ROLE OF ARGYROPHYLIC NUCLEOLAR ORGANIZER REGION STAINING IN IDENTIFICATION OF MALIGNANT CELLS IN EFFUSION

Gill M, Singh U, Mahapatra QS, Gehlot S, Gupta V, Sen R. J Cytol 2011;28:191-5

INTRODUCTION

Pleural, peritoneal and pericardial cavities have a common embryologic mesenchymal origin forming a double layer, namely visceral and parietal layers. Presence of fluid in these cavities constitutes an effusion which can be transudate or exudate. Exudative effusion can be caused by a variety of diseases which could be inflammatory or neoplastic. Malignant tumors give rise to effusion by secondary inflammatory reaction due to circulatory disturbance or direct involvement of serous membranes by tumor invasion. The detection of malignant cells in effusion is important for clinical staging of tumor and deciding the line of treatment.

Recurrence of malignant tumor after treatment of primary tumor is an early indication of residual or resurgent malignancy.
Cytological examination of effusions helps to differentiate between benign and malignant effusions, but fails to allow a definitive diagnosis in a considerable number of cases which are classified as atypical. One of the common problems in effusion cytology is to distinguish reactive mesothelial cells from neoplastic cells Histochemical stains, electron microscopy and immunocytochemical studies supplement cytological evaluation of effusions, but such methods are time consuming and costly, and moreover, some antigens suitable for this purpose are expressed by normal as well as reactively proliferated mesothelial cells.

Argyrophilic nucleolar organizer regions (AgNORs)

Argyrophilic nucleolar organizer regions (AgNORs) are loops of DNA that transcribe ribosomal RNA and are located in the short arm of the acrocentric chromosomes 13, 14, 15, 21 and 22. They can be visualised by silver staining technique that recognizes argyrophilia-associated proteins (AgNORs), which appear as intranuclear black spots. Their frequency within the nuclei is significantly higher in malignant cells than in normal; reactive or benign neoplastic cells. Quantitative and qualitative AgNOR study is more accurate in cytological smears because the whole nucleus can be assessed rather than only part of it as occurs with tissue sections. Derenzini et al. studied AgNORs in cytologic preparations of human serous effusions and found that interphasic NORs in neoplastic cells were more numerous, irregularly distributed throughout the nucleus and had heterogeneity in size.

Inflammatory reactive cells had only a few regularly clustered NORs. Reactive mesothelial cells had regularly clustered distribution of NORs which were less in number and rather uniform in size. Another study done by Mohanty et al. in serous effusion showed that the AgNOR dots were discrete and smaller in benign effusion cases as compared to coarse and aggregated in malignant effusion cases. In benign reactive effusion cases, the mean number of AgNOR dots per nucleus was 2.33 0.71 and 2.83 1.15 by the manual and automated method, respectively, whereas that for malignant effusion cases was 7.48 2.51 and 8.09 1.69 by the manual and automated method, respectively. Thus, Identification of NORs is a reliable method of supplementing conventional cytologic methods to differentiate malignant cells from benign.

MATERIALS AND METHOD

A total of 100 cases of effusion samples were taken up for the present study which was conducted over a period of 2 years. The effusion samples of both indoor and outdoor patients of all age groups were included in the study. Cases in which long-term follow-up or histopathological correlation was not possible were excluded. Samples were centrifuged at a speed of 2000 rpm for 5 minutes. Four smears were prepared from sediment, one each for Leishman and hematoxylin and eosin (H and E) staining and two for AgNOR staining. Smears to be stained with H and E and AgNOR staining were quickly immersed in 95% ethanol before any drying occurred. Smear for Leishman staining was air dried. The cases were classified as benign, malignant and atypical.

AgNOR staining solution was prepared by dissolving 2 g of gelatin in 1% aqueous formic acid to make 100 ml solution at a concentration of 2% (solution A). Fifty percent aqueous silver nitrate solution was prepared by dissolving 5 g of silver nitrate in distilled water to make 10 ml solution (solution B). Working solution was prepared by mixing solution A and B in a proportion of 1: 2 volumes and was poured over the smears and left for 60 minutes at room temperature. The silver colloid formed was washed off with distilled water and the smears were counter stained with neutral red of safranin 0.5%. Smears were again washed with distilled water and dehydrated through ascending grades of alcohol, cleared with xylene and

AgNORs were counted as black dots in the nuclei of all unequivocal malignant cells in cytologically positive effusion and proliferated mesothelial cells in cytologically negative samples. In cytologically atypical effusion, the cells that could be identified as benign mesothelial were omitted and only the atypical cells in each field were studied. AgNORs were counted in 100 such cells from each sample. The pattern of AgNOR dispersion and their shape was compared in benign, malignant and atypical cases and the AgNOR counting was done. The mean number of AgNORs per nuclei was calculated for each sample.

