|Year : 2014 | Volume
| Issue : 1 | Page : 15-20
Evaluation of diagnostic reliability of micronuclei in potentially malignant disorders of oral cavity
Sonal Grover1, Mujib BR Ahmed2, Neethu Telagi2, Anil B Shivappa2, KP Nithin2
1 Department of Oral Pathology and Microbiology, Christian Dental College, Christian Medical College, Ludhiana, Punjab, India
2 Department of Oral Pathology and Microbiology, Bapuji Dental College and Hospital, Davangere, Karnataka, India
|Date of Web Publication||11-Feb-2014|
Department of Oral Pathology and Microbiology, Christian Dental College, Christian Medical College, Ludhiana 141 008, Punjab
Source of Support: None, Conflict of Interest: None
Context: Micronucleus (MN) has been proved to be an important biomarker of genomic damage. MN scoring in oral exfoliated epithelial cells has been used in the past as a biomarker to identify different preneoplastic conditions much earlier than the manifestations of clinical features. However, no attention has been, until now, given to determine the overall accuracy of MN assay in diagnosing potentially malignant disorders (PMDs). Aim: The present study was being conducted to evaluate the diagnostic accuracy of MN assay in PMDs of oral cavity. Materials and Methods: Cytosmears from 30 controls with healthy mucosa and 45 cases as patients with suspicious lesions, clinically diagnosed as PMDs of the oral cavity were taken and separately stained with Papanicolaou (Pap) stain and haematoxylin and eosin (H and E) stain. MN frequency was evaluated and based on the 50 th percentile of all the MN (%) frequency values obtained for both the groups combined; a cut off value was calculated independently for each stain. Results: The sensitivity and specificity with Pap stain was found to be 84% and 93%, respectively, with diagnostic accuracy of 88%, whereas with H and E stain, sensitivity of 89% and specificity of 100% with diagnostic accuracy of 93% was observed. Conclusion: MN assay in oral exfoliated epithelial cells can be conveniently used as a biomarker for the screening of PMDs of oral cavity.
Keywords: Micronucleus, potentially malignant disorder, papanicolaou stain, haematoxylin and eosin stain
|How to cite this article:|
Grover S, Ahmed MB, Telagi N, Shivappa AB, Nithin K P. Evaluation of diagnostic reliability of micronuclei in potentially malignant disorders of oral cavity. CHRISMED J Health Res 2014;1:15-20
|How to cite this URL:|
Grover S, Ahmed MB, Telagi N, Shivappa AB, Nithin K P. Evaluation of diagnostic reliability of micronuclei in potentially malignant disorders of oral cavity. CHRISMED J Health Res [serial online] 2014 [cited 2017 Jul 27];1:15-20. Available from: http://www.cjhr.org/text.asp?2014/1/1/15/126776
| Introduction|| |
Oral cancer is a serious and growing problem in many parts of the globe. In high-risk countries such as Sri Lanka, India, Pakistan, and Bangladesh, oral cancer is the most common cancer in men and may contribute up to 25% of all new cases of cancer.  Many oral cancers are preceded by clinically evident premalignant mucosal changes that give a warning of risk and present an opportunity for detection and preventive measures. The key to diagnosis is the early detection of mucosal changes that may represent disease and which are not variations of normal.  Early detection of a premalignant or cancerous oral lesion promises to improve the survival and the morbidity of patients suffering from these conditions. 
A precancerous lesion consists of morphologically altered tissue that is more likely to be transformed into cancer than its normal counterpart, whereas a precancerous condition is a state associated with a significantly increased risk for cancer. In a recently held World Health Organization (WHO) workshop (2005), it has been recommended to abandon the distinction between precancerous lesions and precancerous conditions and to use the term potentially malignant disorders (PMDs) instead. 
