Colorectal carcinoma and its precursors: role of argyrophilic nucleolar organizer regions (AgNORs).

A silver colloid technique to identify Argyrophilic Organizer Region (AgNOR) was applied to 5 hyperplastic polyps, 5 adenomas with low grade dysplasia, 5 adenomas with high grade dysplasia and 15 adenocarcinomas of the large bowel (5 well differentiated, 5 moderately differentiated and 5 poorly differentiated). The authors suggest the plainness and usefulness of simultaneous application of clumps per cell, AgNORs per clump and total AgNORs counts in the evaluation of neoplastic and preneoplastic lesions of the colon. In fact the results show that the number of clumps per cell is useful to distinguish hyperplastic polyps from adenomas with high grade dysplasia and from all the adenocarcinomas. Using the number of AgNORs per clump there is significant difference between hyperplastic polyps and adenomas with high grade dysplasia and between adenomas with low grade dysplasia and well differentiated adenocarcinomas. Finally the total number of AgNORs can discriminate hyperplastic polyps from adenomas with high grade dysplasia and these from adenomas with low grade dysplasia.     

Hepatocellular carcinoma and preneoplastic lesions of the liver: evaluation of argyrophilic nucleolar organizer regions (AgNORs).

The authors applied a silver colloid technique to identify Argyrophilic Organiser Region (AgNOR) to 8 groups of hepatic lesions: alcoholic hepatitis with dysplasia (3 cases); chronic active hepatitis with dysplasia (4 cases); cirrhosis with dysplasia (5 cases); focal nodular hyperplasia (4 cases) and hepatocellular carcinomas (3 cases of grade I, 3 cases of grade II and 5 cases of grade III of Edmondson). Four cases of non-specific reactive hepatitis were used as control. This work suggests the simplicity and utility of simultaneous application of clumps per cell, AgNORs per clump and total AgNORs counts in the evaluation of neoplastic and preneoplastic lesions of the liver. The results show, in hepatocellular carcinomas, a relationship between the number of clumps, the AgNORs per clump, the total number of AgNORs and the grading of Edmondson. The nodular lesions that can be considered in the differential diagnosis with carcinoma are sufficiently well discriminated using the two parameters AgNORs per clump and total number of AgNORs.     

Expression of argyrophilic nucleolar organizer regions (AgNORs) can be used to assess cellular proliferation as shown in rat thymic sections

Summary Silver-stained nucleolar organizer regions (AgNORs) were studied in thymic sections from 4- and 30-day-old rats. By direct examination under the light microscope cells with a low or high content of AgNORs (type I and type II cells, respectively) were identified and their relative numbers calculated. The mean area of AgNORs per cell was calculated for each type of cell and age group. Additionally, the proportion of cells labelled with bromodeoxyuridine was calculated in sections from the same animals. Visual identification of type I and type II cells was confirmed by a significant (p < 0.01) difference in the mean AgNOR area in both types of cell. Both the proportion of cells with a high expression of AgNORs (type II cells) and that of bromodeoxyuridine-labelled cells were significantly greater (p < 0.01) in 4-day-old rats than in 30-day-old rats. A significant correlation was found between both variables (R² = 0.45;p = 0.002), the relation being best between both variables and age (R² = 0.91;p = < 0.001). These data offer support for an easy interpretation of the AgNOR reaction in which the proportion of cells with a high expression of AgNORs can be used as an index of proliferative activity in a tissue sample.     

Diagnostic value of nucleolar organizer regions (AgNORs) in brush biopsies of suspicious lesions of the oral cavity.

