The study sought a correlation between the number of AgNOR granules and
the degree of cervical intraepithelial neoplasia (CIN). Thirty-five sections (5 normal,
10 CIN1, 10 CIN2 and 10 CIN3) were subjected to retrospective analysis.
The percentage of cells with 1, 2, 3, 4 and more AgNORs was calculated and the
number of granules per 100 cells was counted. The number of cells containing
single granules decreases. However, the number increases with CIN level when
the cells contain 4 and more AgNORs. The number of granules per 100 cells also
increases with the degree of CIN. It can be thus concluded that the number of cells
with 4 and more AgNOR granules can serve as a CIN differentiation exponent

A, Mazurek

J, Dzięcioł

K, Lejmanowicz

M, Oniszczuk

R, Boroń

S, Sulkowski

S, Terlikowski

English

Via Medica Journals

Folia Morphol. Vol. 63, No. 2, pp. 209–212Copyright © 2004 Via MedicaISSN 0015–5659www.fm.viamedica.plS H O R T  C O M M U N I C A T I O N209A morphometric study of nucleolar organiserregions in cervical intraepithelial neoplasiaSławomir Terlikowski1, Janusz Dzięcioł2, Andrzej Mazurek1, Stanisław Sulkowski3,Robert Boroń1, Małgorzata Oniszczuk1, Krzysztof Lejmanowicz11Department of Pathophysiology of Pregnancy, Medical University, Białystok, Poland2Department of Clinical Pathomorphology, Medical University, Białystok, Poland3Department of Anatomy, Medical University, Białystok, Poland[Received 11 September 2003; Accepted 9 January 2004]The study sought a correlation between the number of AgNOR granules andthe degree of cervical intraepithelial neoplasia (CIN). Thirty-five sections (5 nor-mal, 10 CIN1, 10 CIN2 and 10 CIN3) were subjected to retrospective analysis.The percentage of cells with 1, 2, 3, 4 and more AgNORs was calculated and thenumber of granules per 100 cells was counted. The number of cells containingsingle granules decreases. However, the number increases with CIN level whenthe cells contain 4 and more AgNORs. The number of granules per 100 cells alsoincreases with the degree of CIN. It can be thus concluded that the number of cellswith 4 and more AgNOR granules can serve as a CIN differentiation exponent.Key words: cervical intraepithelial neoplasia, argyrophilic nucleolarorganiser regionsAddress for correspondence: Sławomir J. Terlikowski, Department of Pathophysiology of Pregnancy, Medical University,ul.  M. Cure-Skłodowskiej 24A, 15–276 Białystok, Poland, e-mail: ster@amb.edu.plINTRODUCTIONThree grades of dysplasia are defined in the uter-ine cervix, namely cervical intraepithelial neoplasia(CIN) grades 1–3, which represent progressive de-grees of neoplasia. Subjective grading systems usedin classical histopathology are often unable to pre-dict correctly the biological behaviour of these le-sions. Therefore, a simple laboratory method basedon the evaluation of biopsy material that would pre-dict the behaviour of CIN would, be of significantclinical value. Silver staining of nucleolar organiserregion-associated proteins (AgNORs) has becomea widely used method in pathology, mainly for as-sessing the prognosis of malignant tumours. AgNORsare loops of ribosomal DNA, which transcribe to ri-bosomal RNA as the first step of protein synthesis[4]. They are partly located within the fibrillar cen-tres of nucleoli and can be visualised as small blackdots, single or in clusters, within the nucleus. An in-crease in the number of AgNORs is associated withincreased tumour aggressiveness, as the mean num-ber of AgNORs per nucleus is higher in malignantthan in benign tissues, higher in high grade than inlow grade malignancies, and higher in tumours witha poor prognosis compared with those with a goodprognosis [2, 3, 7, 8]. In the present study the roleof AgNOR measurements as a prognostic marker inCIN was evaluated.MATERIAL AND METHODSA total of 35 cervical biopsy specimens were tak-en in retrospect from the case files of the Depart-ment of Clinical Pathomorphology. Five were nor-mal on histological examination, and there were10 of each of the three grades of CIN. Two 4 µmsections were cut from the pretreatment formalin-fixed, paraffin-embedded block. One section wasstained with H&E to verify the histopathological210Folia Morphol., 2004, Vol. 63, No. 2diagnosis; the other was stained by the one-stepsilver colloid technique according to Ploton et al.[4]. The quantitative analysis of the AgNOR expres-sion was performed by means of digital image anal-ysis, using the DP-SOFT PC software. The imageswere generated by a BX41 microscope (OlympusOptical, Poland) connected to a Camedia C3030Zcamera (Olympus Optical, Poland) and fed into thecomputer through a DP-DISPLAY 3.0 (Olympus Op-tical, Poland) for off-line processing. A total of 100cells/case were analysed with the aid of a 1000xmagnification and an oil-immersion lens. The num-bers of nuclei with 1, 2, 3, 4, and > 4 AgNORs withinall the squares in 5 fields were counted and theirpercentages were calculated. The study was ap-proved by the Medical University of Białystok Ethi-cal Committee (protocol no. R-I-003/229/2003). Thedata were stored and analysed by means of CSSStatistica statistical software (Statsoft Inc., Poland).The