Cytological study of oral cells is a nonaggressive technique that is well-accepted by the patient, and its application in the early diagnosis of PMDs is well-established.  Micronucleus (MN) is defined as microscopically visible, round, or oval cytoplasmic chromatin mass next to the nucleus.  Micronuclei (MNi) originate from aberrant mitoses and consist of eccentric chromosomes, chromatid fragments, or whole chromosomes that have failed to be incorporated into the daughter nuclei during mitosis.  MN has been proved to be an important biomarker of genomic damage. A considerable number of studies ,,,,,,, conducted in the past have confirmed a significant increase in MNi frequency in oral exfoliated epithelial cells of PMDs, as compared with normal healthy mucosa. Based on these findings, it was suggested that MN can be used as a biomarker in screening of PMDs. However, no attention has been, until now, given to determine the overall accuracy of MN assay in diagnosing PMDs.
Considering these facts, the present study was carried out to evaluate the diagnostic accuracy of MN assay in PMDs of oral cavity. Cytosmears from cases as patients with PMDs of oral cavity and controls as subjects with healthy normal mucosa were examined for evaluation and comparison of MNi frequency in the two groups.
| Materials and Methods|| |
Study sample consisted of 75 subjects and was divided into two groups as follows
Group I (Controls): Control group comprised of 30 healthy subjects with clinically normal oral mucosa.
Group II (Cases): Comprised of 45 patients [15 oral submucous fibrosis (OSMF), 15 lichen planus, and 15 leukoplakia] clinically diagnosed as having one of the PMDs of the oral cavity.
Relevant history of each patient, including their oral habits, was recorded thoroughly. Age- and sex-matched healthy subjects having no obvious oral lesions or habits of consumption of tobacco, other tobacco-related substances, or other such substances were selected as control group. Cases with confirmed histopathological diagnosis of PMD were only considered for MNi evaluation. Patients with provisional or confirmed diagnosis of any cancer were not included in the study. Written informed consents from these patients were taken for the procedures to be carried out on them. Two cytosmears were taken from each of subject included in the study.
Subjects were asked to rinse their mouth gently with water. Oral mucosal cells were scraped from lesional tissue in cases and from buccal mucosa of control group using a slightly moistened cytobrush (Medscand Medical AB, Malmo, Sweden). The cells were immediately smeared on precleaned microscopic slides. Just prior to drying, the smears were fixed with commercially available spray fixative (available with the RAPIDPAP TM kit) for 15 min.
Cytological staining and evaluation
The cytosmears were separately stained with Papanicolaou (Pap) stain and haematoxylin and eosin (H and E) stain. The slides were mounted with cover glass using DPX mountant. All the slides were observed under light microscope using low magnification (×400) for screening and high magnification (×1000) for counting of MNi.
The most commonly used method, that is, the zigzag method, was followed for screening of slides. One thousand cells with intact nuclei and cell boundaries were counted on each slide. For the purpose of designating an extranuclear body as a MN, the following criteria given by Tolbert et al., , were considered:
- Rounded smooth perimeter suggestive of a membrane
- Less than a third the diameter of the associated nucleus, but large enough to discern shape and color
- Staining intensity similar to that of the nucleus
- Texture similar to that of nucleus
- 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. The same person scored 1000 intact cells blindly in each case to determine the MNi percentage. The data obtained were statistically analyzed. Mann-Whitney test was applied for comparison of MNi frequency (%) between the cases and controls. P value of 0.05 or less was considered for statistical significance. Diagnostic validity test was applied, separately for both the stains. Based on the 50 th percentile of all the MNi (%) frequency values obtained for both the groups combined, a cut off value was calculated and sensitivity, specificity, positive predictive value, negative predictive value, and overall accuracy were calculated independently for each stain. The true and false positives and negatives were based on the following
Cytosmears from cases group with MNi frequency (%) greater than the cut off value.
Cytosmears from control group with MNi frequency (%) greater than the cut off value.
Cytosmears from cases group with MNi frequency (%) lesser than the cut off value.
