Journal of Cytology
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ORIGINAL ARTICLE Table of Contents   
Year : 2008  |  Volume : 25  |  Issue : 3  |  Page : 100-104
Evaluation of AgNOR scores in aspiration cytology smears of breast tumors

1 Department of Pathology, Jawaharlal Nehru Medical College, Aligarh Muslim University, Aligarh, India
2 Department of Radiotherapy, Jawaharlal Nehru Medical College, Aligarh Muslim University, Aligarh, India

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Aim: The aim of this study is to assess the difference in the silver-stained nucleolar organizer regions (AgNOR) scores on aspiration smears from benign and malignant breast tumors, and to determine the feasibility of AgNOR staining as part of the cytological investigation of breast tumors.
Settings: AgNOR scores were evaluated on aspiration smears and tissue sections from 42 breast tumor cases.
Materials and Methods: Smears from 27 breast tumor cases and tissue sections from 15 surgically resected specimens were examined. Counting was performed on a small number of tumor cells (50 cells). Both the mean (mAgNOR) score and the AgNOR proliferative index (pAgNOR index) were assessed for each specimen.
Statistical Analysis: Statistical evaluation was carried out using the Students' 't'-test. The result was considered significant when P < 0.05.
Results and Conclusions: The mAgNOR score and pAgNOR index were significantly higher in smears from malignant tumors than in those from benign tumors (P = 0.01). These results were similar to those obtained for tissue sections and were comparable to established data. The determination of AgNOR scores on cell smears in a limited cell population has practical advantages.

Keywords: AgNOR scores; breast tumors; cytology.

How to cite this article:
Ansari HA, Mehdi G, Maheshwari V, Siddiqui SA. Evaluation of AgNOR scores in aspiration cytology smears of breast tumors. J Cytol 2008;25:100-4

How to cite this URL:
Ansari HA, Mehdi G, Maheshwari V, Siddiqui SA. Evaluation of AgNOR scores in aspiration cytology smears of breast tumors. J Cytol [serial online] 2008 [cited 2023 Mar 23];25:100-4. Available from:

   Introduction Top

In keeping with the current interest in cell proliferation markers, Silver-stained nucleolar organizer regions (AgNOR) scores have been evaluated in a variety of lesions. Studies have shown a significant difference in the AgNOR scores for benign and malignant breast tumors. [1],[2],[3],[4] The utility of AgNOR scores lies in their assistance in the assessment of tumor proliferation rates and in understanding cell and tumour kinetics. In recent times, AgNOR analysis is carried out through standardized morphometry. However, such techniques are not widely available, especially in developing countries.

The manual evaluation of AgNOR scores is a cost-effective alternative to automated methods of evaluation. The staining technique [5] is a relatively simple and rapid one and can be applied to both aspiration smears and tissue sections. On the other hand, it is beset with certain difficulties which preclude its use in routine diagnostic work. [6],[7] Over/understaining of sections and variability in section thickness often lead to difficulties in counting the dots. [6],[7] Another significant hurdle is the tedious process of counting a large number of cells.

Cytology offers a medium for easy application of AgNOR scoring with a reduction in some of these difficulties. Therefore, in this study, we have tried to simplify the problems in counting a large number of tumor cells in tissue sections by applying the process to aspiration smears and by determining AgNOR values in a limited number of cells.

   Materials and Methods Top

The study was conducted on 42 patients who presented in the Surgical Outpatient Department with breast lumps or associated complaints. Twenty-seven cases were subjected to fine needle aspiration (FNA) and tissue sections were obtained from 15 cases who were treated surgically. A concise clinical history, examination, and details of relevant investigations were also obtained. These were helpful in reaching a probable clinical diagnosis as well as in the cytohistological evaluation and formulation of the pathological diagnosis. The AgNOR scores were evaluated after the diagnosis had been established.