STATISTICAL ANALYSIS

Unpaired t-test was applied to analyze the statistical significance of difference of mean AgNOR counts in different cytomorphological categories. P value, confidence interval and degree of freedom were calculated.

A total of 100 cases of pleural, peritoneal and pericardial effusion samples were subjected to conventional Leishman, H and E and AgNOR staining. AgNORs were counted in 100 cells in each case and results were compared among the three cytomorphological categories (benign, malignant and atypical).

Fifty cases of pleural effusion, 47 cases of peritoneal effusion and three cases of pericardial effusion were examined. Cytomorphologically, 57 effusion samples were benign, 28 malignant and 15 samples were labeled as atypical.

Out of 50 pleural effusion samples, 29 were benign, 10 were malignant and 11 were atypical. Among the 47 peritoneal effusion samples, 27 were benign, 16 were malignant and four were atypical. Out of three cases of pericardial effusion samples, one was benign and two were malignant

Cytological findings were non-specific and smears showed varying admixtures of inflammatory cells, mesothelial cells and macrophages. Some of mesothelial cells showed features of activation like nuclear enlargement, coarsely stippled chromatin, prominent nucleoli and multinucleation

Benign effusion-smear showing lymphocytes, macrophages and mesothelial cells. A giant cell is also seen

In the 16 cases, there was an underlying malignancy, but no malignant or atypical cell could be detected in Leishman and H and Estained smears, indicating that serous membranes had not been involved by malignancy and effusions were only reactive effusions revealing mesothelial cells with some of them showing features of activation, along with macrophages and lymphocytes only.

Smears showing a group of mesothelial cells in a benign effusion

AgNOR staining in benign effusion showing 1-2 dots/nucleus in all cell types

1 to 2 AgNOR dots/nucleus with regular borders in activated mesothelial cells

MALIGNANT EFFUSIONS

Out of these 28 effusions categorized as malignant cytologically, 20 were macroscopically hemorrhagic and were proved malignant on FNAC or biopsy. The remaining eight cases were later found to be malignant either by pleural biopsy, FNAC of lungs and FNAC or histopathology of the relevant lymph nodes.

Malignant effusion-smears showing malignant cells

Smear showing irregular 4-5 AgNOR dots/ nucleus in malignant cells

ATYPICAL CASES

These cases were investigated further and were proved to be malignant. Number of AgNOR dots in malignant and atypical cases was higher; moreover, the AgNORs were variably sized, distributed irregularly and had irregular borders

Smear showing atypical cells, Smear showing 4-5 dots/nucleus in atypical cells

Unpaired t-test was applied to analyze the statistical significance of difference of mean AgNOR count in malignant and benign effusions. Here, the 'P' value was less than 0.0001. 95% confidence interval of the difference of two means was from 2.3856 to 2.6144. Unpaired t-test was also applied to analyze the statistical significance of difference of mean AgNOR count in atypical and benign effusions. The 'P' value here was also less than 0.0001. 95% confidence interval of the difference of two means was from 1.6887 to 2.0313. The degree of freedom was 70. As the P value was less than 0.0001 in the different cytomorphological categories, it was considered to be extremely statistically significant.

DISCUSSION

Diagnosis of malignancy is often difficult on pure morphologic grounds and malignant cells cannot always be differentiated from macrophages and particularly reactive mesothelial cells. Interphasic NOR distribution can be used as a parameter to detect nucleolar abnormalities that characterize malignant cells.

SUMMARY
The present study was done to assess the utility of AgNORs in diagnosis of benign and malignant effusion, especially in case where a definite cytological diagnosis was difficult to make on Leishman and H and E smears. In this study, 100 cases of serous effusion were studied comprising of benign effusions, malignant effusions and a third group consisting of atypical cases which could not be classified with certainty as to whether they were reactive mesothelial cells or malignant cells. All effusions were subjected to AgNOR staining, the benign group consisted of cells showing 1 to 2 dots which were regular in size and shape. In malignant group, 3 to 5 dots were observed per cell distributed within the nucleus

The dots had variable size, shape and irregular contours. In atypical group, the reactive mesothelial cells showed 1 to 2 dots and malignant cells showed 3 to 4 irregular dots. In this way, clear separation could be achieved between activated mesothelial cells and malignant cells. A similar study done by Palaoro et al. showed that the assessment of ploidy and the study of AgNOR were better methods than immunocytochemistry for distinguishing between reactive mesothelial cells and adenocarcinomatous cells in serous fluid . Another study done by Asotra and Sharma substantiated the fact that AgNOR counting in fine needle aspiration smears is a simple, sensitive and cost-effective method for

Conclusion
AgNOR can be considered as an extremely useful additional diagnostic tool for cytodiagnosis of effusions. Another advantage of this technique is that it can even be done on Papanicolaou and May-GrunwaldGiemsa-stained smears after destaining, so this technique can be used for cytodiagnosis even when the extra unstained smears are not available.