Silver-binding Nucleolar Organizer Regions Research Articles

Cytogenetics of Two Onychostoma Species in Taiwan by Ag-NOR and 18S rDNA Profiles.

Chiao-Chuan Han, Tsair-Bor Yen, Nian-Cih Chen, and Mei-Chen Tseng (2017) Both Onychostoma barbatulum and O. alticorpus are primary freshwater fish in Taiwan. The former has been developed as an aquaculture species with high economic value, while the latter is a native endemic species in Taiwan. Understanding the cytogenetic information of these two species is necessary for their selected breeding, recovery, and management. In this study, Giemsa staining, silver-binding nucleolar organizer region (Ag-NOR), C-banding, and fluorescence in situ hybridization (FISH) with 18S ribosomal (r)DNA probes were used to analyze the cytogenetic characteristics. Results of Giemsa staining showed that the two Onychostoma species shared the same number of chromosomes, 2n = 50. Respective karyotype formulas of the female and male were 10 m + 22 sm + 10 st + 8 t and 11 m + 22 sm + 10 st + 7 t in O. barbatulum, and 14 m + 18 sm + 8 st + 10 t and 15 m + 18 sm + 8 st + 9 t in O. alticorpus. Karyotypes of both species showed a pair of heteromorphic chromosomes in male fish. Their sex determination should be the XX/XY system. Two pairs of Ag-NORs were found in O. barbatulum, but only one pair occurred in O. alticorpus. C-banding areas were observed on centromeres or telomeres of some chromosomes. FISH revealed different cytogenetic characters between these two species. The above cytogenetic information will contribute to species identification, population recovery, and advantages for breeding and management in the future.

Analysis of silver binding nucleolar organizer regions in exfoliative cytology smears of potentially malignant and malignant oral lesions

Nucleolar organizer regions are nucleolar components that contain proteins that are stained selectively by silver methods; they can be identified as black dots throughout the nucleolus and are known as silver binding nucleolar organizer regions (AgNOR). The number of AgNOR is related to the cell cycle and the proliferative activity of the cells. We investigated AgNOR using exfoliative cytology smears of potentially malignant oral lesions. Eighty individuals were divided into four equal groups: healthy controls, oral leukoplakia, oral submucous fibrosis and oral squamous cell carcinoma. The mean number of AgNOR in each study group gradually increased from control to oral leukoplakia to oral submucous fibrosis to oral squamous cell carcinoma. The proliferative index was increased in the oral premalignant and malignant patients compared to normal subjects. The mean AgNOR size gradually increased from control to oral leukoplakia to oral submucous fibrosis to oral squamous cell carcinoma. Spherical shaped AgNOR were most common in controls, whereas large, clustered and kidney shapes were most common in oral squamous cell carcinoma. Multiparameter analysis of AgNOR in oral exfoliative smears is a simple, sensitive and cost-effective method for differentiating premalignant from malignant lesions and can be used in conjunction with routine cytomorphological evaluation.

Quantitative analysis of Argyrophilic Nucleolar organizer regions in odontogenic cysts and tumor – A comparative study

BackgroundThe nucleolar organizer region (NOR) is by definition part of a chromosome, and nucleolus is a structure containing this chromosomal part and in addition the material which accumulate around the NOR, mostly rRNAs and their precursors as well as specific ribosomal proteins. Argyrophilic Nucleolar organizing region (AgNOR) are silver binding NORs often used to study cell proliferation in various types of tumors. AimQuantitative assessment of Argyrophilic Nucleolar organizing region count and its comparison among dentigerous cyst, keratocystic odontogenic tumor and ameloblastoma. Material and methodForty-five histologically confirmed cases, 15 cases each of keratocystic odontogenic tumor, dentigerous cysts and ameloblastomas were examined for Argyrophilic Nucleolar organizing region. The sections were obtained and Argyrophilic Nucleolar organizer regions staining was done for comparing the proliferative capacity among these lesions. ResultPost hoc analysis for inter-group comparison and one way ANOVA were done in all three groups in this study. P<0.001 was considered significant. The results of AgNOR counts were higher in KCOTs as compared to ameloblastoma and least in dentigerous cysts. The mean AgNOR counts between the study groups were compared using one way ANOVA test and the differences were found to be significant (P<0.001). ConclusionAgNOR counts were significantly higher in KCOT and ameloblastoma as compared to dentigerous cyst suggesting that these lesions have a higher proliferative capacity than dentigerous cyst. The finding of a significantly higher AgNOR counts in KCOT as compared to ameloblastoma represent a difference in proliferative activity and greater growth potential between these two lesions.

