Path. Res. Pract. 188,541-544 (1992)

Evaluation of AgNOR Count in Distinguishing Benign from Malignant Mesothelial Cells in Pleural Fluids M. Colecchia and O. Leopardi Department of Pathology, Ospedale Maggiore Lodi, Italy

SUMMARY The authors have evaluated in eight reactive and eight malignant pleural effusions the number of intranuclear dots representing the nucleolar associated proteins stained with silver colloid technique (interphase nucleolar organizer regions-AgNORs). The mean number per nucleus in benign reactive effusions was 1.56 (SD O.77) while in mesotheliomatous effusions it was 2.81 (SD 1.44). The statistical analysis of values, by Mann- Whitney U Wilcoxon Rank Sum W Test, revealed a significant difference of AgNOR counts in the two cytological samples. The variability of AgNOR areas and morphologies in reactive and mesotheliomatous nuclei in pleural fluids is evaluated applying automatic image analysis.

Introduction The colloidal silver staining of the nucleolar organizer regions (AgNORs) has been used to investigate its diagnostic value in distinguishing benign from malignant lesions 3, 7. Other studies have evaluated the prognostic significance of AgNOR counts correlating it with patients' outcome 16 and with proliferation indices assessed by flow cytometry or immunohistochemically with Ki 67 in histological specimens and in cytological preparations obtained by needle aspiration 8 , imprintlO, or centrifuged cell suspensions from body cavity effusions6 • The authors have examined sixteen pleural effusions eight reactive and eight mesothelioma tons - to evaluate the usefulness of the method and its statistical significance in the two cytological samples utilizing computer-assisted image analysis.

Material and Methods Paraffin-embedded cell blocks of eight mesotheliomatous effusions and eight benign inflammatory pleural effusions were retrieved from the SurgicafPathology of the Ospedale Maggiore di Lodi. Fresh pleural fluids were centrifuged to a cell pellet, then © 1992 by Gustav Fischer Verlag, Stuttgart

used for the preparation of cytologic smears, the remainder of the pellet was fixed in formalin 10 %, processed routinely and embedded in paraffin. Sections were cut at 3 micra thickness and the silver staining for AgNORs was carried out following the procedure reported by Ploton 13.14. The AgNOR staining solution was prepared by dissolving gelatin in 1 % aqueous formic acid at a concentration of 2 %; this solution was then mixed 1 : 2 volumes with 50 % aqueous silver nitrate solution. The final solution was poured over the tissue sections and left for twenty-eight minutes at room temperature. The sections were counterstained for ten seconds with 0.5 % methylgreen. Quantitative· analysis was performed using the densitometry module of an image analyzer (Microanalisi, Italy). The images obtained by videocamera mounted on a light microscope (Axioskop, Carl Zeiss Oberkochen, West Germany) were displayed on a high discrimination TV screen. Analogous video signals were digitalized and stored as 256 x 256 pixels containing 8 bits of intensity values sufficient for 256 levels of gray determination. Digitalized light intensity values were converted by an input look-up table to optical density values, based on previous standardization and calibration of the instrument. We used a green 540 nm. interference filter with a half-width of 10 nm. Measurements were performed with a 40/0.65 objective and a x 10 eyepiece, yielding a final magnification of x 400. We have calculated: a) mean number of AgNORs (50 nuclei/case); b) total AgNOR area (in pixels in 50 nuclei/case). The statistical analysis was performed using MannWhitney U-Wilcoxon Rank Sum W Test to evaluate the differences of the values in reactive and malignant effusions. 0344-0338/92/0188-0541 $3.s 0/0

542 . M. Colecchia and O. Leopardi

Results Table 1 shows the relationship between mean count values obtained for individual cases in the two groups of pleural fluids. The scattergram of AgNOR counts in the two effusion groups shows a wide dispersion of values with superimposition of individual mean data in mesotheliomas and in reactive effusions (Figure 1). The mean AgNOR value for each nucleus in 400 cells is 1.56 (± 0.77) and 2.81 (± 1.44) for mesotheliomas. The value of Mann-Whitney U-Wilcoxon Rank Sum W Test is associated with a 0.00001 significance (2-tailed P test) (Table 2). Therefore, there was a significant difference between the two groups. Table 1. Mean number of nucleolar organizer regions (AgNORs) per cell in each pleural effusion Reactive mesothelium