OBJECTIVE: The aim of this retrospective study was to report on the diagnostic accuracy of AgNOR-analysis as an adjunctive diagnostic tool of conventional oral exfoliative cytology taken from suspicious lesions in our clinic. STUDY DESIGN: Cytological diagnoses obtained from brush biopsies of macroscopically suspicious lesions of the oral mucosa from 75 patients (final diagnoses: 53 histologically proven squamous cell carcinomas, 11 leukoplakias and other inflammatory oral lesions) and from 11 patients with normal mucosa as a negative control group were compared with histological and/or clinical follow-ups. Five smears were doubtful and seven suspicious for tumor cells in the cytologic report. Number of AgNOR's were counted in 100 squamous epithelial cell-nuclei per slide after silver-restaining. RESULTS: Sensitivity of our cytological diagnosis alone on oral smears for the detection of squamous carcinomas was 92.5%, specificity 100%, positive predictive value was 100% and negative 84.6%. The best cut-off value of the mean number of AgNOR dots per nucleus distinguishing benign from malignant cells was 4.8. The percentage of nuclei with more than three AgNORs had a cut-off level of 70%. Applying these methods to twelve doubtful or suspicious cytological diagnoses we were able to correctly establish the diagnosis of malignancy in ten cases of histologically proven cancers and to reveal benignity in two histologically proven cases. Thus we achieved a positive and negative predictive value of 100% each. CONCLUSIONS: Smears from brushings of visible oral lesions, if clinically considered as suspicious for cancer, are an easily practicable, non-invasive, painless, safe and accurate screening method for detection of oral cancerous lesions. We conclude that AgNOR-analysis may be a useful adjunct to other methods in routine cytological diagnosis of oral cancer that can help to solve cytologically suspicious or doubtful cases.     

Gastric carcinoma: histopathology, immunocytochemistry and variations of nucleolar organizer regions (AgNORs).

The true nature of Nucleolar Organizer Regions Argyrophilic related proteins (AgNORs) is still unknown, but there is wide agreement that their number expresses the nuclear activity. We undertook an investigation on fifty cases of gastric adenocarcinomas (previously grouped morphologically into well and poorly differentiated) applying together with the AgNOR technique, histochemical (Alcian Blue/PAS, High Iron Diamine) and immunohistochemical methods (alpha-1-antitrypsin, alpha-1-antichymotrypsin and lysozyme). AB/PAS was more frequently positive in well-differentiated adenocarcinomas. On the contrary HID was prevailingly positive in poorly differentiated adenocarcinomas. alpha 1 ACT was expressed in all poorly differentiated adenocarcinomas and in a few well-differentiated adenocarcinomas, whereas lysozyme and alpha 1 AT were never expressed. The AgNORs were more numerous, larger, clumped and irregular in shape in poorly differentiated adenocarcinomas. Considering that alpha 1 ACT reactivity seems to be well correlated with survival and given that there is a good correlation between the aforementioned characteristics of AgNORs and the expression of alpha 1 ACT, our investigation suggests that the four techniques used in this study could be useful to predict the prognosis.     

Proliferative activity of the thyroid oxyphilic tumor cells estimated by means of quantitative analysis of silver stained nucleolar organizer regions (AgNORs)

Promising results of studies on different neoplasms, by means of morphometric analysis of argyrophilic nucleolar organizer regions (AgNORs), known as proliferative activity marker, made us undertake an attempt to evaluate of proliferative activity in Hürthle Cell Tumors using the same technique. 78 cases including 20 Hürthle Cell Carcinomas (HCC), 32 Hürthle Cell Adenomas (HCA) and 26 hyperplastic nodules with Hürthle Cell Metaplasia (HCM), diagnosed in the Department of Clinical Pathology, Medical Academy of Bialystok in the years 1990-2000, were subjected to analysis. For visualization of the NORs we used the D. Ploton et al. technique. Mean AgNOR count and NORDS (NOR distribution score--the percentage of nuclei with at least 5 argyrophilic granules) were estimated in each case in 100 randomly chosen nuclei. Non-parametric Mann-Whitney U-test was applied to determine statistically significant differences. RESULTS: Mean AgNoRs counts and NORDS values were 5.1 and 52%, 3.4 and 26%, 2.5 and 7% in HCC, HCA and HCM respectively. Statistically significant differences were found between AgNORs counts in carcinomas and adenomas (p<0.01), HCCs and HCMs (p<0.005) and betwveen NORDS in all groups (p<0.001).     

