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People’s Journal of Scientific Research / Volume 16 / Issue 1 / Jan-June 2023
Marking the Micro Details in Potentially Malignant
Disorders of Oral Cavity
Anushree Rathore1, Amit Kumar1, Mohammad Abu Bakr Siddique1, Rani
Mehrotra1, Deepak Singh Choudhary2, Ankit Gupta3
1Department of Oral Pathology and Microbiology, Bhabha College of Dental Sciences, Bhopal,
2Department of Dentistry, All India Institute of Medical Sciences (AIIMS), Bhopal, 3Department of
Oral Pathology and Microbiology, RKDF Dental College and Research Center, Bhopal
ABSTRACT:
Background- The malignancy of the oral cavity constitutes the most important group of malignancies
in South and Southeast Asia.The risk increases with the use of tobacco products.
Materials & Methods- Micronuclei frequency scoring was used as a biomarker to identify different
potentially malignant disorders.
Results- Mean micronuclei index was found higher using Hematoxylin and Eosin stain than
Papanicolaou's stainand May Grunwald's stain.
Conclusion- We concluded that the micronuclei frequencies in oral exfoliated epithelial cells using
three different stains- Hematoxylin and Eosin stain, Papanicolaou's stain, May Grunwald's stain may be
useful in predicting the malignant potential of premalignant lesions.
KEYWORDS: papanicolaou's stain; may grunwald's stain; micronuclei
Address for correspondence :Address for correspondence : Dr. Anushree Rathore, Reader, Department of Oral Pathology &
Microbiology, Bhabha College of Dental Sciences, Bhabha Campus, Jatkhedi, Bhopal-462026, Madhya Pradesh, E-mail:
anushreerahulsingh@gmail.com
Submitted: 14.06.2023, Accepted: 16.06.2023, Published: 26.06.2023
INTRODUCTION:
Oral cancer is still a major global health
concern even after major advance in understanding of
leading to focal growth and morphologic changes in
the early stages in cell transformation.[3] Accumulation
of genetic alterations within oral epithelial
carcinogenesis.[1] Tobacco consumption is the most cells/mucosa, induced by the genotoxins present in
common preventable attribute for oral cancer disease.
Tobacco-related disease kills approximately 6 million
people in world; the figure is estimated to reach around
8 million by 2030. India being one of highest tobacco
producer and consumer of tobacco-related products is
at a verge of risk of being the country with high oral
cancer patients. Tobacco paan masala, and tobacco
with pan and betel quid are the most consumed form of
SmokelessTobacco (SLT) in India.[2] SLT is strongly
associated with precursor lesions of oral cavity and
esophageal cancers.[3] Tobacco contains nicotine and
carcinogens, including nitrosamines (i.e., NNK and
tobacco-related products often lead to oral potentially
malignant disorders (PMDs).
These PMDs includes l eukoplakia,
erythroplakia, lichen planus and oral submucous
fibrosis, are known to have an increased risk of Oral
squamous cell carcinoma (OSCC). These lesions
harbor genomic abnormalities in the form of
m i c r o n u c l e i i n d i c a t i n g e n o t o x i t y i n o r a l
epithelium.The micronuclei index has become one of
the standard cytogenetic biomarkers used in cancer
biology. Many studies have indicated that there is an
increased micronuclei index in PMD which are
NNN). Nitrosamines cause epithelial cells' division procurers of OSCC.[4] Identifying the presence of
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How to cite this article: Rathore A, Kumar A, Siddique MAB, Mehrotra
|R, Choudhary DS, Gupta A. Marking the Micro Details in Potentially
Malignant Disorders of Oral Cavity. PJSR. 2023;16(1):20-25.
People’s Journal of Scientific Research / Volume 16 / Issue 1 / Jan-June 2023
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Rathore A et al. Micronuclei in PMDs
micronuclei in oral epithelial cells in early stage can
help clinicians in the initiation of preventive measures.
Exfoliative cytological study of oral cells is a
non-aggressive technique and its application in the
early diagnosis of PMDs is well-established. The assay
is reliable and technically easy to perform. Early
detection and early intervention of oral cancer can
prolong life expectancy and reduce the years of life lost,
indicating the importance of proactive screening and
oral hygiene.
Numerous DNA specific stains available are
used to study the micronuclei and chromosomal
abnormalities within the cells. Many investigators
investigated a variety of stains including DAPI,
acridine orange (AO), Hoechst, and propidium iodide,
Feulgen-Fast Green (FFG), May-Grunwald Giemsa
(Giemsa) and Papanicolaou (Pap). Feulgen-Fast Green
(FFG) which enable easy identification of Micronuclei,
making it clearly stand among other stains. However,
relatively lengthy procedures may result in theunder-
scoring of Micronuclei. Many comparative studies of
micronuclei using stains were performed in the past.
