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  Table of Contents  
Year : 2015  |  Volume : 52  |  Issue : 1  |  Page : 75-79

Efficacy of argyrophilic nucleolar organizing region analysis using computer-assisted and manual in oral leukoplakia: A comparative study

1 Department of Oral Pathology, Chandra Dental college and Hospital, Barabanki, Lucknow, Uttar Pradesh, India
2 Department of Oral Pathology, Saraswati Dental College and Hospital, Lucknow, Uttar Pradesh, India
3 Department of Oral Pathology, Erstwhile KGMC- King Gorge Medical College, Lucknow, Uttar Pradesh, India

Date of Web Publication3-Feb-2016

Correspondence Address:
Kavita Nitish Garg
Department of Oral Pathology, Chandra Dental college and Hospital, Barabanki, Lucknow, Uttar Pradesh
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/0019-509X.175589

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 » Abstract 

Objective: Nucleolar organizer regions are loops of DNA containing ribosomal RNA genes and presumably are associated with ribosomal RNA activity, protein synthesis, and cell proliferation. Argyrophilic nucleolar organizer region (AgNOR) count has been suggested as an objective method in differentiating dysplastic lesions from non-dysplastic lesions. Materials And Methods: This descriptive study was done on archival paraffin blocks (n = 60), consisting of 10 normal human oral epithelium, 22 cases of non-dysplastic leukoplakia (NDLK), and 28 cases of dysplastic leukoplakia (DLK). The AgNORs were counted with the aid of a manual using conventional light microscopy and photographs of the same were taken and analyzed using Image Pro Express 6.0 (Media Cybernetic Inc., USA) for windows. Results: The mean AgNOR count per nucleus was found to be higher in patients with DLK as compared to NDLK and controls using both manual counting and image analysis method and on comparing both the techniques, image analysis provide a more accurate reflection of AgNOR counts than manual counting. Conclusion: To conclude, reliability of computerized image technique of AgNOR count is the most appropriate marker to differentiate between dysplastic and NDLK. Computer-assisted image analysis system was found to be an effective tool in achieving high reproducibility as compare to manual.

Keywords: Argyrophilic nucleolar organizer region, computerized image analysis, leukoplakia, nucleolar organizer regions

How to cite this article:
Garg KN, Raj V, Chandra S. Efficacy of argyrophilic nucleolar organizing region analysis using computer-assisted and manual in oral leukoplakia: A comparative study. Indian J Cancer 2015;52:75-9

How to cite this URL:
Garg KN, Raj V, Chandra S. Efficacy of argyrophilic nucleolar organizing region analysis using computer-assisted and manual in oral leukoplakia: A comparative study. Indian J Cancer [serial online] 2015 [cited 2022 May 17];52:75-9. Available from:

 » Introduction Top

Cancer is a major cause of disease and death throughout the world. Oropharyngeal cancer is the fifth most common cancer worldwide in men and seventh in women.[1] Oral cancer is one of the 10th most common cancers in the world and shows marked geographic differences in occurrence.[2]

The terms “precursor lesions,” “pre-cancer,” “pre-malignant,” “intraepithelial neoplasia,” and “potentially malignant” have been used in the literature to broadly describe clinical presentations that may have a potential to become cancer.[3] In present, WHO has recommended the term “potentially malignant disorders (PMD),” as it describes that not all lesions or conditions may transform to cancer, rather that there is a family of morphological alterations among which some may have an increased potential for malignant transformation.[4] The rate of malignant transformation of PMD in India ranges from 0.13% to 2.2% per year.[5]

Many oral squamous cell carcinomas (OSCCs) are preceded, or accompanied by oral potentially malignant lesions (PML), especially leukoplakia.[6]

Warnakulasuriya et al.,[4] in 2007 stated leukoplakia should be used to recognize as white plagues of questionable risk having excluded (other) known diseases or disorders that carry no increased risk for cancer.