Silver binding nucleolar organizer regions dots in oral leukoplakia with epithelial dysplasia and oral squamous cell carcinoma: an in vivo study.

Silver binding nucleolar organizer regions (AgNOR) in normal oral mucosa (NOM), oral leukoplakia with epithelial dysplasia (ED), and oral squamous cell carcinoma (OSCC) were studied. The mean AgNOR count per nucleus increased from NOM to ED to OSCC. Tissue showing ED in oral leukoplakia and OSCC cases showed higher counts, wider scatter, and smaller size of AgNOR dots in the nuclei. The study seems to suggest that time method has some potential in distinguishing between NOM and oral leukoplakia with ED and OSCC. Studies of larger numbers are needed to arrive at more substantial conclusions.

Histologic grading and nucleolar organizer regions in oral squamous cell carcinomas

ObjectiveThe purposes of this study were to histologically assess different types of oral squamous cell carcinoma and the silver-binding nucleolar organizer region (AgNOR) morphology in neoplastic cells, as well as to quantify the number of AgNORs in each type of carcinoma in order to relate AgNOR count and histologic grading.Material and MethodsTwenty-eight cases of oral squamous cell carcinoma were divided into 4 groups, namely well-differentiated, moderately differentiated, poorly differentiated, and undifferentiated. For NOR study, 3-µm-thick sections were stained with 50% aqueous silver nitrate solution. The predominant microscopic pattern of NORs was determined. Quantitative analyses of NORs were obtained of all cells present on each histological field using a 0.025 mm2 eyepiece graticule. Different histological fields were analyzed until the total number of NORs was 120 cells for each tumor. Kruskall-Wallis test was applied to compare the groups of sample data at a significance level of p=0.05.ResultsThe mean number of AgNORs per nucleus was 3.20 for the well-differentiated group, 5.33 for the moderately differentiated one, 8.27 for the poorly differentiated one, and 10.08 for the undifferentiated one. AgNOR count was significantly different (p<0.05) among all of the studied groups.ConclusionAgNOR staining technique seems to be a useful diagnostic tool since differences in AgNOR numeric values can be identified in the different types of oral squamous cell carcinoma. This technique is easy to handle and inexpensive, thus justifying its large use in histopathology.

A study of HER-2/neu expression and silver binding nucleolar organizer regions (Ag NORs) in perilesional normal salivary gland tissue

Salivary gland neoplasms comprise phenotypically and biologically diverse lesions of uncertain histogenesis. The present study was carried out to assess the immunohistochemical expression of HER-2/neu protein and of silver binding nucleolar organizer regions in normal salivary tissue. Twenty paraffin blocks of salivary tumors containing surrounding normal salivary tissue were used in the study. One section was processed to investigate HER-2/neu protein expression and another section was stained to assess the mean Ag NOR counts in normal salivary tissue. Only two cases of the studied normal salivary tissue showed HER-2/neu expression (10%), which was expressed only in ductal cells with 4.2±2.1 mean number of reactive cells per five high power fields (HPFs). Statistically significant (P=0.01) higher mean Ag NOR counts in ductal (2±0.83) than in acinar cells (1.2±0.01) of normal salivary tissue were found. It was concluded from the data of the present study that ductal cells of normal salivary tissue are a proliferative pool and an oncogenic target which plays an important role in the histogenesis of salivary gland tumors.