Casartelli et al. evaluated Hoechst, PI, and Giemsa, and
found Hoechst was the more reliable for staining and
identifying micronuclei.Giemsa, FFG, DAPI, and
acridine orange stains' comparison show that Giemsa is
more associated with falsification of micronuclei and
FFG is better than other stains.The Papanicolaou (Pap)
stain was the preferred method of detecting micronuclei
in oral epithelial cells when compared to Giemsa stain.
The fluorescent staining (acridine orange) was more
sensitive for micronuclei detection than FFG in oral
exfoliated cells in subjects of leukoplakia and
squamous cell carcinoma. Studies are needed to
determine whether some micronuclei and nuclei may
lose DNA through karyolysis while maintaining the
protein structure of chromatin and the nuclear
envelope, so that they would still be detectable by stains
that are not DNA-specific.
Looking at the present scenario, we decided to
evaluate micronuclei index in exfoliated cells using
Hematoxylin and Eosin stain (H&E), Papanicolaou's
stain (PAP), May Grunwald's stain (MGG) among the
OPMD's cell samples. The objective of study was: (a)to
determine micronuclei index among OPMD's and
OSCC, (b)to determine efficacy of H & E, PAP, MGG
stains for staining and identifying micronuclei, (c)
comparisons of H&E, PAP, MGG in OPMD's.
MATERIALS &METHODS:
The study was approved by the Research
ethical committee, PCDS & RC and study was
Microbiology. The subjects were pooled from the
outpatient department based on criteria and divided
into four groups as follows: (a) subjects withoral
s u b m u c o u s f i b r o s i s a n d l a t e r c o n f i r m e d
histopathologically as dysplastic lesions, (b) subjects
with clinical leukoplakia and later confirmed
histopathologically as dysplastic lesions, (c) subjects
with clinical lichen planus and later confirmed
histopathologically as dysplastic lesions(d) healthy
subjects without history or habit of SLT chewing or any
other form of tobacco consumption. Each group was
comprised of 20 subjects. Written consent was
obtained from subjects after explaining the purpose of
study.
Collection of sample and processing:
Subjects were asked to rinse the oral cavity
with saline before obtaining the cytology sample.
Standard exfoliative cytology procedure was used to
collect samples from site. The scrapes were taken with
the help of a sterile metal spatula. The sample was
transferred to a clean glass slide and fixed with alcohol.
These slides were dried for 5-10 minutes and stained
with H&E, PAP and MGG. All stained slides were
examined at 1000X magnification using oil immersion
objective under Binocular research Microscope
(Olympus CH 20i, Olympus, India). Minimum 5 slides
were prepared and screened for cell yield. Those with
greater cell yield were selected for evaluation and
counting of micronuclei. Minimum two slides for each
stain per subject were counted. Micronuclei were
counted as per Tolbert's criteria: (a) Rounded smooth
perimeter suggestive of a membrane. (b) Less than a
third the diameter of the associated nucleus, but large
enough to discern shape and color, (c) Staining
intensity like that of the nucleus, texture like that of
nucleus, (d) Same focal plane as nucleus and absence
of overlap with, or bridge to, the nucleus. Only those
structures fulfilling the above-mentioned criteria were
recorded as MNi (Figure 1). 720 slides were prepared,
and 1000 cell/slide were counted for micronuclei. 7,
20,000 cells were counted in total. The gathered data
was analyzed using unpaired t- test and chi-square test.
RESULTS:
The mean number of micronuclei were
observed using H&E, PAP and MGG stain in subjects
with OSMF. Mean micronuclei index was found high
(p<0.001) using H &E stain (12.20) than PAP (8.20)
and MGG (5.40)[Graph 1].The mean number of
micronuclei were observed using H&E, PAP and MGG
stain in subject with leukoplakia. Mean micronuclei
conducted at Department of Oral Pathology & index was found high (p<0.001) using H &E stain
Rathore A et al. Micronuclei in PMDs
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People’s Journal of Scientific Research / Volume 16 / Issue 1 / Jan-June 2023
Figure 1: Photomicrographs showing micronuclei (circle) in oral exfoliated epithelial cells (a: H and E, b: Pap, c: MGG, ×1000)
(18.00) than PAP (12.20) and MGG (5.80)
[ Graph 2].
The mean numbers of micronuclei were observed using
H&E, PAP and MGG stain in subject with Lichen
planus. Mean micronuclei index was found high
(p<0.001) using H &E stain (8.0) than PAP (5) and
MGG (2.2)[ Graph 3].The mean number of
micronuclei were observed using H&E, PAP and MGG
stain in subjects with Control subjects. Mean
micronuclei index was found high (p<0.001) using H
&E stain (2.40) than PAP (1.60) andMGG (0.40)[
Graph 4].The mean number of micronuclei were
observed using H&E stain in subjects with OSMF,
Leukoplakia, Lichen planus. Control Mean
micronuclei index was found high (p<0.001) in
Leukoplakia (12.20) when observed using H &E
stain [Graph 5].