Leukoplakia denotes a white lesion not attributable to any other diagnosis. Leukoplakia as a clinical entity may have varied histological presentation ranging from mildly hyperkeratotic lesions to those exhibiting severe dysplastic features. Many oral leukoplakias regress or stay quiescent, but many progress and are eventually excised, whereas between 3% and 6% turn into squamous cell carcinoma (SCC) in the future.[7]

Oral leukoplakia is a clinical term and the lesion has no specific histopathology. It may show atrophy or hyperplasia (acanthosis) and may or may not demonstrate epithelial dysplasia. It has a variable behavioral pattern but with an assessable tendency to malignant transformation. It must be noted that oral epithelial dysplasia has no specific clinical appearance and the term should not be used as a clinical descriptor of a white lesion.[4]

Nucleolar organizer regions (NORs) are loops of DNA containing ribosomal RNA genes. In man, they are located on the short arm of the five acrocentric chromosomes, i.e., chromosome numbers 13, 14, 15, 21, and 22[7] that presumably are associated with ribosomal RNA activity, protein synthesis, and cell proliferation.[8] NORs have recently been demonstrated in tissue sections by a silver colloid technique, and can be visualized as brown to black dots (argyrophilic nucleolar organizing region [AgNOR]).[9] It has, in general, been observed that the number of AgNOR sites in malignant cells are significantly greater than those in their normal or benign counterparts.[10] Several investigators suggest that quantification of AgNORs was associated with increased tumor aggressiveness; i.e., the mean number of AgNORs per nucleus was higher in malignant than in benign tissues and higher in high-grade than in low-grade malignancies.[8]

This study was designed to assess reliability of AgNOR counts using manual as well as image analysis in differentiating non-dysplastic leukoplakia (NDLK) and dysplastic leukoplakia (DLK).

 » Materials and Methods Top

The pathologist's assessment of dysplasia is still subjective. The terminology of dysplasia has been used for many years and has been adopted again by WHO in the latest version of Classification of tumors of the oral cavity and oropharynx.[3] However, dysplasia as a concept represents a spectrum of change rather than discrete identifiable stages.

The study group includes biopsy specimens from 50 cases of oral leukoplakia (22 non-dysplastic and 28 dysplastic) and 10 specimens from normal oral epithelium were taken as a control. Ethical clearance was taken from ethical committee of the institution. All specimens were collected from biopsy tissues in the archival of the Department of Oral Pathology and Microbiology, Saraswati Dental College and Hospital, Lucknow, India. The cases were diagnosed using routine hematoxylin and eosin-stained sections and these diagnoses were considered the correct/”gold standard” diagnosis for comparing with the AgNOR-based diagnoses.

The cases were divided into two groups. The first group, named as NDLK, consists of lesions diagnosed as hyperkeratosis, epithelial hyperplasia, and mild dysplasia. The second group, named as DLK, consists of moderate epithelial dysplasia, severe dysplasia, and carcinoma in situ.

Following this, the tissue sections were stained for AgNOR count which was done for each case.

AgNOR staining and counting

Method of AgNOR staining and counting

We used a method introduced by Ploton et al.,[11] in which 3 µm tissue sections were cut, deparaffinized in xylene, and dehydrated by 70%, 80%, and 90% alcohol consecutively. After being rinsed in deionized water, all slides were simultaneously placed in fresh silver nitrate solution and incubated in the dark for 30 min at room temperature. The silver nitrate solution was prepared by mixing two parts of 2% gelatin in 1% formic acid with one part of 50% aqueous silver nitrate solution. No counterstains were applied. The sections were then washed in running deionized water, dehydrated in ascending alcohol concentrations cleared in xylene, and mounted in Dibutyl Phathalate Xylene (DPX). The AgNORs were visualized as intranuclear brown to black dots of different sizes under light microscopy.

An oral and maxillofacial pathologist randomly assessed 100 cells in basal and parabasal regions of tissue sections on each slide under ×100 magnification. The AgNORs were counted with the aid of a manual using conventional light microscopy and photographs of the same were taken using Olympus live view digital SLR camera E-330. The photographs were analyzed using Image Pro Express 6.0 for windows (Media Cybernetics Inc. USA). Individually, discernible and separate black dots/blebs were recorded and the average number of dots in each nucleus was calculated. Where two or more dots are closely associated and are non-resorbable, they were counted as single AgNOR dot. The results were statistically analyzed for relationship between AgNOR count in normal oral epithelium, NDLK, and DLK using both manual and computerized analysis.

Statistical analysis

One-way analysis of variance (ANOVA) was performed as a F-test (df - degree of freedom) to compare the mean AgNOR counts between normal epithelium, NDLK, and DLK to determine whether there were any differences between the mean AgNOR counts of three groups. For statistical evaluation, a priori significance level was set at 0.05. Owing to unequal sample size, pairwise comparison test was performed. The statistical analysis was done using Statistical Package for Social Sciences (SPSS Inc. Chicago) Version 15.0 statistical analysis software.

