| Abstract|| |
Background: Nucleolar organizer regions (NOR) are associated with proliferative activity and represent a diagnostic and prognostic marker.
Materials and Methods: Smears were taken from smokers, tobacco chewers, oral squamous cell carcinoma patients, and normal subjects and evaluated by 2 silver-staining nucleolar organizer region (AgNOR) counting methods: (1) mean number of AgNORs per nucleus (mAgNOR); and (2) percentage of nuclei with >3 and >5 AgNORs (pAgNOR).
Results: A statistically significant difference was observed between normal subjects, smokers, tobacco chewers, and oral cancer patients and between tobacco chewers with and without lesion. No significant difference was observed between tobacco chewers and smokers except in the percentage of >5 criteria.
Conclusions: AgNOR enumeration using noninvasive methods, such as the cytobrush appears to be useful technique in distinguishing between normal mucosa, mucosa with and without lesions exposed to carcinogens, such as tobacco and frank oral carcinoma.
Keywords: AgNOR method, cell proliferation rate, cytobrush, nucleolar organizer regions, tobacco
|How to cite this article:|
Sharma A, Saxena S. Quantification of AgNOR expression in exfoliated oral mucosal cells of tobacco chewers with and without lesion. Indian J Dent Res 2012;23:251-6
Squamous cell carcinoma of the head and neck is one of the most common cancers, currently holding the sixth position in the worldwide cancer statistics; and this could be even more common in the Indian scenario because of the common use of tissue abuse habits, such as smoking, smokeless tobacco, alcohol, gutkha, and others.  Oral cancer accounts for one third of the total cancer cases, and 90% of the patients are tobacco chewers and smokers.  Screening can reduce morbidity and mortality associated with oral cancers. The visual detection of oral cancers at initial stages is sometimes hindered by difficulty in differentiating the premalignant and malignant lesions from similar looking benign lesions. The most practiced technique for diagnosing oral squamous cell carcinoma (OSCC) is biopsy. But biopsy is an invasive technique compounded with the patient's reluctance for the procedure. On the other hand, exfoliative cytology is an easy, painless technique that could be beneficial for early diagnosis of oral cancers. Cytologic examination with cytobrush is a useful, noninvasive technique for detecting the early dysplastic changes and suspicious oral lesions.
|How to cite this URL:|
Sharma A, Saxena S. Quantification of AgNOR expression in exfoliated oral mucosal cells of tobacco chewers with and without lesion. Indian J Dent Res [serial online] 2012 [cited 2014 Nov 26];23:251-6. Available from: http://www.ijdr.in/text.asp?2012/23/2/251/100436
The behavior of a tumor is generally reflected by its proliferation rate. One such method of measuring the proliferation rate of a cell is the AgNOR, the silver-staining nucleolar organizer region technique. 
Considering this, the present study was contemplated to carry out a quantitative assessment of AgNORs in exfoliated cells from the oral mucosa of smokers and tobacco chewers, using cytobrush of brush biopsy and to compare the number of AgNORs in the mucosa of smokers, tobacco chewers with and without lesion, with OSCCs, and normal subjects.
| Materials and Methods|| |
This exfoliative cytologic study was carried out in the Department of Oral Pathology and Microbiology, Subharti Dental College, Meerut, India.
The study consisted of 55 subjects, 40 patients with a history of tobacco chewing/smoking, 10 normal healthy individuals, and 5 OSCC cases. Only male subjects ranging from 35 to 65 years of age were included to avoid any hormonal discrepancies. Smears were obtained from 3 different sites for each individual, thus comprising a total of 165 slides. The groups were as follows:
The smoker group comprised those who had smoked 10 or more bidis daily for at least 10 years or more. The tobacco chewer group comprised subjects who chewed more than 5 packets of tobacco daily for at least 5 years or more.
- Group I-20 were tobacco chewers, of whom 10 had tobacco pouch keratosis with dysplasia diagnosed by routine histopathology and 10 had normal oral mucosa.
