International Journal of Oral & Maxillofacial Pathology. 2012;3(2):15-20 ISSN 2231 – 2250

Available online at http://www.journalgateway.com or www.ijomp.org

Research Article

Comparative Study of Exfoliated Oral Mucosal Cell Micronucleus Frequency in Potentially

Malignant and Malignant Lesions

Sarika Laxman Dindgire, Suchitra Gosavi, Ramniwas M. Kumawat, Sindhu Ganvir, Vinay Hazarey

Abstract

Objectives: 1. To observe and compare the micronuclei (MN) index in potentially malignant lesions / conditions and malignant cases. 2. To compare Papanicolaou (Pap) and May-Grunwald Giemsa (MGG) stain as two techniques to detect micronuclei in potentially malignant and malignant cases. Study design: Cytological smear of ten patients with habit of tobacco using in any form without any lesion, 20 smokers with potentially malignant lesions / conditions and 20 smokers with oral squamous cell carcinoma were studied using Pap and MGG staining method. The frequency of MN was determined under 40X. The mean MN count was compared using the ANOVA with Bonferroni test for statistical analysis. Results: Average MN frequencies were increased in potentially malignant lesions / conditions as compared to tobacco users without any lesion and further increased in oral squamous cell carcinomas. Pap stain is the preferred method in the field studies for scoring and detecting MN compared to MGG. Conclusion: The MN count can be used as a noninvasive early detection tool, for mass screening, patient education and to check for the efficacy of treatment.

Keywords: Micronuclei;Cytology;Papanicolaou;May-Grunwald Giemsa Stain;Preneoplastic

Conditions; Oral Squamous Cell Carcinoma.

Sarika Laxman Dindgire, Suchitra Gosavi, Ramniwas M. Kumawat, Sindhu Ganvir, Vinay Hazarey. Comparative Study of Exfoliated Oral Mucosal Cell Micronucleus Frequency in Potentially Malignant and Malignant Lesions. International Journal of Oral and Maxillofacial Pathology; 2012:3(2):15-20. ©International Journal of Oral and Maxillofacial Pathology. Published by Publishing Division, Celesta Software Private Limited. All Rights Reserved.

Received on: 14/11/2011 Accepted on: 27/02/2012

Introduction

Cancer, modern epidemic among non- communicable diseases is the second commonest cause of mortality in developed countries and remains one of the ten commonest causes of mortality in developing countries like India, is a complex disease with altered expression, abnormal growth and disruption of normal function of cells caused by genotoxic effects of chemical carcinogens or environmental pollutants resulting in genomic instability at an early stages of cancer, which is reflected often as leukoplakia, erythroplakia, Lichen planus, and submucous fibrosis. Oral cancer
is the 11th most common cancer worldwide and the most common in India.1
To evaluate the genotoxic risks / effects in the tobacco users on buccal mucosa, deoxyribo nucleic acid (DNA) damages can be assessed by chromosomal aberrations, sister chromatid exchanges and micronuclei
(MN) test.2 Exposure of a tissue to genotoxic carcinogens leads to an increase in chromosomal aberrations.3-6 Consistent with
this hypothesis, karyotypic anomalies and
elevated DNA content have been observed
in various preneoplastic lesions. Before particular karyotypic anomalies can be established, a long period of breakage and translocation of chromatid must occur. Such chromatid anomalies or chromosomal deletions can lead to formation of MN, which are DNA containing bodies in the cytoplasm without any structural connection to the main
nucleus.7 MN are small, extra-nuclear bodies separated from the main one, generated
during cellular division by late chromosomal fragments because of their association with chromosomal aberrations.6-8 The MN test is one of the current rapid, efficient and
economical techniques used as a indicator
of genotoxicity, as it provides a quantitative measures of the genotoxic action of carcinogens and mutagens.9-14
Micronuclei have been defined as a microscopically visible; round to oval cytoplasmic chromatin mass next to the nucleus.15 MN formation is the result of
segregation defects due to chromosomal instability causing chromatin to be excluded from the reforming nucleus.16 MN count has been proven to be a reliable biomarker for oral cancer risk.17-19

©2012 International Journal of Oral and Maxillofacial Pathology. Published by Publishing Division, Celesta Software Private Limited. All Rights Reserved

16 Sarika Laxman Dindgire et al., ISSN 2231 - 2250

Materials and Methods

Study sample: The study sample was collected from the outpatient department. All individuals gave a written consent for participation. All individuals were interviewed for type of habit, duration and intensity of habit, dietary habits, systemic and local disease history and family history.