Mesothelioma

1.6 1.8 1.5 1.9 1.7 2.1 1.2 1.3

3.3 2.9 3.1 1.9 2.0 3.3 3.8 2.6

•

Mean N

AgNOR" per cell

Table 2. Statistical evaluation of AgNOR counts in reactive and mesotheliomatous cells by Mann-Whitney U-Wilcoxon Rank Sum W Test Group 1 Reactive mesothelium Mean Std Dev Kurtosis Skewness Range Maximum Mode

1.569 .776 2.071 1.435 4.000 5 1.000

.037 .0.62 .230 .115

S.E. Mean Variance S.E. Kurt S.E. Skew Minimum Sum Median

706.000 1.000

.068 2.076 .230 .115 1 1266.000 3.000

1

Group 2 Mesothelioma Mean Std Dev Kurtosis Skewness Range Maximum Mode

2.813 1.441 3.887 1.451 9.000 10 2.000

S.E. Mean Variance S.E. Kurt S.E. Skew Minimum Sum Median

U 43687.0

W 145162.0

Corrected for ties 2-tailed P -15.4449 .00001 Z

Two peaks in the reactive effusions, one at 8 pixels and the other slightly above twenty, indicate an oscillating number of dots in reactive mesothelial cells (Fig. 2); likewise mean dot count values in single reactive pleural effusions show analogous differences, ranging from a minimum of 1 dot to a maximum of 5 dots. No cells

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Fig. 1. Scattergram of mean number of AgNORs per cell in reactive mesothelium (R) and mesothelioma (M). Each dot corresponds to 11 of the 16 cases studied, with 50 cells evaluated for each case.

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NOR Areas (pixeis)

Fig. 2. Diagram of the distribution of areas of AgNORs in reactive pleural fluids and in mesotheliomata (outlined). Scalling corresponding to number of cells in linear, scalling on the horizontal axis (areas of AgNORs) is logarithmic.

AgNORs in Mesothelial Cells· 543

Discussion The cytogeneticist's identification and use of nucleolar organizer regions in the diagnosis of trisomies 2,5 led pathologists, thanks to the development of the easily performed one-step method of Ploton, to investigate the diagnostic usefulness of AgNOR counts. Many reports indicated that malignant cells from tumours of various organs have higher AgNOR counts than normal cells from the tissues of origin 14. Discrepancies do occur such as those found in studies on mammary cancer8 and in comparisons between naevi and melanomas 11 ,12. In previous studies about reactive and malignant mesothelial cells there are different evaluations of usefulness of this technique 1, 6, 18. Ayres compared ten normal and ten reactive mesothelial and twenty-five mesotheliomatous tissues, including four types - tubulopapillary, undifferentiated, squamous and mixed - finding a significant difference bet\veen each group with regard to mean AgNOR values. Derenzini et al. have effected the counting of AgNOR dots in mesothelial cells of pleural fluids 6 • The distinction between benign and malignant cells or between malignant cells and macrophages, lymphoblasts and active lymphocytes is difficult. The argyrophilia of nucleolar associated proteins produces black nucleolar dots of AgNORs in yellow nuclei. Active lymphocytes show a single dot, while macrophages and neutrophils have one or two. Mesothelial cell clusters contain a variable number of dispersed dots, whereas in mesotheliomatous cells th,ey are irregular, numerous and irregularly distributed occurring in two or three nucleoli (Figure 3). These differences can easily be appreciated on light microscopy with 100x magnification, but quantitative objective assessment is possible using a semiautomatic analyzer as described by others l5 . Mean interphase AgNOR values for mesotheliomatous and benign reactive mesothelial cells are distinct whilst single case analysis is difficult due to overlap. The presence of proliferating and non-proliferating cells probably accounts for the double

Fig. 3. Mesothelioma. Many dots are localized within two or three nucleoli. They are irregularly distributed and do not exhibit an uniform size (AgNOR counterstained with methyl green, X 100).

peak in the relative AgNOR area curves for reactive mesothelial cells (Figure 2). Dot counting criteria are not uniform: in our study mean values for benign and reactive mesothelial cells are not dissimilar (1.56 ± 0.77) to those of Ayres (1.75 ± 0.55), but our values for mesotheliomas differ (2.8 ± 1.44) consistently from Ayres (>5/nucleus). The causes for these findings are many: the variety of methods in preparation, fixation and embedding of tissues 9,17, their sources - whether biopsy or cytological prepations - and lastly the different criteria used in assessing positivities4, 17. We found in the literature great variability in incubation times for the silvering method from 10 minutes (Derenzini) to 30 minutes (Ayres). Therefore using a standard of single dots in lymphocytes we obtained a satisfactory staining time of twenty-eight minutes for mesothelial cells in effusions. The authors sustain that computer-assisted image analysis instead of obsolete optic quantitative criteria is useful in standardizing the silvering method for AgNORs.