DISCUSSION:
Oral cancer is still a health burden in the world
Graph 1: Mean no. of nuclei among three stains in OSMF patients.
even in the 20th century. Even though there are many
advances in detection and treatment, mortality and
morbidity rates are high in many body cancers.
Prevention is still the most effective way to reduce
mortality and morbidity rate in the world. However, to
detect and predict early premalignant changes in the
tissue is still a dilemma among the pathologists,
clinicians, and oncologists. The Etiology factor-based
People’s Journal of Scientific Research / Volume 16 / Issue 1 / Jan-June 2023
23
Rathore A et al. Micronuclei in PMDs
Graph 2: Mean no. of nuclie among all three stains in LEUKOPLAKIA
Patients.
Graph 3: Mean no. of nuclei among all three stains in LICHEN PLANUS
Patients.
approach is vague since carcinogenesis is a
multifactorial and continuous evolving process.
Oral cancer is preceded by OPMD's and has
variable rate of malignant transformation. Oral
Leukoplakia is a common potentially malignant
disorder affecting oral mucosa. The annual malignant
transformation ranges of leukoplakia, Lichen planus
and OSMF are 2% to 3%, 0.5%, 7.6 % respectively %
.[5,6]
Currently, i t ' s difficult to anticipate
transformation of OPMD's that can be implemented to
prevent such transformation.Cytology has been
reliable, convenient, low cost, effective primary
diagnostic testutilized all over the world. Cytological
changes such as cytoplasmic granulation, cellular
enlargement, vacuolization, pyknosis, binucleation,
karyorrhexis, karyolitic, micronucleation, nuclear
budding, nuclear enlargement is easily identified within
exfoliated cell samples. Recently, the Micronuclei
index has been suggested to be effective in cancer risk
prediction, screening, diagnosis, and monitoring
treatment progress[7].
Cytology for micronuclei index has been used
before to determine the genotoxicity due to SLT among
PMD's. Various environmental, chemical, occupational
Graph 4: Mean no. of nuclei among all three stains in CONTROL Patients.
Graph 5: Mean no. of nuclei among all four lesion by H & E STAINING.
factors, lifestyle habits, can produce these changes
among the oral mucosal cells.[8] Among them tobacco-
specific nitrosamines have been reported to be potent
mutagenic agents which are thought to be responsible
for the induction of chromosomal aberrations resulting
in production of micronuclei.
Tobacco users have high micronuclei index [9],
which also plays role in development of OSCC.[4] The
effect of tobacco on buccal cells shows that there is
chromosomal breakage and increase in DNA damage.
Buccal micronucleus cytome assay has been suggested
as a tool for biomonitoringDNA damage under HUMN
project.[10]
Our study has tried to resolve the issue of
micronuclei counting among OPMD's, viz. OSMF,
Leukoplakia, and Lichen planus. The present study
found that leukoplakia has higher (p<0.001)
micronuclei index as compared to the others. The
results were significant. This is in accordance with
what was observed by other researchers previously. [11,
12] The tobacco-related DNA damage, which was more
closely correlated with OSCC than OSMF and lichen
planus, is due to tobacco smoking.[13]
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People’s Journal of Scientific Research / Volume 16 / Issue 1 / Jan-June 2023
Rathore A et al. Micronuclei in PMDs
Areca nut, autoimmunity is usually associated with
OSMF and l ichen planus respectively. Our
observations are like Mahimkar et al.[14], indicating
increased DNA damage in leukoplakia.
Various specific stains and non-specific DNA
stains were used in evaluation of micronuclei in
Pathol. 1993;75:323-32.
4. Bradley G, Magalhaes MA, Hyrcza M. Mutational
signatures in oral cancer indicate a complex role for
tobacco smoke carcinogens. Oral Diseases. 2017.
5. Van der Waal I. Oral potentially malignant disorders: Is
malignant transformation predictable and preventable?
exfoliated buccal cells.[11,15] Our results were in Medicina Oral, Patología Oral Y CirugíaBucal, 2014;
accordance with Grover et al and Katarkar et al that
OPDM's show higher micronuclei counts than normal
subjects as well as higher micronuclei count was seen
with H &E stain. The micronuclei count in OPMD's
was 5-fold as compared to control whereas Bloching et
al.[16] and Katarkar et al[11] showed there was a 2.2 and
3.6-fold increase in the micronuclei frequency in
leukoplakia as compared to normal subjects.
CONCLUSION:
The micronuclei count assay has potential to
predict genotoxicity as well as malignant potential
among the premalignancies since there are no other
parameters available as of now. Our study concludes
that Micronuclei cont. index can be helpful in
predicting the malignant potential among the subjects
even with the help of Non-specific H & E stain. H&E
stain is routinely used, available and have easy
practicability. However, one needs to train themselves
in micronuclei counting procedure and acquire skills
for accurate identification.
Financial Support and sponsorship
Nil.
Conflicts of interest
There are no conflicts of interest.
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