The study sample was divided into three categories as follows:

Group I: Ten patients of tobacco users in

any form without any lesion.

Group II: Twenty patients of potentially

malignant lesions / conditions like submucous fibrosis, lichen planus, leukoplakia and erythroplakia. Out of 20 potentially malignant lesions / conditions, 9 (45%) patients had submucous fibrosis, 6 (30%) had leukoplakia, 3 (15%) had erythroplakia and the rest 2 (10%) had lichen planus. (Graph 1)

Group III: Twenty patients suffering from

different stages of oral squamous cell carcinoma confirmed by histopathological examination were included in our study.
and then stained with MGG stain (Fig 2).
500 cells per slide were counted under high power magnification (40X). Only cells which were not fragmented and not overlapping were counted. Five blind examiners carried out the count. Observations were recorded and tabulated. Collected data was subjected to ANOVA with Bonferroni test (Multiple comparison tests).

Figure 1: PAP stain showing MN (40X)

Erythropl

akia

15%

Lichen

planus

10%

Submuco us fibrosis

Leukoplak ia

30%

45%


Figure 2: MGG stain showing MN (40X)

Graph 1: Distribution of cases among potentially malignant lesions/conditions.

Procedure: Before sampling all individuals were asked to rinse the mouth thoroughly with tap water. Exfoliated cells were collected from the buccal mucosa on affected side using a clean premoistened wooden spatula. Scraped material was spread on the precleaned slides and smeared for each individual, two slides were prepared by smearing the oral scraping on the slides for a total of 100 (50 slides stained with Pap and 50 with MGG stain). For Pap stain, the smears were allowed to air dry and fixed with Biofix spray and then stained with Pap stain (Fig 1) and for MGG stain the slides were kept in freshly prepared fixative in the proportion of 3 parts of methanol and

1 part of glacial acetic acid for 20 minutes
Figure 3: MGG stain showing bacterial colonies and cell debris masking the MN completely (40X).

Results

Out of 40 referred cases of lesions, 20 were in the age group of 10-60 years presenting with various potentially malignant lesions / conditions and the rest 20 were in the similar

ISSN 2231 – 2250 Comparative Study of Exfoliated Oral Mucosal Cell..... 17

age group, presenting with oral squamous cell carcinoma. (Table 1) Mean MN index was 8.66 in erythroplakia cases and it was more in comparison with other potentially malignant lesions / conditions. (Table 2) There was a statistically significant difference in the mean MN count between the tobacco users without any lesion to potentially malignant lesions/ conditions to malignant cases. (Table 3)
condition (PMC) was statistically non- significant. (Table 5) Difference between mean MN count between group III with premalignant lesion and premalignant condition was statistically significant. (Table
6) There was statistically significant difference between group III with PML and PMC, but in difference between PML and PMC is non- significant. (Table 7)

Group I and II

P = 0.001

Significant

Group II and III

P = 0.017

Significant

Group I and III

P = 0.000

Highly

Significant

Table 4: The intercomparison analysis between different groups.
Table1: Age and Sex wise distribution of potentially malignant and malignant cases.

Group N Mean S.D.

Group I 10 2.8 1.229

PML 9 4.89 3.1

PMC 11 4.82 1.328

ANOVA

F p-value

3.501 0.058


Table 5: Comparison between Group I with
PML and PMC.
Table 2: The comparison of mean micronuclei index in potentially malignant lesion.
Table 6: Comparison between Group III with
PML and PMC.

(I)

(J)

P -

value

Significance

Group III

PML

0.001

Significant

Group III

PMC

0.001

Significant

PML

PMC

1

Non -

Significant

Table 3: Comparison of mean MN index between premalignant and malignant cases.
There was statistically significant difference between group I and group II, group II and III and highly significant difference between group I and III. (Table 4) Difference between mean MN count among group I with premalignant lesion (PML) and premalignant
Table 7: Statistical analysis between Group
II and Group III.