FNA smears were stained with Papanicolaou stain while hematoxylin and eosin and AgNOR silver staining were applied to both smears and sections. Smears were examined using a 40X objective while a 100X objective was employed for sections. AgNOR scores were counted for 50 tumor cells in each smear and tissue section. Care was taken to ensure that only tumor cells were selected for this purpose and other accompanying cells ( e.g ., stromal and inflammatory cells) were excluded. Areas of necrosis, heavy inflammation, and marked cellular overlap were not evaluated.

AgNORs were identified as dark brown / black intranuclear dots, and the number and morphology of dots was determined in each nucleus. Clusters of several small AgNORs were counted as a single unit if the dots could not be discerned separately. The AgNOR score was expressed as the mean AgNOR count (mAgNOR) and pAgNOR (AgNOR proliferative index). The pAgNOR index was taken to be the percentage of cells with ≥ 5 AgNORs per nucleus. Statistical analysis of the observed values was carried out with the help of the Students' 't'-test. The result was considered significant when P < 0.05.

   Results Top

The study examined 25 benign breast tumors and 17 malignant tumors (Infiltrating ductal carcinoma, not otherwise specified). All patients were females except one male patient who had been diagnosed with gynecomastia. The peak incidence of benign tumors was in the second decade of life while malignancy was more frequent in the fourth decade. Benign tumors included fibroadenomas (15 cases), fibrocystic disease (nine cases), and one case of gynecomastia [Table 1]. AgNORs were visible as dark brown or black dots in the nucleus, either singly or in clusters. The dots were fine, round, discrete, and singly dispersed in benign tumors [Figure 1a] whereas the dots were larger, coarse, irregularly distributed, and tended to form clusters in malignancy [Figure 1b].

Benign lesions yielded a significantly lower mAgNOR count of 2.96 ± 0.72 as compared to a value of 4.0 ± 1.42 in malignant tumors ( P = 0.01). There was also a marked difference in pAgNOR values between the benign (14.1%) and malignant (36.6%) categories [Table 2]. The difference in the number and morphology of AgNOR dots in fibrocystic disease and infiltrating ductal carcinoma (NOS) is depicted in [Figure 2a] and [Figure 2b]. A similar observation was made with respect to mAgNOR values for tissue sections from benign (2.7 ± 0.47) and malignant tumors (6.1 ± 2.16). The difference in the pAgNOR indices was much more striking, with values of 10 and 70.9% being observed in benign and malignant conditions respectively [Table 2].

Among benign tumors, both fibroadenomas and fibrocystic disease yielded similar mAgNOR and pAgNOR values both for smears and tissue sections while the pAgNOR scores were found to be slightly higher in histopathological investigations for fibrocystic disease.

   Discussion Top

Breast tumors are one of the few pathological lesions where statistically significant differences have been found in AgNOR scores between benign and malignant tumors. [1],[2],[3],[4] Several older studies utilized mean AgNOR counts, alone [1],[2] or in conjunction with the AgNOR proliferative index. [8],[9] The assessment of AgNOR patterns has also been used in the evaluation of AgNOR counts in breast neoplasms. [10] Standardized morphometric analysis has enabled precise and rapid interpretation of NOR counts in recent times. [11],[12],[13]

Our study took into consideration both mean AgNOR scores and AgNOR proliferative indices besides the morphological appearance of AgNOR dots. The AgNORs were clearly visible in smears as brown/black intranuclear dots upon 500X magnification. This is an advantage over the use of tissue sections where clarity is achieved only on 1000X magnification and thin sections are essential. In order to simplify the tedious process of counting a large number of cells, we have evaluated the AgNOR scores in a limited number of tumor cells (50 cells), both for smears and sections. The results are similar to those determined on a large cell population. [1],[2],[14]