- Group II-20 smokers were taken of whom 10 had leukoplakia diagnosed as hyperkeratosis with dysplasia by routine histopathology and 10 had normal oral mucosa.
- Group III-10 normal healthy individuals were taken after a thorough questionnaire and examination. These were subjects who had never smoked or chewed tobacco and had no other oral habits.
- Group IV-5 patients, diagnosed for OSCC.
A detailed case history was taken for each patient, including any serious illness or hospitalization in the last 2-3 years, and patients with any systemic disease were excluded from the study.
After obtaining detailed records and taking informed consent, the exfoliative cytology was performed for each subject. Smear was taken with a cytobrush (CDx Laboratories, USA) from 3 sites-the buccal mucosa, the floor of the mouth, and the palate, following which they were fixed with a biofixative spray. The fixed slides were subjected to AgNOR staining according to the method of Ploton et al and counted by the method of Crocker et al.
Principle of staining
Figure showing initial binding of silver to carboxyl group followed by continuing nucleation around sulfhydryl groups
- Nucleolar organizer regions are associated with histone and nonhistone proteins.
- The negatively charged carboxyl groups in the nonhistone proteins give the characteristic AgNOR reaction.
- In this reaction, the carboxyl groups initially bind to the silver ions.
- This binding reduces the silver ions to metallic silver submicroscopic deposits.
- This initial deposit acts as a focus for further deposition of silver, drawn from these electronegative silver groups.
- The sulfhydryl groups cause the development of microdeposits of silver into characteristic black dots visible at the light microscopic levels.
- The active sites formed by sulfhydryl groups and carboxyl groups are blocked by formalin due to the formation of methylene bridges, which are broken by alcohol. Thus the active groups are made available to the silver ions again.
AgNORs were counted in the first 50 non-overlapping cells in each slide, in a blinded fashion by 3 observers and an average score was calculated. The percentage of cells with more than 3 and more than 5 nuclei was also calculated. Significance of mean values of AgNOR and % AgNOR (>3, >5) between sites, habits, and lesions were tested using one-way analysis of variance after ascertaining the homogeneity of variance between the comparative groups. Tukey test was used where the variance between the groups was heterogenous. Student's t test was used where there were only 2 groups and Chi-square test was applied for the presence of lesions (%) with respect to site.
| Results|| |
AgNOR dots were seen as dark brown to black dots inside a brownish nucleus within a yellow cytoplasm. Comparisons were made between the different groups, between tobacco chewers with and without lesion, and among the different sites in the various groups with lesion.
A highly significant increase in the mean AgNOR values were observed in the OSCC group, smokers, and tobacco chewers as compared with normal (P<0.001). In the intergroup comparison, the cases of OSCC group exhibited a significantly higher mean AgNOR value as compared with smokers (P<0.001) and tobacco chewers (P<0.001). No significant change in the mean AgNOR was, however, observed in smokers as compared with tobacco chewers (P>0.001) [Table 1].
The data were also analyzed with respect to the percentage of cells with >3 and >5 AgNOR values in different groups by 3 different observers. A significant increase (P<0.001) in the percentage was observed in the OSCC patients, smokers, and tobacco chewers as compared with normal in both the percentage groups (>3 and >5). The OSCC cases exhibited a significant increase (P<0.001) in percentage in both >3 and >5 criteria as compared with smokers and tobacco chewers. No significant change (P>0.001) in the percentage AgNOR was observed in smokers as compared with tobacco chewers except in >5 category (P<0.05). It was interesting to note that the values were comparatively higher in tobacco chewers than smokers, although there was no statistical significance between the two [Table 2].
Comparison between tobacco chewers with and without lesions showed that the tobacco chewers with lesions had significantly higher mean AgNOR as compared with those without lesions (P<0.001). The increase in AgNOR counts in tobacco chewers with lesions was even more marked as compared with those without lesions in percentage of cells with >3 and >5 AgNORs (P<0.001) [Table 3].