 » Results Top

The number of AgNOR dots in each group via both techniques is illustrated in [Table 1]. The mean number of AgNOR for each group was as follows: Control (0.927 ± 0.870, 0.99 ± 0.08), NDLK (1.281 ± 0.133, 1.47 ± 0.20), and DLK (1.605 ± 0.321, 1.91 ± 0.53) via image analysis and manual technique, respectively. One-way ANOVA indicated a significant difference [P < 0.001; [Table 2] for both the techniques. The intergroup difference using Student 't' test and 'P' value also showed statistically significant differences between the following groups: Control versus leukoplakia groups and NDLK versus DLK (P < 0.001) via image analysis, whereas control versus leukoplakia groups (P < 0.001) and in NDLK and DLK (P = 0.001) through manual counting [Table 3]. With the correlation, overall good correlation between mean AgNOR and mean manual count was seen (r = 0.56; P < 0.001), this correlation was maximum (r = 0.93; P < 0.001) for normal group, while for dysplastic group, it was very poor (r = 0.086; P = 0.662) [Table 4].
Table 1: Mean argyrophilic nucleolar organizing region count/nucleus

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Table 2: One-way analysis of variance (ANOVA “F”) of nucleolar organizer regions dots quantification

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Table 3: Intergroup differences for argyrophilic nucleolar organizing region count

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Table 4: Correlation between mean argyrophilic nucleolar organizing region count (image analysis) and mean manual count

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 » Discussion Top

Oral cancer is one of the 10th most common cancers in the world and shows marked geographic differences in occurrence.[2] It is well established that virtually all SCCs are preceded by visible changes in oral mucosa, usually by the way of white (leukoplakia) to red patches (erythroplakia). It is believed that identification and monitoring of these PML and conditions allow clinician to detect and treat early intraepithelial stages of oral carcinogenesis such as epithelial dysplasia or carcinoma in situ, which usually precede the development of invasive OSCC.

Determination of dysplasia in leukoplakia is an extremely important issue in determining the treatment threshold. Clinical innocuousness of the lesion does not necessarily imply a histologically benign lesion. Criteria for determining the degree of maliciousness of the lesion has traditionally been subjective based on the dysplastic features. However, subjectivity plays a role because the features describing dysplasia, ranging from hyperplasia to multiple mitotic figures, all are treated evenly though they reflect significant different levels in the pathophysiology of the lesion. This ambiguity results in difference between oral pathologists in agreeing upon/diagnosing dysplastic lesions.[12]

The assessment of the potential malignancy of oral lesions clinically or from light microscopic examination of hematoxylin and eosin-stained tissue sections is not totally satisfactory. The epithelial dysplasia as judged by conventional criteria does not always signify that malignancy will result. The diagnosis and prognosis of such lesions are likely to be improved by examination of more specific cellular changes. One such method is the AgNOR technique, which is silver staining method, demonstrating NOR-associated proteins.[13] NORs are loops of DNA on the short arm of acrocentric chromosomes that presumably are associated with r-RNA activity, protein synthesis, and cell proliferation.[14] Quantitative changes of NORs can imply the degree of cell nucleolar activity in hyperplastic and neoplastic conditions.[15]