Cytosmears from control group with MNi frequency (%) lesser than the cut off value.
| Results|| |
The present study comprised of total 75 subjects [Table 1] divided as 45 cases of clinically diagnosed PMDs of oral cavity (OSMF, lichen planus, and leukoplakia) and 30 healthy control subjects without any habits of consumption of tobacco, other tobacco-related substances, or other such substances.
The mean age of the controls was found to be 36.9 ± 10.1 years, whereas for PMDs it was 39.1 ± 15.1 years. The mean age of the subjects with OSMF was found to be 29.9 ± 10.0 years, for lichen planus it was 40.5 ± 15.6 years, and for leukoplakia it was 46.8 ± 14.9 years.
Among the group I (control), out of 30 subjects, 15 (50%) were males and 15 (50%) were females. In group II (cases), out of 45 subjects, 35 (77.8%) were males and 10 (22.2%) were females. Among the different PMDs, except for lichen planus (F-60%, M-40%), the same pattern of males predominance was seen in OSMF (M-100%, F-0%) as well as in leukoplakia (M-93.3%, F-6.7%).
|Table 1: The distribution of 75 subjects among controls and different potentially malignant disorders|
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In both the stains, statistically significant results (P < 0.05) were obtained, using Mann-Whitney test for comparison of MNi frequency between cases taken together and controls, as well as for separate comparison between each of the PMDs and controls [Table 2].
|Table 2: Comparison of micronuclei frequency (no. of micronuclei/1000 cells) between the cases and controls|
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The sensitivity and specificity with Pap stain [Figure 1] was found to be 84% and 93% respectively with diagnostic accuracy of 88%, whereas with H and E stain [Figure 2], sensitivity of 89% and specificity of 100% with diagnostic accuracy of 93% was observed [Table 3].1 and 3.2].
|Figure 1: Photomicrograph showing micronuclei (arrow) in oral exfoliated epithelial cell stained with Papanicolaou stain (×100)|
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|Figure 2: Photomicrograph showing micronuclei (arrow) in oral exfoliated epithelial cell stained with haematoxylin and eosin stain (×100)|
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| Discussion|| |
The global burden of cancer has continued to increase over the centuries. It is estimated that more than one million new cases are being detected annually in the Indian subcontinent. Ninety percent of all oral malignancies are oral squamous cell carcinomas (OSCCs). The 5-year survival rate for cancer is directly related to the stage at which the diagnosis is made. Prevention and early detection of a premalignant or cancerous oral lesion promises to improve the survival and the morbidity of patients suffering from these conditions. 
Virtually, all OSCCs arise from a premalignant precursor, but it is difficult to specifically define the term premalignant.  In a recently held WHO workshop (2005), a new terminology was introduced, to refer to all clinical presentations that carry a risk of cancer as ''potentially malignant disorders.''  In the present study, the most common of all the PMDs of the oral cavity was included as cases.
Biopsy is an invasive technique with surgical implications, technique limitations for professionals, and psychological implications for most patients. It also presents limitations when the lesions are large and in those cases it is important to select the most appropriate site for the biopsy. These issues underline the importance of discovering and developing new diagnostic methods, improving the existing ones, and discovering new therapeutic targets for oral neoplastic diseases. 
Exfoliative cytology was first designed for early detection of cervical cancer and it has been primarily applied in oral medicine practice to detect early changes in oral mucosa related to malignancy.  MN is the name given to the small nucleus that forms whenever a chromosome or a fragment of a chromosome is not incorporated into one of the daughter nuclei during cell division. There are two predominant mechanisms leading to the formation of MN in a mitotic cell: (1) chromosomal breakage (clastogenic effect) and (2) dysfunction of the spindle apparatus (aneugenic effect). ,
As a biomarker of genomic damage, MN has been proved to be an important upcoming marker of tumorogenesis.  Role of MNi in oral exfoliated epithelial cells in screening as well as grading of oral cancer is well-established by many investigators. ,,,,
According to literature, there are studies ,,,,,,, being conducted in the past, in which statistically significant difference was found, between mean percentage of MNi in PMDs and healthy subjects as controls. The same results were also observed in our study. This study is unique in itself, as this is the first study, according to the best of authors' knowledge, ever being conducted to evaluate the diagnostic accuracy of MNi frequency in PMDs.
