Evaluation of AgNoR scores in aspiration cytology smears of breast lesions and their correlation with histopathology

Correspondence: Dr Niraj Nepal, MD Department of Pathology, Nobel Medical College, Biratnagar, Nepal Email: niraj_nepal2000@yahoo.com ,nepalniraj@gmail.com. Background: Breast cancer is the leading cause of cancer-related deaths in Asia. The number of intranuclear silver stained structures, termed AgNORs, is significantly higher in malignant cells than in normal, reactive or benign cells. The main purpose of this study was to evaluate the AgNOR scores in aspiration cytology smears of breast lesions and their correlation with histopathology.

malignant cells than in normal, reactive or benign cells.
The development and continued growth of cancers involves altered rates of cell proliferation.In early breast cancer, measurement of proliferation can be used in conjunction with tumor size, grade, nodal status and steroid receptor status as a prognostic indicator.Proliferation rates can provide useful information on prognosis and aggressiveness of individual cancers and can be used to guide treatment protocols in clinical practice.Various techniques have been developed to evaluate and quantify proliferation rates in the laboratory such as mitotic activity, the S-phase fraction (SPF), Ki67 labeling index, MIB, proliferating cell nuclear antigen (PCNA), and other methods such as argyrophilic nucleolar organiser regions. 1 The main purpose of this study was to evaluate the AgNOR scores in aspiration cytology smears of breast lesions and their co-relation with histopathology.

MATERIALS AND METHODS
This was a prospective cross-sectional hospital based study conducted in the department of Pathology, Manipal Teaching Hospital, Pokhara over a period of 24 months from October, 2009 to 2011.A total of 40 cases were included in the study, which included fine needle aspiration and biopsy.All women above the age of 15, with proper clinical history and adequate material on fine needle aspiration cytology (FNAC) and biospy (lumpectomy/mastectomy) were included in the study.In all cases of FNAC samples, the slides were stained with Leishman stain and silver staining for AgNORs.Similarly histological specimens were processed routinely and stained with H&E and silver staining for AgNORs.AgNOR stained slides were analyzed for AgNOR dots.
Comparisons of the cytological and histopathological findings were made with AgNOR dots and were evaluated and counted in the slides taken as 50 cells' mean number and pattern.The mean number of dots was counted in both malignant and benign cells of both FNA and biopsy slides.The AgNOR dots morphology were divided into four groups: a) Spherical, irregular, b) aggregate and c) scattered.SAPA score was also performed and every case was scored according to the scoring system. 2 Scores were assigned by keeping in mind the estimated number of dots, the size, and shape of dots, clusters and their variations form cell to cell.SAPA was estimated and found to be higher in malignant cases as compared to benign breast lesions.

RESULTS
A total 40 cases of breast lesions were included in the study.The age range was from 17 to 90 years.Highest number of breast cancer was observed in the group of 40-49 years, followed by 50-59 years.There were 29 cases of malignant breast tumors and 11 cases benign breast tumors.Benign cases included fibroadenoma (n=7, 73%), fibrocystic disease (n= 3, 20%) and acute mastitis (n= 1, 7%).
Of the 29 malignant cases diagnosed in FNAC, 20 cases were operated and sent for histopathological evaluation.Amongst the 20/29 cases of malignant breast tumors, 18 cases were of Invasive carcinoma, No special type, 1 case of mucinous    Nepal N et al.  carcinoma and 1 case was of medullary carcinoma.In all 18 cases 100 % correlation was observed in both histological and FNAC.Amongst the 19 histopathological graded cases, grade 1 was the highest (n= 11), followed by grade 2 (n= 7) grade 3 (n=1).
AgNOR counts were performed in 40 cases of FNAC out of which 29 cases of malignant breast lesions and 11 cases of benign breast lesions.AgNOR counting's were also performed in 7 cases of benign breast lesions (fig. 1) and 20 cases of biopsy slides of malignant breast (fig.2).
The mean AgNOR counts were The AgNOR dots morphology were homogenous symmetric and had regular contours in both FNAC and histopathology slides of benign breast lesion.In the case of malignant breast lesions, the dots were asymmetric and had irregular contours.They were aggregated, smaller and more scattered.SAPA score was also performed and every case was scored according to the scoring system and was found to be significant in differentiating benign and malignant lesions (Table 6).

