolar area.[8]. Intraorally, a yellowish

white, thick lesion is appreciable occasionally associated with loose tissue tags. It has been

suggested that the scraping off the surface layers of buccal mucosal cells may be possible

because of the caustic action of the mixture used which is approximately 8.3.They concluded that

tobacco pouch keratosis was four times more common than leukoplakia in central Maharashtra

rural population. During the transformation of a normal mucosal lesion into potentially

malignant disorder or cancer, the molecular changes or the microscopical changes appear earlier

than the appreciable clinical changes. Early diagnosis and prompt management at premalignant

stages could help us limit the debilitating status of the individual, morbidity and mortality rates

associated with OSCC. Screening of high risk patients, by clinical examination can be done at

early-stage disease, as it is curable and thereby improves the prognosis of the patient.[12].

The smear procured by exfoliative cytology can be qualitatively and quantitatively analysed.[4].

Accurate diagnosis can be made on the basis of different cytological parameters such as nuclear

diameter, cellular diameter, nuclear-to-cellular diameter ratio, nuclear shape, nuclear continuity

variations, texture of the nucleus .[17]. Nuclear diameter, cellular diameter and their ratio prove

to be the most accurate parameters for assessment of oral epithelial lesions [5, 6]. Exfoliative

cytology can also be used for assessment of post treatment malignant lesions and clinically
suspicious lesions.[3, 18]. Ramesh T et al. (1998) evaluated the nuclear and cellular diameters of

normal mucosal, dysplastic and malignant epithelial cells. “They found that cellular diameter in

normal mucosal cells was highest, lower in dysplastic lesions and lowest in malignant cells. On

the contrary, nuclear diameter was lowest in normal mucosa, higher in dysplastic lesions, and

highest in malignant cells”. It was suggested that alterations seen in cellular and nuclear

dimensions such as decreased cellular dimension and increased nuclear dimension are indicative

of early malignancy. Our study samples showed increased nuclear dimension, decreased cellular

dimension and also a fall in ratio of nuclear dimension to cellular dimension in groups B and C

compared to group A was observed. DNA content of the nucleus and the nuclear diameter

exhibit a direct relationship with each other. This is why nuclear diameter increases. These

observations suggested that smokeless tobacco may be the factor which produced significant

nuclear and cellular alterations in the B and C groups. In group B, that is tobacco users with no

buccal mucosal lesion, the oral mucosa seemed to be normal on clinical examination, the mean

difference of cellular size showed statistically significant variations in comparison with group C.

This study confirmed only the etiology–effect relation between smokeless tobacco consumption

and quantitative nuclear and cellular changes.

CONCLUSIONS:

This study emphasizes on the early recognition of cellular alterations in order to provide early

intervention so as the early transformation of OPMDs to OSCC is limited. Applying

quantitative techniques to the smears obtained from oral mucosal lesions showing premalignancy
and lesions which appear to be suspicious on clinical examination should be encouraged to

improve the overall quality of exfoliative cytology in making accurate diagnosis. Thus,

cytomorphology proves to be an invaluable parameter for the assessment of influence of tobacco

on buccal epithelial cells..

SUMMARY:

It was found that smokeless tobacco in any form proves to be one of the important etiologies in

causing precancer and cance as it produces significant changes on oral mucosa, which can be

evaluated by studying the cytomorphometric parameters of the oral epithelial cells. These

parameters show notable variations with respect to nuclear size, cellular size and their ratios

when compared with normal mucosa. Molecular changes start to occur comparatively earlier

than clinical changes. These molecular changes can be detected accurately using

cytomorphometry aiding in correct and early diagnosis. Cytomorphometric measurements

provide an objective method for evaluation of epithelial dysplasia and to predict their potential of

malignant transformation. If oral mucosal premalignant lesions are detected at earlier stages,

their potential of malignant transformation reduces. Early intervention of precancerous lesions

improve the prognosis of the condition limiting the morbidity and mortality rates.

Cytomorphometry is comparatively easy, less time consuming and cost effective.

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