Casartelli et al.,  concluded that the gradual increase in MNi frequency from normal mucosa to precancerous lesion to carcinoma suggested a link of this biomarker with neoplastic progression. According to Samanta and Dey,  the various possible explanations for MNi formation in preneoplastic conditions include chromosomal aberrations, chromosome loss/breakage, mitotic apparatus dysfunctions, aneuploidy, and genetic instability.
The exact role of MN in detecting the PMDs is probably the most challenging issue. Samanta and Dey,  explained several reasons for this. First, MN formation is generally considered as a manifestation of genetic damage or chromosomal breakage. However, many conditions like radiation, drugs, pollutants, even normal aging process may be responsible for MN formation. Of course, malignancy is both a cause as well as effect of genetic damage. So increased MN is suggestive but not diagnostic of preneoplastic condition and caution should be exercised in the form of meticulous clinical history and examination. Second, lack of standardization and information about the baseline MN frequency. It is a manifestation of day to day exposure to environmental pollutants, infections, nutrition, radiation, foods, and the genetic make-up or ethnicity which again varies around the globe. So, there must be an upper limit of the baseline MN frequency only beyond which we can label it as increased MN frequency. This calibration of the upper limit for a given population is one of the toughest issues.  Keeping these views in mind, the authors of the present study statistically found a cut off value and based on that made an attempt to calculate the diagnostic accuracy of MN assay in PMDs.
A considerable number of studies ,, have been done in the past, using Pap stain for evaluation of MNi, but there is hardly any optimal information available in the literature in context to the use of H and E stain for the same. Thus in this study, along with Pap stain, H and E stain was also used to explore, whether the latter can be used for precise evaluation of MNi frequency. To our surprise, the sensitivity, specificity, and diagnostic accuracy values obtained with H and E stain (89%, 100%, and 93%, respectively) were higher as compared with the ones seen with Pap stain (84%, 93%, and 88%, respectively). The explanation for a relatively higher diagnostic accuracy observed with H and E stain as compared with Pap stain warrants further detailed evaluation, which is beyond the scope of this study.
As established by many investigators, for confirmation of diagnosis of cancerous and precancerous lesions, a surgical excision followed by a histopathological diagnosis remains the gold standard.  Since optimal sensitivity and specificity values were obtained in this study, the authors hypothesize that MN assay in oral exfoliated epithelial cells might serve an appropriate diagnostic test for PMDs in future. However, until today, as this is the only study being done so far to evaluate its diagnostic accuracy, the authors are constrained to make any conclusion at this point of time. This study lays a foundation for other similar studies to be carried out in future, on larger populations to establish the definite significance of MN assay in diagnosis of PMDs. At the same time, authors also realize its limitation when compared with a biopsy, that this diagnostic test does not differentiate between the different PMDs but a histopathological examination does. It can only specify whether a particular lesion is a PMD or not. The important role of MN assay comes into play in screening of doubtful lesions, primarily misdiagnosed clinically as variations of normal mucosa. In case of such lesions, the clinicians often find themselves entrapped in dilemma to keep the patient under observation or to take a biopsy. Based on an increased MNi count observed in such lesions, a decision can be made to go ahead with biopsy for the confirmation of diagnosis.
PMDs of oral cavity are associated with an increased number of MNi in oral exfoliated epithelial cells. The diagnostic accuracy was found to be 88% with Pap stain and 93% with H and E stain, thus MN assay in oral exfoliated epithelial cells can be conveniently used as a biomarker for the screening of PMDs of oral cavity.
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[Figure 1], [Figure 2]
[Table 1], [Table 2], [Table 3]