The diagnostic and prognostic implications of silver-binding nucleolar organizer regions in periodontal lesions

The periodontal lesions with cellular proliferation can be assessed by various methods. One of the most recent methods to determine the proliferative activity is silver-staining nucleolar organizer region (AgNOR) staining. The purpose of the present study was to evaluate, if AgNOR count can act as a proliferative marker and can aid in the diagnosis and prognosis of periodontal lesions. For this study, subjects with healthy gingival status, non-neoplastic lesions, neoplastic lesions, and plaque-induced gingivitis were included. Following the provisional diagnosis of clinical entity, biopsies were taken from the respective selected sites for histopathological diagnosis. In plaque-induced gingivitis cases, a second biopsy was taken from the selected sites 3 weeks following scaling. After histological confirmation, one more section was prepared, which was subjected to AgNOR staining, and AgNOR numbers were counted by individual and cluster counts and statistically analyzed. Results showed the highest AgNOR count in neoplastic lesions. Non-neoplastic lesions showed a higher AgNOR count as compared to clinically healthy gingiva. Plaque-induced gingivitis showed a considerable reduction in AgNOR count after treatment. Results of this study confirmed that AgNOR count reflects the cellular proliferation and has a limited diagnostic value. However, the prognostic value of AgNOR for periodontal lesions is dependable.

Treatment with Valproic Acid Restores Differentiation in AML1/ETO-Positive Leukaemic Cells.

In t (8, 21) acute myeloid leukaemia (AML), the leukaemogenesis is supposed to be promoted by interference with expression of AML1 target genes. Repressor complex associated with AML1/ETO fusion protein recruits class I histone deacetylases (HDAC). Valproic acid (VPA) was found to have an extensive effect on AML blasts, via inhibition of class I HDAC. It was shown previously that VPA treatment disrupts the AML1/ETO-HDAC1 complex from AML1 promoter thus leading to apoptosis at different cell lines. However, there is still lack of in-depth morphological and immunophenotypical proof of the hypothesized restoration of differentiation after treatment with VPA. We aimed to characterize the differentiation effect of VPA on AML1/ETO-positive leukaemic cells. Kasumi-1 (AML1/ETO-positive) cell line and MV4-11 (MLL/AF4-positive) cells were treated with VPA and Trichostatin-A (TSA) another HDAC inhibitor. The effect of VPA on cell cycle and differentiation was examined by flowcytometry. DNA, RNA and AgNORs staining of nucleus and nucleolus were used for morphological evaluation. VPA treatment of Kasumi-1 cells resulted in decreased expression of early myeloid progenitor antigens [CD33 (p<0.001), CD34 (p=0.003), CD117 (p<0.001)] and increased expression of antigens typical for more differentiated myeloid cells [CD11a (p=0.001), CD11b (p=0.007)]. Morphological evaluation revealed nucleus fragmentation without chromatin condensation, which evoked monocytoid look of the nucleus in VPA-treated Kasumi cells. The nucleolar morphology and cytochemistry indicated that these cells entered the ageing process and decreased proliferation activity. Large nucleoli characterised by a reduced number of silver-binding nucleolar organizer regions (AgNORs) as well as by their translocation to the nucleolar periphery represented a base for such interpretation. VPA-treated Kasumi cells exhibited accumulation in G1/G0 phase and decreased proliferation (VPA 0.5mM - G1/G0 70%, S/G2/M 16%; control cells - G1/G0 62%, S/G2/M 28%). Conversely, MV4-11 cell line (representing myeloid leukaemic cells of non-CBF origin) did not show any statistically significant differences in imunophenotype after VPA treatment (CD15, CD11b, CD117, CD45, HLA DR, CD14). VPA treated MV4-11 cells did not show accumulation in G1/G0 phase (VPA 0.5mM G1/G0 63%, S/G2/M 13%; control G1/G0 70%, S/G2/M 22%). We show that treatment with VPA restores differentiation of AML1/ETO positive cells as presented by both changes in immunophenotype and morphological analysis. This observed differentiation correlates with decrease in cell cycle. We demonstrate that the effect on differentiation is specific for AML1/ETO-positive cells as it is not noticeable in leukaemic cells of non-CBF origin.

Effects of dexamethasone on pulp cells in cultured human teeth.