Comparison of mean AgNOR between the 3 different sites, that is, floor of the mouth, buccal mucosa, and palate was done. A significant increase in the mean AgNOR was observed in buccal mucosa in smokers with lesions (P<0.001) as compared with floor of the mouth and palate. No significant difference in the mean AgNOR was observed in OSCC patients and tobacco chewers with leukoplakia, among the different sites (P>0.001) [Table 4].
|Table 4: Comparison of mean AgNOR at different sites among groups with lesion|
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Comparison of percentage of AgNOR (>3 and >5) at 3 different sites, floor of the mouth, buccal mucosa, and palate were also done. A highly significant increase in the percentage of AgNOR in >3 criteria was observed in the buccal mucosa in smokers with lesions as compared with those in floor of the mouth and palate (P<0.001). However, no such difference was observed in percentage at >5. Also, no significant difference in percentage was observed in the palate, buccal mucosa, and floor of the mouth in the OSCC patients and tobacco chewers with lesions (P>0.001). It was interesting to note that the values were highest in the buccal mucosa in all the groups [Table 5].
|Table 5: Comparison of percentage of cells with >3 AgNORs at different sites among groups with lesion|
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| Discussion|| |
Clinical examination and histopathologic studies of biopsied material are the classical diagnostic methods used for the diagnosis of oral cancerous lesions. Biopsy is an invasive technique, needs professional competence, and may cause psychologic implications for some patients. On the other hand, "exfoliative cytology" is an easy, painless technique that could also be beneficial for early diagnosis of oral cancer.
The reliability of oral exfoliative cytology has been questioned in many studies, but due to the advent of newer modifications it is now comparatively more reliable. Obtaining and sampling cells within the basal and parabasal region of the epithelium is central to the consistent and accurate diagnosis of early oral cancer, oropharyngeal cancer, and precancer. The superiority of the cytobrush with regard to sample collection from the basal layer of a given lesion has been proved by many studies as compared with other methods of exfoliative cytology. , In this study, all the smears collected by cytobrush of brush biopsy (procured by CDx Laboratories, USA) contained more than sufficient number of cells required for microscopic analysis. The behavior of a tumor is generally thought to reflect its growth rate, focusing interest on seeking a reliable guide to a patient's prognosis by measuring cell proliferation, and correlating these data with the tumor's known potential for metastasis and recurrence.
One such method of measuring the proliferation rate of a given cell is the AgNOR technique, which is a silver staining method, demonstrating NOR-associated proteins. 
Nucleolar organizer regions are parts of the genetic apparatus of the cell that aid toward the formation of the nucleoli. They are segments of chromosomes encoded for ribosomal RNA and are present on specific loops of DNA, located in the secondary constrictions of acrocentric chromosomes 13, 14, 15, 21, and 22. 
Nucleolar organizer regions have attracted much attention because of the claims that their frequency within the nuclei is significantly higher in malignant cells than in normal, reactive, or benign neoplastic cells. ,
The advantage of using exfoliated cells for AgNOR counting is that the whole cell can be examined, reducing the possibility of underestimating the AgNOR counts per nucleus. The risk of obscuring some AgNORs by superimposition and coalescence is minimal. 
The smears obtained for this research presented enough quantity and quality for counting, but obtaining a clear field was made difficult by what is believed to be a silver affinity to mucus, food residues, and other debris frequently present in the oral cavity, in spite of definite measures taken to avoid the above artifacts. Similar background staining has also been reported by Cancado et al in their evaluation of the NOR-associated proteins in exfoliative cytology of normal buccal mucosa. 
Many papers have reported about the diagnostic values of AgNOR staining in OSCCs using biopsy specimens. ,, However, only a few investigators have dealt with AgNOR on cytologic material (scrapings or brushings) of the oral cavity.
