Stereological estimation of nucleolar organizer regions in prostatic tissue with an optical disector.

Silver-stained nucleolar organizer regions (AgNORs) were studied in 10 hyperplastic, 10 inflamed, nine prostatic intraepithelial neoplastic (PIN) and 20 malignant prostatic lesions. Optical disector measurement on 15 micron sections showed that upper frequency limits for 1.5 microns and 2.0 microns AgNOR particles were 10% and 2% in benign lesions and 18% and 9% in PIN. Using these cut-off values for diagnosis we found a low sensitivity and high specificity in distinguishing benign lesions from PIN and PIN from carcinoma. A high sensitivity and high specificity were obtained in separating benign lesions from carcinoma. AgNOR typing on routine "two-dimensional" sections showed that upper frequency limits for types B2, B3 and C2 were 25%, 3% and 0% in benign lesions and 38%, 23% and 11% in PIN. Using these cut-off values for diagnosis we found lower sensitivity and specificity values among all the differential diagnostic categories. We conclude that although both methods may contribute to the differential diagnosis between benign and malignant prostatic lesions, stereological estimation was a technically simple method, ensuring unbiased sampling and resulting in higher sensitivity and specificity. It may thus prove helpful in the differentiation of borderline cases in routine diagnostic pathology.     

Argyrophilic nucleolar organizer regions and alpha-fetoprotein in adenomatous hyperplasia in human cirrhotic livers.

Recently, adenomatous hyperplasia (AH) of the liver has been suspected as a precancerous lesion in human hepatocarcinogenesis. The authors examined 75 cases of AH from 42 cirrhotic livers, using staining of argyrophilic nucleolar organizer regions (AgNORs). These reflect proliferative cell activity. Findings in AH were compared with those seen in hepatocellular carcinoma (HCC) and other chronic liver diseases. Expression of alpha-fetoprotein (AFP) was also examined immunohistochemically. The authors classified AH into three types: ordinary (OAH), atypical (AAH), and AH with focal malignancy (FM). OAH implies a lack of atypia; AAH represents AH with structural and cellular atypia but without the features of overt carcinoma; and FM denotes AH with foci of overt HCC. Forty of the 75 cases of AH were categorized as OAH, 19 as AAH, and 16 as FM. The noncancerous areas of FM had features of AAH. The mean number of AgNORs in AH was intermediate between that seen in cirrhosis (2.93) and HCC (6.18) and showed a step-wise increase in the following order: OAH (2.95), AAH (3.89), noncancerous areas in FM (4.58), and malignant foci in FM (5.71). There was no significant difference in AgNOR counts between OAH and cirrhosis. AgNOR counts in AAH and FM were significantly higher than those of OAH, and lower than those of HCC. AFP was positive in 12 of 25 HCCs and in malignant foci of 3 FM lesions, but it was absent in OAH and AAH. These data suggest that OAH has a limited capacity for proliferation but that AAH and FM are much more replicative. The latter two conditions are probably preneoplastic lesions or early forms of HCC.     

Nucleolar organizer regions in cutaneous tumours

A silver colloid technique to identify nucleolar organizer region associated protein (AgNOR) has been applied to 68 cutaneous tumours. Basal cell carcinomas, eccrine tumours, apocrine tumours, and hair follicle tumours had differences in their numbers of AgNORs; these appeared as small black dots in their nuclei. Dermatofibromas and squamous cell carcinomas showed a degree of variability in the number of AgNORs depending on the cellularity of the former and differentiation of the latter. Basal cell carcinomas possessed significantly many more AgNORs per nucleus than the other neoplasms. It is suggested that this technique, previously the province of the cytogeneticist, may be of use in the diagnosis of cutaneous neoplasms.     

Diagnostic significance of mean numbers of nucleolar organizer regions in benign and malignant transitional tumours of nose

On the basis of a silver colloid technique to identify nucleolar organizer region-associated protein (AgNOR) counts, transitional papillomas of the nose showing malignant change could easily be distinguished from their benign counterparts and both could be distinguished from invasive carcinomas.     

Nucleolar organizer regions in smooth muscle and breast tumours.