DISCUSSION
AgNOR technique is implicated to show the difference between benign and malignant cells of the particular lesions as they can be easily demonstrated in routinely processed cytological and histological sections. 3AgNOR staining in aspiration smears of the breast is simple and feasible technique.There is very good discrimination between benign and malignant lesions. 4jority of the dots were homogenous symmetric and had regular contours in both FNA and HP slides.The mAgNOR value obtained in our study was 1.734.There was not much difference in AgNOR counts of different types of benign lesions.We observed a slight increase in the AgNOR scores in cases of fibrocystic disease as compared to fibroadenoma and acute mastitis.The mAgNOR values obtained in our study are in concordance with the observations of other authors. 5,6 a study done by Ansari H et al observed a slight increase in cases of fibrocystic breast disease in comparison to fibroadenoma. 5,6Dube MK and Govil A 7 , in their study have also reported a similar AgNOR count of 1.2 with the range of 1.0-1.5, whereas Rajeevan K et al 4 , have reported AgNOR count of 2.8 with the range of 0.7-3.0.Their study stated that distribution of AgNOR counts in different types of benign lesions did not show significant differences.
In the case of malignant breast lesions, the dots were asymmetric and had irregular contours.They were aggregated, smaller and more scattered.The AgNOR counts tend to increase with increasing nuclear grades of malignancy.In our study the AgNORs were significantly higher in the malignant cases, being 4.508 with the range of 3.3 -6.04.
Ansari et al 8 also have reported mean AgNOR count of of 4.0.Simha A et al 9 , Kim A et al 10 and Kumar A et al 11 , have also reported a mean AgNOR count for malignant lesion as 3.5, 5.09 and 6.57 respectively.These studies show a definite discrimination between benign and malignant lesions, but some studies have reported the overlap of the values. 4The overlap was reported to be due to occasional malignant cases with low AgNOR counts and occasional benign cases with high counts. 4However we had no overlap of AgNOR count in our study.
Amongst the different histological types of carcinoma in our study, medullary carcinoma showed lower mAgNOR of 3.5 as compared to IDC showing highest of 5.68.
In a comparative study of benign and malignant breast lesions in FNAC, there was a significant difference of mAgNOR with a p value of less than 0.001 which was statistically significant.Similar finding was observed by other study. 12milarly in histopathology specimens the mean AgNOR counts for benign cases were 2.16 whereas it was 5.84 for malignant breast lesions.Similar findings were observed by other studies. 4,12In this present study, AgNOR counts were correlated with tumor grade in malignant breast lesions.The mean AgNOR count for grade-I was 4.2079 and grade-II was 4.6971.
Kazuhiko H et al 13 ,carried out a study and analyzed AgNOR in a total of 76 patients with breast carcinoma that included 33 cases of IDC in which the mean AgNOR count for grade-I was 4.71 +/-1.17, in grade-II it was 4.38 +/-1.41 and in grade-III it was 5.42 +/-1.63.No significant difference between the various grades was in his and our studies.
Regarding the polymorphism of AgNOR dots, in our study, the shape of AgNOR dots in benign and malignant lesions showed different patterns.Dots were homogenous, symmetric and had regular contours in the cases of benign breast lesions.Whereas, in the case of malignant breast lesions, the dots were asymmetric and had irregular contours, they were aggregated, smaller and more scattered.
Khanna AK et al studied 24 cases of carcinoma and found that SAPA as an important diagnostic tool to differentiate between benign and malignant lesions.SAPA scoring for normal was 5.2 whereas for carcinoma it was 8.0. 2 In another study done by Akhtar GN, SAPA score was helpful in differentiating benign from malignant cells. 14e average area and distribution of the dots were more discriminating between the benign and malignant lesions than the simple dot count.In benign lesions, there was uniform small compact centrally placed dot and large irregular marginally located dot in infiltrating ductal carcinoma. 15In contrast, counting of every single AgNOR dot is laborious and less reproducible.Analysis of the AgNOR dots using computer assisted image analysis has been introduced.Image analysis provides information about the size and distribution of AgNOR.It seems that, the addition of size or area measurements using image analysis gives improved diagnostic and prognostic specificity.[18]

CONSLUSION
The mAgNOR counts were significantly higher in smears from malignant breast lesions than in those from benign.These results were similar to those obtained for tissue sections and were comparable to established data.Both AgNOR counts and SAPA score gave similar results done in this study, indicating that SAPA is also as convenient, reproducible and rapid method of AgNOR evaluation.

Figure 2 :
Figure 2: Photomicrograph showing FNAC and biopsy slide (inset) of invasive ductal carcinoma.Note the increase in numbers of AgNOR dots with scattered and irregular contour.(AgNOR stain, X100).

Table 6 : AgNOR count and SAPA score in benign and malignant breast lesion
1.734 for benign cases and 4.508 for malignant cases.The descriptive values of AgNoRs dots in FNAC and Biopsy slides are shown in table 1 and table 2 respectively.Statistically significant 'P' value < 0.05 for both benign and malignant cases in FNAC as shown in table 3.
possible for grade-3 because only 1 case was included in the study.Comparing the grades, we can see that in grade-1 the mean AgNOR counts were 4.2709 with 4.6971 in grade-2 which shows that in higher grade the mean AgNOR count was higher.