As seen from the results of this study, the mean AgNOR count per nucleus was found to be higher in patients with leukoplakia as compared to controls both with manual counting method and image analysis [Table 1], [Figure 1]. Within the leukoplakia group, the AgNOR counts were greater in those patients diagnosed having DLK than those having NDLK [Tables 1 and 2, P < 0.001]. These findings are in concordance with previous studies where a similar finding of increased AgNOR count has been reported in OSCC as compared to normal oral epithelium.[16] Our results are also very similar to those reported by Warnakulasuriya and Johnson,[17] Cano Montoya et al.,[18] and Chattopadhyay et al.,[19] who stated that mean AgNOR count increased gradually from normal epithelium to NDLK to DLK to SCC [7] and higher counts found in many carcinomas were due to dispersion and disaggregation of AgNORs within the nucleoplasm, and dysplastic lesions with higher and more dispersed counts represent those at greater risk of malignant transformation.[17],[19] The results of this study support the view that in dysplastic tissue, chromosomal disarray with multiple nucleoli appears to result in an increase in AgNORs, and higher AgNOR counts suggest poor prognosis in oral cancer.[18] Another finding evident in this study was that the mean counts obtained by image analysis were comparatively lower than those obtained by manual counting [Table 1] and [Table 2], [Figure 2], P < 0.001]. When correlation was derived between the two methods used for evaluating AgNOR counts, over all good correlation was found [Table 4], [Figure 2], P < 0.001]. The best correlation was found within the control group, whereas the correlation within the dysplastic group was poor (P = 0.662). The reasons for low counts obtained in image analysis could be due to the fact that non-specific silver particles could be counted as AgNOR dots in manual counting but the advantage of magnification of image and better resolution would lead to exclusion of these in an image analysis system. This option of image enhancing and magnification in image analysis helps in differentiating between specific and non-specific staining and hence small specific dots could also be counted. The advantage of high magnification of enhance resolution also helped in differentiating closed placed dots which on manual observation might have be counted as single dots [Figure 3] and [Figure 4].[20] Hence, image analysis may provide a more accurate reflection of AgNOR counts than manual counting. The lack of correlation between the manual counts and image analysis counts in dysplastic groups may be due to smaller and more numerous AgNOR dots in dysplastic cells, as found by Pandit and Aithal [14] which will increase the probability of mistaking between AgNOR dots and non-specific silver precipitate. Another finding which is evident in this study is that the dysplastic cells contain more AgNORs compared to non-dysplastic cells. These findings are in support of previous findings by Lo Muzio et al., who suggested that high-degree malignant lesions often contain more AgNORs than the corresponding low-degree forms.[21] Other authors have also suggested that the number of AgNORs may be related with the degree of malignancy of the lesion rather than simply representing cellular proliferation. The increase in AgNOR number could be the expression of an alteration of the mechanism controlling cellular proliferation and perhaps cellular differentiation. Hence, the mean number of AgNOR count in the oral mucosa could be indicative of the degree of malignancy and the absolute number of AgNORs could allow discrimination between lesions with low- and severe degree dysplasia.[21]
Figure 1: Comparison of argyrophilic nucleolar organizing region count via image analysis and manual method

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Figure 2: Correlation between mean argyrophilic nucleolar organizing region count and mean manual count

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Figure 3: Histological section stained with argyrophilic nucleolar organizing region (AgNOR) technique. AgNOR dots are seen as brown to black dots within the nuclei

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Figure 4: Computer aided analysis showing counting of argyrophilic nucleolar organizing region dots

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When the pairwise intergroup comparison in mean AgNOR counts per nucleus by manual as well as image analysis technique [Table 3] was made, it revealed statistically significant difference between the control and the leukoplakia patients (P < 0.001) as well as between NDLK and DLK (P < 0.001). These finding are consistent with those reported by Chottapadhay et al.,[7] who found an increase in counts of AgNOR from normal epithelium to leukoplakia without dysplasia to leukoplakia with dysplasia to SCC.[22] They suggested that higher AgNOR count in leukoplakia and more widely scattered dots in dysplastic lesions indicate a greater potential for malignant transformation.[7] Neoplastic cells generally exhibit a rise in the synthesis of normal and abnormal products and thus frequently feature of significant rise in AgNOR material. AgNOR counts rise with increase cell ploidy, increased transcriptional activity, and in stages of active cell proliferation. Moreover, AgNOR counts may be a good indicator of cell proliferation but rather represent variation in metabolic or transcriptional activity.[23]

In summary, AgNOR count manually as well as via image analysis increases as dysplastic changes increases. Also there is significantly greater increase in AgNOR count of dysplastic and NDLK as compared to normal epithelium. Hence, it was found that mean AgNOR count was the most reliable individual AgNOR-related parameter to differentiate DLK from NDLK. The results of the study also revealed a good correlation between AgNOR counts obtained by manual counting method and those obtained by image analysis.

To conclude, the AgNOR counts could be valuable tool for defining objective criteria for diagnosis/determination of dysplasia in oral leukoplakia. Computer-assisted image analysis system can be an effective tool in achieving high reproducibility in improving the diagnostic efficacy of AgNOR technique leading to early diagnosis and proper management of oral PML.

Computerized image analysis is more reliable method since the low counts of AgNOR obtained in image analysis could be due to the fact that non-specific silver particles could be counted as AgNOR dots in manual counting but the advantage of magnification of image and better resolution would lead to exclusion of these in an image analysis system and it also reduce the interobserver variability.