Paraffin sections of formalin fixed tissues, obtained from patients with smooth muscle and breast tumours, were studied by the silver staining technique to quantitate the Nucleolar Organizer Regions (AgNORs) per nucleus and to assess its significance as an independent variable in predicting the behaviour of these neoplasms. Five benign and five malignant tumours of smooth muscle along with ten benign and ten malignant epithelial tumours of breast were studied. Normal myometrium and breast tissue served as controls. Control, benign and malignant tumours of smooth muscle showed mean AgNOR scores of 2.68, 3.89 and 12.50 per nucleus respectively. Control, benign and malignant tumours of breast showed mean AgNOR scores of 1.75, 7.45 and 12.72 per nucleus respectively. These results suggest that quantitative analysis of AgNORs per nucleus is capable of differentiating benign from malignant lesions of smooth muscle and breast.     

Silver staining of nucleolar organizer regions in prostatic lesions

Variations in the number of silver-stained nucleolar organizer region-associated proteins (AgNORs) were studied in paraffin sections of 42 benign prostatic lesions, comprising four cases of granulomatous prostatitis, five of squamous or transitional metaplasia, eight of atypical and 25 of regular hyperplasia, and 37 of prostatic adenocarcinoma, with their metastases. There was a significant difference between the mean AgNOR counts of the benign and malignant prostatic lesions (1.58 +/- 0.26 v. 4.34 +/- 1.53; P less than 0.01). The mean AgNOR counts significantly increased with increasing Gleason's grade (P less than 0.01) and clinical stage (P less than 0.05) of the tumours. AgNOR counting may contribute to the conventional diagnostic and prognostic indices of cancer of the prostate.     

Comparative study of dma content and interphase nucleolar organizer regions in human pituitary adenomas

DNA content and the number of nucleolar organizer regions (NORs) are claimed to be valuable markers of biological behavior in many tumors. Forty-three human pituitary adenomas removed by surgery were embedded in paraffin and studied by flow cytometry in order to establish DNA index and percentage of cells in different phases of cell cycle. In 31 of these tumors, silver staining showing argyrophilic proteins bound to NORs (AgNORs) was applied. DNA analysis revealed DNA aneuploidy in 32% of the adenomas. The highest number of aneuploid tumors was found in silent corticotroph adenomas (5 of 9), followed by prolactin cell adenomas (3 of 6). The lowest number of aneuploid tumors was noted in null cell adenomas and oncocytomas. The presence of an aneuploid peak could not be correlated with the size of adenomas or with invasiveness. The number of cases with S fraction higher than median was not increased in large or invasive tumors. No close correlation was found between DNA index and AgNOR reading or between S fraction and AgNOR number. These results show that DNA index and AgNOR counting must be interpreted with caution as proliferation markers in pituitary adenomas.     

Argyrophilic nucleolar organizer regions in Helicobacter pylori-associated gastric lesions

Three hundred and fifty biopsies from patients undergoing upper gastrointestinal endoscopy were studied for histopathological changes, H. pylori infection and argyrophilic nucleolar organizer region (AgNOR) counts. Histopathological examination revealed normal gastric mucosa in 10 (2.85%), gastritis in 254 (72.56%), intestinal metaplasia in 12 (4.0%), dysplasia in 13 (3.7%) and adenocarcinoma in 61 (17.4%). The mean (SD) AgNOR count was 1.66 (0.20) in normal, 2.43 (0.64) in gastritis, 3.09 (0.52) in intestinal metaplasia, 4.17 (0.31) in dysplasia, and 6.57 (0.98) in carcinoma. A statistically significant difference was observed between the AgNOR count of normal gastric mucosa and gastritis (p<0.001), gastritis and dysplasia (p<0.001), and dysplasia and adenocarcinoma (p<0.001). A statistically significant increase in mean AgNOR count was found with increase in H. pylori density in gastric biopsies (p<0.001) with gastritis. No significant difference was observed between mean AgNOR count of intestinal and diffuse type carcinomas. The AgNOR count in gastric biopsies with adenocarcinoma and H. pylori infection was 7.03 (0.85) as compared to 6.89 (0.73) in gastric biopsies with evidence of adenocarcinoma but without H. pylori infection. The difference was not statistically significant. The findings support the role of H. pylori as a promoting agent in gastric carcinogenesis by stimulating gastric epithelial cell proliferation at the stage of chronic inflammation, thereby making the cells more susceptible to endogenous or exogenous carcinogenic agents.     