The mean number of AgNORs per nucleus (mAgNOR count) has been shown to represent changes in ploidy. [8] The AgNOR proliferative index (pAgNOR index) is the percentage of cells having more than a certain number of AgNORs per nucleus and it is considered to be a reflection of cell proliferation. [8] It therefore follows that the pAgNOR score should be a better and more consistent indicator of changes in the proliferative capacity of a breast tumor. This is evident from our observations. We determined the percentage of cells having ≥ 5 AgNORs per nucleus in both benign breast tumors as well as in infiltrating ductal carcinomas. This criterion has been utilized in other studies as well. [9],[15]

Benign breast tumors generally showed fine, round, and singly dispersed dots in the nucleus. The mAgNOR values obtained in our study are in concordance with the observations of other authors. [14],[16] There was no statistically significant difference in the mAgNOR values among the benign tumors, which is also in agreement with published data. [1],[2] We observed a slight increase in the pAgNOR scores in cases of fibrocystic disease as compared to fibroadenoma (in tissue sections). However, an overlap in individual values and high AgNOR counts in the single case of gynaecomastia in our study indicates that an absolute distinction cannot be made on the basis of AgNOR scores.

The morphology of silver-stained nucleolar regions (dots) in malignancy was at variance with benign lesions. Dots were large, coarse, often deeply stained, and had variable size and distribution; clusters were frequently observed.

Both mAgNOR and pAgNOR values were higher in aspiration smears as well as tissue sections from breast malignancies in our study. Similar observations have been made in previous studies (with reference to mAgNOR counts). [1],[2],[14] Although a degree of overlap was again present in the scores between benign and malignant lesions, the pAgNOR indices showed less overlap in contrast to mAgNOR values. Possibly, pAgNOR scores can serve as a better method of differentiating a malignant lesion from a benign tumor.

With regard to prognosis, an important observation has been made in a recent study that showed that the prognostic role of AgNORs in breast carcinoma is linked to the tumor suppressor genes, p53 and RB and not to the association of AgNORs with cell proliferation rates. [13]

The quality of AgNOR staining in tissue sections is dependent on the section thickness, which in turn, influences the incubation period. Section thickness also influences the accuracy in counting of dots. These problems can be overcome by the use of aspiration smears, wherein staining time is better controlled and the counting of dots becomes easier due to cellular dispersal. Moreover, the results are comparable to those obtained on tissue sections, and are in agreement with established data, even when a small number of cells are counted. This makes the process less cumbersome and is easily adapted to cytological investigations, although the counting of only a small number of cells may not be appropriate for sections. However,cytology has its own drawbacks. It is sometimes difficult to identify the cell type. Another problem is the clumping of cells into clusters and difficulty in counting clustered AgNOR dots. [16] More importantly, adequate sampling of representative areas is needed to ensure an accurate estimation, which is better achieved by the examination of tissue sections.

   Conclusions Top

Whereas an absolute distinction between benign and malignant breast tumors is not possible based on AgNOR scores alone, high values, especially of the pAgNOR index, are suggestive of malignant change. Based on its consistent association with cellular proliferative activity, the pAgNOR score can specifically be put to use as a cell proliferation marker. In addition, the overlap in the pAgNOR scores between benign and malignant lesions is less than that observed in the mAgNOR scores. Useful ancillary diagnostic support can be provided when AgNOR scores are studied in association with the AgNOR dot pattern. Nevertheless, cautious interpretation is essential due to the presence of overlap in the values of benign and malignant breast tumors. Where automated morphometric methods are not accessible, the manual evaluation of AgNOR scores can thus be considered as a cost-effective alternative. As shown in our study, the assessment of a limited number of cells in smears helps to simplify the process. Thus, cytology may be used as a rapid, relatively simple, and cost-effective method for AgNOR staining in breast tumors.

Whereas AgNOR scores do not independently provide definite information in relation to prognosis, it is proposed that they could contribute towards prognostication if considered in combination with specific clinico-morphological parameters. In this regard, further studies are required to evaluate the variation in AgNOR scores in breast malignancy cases after treatment, with special emphasis on cases with recurrence of the malignant lesion.