Age-related changes in cellular activity in human submandibular glands as evaluated by argyrophilic nucleolar organizer regions

Objective: To examine the age-related changes in cellular activity of epithelial components of human submandibular glands, evaluated on the basis of argyrophilic nucleolar organizer regions (AgNORs). Design: Epithelial components of human submandibular glands were divided into serous acinar cells, mucous acinar cells, intercalated duct cells, striated duct cells, and interlobular duct cells. The mean AgNOR number of each cell type was compared among six age groups. Setting: The study was conducted at the Department of Oral Pathology, Tohoku University School of Dentistry, Japan. Subjects: Necropsy specimens from 66 males and 57 females 1 to 97 years old. Results: In all cell types except for intercalated duct cells, the mean AgNOR number was lowest in the 0-14 year-old group and highest in the 15-29 year-old group. The value then gradually decreased with advancing age and ultimately reached a similar level to that in the 0-14 year-old group. In intercalated duct cells, the mean AgNOR number did not differ significantly between any age group. There were no significant sex-related differences. Conclusions: The cellular activity of almost all components of human submandibular glands rises in adolescence and young adulthood and then decreases with aging. These results suggest that intercalated duct cells are capable of not only proliferation but also division into other components; these cells may thus compensate for the reduced activity of other components in elderly subjects.     

Micronuclei,nuclear lesions and interphase silver-stained nucleolar organizer regions (AgNORs) as cyto-genotoxicity indicators in Oreochromis niloticus exposed to textile mill effluent

In this study, cyto-genotoxic effects of a textile mill effluent on fish Oreochromis niloticus were investigated using the micronucleus (MN) test and methods to analyze interphase silver-stained nucleolar organizer regions (AgNORs). Fishes were exposed to three different concentrations of textile mill effluent (5, 10 and 20%(v/v)) for 3, 6 and 9 days. Cyclophosphamide (2mg/l) was used as a positive control. Micronucleus frequencies were examined in peripheral blood erythrocytes and gill cells. Nuclear abnormalities (NA) other than micronuclei such as binuclei, lobed nuclei, blebbed nuclei and notched nuclei were also evaluated in peripheral erythrocytes. Interphase AgNOR parameters were examined in epithelial cells obtained from the edge of caudal fins after 90 and 180min of exposure. As a result, dose-dependent increases in the frequencies of micronuclei and other NA in erythrocytes were observed. MN frequencies in gill cells also significantly increased, while the interphase AgNOR parameters in fin cells decreased, as a result of textile effluent and cylophosphamide treatments.     

Nucleolar organiser regions in colonic dysplasia. A preliminary study

As a preliminary investigation in the evaluation of the argyrophilic nucleolar organiser region (AgNOR) technique in colonic dysplasia, quantitation of AgNORs was carried out in biopsies of normal rectal mucosa and tubulovillous adenomas. The AgNOR counts in the lower third of the normal crypts were approximately twice those in the surface mucosa but there was no significant difference between counts in normal crypt bases and adenomas. It is concluded that the AgNOR technique is unlikely to be of value in the assessment of colonic dysplasia.     

Argyrophilic nucleolar organizer regions (AgNORs) in interphases and metaphases of normal and neoplastic gill cells of Macoma balthica (Bivalvia: Tellinidae) from the Gulf of Gdansk, Baltic Sea

Chromosome analysis of gill cells of different populations of Macoma balthica (L.) from the Bay of Gdansk (Baltic Sea) revealed 2 clam categories, 1 with neoplastic features and 1 without. Silver-staining was performed on interphase and metaphase cells of both categories. The mean argyrophilic nucleolar organizer region (AgNOR) count per abnormal interphase cell was significantly higher than in normal interphase cells. Normal silver-stained metaphases had 3 nucleolar organizer region (NOR) chromosome phenotypes. The location of the NORs in the most frequent phenotype (55.6% in 54 metaphases scored) was interstitial on the largest metacentric chromosome pair, Pair No. 1. Abnormal silver-stained metaphases had a higher number of active NOR sites. Different phenotypes were observed (frequency greater than 10% for 67 metaphases scored); 2 were similar to those in normal metaphases and 5 were ectopic. The higher activity of AgNORs observed in abnormal cells confirmed the diagnosis of malignant neoplasia.     

Expression of nucleolar organizer regions (NORs) in ovarian epithelial tumors

AgNOR staining technique was tested in ovarian epithelial tumors to evaluate its diagnostic potential in distinguishing between borderline tumors and well-differentiated carcinomas. In our opinion, the AgNOR count appears useful for assessing differences only between borderline and well-differentiated serous ovarian tumors at stage I of FIGO clinical advancement.     