This study set out to examine in vitro the action of dexamethasone (Dex), a synthetic glucocorticoid on the pulp cells of human teeth, particularly in the cell-rich zone of the pulp where cell density is especially high. The present experiment was performed on 10 third molars extracted from patients aged between 16 to 20 years for orthodontic reasons under local anesthesia. The teeth were otherwise found to be sound and free of dental caries. Upon extraction, each tooth was divided into two parts mesio-distally. Each part was then cultured separately in Dulbecco's modified Eagle medium supplemented with 10% fetal bovine serum for a period of 7 days. 100nM Dex, 1mM sodium-β-glycerophosphate and 0.2mM ascorbic acid were added to one half of the total cultures which served as the experimental group. Four areas of the pulp tissue, the roof, the root, the lateral wall and the base were then each divided into three layers of equal thickness measuring 150×33.0±8.26 (mean±S.D.) μm to correspond to the average thickness of the cell-rich zone and observed histological examinations of the cells were performed by hematoxylin-eosin (H.E.) staining and silver-binding nucleolar organizer region (AgNOR) staining. Results obtained from the test group cells showed a significant increase in the mean number of AgNOR in the roof and root areas in the cell-rich zone in addition to a slightly larger mean cell size in almost every zone. This shows that culture of 7 days in dexamethasone-added medium can lead to a heightened level of cell activity in the roof and root areas of the cell-rich zone.

AgNOR counts and their combination with flow cytometric analyses and clinical parameters as a prognostic indicator in breast carcinoma.

Silver binding nucleolar organizer regions (AgNORs) were counted in tissue sections and were shown to assist in the distinction between benign and malignant breast lesions. The mean AgNOR counts in fibroadenomas were 1.05 +/- 0.85 (n = 18), in lobular carcinoma 3.55 +/- 0.56 (n = 35), and in intraductal carcinoma 4.83 +/- 1.20 (n = 45). AgNOR counting may provide information on breast cancer prognosis supplementary to that obtained with other methods such as flow cytometric analysis. The AgNOR values in carcinomas were also compared with ploidy and S-phase fractions (S) by flow cytometry. More than 75% of the total cancers with counts above 3 dots per nuclear profile contained aneuploid cells, whereas 20% with counts below 3 contained diploid cells (P < 0.05). Similar trends were noticed with regard to S-phase fractions, which were 45% +/- 12.5 and 14% +/- 4.5, respectively, for the two groups (P < 0.05). AgNOR counting, flow cytometry and clinical parameters may provide complementary information on breast cancer prognosis.

Oral submucous fibrosis. A review.

Oral submucous fibrosis (OSF) affects an estimated 2.5 million people, mostly in the Indian subcontinent. Limitation of oral opening resulting in difficulty in eating is the main presenting feature. Although nutritional deficiencies and immunological processes may play a part in the pathogenesis, the available epidemiological evidence indicates that chewing betel quid (containing areca nut, tobacco, slaked lime or other species) is an important risk factor for OSF. Genetically determined susceptibility could explain why only a small fraction of those using betel quid develop the disease. In OSF there is an incidence of oral cancer of 7.6 per cent for a median 10-year follow-up period. Risk markers for malignant transformation in OSF include epithelial dysplasia, silver binding nucleolar organizer region counts, and sister-chromatid exchange frequencies; p53 tumour suppressor gene mutations may be involved in these potentially malignant changes.

Number of Nucleoli and Nucleolar Organizer Regions per Nucleus and Nucleolus — Prognostic Value in Canine Mammary Tumors

Twenty-eight canine mammary tumors were evaluated for histopathologic classification as recommended by the World Health Organization and silver-binding nucleolar organizer region (AgNOR) and nucleolus counts. Samples of surgically excised tumors and tumors taken at necropsy were fixed in neutral formalin, embedded in paraffin, and cut into 1-3-microns-thick sections. Two sections were taken from each tumor: one was stained with hematoxylin and the other was treated with the silver staining technique for the demonstration of AgNORs. After histopathologic classification, the number of nucleoli and the number of AgNORs/nucleus and AgNORs/nucleolus were determined. Statistical analysis (Student's t-test) showed a significant difference in the mean number of nucleoli (P < 0.005), mean number of AgNORs/nucleolus (P < 0.001), and mean number of AgNORs/nucleus (P < 0.005) between benign and malignant canine mammary tumors. There was no significant differences between metastatic and nonmetastatic malignant tumors.

Biological behavior of human dental pulp cells in response to carious stimuli analyzed by PCNA immunostaining and AgNOR staining.