References 1 AyresJG, Crocker JG, Skilbeck NQ (1988) Differentiation of malignant from normal and reactive mesothelial cells by the argyrophil technique for nuclear organizer region associated proteins. Thorax 43: 366-370 2 Busch H, Daskal Y, Gyorkey F, Smetana K (1979) silver staining of nucleolar granules in tumor cells. Cancer Res 39: 857~863

, Crocker JG, Paramjitnar (1987) Nucleolar organizer regions in lymphomas. J Path 151: 111-118 4 Crocker JG, Boldy DAR, Egan HJ (1989) How should we count AgNORs? Proposals for a standardized approach. Journal of Pathology 158: 185-188 5 De La Cruz FF, Gerald PS (1981) Trisomy 21. 165-167. University Park Press, Baltimore 6 Derenzini M, Nardi F, Farabegoli F, Ottinetti A, Roncaroli F, Bussolati G (1989) Distribution of silver-stained interphase

544 . M. Colecchia and O. Leopardi nucleolar organized regions as a parameter to distinguishing neoplastic from non-neoplastic reactive cells in human effusions. Acta Cytol4: 491-498 7 Egan MJ, Raafat F, Crocker J, Williams D (1988) Comparative study of the degree of differentiation of neuroblastoma and mean numbers of nucleolar organizer regions. J Clin Pathol 41 : 527-531 8 Giri DD, Nottingham JF, Lawry J, Dundas JAC, Underwood JCE (1989) Silver-binding nucleolar organizer regions (AgNORs) in benign and malignant breast lesions: correlations with ploidy and growth phase by DNA flow cytometry. J Path 157: 307-313 9 Griffith AP, Butler CW, Roberts P, Dixon MF, Quirke P (1989) Silver-stained structures (AgNORs), their dependence on tissue fixation and absence of prognostic relevance in rectal adenocarcinoma. J Pathol 159: 121-127 10 Hall PA, Crocker J, Watt A, Stansfeld AG (1988) A comparison of nucleolar organizer region staining and Ki-67 immunostaining in non-Hodgkin's lymphoma. Histopathology 12: 373-391 11 Howat AJ, Giri DD, Cotton DWK, Slater DN (1989) Nucleolar organizer regions in Spitz nevi and malignant melanoma. Cancer 63: 474-478 12 Leong ASY, Gilham P (1989) Silver staining of nucleolar organizer regions in a malignant melanoma and melanotic nevi. Human Pathology 20: 257-262

13 Ploton D, Menager M, Jeannesson P, Himber G, Pigeon F, Adnett JJ (1986) Improvement in the staining and in the visualization of the argirophilic proteins of the nucleolar organizer region at the optical level. Histochem J 8: 5-14 14 Ploton D, Menager M, Lechki C, Jeannesson P, Visseaux S, Adnett JJ (1988) Coloration des organizateur nucleoraires (NORs) par l'argent. Application a l'etude de la structure du nucleole et interets en pathologie. Ann Pathol 3: 248-252 15 Ruschoff J, Plate HK, Contractor H, Kern S, Zimmermann R, Thomas C (1990) Evaluation of nucleolar organizer regions (NORs) by automatic image analysis: a contribution to standardization. J Path 161: 113-118 16 Ruschoff J, Bittinger A, Neumann K, Schmitz-Moormann P (1990) Prognostic significance of nucleolar organizer regions (NORs) in carcinomas of the sigmoid colon and rectum. Path Res Pract 186: 85-91 17 Smith PJ, Skilbeck N, Harrison A, Crocker J (1988) The effect of a series of fixatives on the AgNOR technique. J Pathol 155: 109-112 18 Soosay G, Happerfield L, Papadaki L, Griffiths, Bobrowls (1989) Evaluation of immunohistochemistry electron microscopy and silver impregnation of nucleolar organizer regions in the differentiation between malignant mesothelioma, metastatic adenocarcinoma, and reactive mesothelial hyperplasia. Abstract Book 157 A Meeting of Pathological Society of Great Britain and Ireland, London

Received September 27, 1991 . Accepted November 4, 1991

Key words: Nucleolar Organizer Regions - Pleural fluids Dr. Maurizio Colecchia, Department of Pathology, Ospedale Maggiore, Piazza Ospedale, 20075 Lodi (Mi), Italy