A comparison of within-plant karyological heterogeneity between agamospermous and sexualTaraxacum (Compositae) as assessed by the nucleolar organiser chromosome

Morphological variation for the NOR chromosome was studied for four half-siblings of a sexual outbreedingTaraxacum, for three siblings of the obligate agamospermT. pseudohamatum, and for two individuals of the agamospermT. brachyglossum. No rearrangement was detected for the 113 chromosomes of sexuals, or for 41 chromosomes of two agamospermous individuals. In the other three agamospermous individuals, 3/16, 5/50, and 5/20 chromosomes showed evidence of chromosomal rearrangement. The majority of rearrangement events (10/13) occurred to the satellite rather than to the body of the NOR-chromosome. It is considered that such high levels of somatic chromosomal rearrangement in agamospermousTaraxacum may be the result of activity by transposable genetic elements. This recombination may be of selective advantage to asexual plants which cannot generate genetic variability through the sexual process.     

Silver staining of the nucleolar organizer regions (NORs) in semithin Lowicryl sections

The one-step silver technique was applied to semithin Lowicryl sections of root meristem cells of Allium cepa and a human tumor cell line (TG cells). In vegetal cells, after 5 min of staining reaction, the Ag-NOR proteins formed ring-shaped structures peripherally within the nucleolus. In animal cells silver granules were distributed over the entire nucleolus. The specificity of the staining reaction was increased by incubation of the sections in NH4Cl and Schiff's reagent prior to Ag-NOR silver staining.     

Melanin bleaching in argyrophilic staining of AgNORs in pigmented lesions: a morphometric evaluation

OBJECTIVE: To establish a procedure that can effectively bleach melanin from pigmented lesions without affecting quantification of argyrophilic staining of nucleolar organizer regions (AgNORs). STUDY DESIGN: Twenty banal compound nevi, five from each of nonpigmented, slightly pigmented, moderately pigmented and heavily pigmented groups, were bleached by 10% H202 for periods of 0 (nonbleached controls) and 24 hours. AgNOR size and count parameters of nevomelanocytic nuclei were measured by video image analysis. Melanin bleaching using KMnO4 was also investigated. RESULTS: In all lesions treated with 10% H202 for 24 hours, the melanin was bleached effectively, with no qualitative change in AgNOR appearance. There were no significant differences in mean AgNOR number per nucleus (AgNOR number), mean individual AgNOR size (AgNOR size) or mean percentage of AgNOR area per nucleus (% nuclear area) between nonbleached and bleached sets in both the nonpigmented and slightly pigmented groups. However, disintegration of AgNOR dots was observed in those treated with 1% KMnO4 for 5, 10 and 15 minutes. There were significant decreases in AgNOR size (P = .002) and % nuclear area (P = .003) and increase in AgNOR number (P = .05) in the slightly pigmented group evaluated when treated with 1% KMnO4 for five minutes. CONCLUSION: Melanin in pigmented lesions can be bleached effectively with an H202 procedure without significantly affecting AgNOR staining properties in contrast to bleaching with KMnO4.     

A combined staining method for argyrophilic nucleolar organizer regions and for glial fibrillary acidic protein in astrocytes of human brain

Summary Different protocols are described for the combined staining method by which argyrophilic nucleolar organizer region sites can be evaluated in human astrocytes that are immunoreactive for glial fibrillary acidic protein. Among the four protocols studied, the following method was superior to others in terms of unambiguous visualization of the regions in glial fibrillary acidic protein-positive astrocytes; the first step was immunostaining for the protein with a blue colour reaction of alkaline phosphatase, followed by sequential colloidal silver staining for the regions. By this double staining method, we have demonstrated that the reactive astrocytes found in white matter around the metastatic lesion of carcinoma and the infarction, contain more argyrophilic nucleolar organizer regions in terms of the count as well as the area than glial fibrillary acidic protein-positive astrocytes present in the white matter of the normal brain. In conclusion, the double staining may provide valuable information on the cellular activity of astroglia when performed on routine formalin-fixed paraffin sections of the human brain.