The change in proliferative and metabolic activities of human dental pulp cells responding to carious stimuli was studied by means of immunohistochemical staining for proliferating cell nuclear antigen (PCNA) and silver-binding nucleolar organizer region (AgNOR) staining. We classified the pulp tissues into five groups according to the progression of dental caries, ranging from grade 0 (the pulp of noncarious teeth) to grade 4 (the pulp of perforated carious teeth). PCNA-positive pulp cells were detected only in advanced dental caries (grades 3 and 4), and the difference in immuno-positive rate was significant between the two grades (p <0.001). However, the mean number of AgNORs per nucleus increased even in the early phase of dental caries, significant differences being detected between grades 1 and 2 (p<0.005), 2 and 3 (p<0.005), and 3 and 4 (p<0.001). Our data suggested that the metabolic activity of dental pulp cells was enhanced in the early phase of dental caries. However, proliferation of pulp cells occurred later in small degrees during fully developed caries such as grades 3 and 4. The slow and weak response in cellular proliferation might contribute to the usual fragility of the pulp to various assaults including caries or pulpitis.

Different expression of nm23-H1 and nm23-H2 protein with proliferative activities in various sizes of human renal cell carcinoma.

Ninety-five paraffin blocks of renal cell carcinomas (RCCs) and background normal tissue were obtained from patients in Nara Medical University Hospital and used for studying silver-binding nucleolar organizer regions (AgNORs), proliferating cell nuclear antigen (PCNA) and nm23 oncogenes. The RCCs were grouped by size (< or = 30, > 30 and < or = 50, > 50 mm of average diameter) and grade according to the General Rules for Clinical and Pathological Studies of Renal Cell Carcinoma, Japan. Numbers of cases were 17 in the < or = 30-mm group, 36 in the > 30- and < or = 50-mm group, and 42 in the > 50-mm group. AgNOR scores were 4.68 +/- 1.22, 3.91 +/- 1.31 and 3.01 +/- 1.05, respectively, with significant intergroup differences. Frequencies of PCNA-positive cells per 500 cells were 33.8 +/- 23.5, 39.4 +/- 13.8 and 45.9 +/- 38.8. nm23-H1 immunohistochemical staining proved strongly positive in 21 of 24 areas of normal proximal convoluted tubules, 4 of 8 dysplasia cases, 2 of 17 in the < or = 30-mm group RCCs, 1 of 36 in the > 30- and < or = 50-mm group RCCs and none of 42 in the > 50-mm RCCs. nm23-H2 in contrast was weakly positive in 3/24, 2/8, 12/17, 30/36 and 20/42 of the cases, respectively. The results indicated an inverse relationship between tumor size and score of AgNOR without any variation in PCNA labeling and contrasting expression of nm23-H1 and nm23-H2 linked to the size of RCCs.

THE ESTIMATION OF PROLIFERATIVE ACTIVITY BY PCNA AND AGNORS IN LEUKOPLAKIA AND SQUAMOUS-CELL CARCINOMA OF THE ORAL CAVITY

The proliferating activity of leukoplakia and squamous cell carcinoma (SCC) was estimated using proliferating cell nuclear antigen (PCNA) immunostaining and silver-binding argyrophilic nucleolar organizer region (AgNOR) staining. Twenty-eight leukoplakias and 15 SCCs of the oral cavity were used in this study. The mean+/-S.D. of PCNA LI and AgNOR counts were 7.7+/-6.0% and 2.43+/-0.68/nucleus in leukoplakias, and 22.3+/-11.6% and 4.77+/-1.49/nucleus in SCCs. Both of PCNA LI and AgNOR counts were significantly higher in SCCs than in leukoplakias. There was a significant linear correlation between PCNA LI and AgNOR counts (p=0.0022) in leukoplakias, but not in SCCs. In the series of leukoplakias, PCNA LI apparently increased in leukoplakias with dysplasia and malignant transformed cases. Our data suggest that PCNA LI and AgNOR counts are useful markers of proliferating activity, and PCNA LI might be a prognostic factor of leukoplakias.