two-tailed Student’s t-test was used for com-parison of group means. A P value of 0.001 wastaken as significant.RESULTSAfter incubation with AgNO3 solution, proteinsassociated with nucleolar organiser regions wereseen as tiny granules grouped mainly within thenucleoli and sometimes in the nuclear caryoplasm.The normal squamous epithelium showed singlegranules mainly in the cell nuclei of the reproduc-tive layer (Fig. 1). In early lesions (CIN1) AgNOR gran-ules were found in the cells of the basement andparabasement layer (Fig. 2). In CIN3 numerous Ag-NOR granules were observed throughout the epi-thelium (Fig. 3). Mean AgNOR counts per cell andthe percentage of cells with 1, 2, 3, 4 and moregranules are presented in Table 1.DISCUSSIONThe results of our study are in accordance withprevious studies and confirm that AgNOR counts al-low for discrimination between normal cervical epi-thelium and CIN, and between CIN and squamouscell carcinoma (SCC) [1–3, 5]. Mean AgNOR countsand the percentage of cells with more than 4 Ag-NORs were significantly higher in malignancy thanin benign epithelium, thus confirming previous re-ports by Sakai et al. [6]. Our analysis has demon-strated that the number of cells containing singlegranules decreases, increasing with CIN degree whenthe cells contain 4 and > 4 AgNORs. The number ofgranules per 100 cells also increases with CIN. It canthus be concluded that the number of cells contain-ing 4 and more AgNOR granules can serve as a CINdifferentiation exponent. The presence of a largenumber of nucleolar organiser regions that expressan increase in the cellular proliferative activity canbe treated as an additional index of neoplastic trans-formation risk in correlation with routine histopatho-logical evaluation.Figure 1. Nucleolar organizer regions stained by argyrophilic technique in normal cervical squamous epithelium. Silver-binding black dotsare evident in the nuclei of epithelial cells. Mag. 250 ¥.211Sławomir Terlikowski et al., A morphometric study of nucleolar organiser regions in cervical intraepithelial neoplasiaTable 1. Mean AgNOR counts and percentages of nuclei with 1, 2, 3, 4 and more AgNORs in cervical intraepithelial neoplasiaMean p 1 p 2 p 3 p 4 and > pAgNORa AgNORb AgNORsb AgNORsb AgNORsbNormal 1.3 ± 0.6 65 ± 7 > 0.001 38 ± 8 11 ± 6 12 ± 5(n = 5) (0.6–1.9) (57–73) vs. CIN 1, (29–47) (5–16) (6–18)CIN 2 andCIN 3CIN 1 1.4 ± 0.2 NS 54 ± 6 > 0.05 41 ± 4 NS 14 ± 8 NS 14 ± 5 NS(n = 10) (1.1–1.7) (47–61) vs. CIN 2 (36–46) (6–22) vs. normal (8–19) vs. normaland CIN 3CIN 2 1.5 ± 0.2 NS 49 ± 6 > 0.03 38 ± 7 NS 16 ± 5 NS 19 ± 7 > 0.001(n = 10) (1.2–1.7) (42–55) vs. CIN 3 (31–45) (13–24) vs. normal (12–25) vs. normaland CIN 1 and CIN 1CIN 3 2.3 ± 0.6 > 0.001 45 ± 7 36 ± 6 NS 24 ± 8 > 0.001 32 ± 6 > 0.001(n = 10) (1.6–2.9) vs. normal (38–51) (29–42) (16–32) vs. normal (25–39) vs. normalCIN 1 and CIN 1 and CIN 1 andCIN 2 CIN 2 CIN 2amean ± standard deviation/cell, bmean ± standard deviation (%)Figure 3. Nucleolar organizer regions stained by argyrophilic tech-nique in cervical intraepithelial neoplasia (CIN3). Mag. 250 ¥.Figure 2. Nucleolar organizer regions stained by argyrophilic tech-nique in cervical intraepithelial neoplasia (CIN1). Mag. 250 ¥.212Folia Morphol., 2004, Vol. 63, No. 2REFERENCES1. Calore EE, Maeda MY, Cavaliere MJ, Pereira SM, Shih LW,Pereira GM, de Melo JR (1997) Study of organizernucleolar regions by the argyrophil technique in cer-vical intraepithelial neoplasias. Minerva Ginecol, 49:59–62.2. Lahshmi S, Nair SA, Jayasree K, Jayaprakash PG, Ra-jalekshmy TN, Kannan S, Pillai R (1993) Argyrophilicnucleolar organizer regions (AgNORs) in inflammato-ry pre-malignant and malignant lesions of the uterinecervix. Cancer Lett, 71: 197–201.3. Pelusi G, Tere D, Formelli G, Rinaldi AM, DerenziniM (1997) AgNOR protein quantity of cervical smearscorrelates with that of histological sections in cervi-cal intraepithelial neoplasia. Eur J Histochem, 41:105–110.4. Ploton D, Menager M, Jeanneson P, Himber G, Adnet JJ(1986) Improvement in the staining and in the visu-alization of the argyrophilic proteins of the nucleolarorganizer region at the optical level. Histochem J, 18:5–14.5. Rowlands DC (1988) Nucleolar organising regions incervical intraepithelial neoplasia. J Clin Pathol, 41:1200–1202.6. Sakai YI, Sakai AT, Isotani S, Cavaliere MJ, de AlmeidaLV, Calore EE (2001) Morphometric evaluation of nu-cleolar organizer regions in cervical intraepithelial neo-plasia. Pathol Res Pract, 197: 189–192.7. Terlikowski S, Lenczewski A, Famulski W, Sulkowski S,Kulikowski M (2001) Expression of nucleolar organiz-er regions (NORs) in ovarian epithelial tumors. FoliaHistochem Cytobiol, 39: 161–162.8. Terlikowski S, Lenczewski A, Sulkowski S, Kulikowski M(1999) Nucleolar organizer regions in differentiatedpreneoplastic and neoplastic endometrial lesions. GynecolObstet Invest, 47: 205–209.

A morphometric study of nucleolar organiser regions in cervical intraepithelial neoplasia

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A morphometric study of nucleolar organiser regions in cervical intraepithelial neoplasia

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