Silver-stained nucleolar organizer regions and immunoglobulins in cutaneous squamocellular tumors

This study was conducted to elucidate the biological activity of epidermal cells in cutaneous squamocellular tumors by counting the number of silver-stained nucleolar organizer regions (AgNORs), to estimate the quantity of Ig-producing cells and the inflammatory cellular infiltrate (ICI), and to achieve a comparative evaluation.

Twenty cases of actinic keratosis (AK), 20 in situ squamous cell carcinomas (ISC), and 20 invasive squamous cell carcinomas (SCCs) were assessed using a silver colloid technique. Ig-producing or binding cells and ICI were also investigated immunohistochemically.

In all samples, AgNORs, Ig-producing cells, and ICI increased in proportion to the degree of malignancy. With regard to AgNORs values, a statistically significant difference was confirmed between AK and ISC (p<0.01), AK and SCC (p<0.001), and ISC and SCC (p<0.05). IgG-producing cells predominated in each case. Furthermore, a linear correlation was detected between ICI and AgNORs in AK and ISC.

The significant difference in the number of AgNORs among the 3 stages of involution of SCC reinforces the value of AgNORs as a marker for malignant potential. Despite the absence of a correlation between AgNORs and the proportion of Ig-producing cells, the association between ICI and AgNORs in AK and ISC was obvious.     

Interphase argyrophilic nucleolar organiser regions and nucleolar counts in transitional cell bladder tumours.

AIMS: To see whether a correlation exists between clinicopathological parameters, argyrophilic nucleolar organiser regions (AgNORs), and nucleolar counts in the nuclei of tumour cells in patients with transitional cell bladder carcinoma. METHODS: Paraffin wax embedded sections from a total of 62 cases of primary transitional cell bladder carcinoma were stained with the silver colloid method. The numbers of individual silver grains (AgNORs) in nucleoli and the numbers of nucleoli were counted in 100 nuclei. The correlation between AgNORs and nucleolar counts and patients' sex, tumour grade, disease stage, recurrence pattern, and tumour related survival was analysed. RESULTS: The numbers of nucleoli in tumour cells were higher in male patients (p < 0.032). AgNOR numbers correlated with tumour grade (p = 0.017) and recurrence (p = 0.046). In multivariate analysis, the variation coefficient of AgNOR scores was found to be the only independent predictor of the duration of tumour free period in patients with recurrent disease (p < 0.002). AgNOR scores and nucleolar counts were of no value in distinguishing superficial and invasive tumours or in predicting tumour related survival. CONCLUSIONS: AgNOR scores in transitional cell bladder carcinoma reflect variations in tumour biological behaviour; however, the clinical value of this technique in patients with urinary bladder carcinoma is limited.     

Morphometric evaluation of nucleolar organizer regions in cervical intraepithelial neoplasia

The objective of this study was to determine whether some morphometric parameters and two different methods of counting AgNOR dots were correlated with the grade of cervical intraepithelial neoplasia. Thirty uterine cervix biopsies (8 cases of cervicitis, 9 CIN I, CIN II and 6 CIN III) were studied. Two methods were used to count AgNOR dots. The first one consisted of counting the number of epithelial cells with 1, 2, 3, 4, or more dots. The second method, based on a computer analysis system, consisted of counting the total number of dots in 100 cells, without considering the number of dots per cell. Using the same computer analysis system, the following parameters were measured: area, diameter, perimeter, roundness and length of each dot. The following parameters were found to be correlated with the grade of intraepithelial neoplasia: 1) number of cells with 1 dot, which decreased with increasing grade of cervical intraepithelial neoplasia; 2) number of cells with 4 dots or more, which increased with increasing grade of cervical intraepithelial neoplasia; 3) total number of dots per 100 cells, which progressively increased with increasing grade of intraepithelial neoplasia. We conclude that counting cells with 4 or more dots is the more trustworthy parameter for distinguishing the grade of cervical intraepithelial neoplasia.     