In the present study, the mean and percentage AgNOR value for normal subjects differed significantly (P<0.001) from OSCC patients, smokers, and tobacco chewers. The results of this study are in accordance with previous studies of Ghosh et al,  Cabrini et al,  Chattopadhyay et al,  Xin Xie et al,  and Derenzini et al,  that showed that the mean values of AgNOR for cancerous lesions were significantly higher than those of normal mucosa [Figure 1] and [Figure 2].
|Figure 1: Photomicrograph of normal mucosa showing well-defined 1-2 AgNOR dots per nucleus (×100)|
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|Figure 2: Photomicograph of oral squamous cell carcinoma patient showing bizarre multiple AgNOR dots per nucleus|
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On the other hand, Schulze et al,  observed in their study that there was no detectable increase in the number of NORs in cells of patients with malignant disease as compared with healthy adults. 
Leek et al,  in their study, found an increase in the size of AgNORs cluster rather than their number in proliferating tissues. To some extent it is likely that these parameters are different expressions of the same thing, increased expression of AgNOR proteins, larger AgNOR cluster simply represent coalescence of silver staining NORs that cannot be discerned individually. 
A significant difference was also observed between tobacco chewers/smokers and OSCC patients (P<0.001), but no significant difference was noted between smokers and tobacco chewers (P>0.001) [Figure 3] and [Figure 4]. This is the first time an exfoliative cytology has been done to compare AgNOR values between tobacco chewers and smokers, and tobacco chewers with and without lesion. Also, there is little reference in the literature using AgNOR technique in tobacco chewers.
|Figure 3: Photomicrograph of chewer with lesion showing 3-4 AgNOR dots per nucleus (×100)|
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|Figure 4: Photomicrograph of smoker with lesion showing 3-4 AgNOR dots per nucleus (×100)|
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No significant difference was noted between smokers and chewers, except in the >5 category where the values were statistically higher in tobacco chewers, which could be attributed to the direct contact of the carcinogens with the oral mucosa in case of chewers. Thus this study also attempts at proving the deleterious effects of tobacco on the oral mucosa.
Sampiao et al used AgNOR count in exfoliative cytology of normal buccal mucosa of smokers and nonsmokers and concluded that cigarette smoking influences proliferative activity in the cells of normal buccal mucosa. 
Comparison between tobacco chewers with and without lesions was also carried out, and a significant difference was noted (P<0.001) [Figure 5] and [Figure 3]. No correlation between the counts and the number of tobacco packets chewed or the number of cigarettes smoked was drawn in the present study.
|Figure 5: Photomicrograph of chewer without lesion showing 2-3 AgNOR dots per nucleus (×100)|
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This study also aimed at finding any significant difference between the 3 sites, namely, buccal mucosa, palate, and floor of the mouth among the different groups with lesions. A significant difference was observed in the buccal mucosa of smokers as compared with the palate and floor of the mouth (P<0.05). The only probable explanation drawn for this was that the buccal mucosa may be more exposed to the action of carcinogenic agents as compared with the palate or floor of the mouth.
A similar study was carried out by Paiva et al, using AgNOR in exfoliated cells from smokers and alcoholics from the mucosa of the lower lip, border of the tongue, and floor of the mouth. In his study, the border of tongue showed the least proliferative activity in comparison with the lower lip and floor of the mouth. 
Another observation in the present study was that the AgNOR dots in OSCC patients were bizarre in shape and more in number as compared with those in normal subjects. Similar findings were also obtained in the studies by Ghosh et al and Derenzini et al.
| Conclusion|| |
The prevalence of cancer would increase with habits, such as smoking and tobacco chewing. Early detection of premalignant and malignant lesions can significantly reduce the morbidity and mortality associated with oral cancer.
Exfoliative cytology coupled with AgNOR offers the Oral Pathologist a simple, rapid, and inexpensive method for early detection of premalignant and malignant lesions.
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Department of Oral Pathology, DJ Dental College, Modinagar
[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5]