Discussion

Squamous cell carcinoma of the oral mucosa accounts for 90 - 95% of all oral malignancies.20 Oral exfoliative cytology has been used extensively for screening cellular
alteration. An accuracy of 95% and a reliability of more than 96% in detection of squamous cell carcinoma in mass screening

18 Sarika Laxman Dindgire et al., ISSN 2231 - 2250

have been reported in the literature.21 Oral exfoliative cytology can reveal various cellular alterations in squamous cell carcinoma. It includes karyorrhexis, karyolysis, micronucleus formation, pyknosis, binucleation, broken-egg nucleus,
anucleation, etc.22,23
Micronuclei in oral exfoliated cells is a marker of chromosomal damage caused by genotoxic agents from tobacco and tobacco- related substances, alcohol, etc.24 The
micronucleus assay has been used to assess the genotoxic damage in oral squamous cell carcinoma and oral
premalignancies.19,25
The present study was undertaken to identify a feasible and economical method which could be used as a screening test in high risk population for identifying the effects of genomic instabilities and to introduce timely interventional strategy in order to treat and control the epidemic. The present study shows both premalignant and malignant cases were found to be more prevalent in males which are in order with the findings
given by Butterworth26 in 2000. Maximum numbers among the males were in the age
group of 30-50 years and in the females in the age group of 31-40 years. In the present study, among patients with oral carcinoma
70% had consumed tobacco either in the
form of chewing/ smoking. Our findings are in accordance with the study conducted by Scully et al27 in 2000 who reported that 75% of the patients of oral carcinoma were tobacco users. Similar results were also observed by Matsui28 in 2006. Hence,
tobacco can be considered as a leading carcinogenic agent for causing DNA damage by its genotoxicity which leads to cancerous proliferation.
In the present study amongst all premalignant conditions, the erythroplakia cases had highest number of micronuclei because it has highest degree of epithelial dysplasia which, probably explains the observation made by many researchers as to why majority of erythroplakic lesions finally progressed to malignancy.
Mean micronuclei index shows count in tobacco users without any lesion was 2.230, in potentially malignant cases its 4.85 which to a large extent confirms the findings given
by Buajeeb et al29 in 2007 and in malignant cases it is 8.3. It suggest mean MN index was less in potentially malignant cases
compared with malignant cases, this might be due to the minimal dysplastic changes occurring in SMF and lichen planus.
In the present study comparison between Pap stain and MGG stain was done. The study showed that under field conditions Pap stain is more practical than MGG stain for staining MN. Regarding Pap stain, it is easier to read, easier to process and transport in the field, it is more practical, and the 95% ethyl alcohol used for fixation which is bactericidal effect and it also maintains the morphological integrity of cell. PAP consists of nuclear stain i.e. Haemotoxylin that stains all nuclear DNA, both intranuclear and extra- nuclear. It also consists of two counter stains that make the cytoplasm transparent and
cell boundaries well demarcated.30
However, there are certain disadvantages of MGG stain over PAP stain i. e. it is difficult to read, had bacterial colonies and cell debris masking the oral cells completely, so mean MN could not be detected properly (Fig 3). MGG stain requires slides to be air dried and
then placed in methanol (80%) at 00C, this was difficult to apply in the field and resulted
in difficulty in transport. So this study showed PAP stain is better than MGG stain for staining MN, this conclusion is consistent with studies by Roberts31 and Guzman et al.32

Conclusion

1. Micronucleus index was observed to be two folds more in malignant cases when compared with potentially malignant cases.
2. Obtained data shows significant number of micronuclei in potentially malignant lesions / conditions which can be used in
high risk populations as a screening test.
3. Pap stain is the preferred method in the field studies for scoring and detecting MN as compared to MGG.

Author Affiliations

1.Dr.Sarika Laxman Dindgire, Assistant Professor, 2.Dr.Mrs.Suchitra Gosavi, Associate Professor, 3.Dr.Ramniwas M Kumawat, Assistant Professor, 4.Dr.Mrs.Sindhu Ganvir, Professor and Head, 5.Dr.Vinay Hazarey, Dean and Professor, Department of Oral Pathology and Microbiology, Govt. Dental College and Hospital, Nagpur, Maharashtra, India.

Acknowledgement

We would like to thank the staff members of the oral pathology department for their support & cooperation.

ISSN 2231 – 2250 Comparative Study of Exfoliated Oral Mucosal Cell..... 19

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Author Correspondence

Dr. Sarika Laxman Dindgire, Assistant Professor,

Department of Oral Pathology and

Microbiology,

Govt. Dental College and Hospital, Nagpur, Maharashtra, India.

Email: sagarika1305@gmail.com

Ph: +91 9096464702

Source of Support: Nil, Conflict of Interest: None Declared.