Studies an morphological typing of argyrophilic nucleolar organizer regions

Morphological typing of argyrophilic nucleolar organizer regions (AgNOR) in 300 caws of malignant tumors and 140 cams of benign lesions was analyzed and five morphological types of ApNOR were described in detail. In malignant tumors, the diffuse type (78%) was the most frequently seen, and in benign lesions, the nucleolar type (92.85%); the difference was thus highly significant (P < 0.001). The intranucleolar and aggregate types were not observed in benign lesions. There was no obvious difference in the proportion of the mixed type in benign and malignant lesions (P > 0.05). The relationship between grade of malignancy and morphological typing of AgNOR and its clinical significance are discussed.     

Silver staining method for nucleolar organizer regions in cervical smears

The distribution of nucleolar organizer regions (NORs) was studied in Papanicolaou preparations of cervical smears in order to distinguish benign from preneoplastic lesions. Destained smears (six defined as normal, six as inflammatory with squamous metaplasia, six as CIN I, six as CIN II, and five as CIN III) were submitted to the Ag-NOR method after staining with Orange G and EA36. Ag-NOR count was performed in previously outlined fields on the smears. Statistically significant differences (P < .05) were found between the normal smears, inflammatory smears with squamous metaplasia, and each grade of CIN. We conclude that the Ag-NOR technique could be useful to evaluate cervical smears of doubtful interpretation, using previous demarcation of the abnormal fields/cells. Diagn. Cytopathol. 16:497–499, 1997. © 1997 Wiley-Liss, Inc.     

Thymic epithelial tumours: Evaluation of malignant grade by quantification of proliferating cell nuclear antigen and nucleolar organizer regions

Cellular proliferation was studied by quantification of proliferating cell nuclear antigen (PCNA) and argyrophilic nucleolar organizer regions (AgNORs) of cells in 29 thymic epithelial tumours: 8 noninvasive (7 cortical and 1 mixed) thymomas, 11 invasive/metastatic (all cortical) thymomas, and 10 thymic carcinomas. Thymic carcinoma showed the highest percentage of cells positive for PCNA (14.73±5.419%) and the largest mean number of AgNORs per nucleus (4.89±0.756). The mean percentage of PCNA-positive cells and number of AgNORs in thymoma groups were as follows: in noninvasive thymoma 2.96±1.256% and 2.73±0.647, respectively, and in invasive/metastatic thymoma 4.41±1.823% and 3.68±1.148, respectively. The differences in PCNA and AgNORs were statistically significant between thymic carcinoma and each of thymoma groups. The overlap of the values between these tumours was minimal in the PCNA stains, although it was considerable in AgNOR counts as previously noted. However, there was no statistically significant difference in these markers between noninvasive and invasive/metastatic thymomas. These results indicate that thymoma in general is a slow-growing tumour compared with thymic carcinoma and that noninvasive thymoma is similar to invasive/metastatic thymoma with regard to proliferative activity; these latter two tumours may represent an essentially identical type in different stages of progression.Part of this work was presented at the 80th General Meeting of the Japanese Society of Pathology held in Osaka, April 1991     

Silver staining of nucleolar organizer regions in malignant melanoma and melanotic nevi

The recently described method of staining nucleolar organizer regions (NOR) with colloidal silver nitrate was applied to the paraffin sections of five junctional nevi, 13 compound nevi, seven Spitz nevi, nine cellular blue nevi, 11 dysplastic nevi, seven malignant lentigines, 12 superficial spreading melanomas, and 14 secondary melanomas. There was a significant difference between the pooled silver-NOR (AgNOR) numbers of the 45 benign lesions (mean, 1.22; SD, 0.51) and the 33 melanomas (mean, 9.18; SD, 4.05) by t test analysis (P less than .01). The difference was striking enough to be recognized on casual microscopic examination, suggesting that AgNOR staining may be a useful technique to help separate melanocytic nevi from malignant melanomas.