   References Top

1.Meehan SM, Magee H, Carney DN, Dervan PA. The diagnostic value of silver nucleolar organizer region assessment in breast cytology. Am J Clin Pathol 1994;101:689-93.  Back to cited text no. 1  [PUBMED]  
2.Rath-Wolfson L, Hamel I, Halpern M, Klein B, Gal R. Nucleolar organizer regions in breast cytology material. Acta Cytol 1995;39:852-7.  Back to cited text no. 2  [PUBMED]  
3.Kesari AL, Chellam VG, Nair PP, Madhavan J, Nair P, Nair MK, et al . Tumor proliferative fraction in infiltrating duct carcinoma. Gen Diagn Pathol 1997;143:219-24.  Back to cited text no. 3  [PUBMED]  
4.Bankfalvi A, Öfner D, Schmid KW, Schmitz KJ, Breukelmann D, Krech R, et al . Standardized in situ AgNOR analysis in breast pathology: diagnostic and cell kinetic implications. Pathol Res Pract 1999;195:219-29.  Back to cited text no. 4    
5.Chiu KY, Loke SL, Wong KK. Improved silver technique for showing nucleolar organizer regions in paraffin wax sections. J Clin Pathol 1989;42:992.  Back to cited text no. 5  [PUBMED]  [FULLTEXT]
6.Crocker J, Boldy DAR, Egan MJ. How should we count AgNORs? Proposals for a standardized approach. J Pathol 1989;158:185-8.  Back to cited text no. 6    
7.Underwood JCE, Giri DD. Nucleolar organizer regions as diagnostic discriminants for malignancy. J Pathol 1988;155:95-6.  Back to cited text no. 7  [PUBMED]  
8.Mourad WA, Erkman-Balis B, Livingstone S, Shoukri M, Cox CE, Nicosia SV, Rowlands DT Jr. Argyrophilic nucleolar organizer regions in breast carcinoma. Correlation with DNA flow cytometry, histopathology and lymph node status. Cancer 1992;69:1739-44.  Back to cited text no. 8    
9.Mourad WA, Setrakian S, Hales ML, Abdulla M, Trucco G. The argyrophilic nucleolar organizer regions in ductal carcinoma in situ of the breast. The significance of ploidy and proliferative activity analysis using this silver staining technique. Cancer 1994;74:1739-45.  Back to cited text no. 9    
10.Khanna AK, Ansari MA, Kumar M, Khanna A. Correlation between AgNOR count and subjective AgNOR pattern assessment score in cytology and histology of breast tumours. Anal Quant Cytol Histol 2001;23:388-94.   Back to cited text no. 10  [PUBMED]  
11.Bellotti M, Elsner B, Kahn A, Bezodnick L, Pisilli L, Greco P. Morphometric determination of AgNORs in breast carcinoma. Correlation with flow cytometry and proliferating cell nuclear antigen. Anal Quant Cytol Histol 1997;19:139-44.  Back to cited text no. 11    
12.Bankfalvi A, Giuffrθ G, Öfner D, Diallo R, Poremba C, Buchwalow IB, et al . Relationship between HER2 neu status and proliferation rate in breast cancer assessed by immunohistochemistry, fluorescence in situ hybridisation and standardised AgNOR analysis. Int J Oncol 2003;23:1285-92.  Back to cited text no. 12    
13.Derenzini M, Ceccarelli C, Santini D, Taffurelli M, Trerι D. The prognostic value of the AgNOR parameter in human breast cancer depends on the pRb and p53 status. J Clin Pathol 2004;57:755-61.   Back to cited text no. 13    
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Correspondence Address:
Hena A Ansari
Bait-Ul-Anwaar, Ahmad Gali, Medical Road, Aligarh Muslim University, Aligarh - 202 002, UP
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/0970-9371.44044

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  [Figure 1a], [Figure 1b], [Figure 2a], [Figure 2b]

  [Table 1], [Table 2]

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