 » References Top

Bánóczy J, Gintner Z, Dombi C. Tobacco use and oral leukoplakia. J Dent Educ 2001;65:322-7.  Back to cited text no. 1
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World Health Organization. World Health Organization classification of tumours. In: Barnes L, Eveson JW, Reichart P, Sidransky D, editors. Pathology and Genetics Head and Neck Tumours. Lyon: International Agency for Research on Cancer (IARC) Press; 2005. p. 177-9.  Back to cited text no. 3
Warnakulasuriya S, Johnson NW, van der Waal I. Nomenclature and classification of potentially malignant disorders of the oral mucosa. J Oral Pathol Med 2007;36:575-80.  Back to cited text no. 4
Napier SS, Speight PM. Natural history of potentially malignant oral lesions and conditions: An overview of the literature. J Oral Pathol Med 2008;37:1-10.  Back to cited text no. 5
Van der Waal I, Schepman KP, van der Meij EH, Smeele LE. Oral leukoplakia: A clinicopathological review. Oral Oncol 1997;33:291-301.  Back to cited text no. 6
Chattopadhyay A, Chawda JG, Doshi JJ. Silver-binding nucleolar organizing regions: A study of oral leukoplakia and squamous cell carcinoma. Int J Oral Maxillofac Surg 1994;23:374-7.  Back to cited text no. 7
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Howat AJ, Giri DD, Cotton DW, Slater DN. Nucleolar organizer regions in Spitz nevi and malignant melanomas. Cancer 1989;63:474-8.  Back to cited text no. 9
Smith PJ, Skilbeck N, Harrison A, Crocker J. The effect of a series of fixatives on the AgNOR technique. J Pathol 1988;155:109-12.  Back to cited text no. 10
Ploton D, Menager M, Jeannesson P, Himber G, Pigeon F, Adnet JJ. Improvement in the staining and in the visualization of the argyrophilic proteins of the nucleolar organizer region at the optical level. Histochem J 1986;18:5-14.  Back to cited text no. 11
Warnakulasuriya S, Reibel J, Bouquot J, Dabelsteen E. Oral epithelial dysplasia classification systems: Predictive value, utility, weaknesses and scope for improvement. J Oral Pathol Med 2008;37:127-33.  Back to cited text no. 12
Schwarzacher HG, Wachtler F. Nucleolus organizer regions and nucleoli. Hum Genet 1983;63:89-99.  Back to cited text no. 13
Pandit S, Aithal D. A qualitative and quantitative estimation of AgNORS in dysplastic and non-dysplastic leukoplakias. Indian J Dent Res 2002;13:27-30.  Back to cited text no. 14
Allison RT, Spencer S. Nucleolar organiser regions in odontogenic cysts and ameloblastomas. Br J Biomed Sci 1993;50:309-12.  Back to cited text no. 15
Cabrini RL, Schwint AE, Mendez A, Femopase F, Lanfranchi H, Itoiz ME. Morphometric study of nucleolar organizer regions in human oral normal mucosa, papilloma and squamous cell carcinoma. J Oral Pathol Med 1992;21:275-9.  Back to cited text no. 16
Warnakulasuriya KA, Johnson NW. Nucleolar organiser region (NOR) distribution as a diagnostic marker in oral keratosis, dysplasia and squamous cell carcinoma. J Oral Pathol Med 1993;22:77-81.  Back to cited text no. 17
Cano Montoya LC, Alvarez Gómez GJ, Valencia Londoño WA, Ramírez España JA, Prada Navas CA. Analysis of the tissue marker AgNOR in leukoplaquia and oral squamous cell carcinoma. Med Oral 2002;7:17-21, 22-5.  Back to cited text no. 18
Chattopadhyay A, Ray JG, Caplan DJ. AgNOR count as objective marker for dysplastic features in oral leukoplakia. J Oral Pathol Med 2002;31:512-7.  Back to cited text no. 19
Raj V, Garg KN, Chandra S, Khare A, Bose A. Comparison of inter-observer variability between computer aided image analysis and manual counting of AgNORs in grading of oral leukoplakia: A double blind study. Asian J Oral Health Allied Sciences 2011;1:110-1.  Back to cited text no. 20
Lo Muzio L, Mignogna MD, Staibano S, de Vico G, Salvatore G, Damiano S, et al. Morphometric study of nucleolar organiser regions (AgNOR) in HPV-associated precancerous lesions and microinvasive carcinoma of the oral cavity. Oral Oncol 1997;33:247-59.  Back to cited text no. 21
Karabulut A, Reibel J, Therkildsen MH, Praetorius F, Nielsen HW, Dabelsteen E. Observer variability in the histologic assessment of oral premalignant lesions. J Oral Pathol Med 1995;24:198-200.  Back to cited text no. 22
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  [Figure 1], [Figure 2], [Figure 3], [Figure 4]

  [Table 1], [Table 2], [Table 3], [Table 4]


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