Progressive growth of rat bladder carcinomas after exposure to prolonged uracil‐induced urolithiasis

Dietary uracil at the 3% level induces urinary bladder tumors in rats through urolithiasis-dependent mechanical irritation. In the present study, comparison of lesions induced by uracil administration over the different periods of 36 weeks (middle-term) and up to 103 weeks (long-term) revealed significant elevation of both incidences and multiplicity of transitional cell carcinomas (TCCs) in the long-term group. Histopathological assessment in terms of tumor biology further demonstrated significantly higher grading on the basis of the degree of cellular and structural atypia, and greater depth of invasion in the long-term group. Application of markers for cell proliferation activities including proliferating cell nuclear antigen (PCNA) and silver-binding nucleolar organizer regions (AgNORs) also revealed significantly elevated AgNOR counts in the long-term group TCC. AgNOR counts and PCNA rates in TCCs showed relation to the histological grades. Thus the present study demonstrated that prolonged uracil-induced urolithiasis causes more biologically aggressive bladder carcinomas with invasive potential. Continuous stimulation of cell proliferation presumably has the potential to facilitate multiple genetic alterations leading to development of more malignant carcinomas. However, it should be borne in mind that the difference in bladder cancer development might also be related to the fact that the animals survived longer and that the early lesions therefore had more time to progress to more advanced stages.

Silver-binding argyrophilic nucleolar organizer regions (AgNOR) in gall-bladder cancer: correlations with malignant grading and prognosis.

Silver-binding nucleolar organizer regions (AgNOR) were investigated and compared in 43 cases of gall-bladder cancers and 10 normal gall-bladder samples using an image analyzer. The mean numbers of AgNOR per nucleus (AgNOR number) were 3.28 +/- 1.38 in the gall-bladder cancers and 1.86 +/- 0.20 in the normal gall-bladder cases. The respective mean areas of AgNOR per nucleus (AgNOR area) were 6.96 +/- 3.78 microns2 and 1.89 +/- 0.21 microns2. The differences were statistically significant (P < 0.0001) for both parameters. In addition, increased frequency and enlargement were both apparently correlated with poor prognosis (P < 0.011 and P < 0.046, respectively), with AgNOR number and AgNOR area showing tendencies for increase in cases of histologically high grade malignancy, advanced cancer and regional lymph node involvement. In conclusion, AgNOR number and AgNOR area appear to be useful indicators for the grading of malignancies and for the prediction of gall-bladder cancer prognosis.

Silver-binding nucleolar organizer region proteins as a possible prognostic indicator in oral submucous fibrosis

Silver-binding nucleolar organizer region proteins (AgNORs) were counted in sections from formalin-fixed, paraffin-embedded tissue blocks of oral submucous fibrosis (OSMF) and oral squamous cell carcinoma (SCC). Moderately advanced and advanced cases OSMF, and well-differentiated and poorly differentiated cases of SCC, were studied with respect to the relation between AgNOR counts and histologic grading. Normal oral mucosa collected from age- and sex-matched subjects constituted the control group. The pooled mean (±SD) AgNOR counts were as follows: normal mucosa, 2.50 ± 0.52 ( n = 5); moderately advanced OSMF, 5.24 ± 1.23 ( n = 10); advanced OSMF, 7.26 ± 1.12 ( n = 10); well-differentiated SCC, 8.29 ± 1.47 ( n = 5); and poorly differentiated SCC, 9.49 ± 0.74 ( n = 5). The pooled mean AgNOR counts in advanced OSMF were higher than in moderately advanced cases ( t = 3.84; p < 0.005) and those in poorly differentiated SCC were higher than those of well-differentiated SCC, although the comparison was not significant in the latter ( t = 1.63, p > 0.10). AgNOR counts could hold promise for predicting the biologic behavior of OSMF because this study demonstrated a correlation with clinical and histologic grading.

Silver binding nucleolar organizer regions in adrenocortical neoplasia.

The possible contribution of the silver colloid technique for staining nucleolar organizer regions in the distinction between benign and malignant adrenocortical neoplasms was investigated. Nine cases of adenoma, eight cases of carcinoma, nine cases of hyperplasia and four normal adrenal cortex specimens were examined. The mean silver binding nucleolar organizer region (Ag-NOR) value in adenoma was 4.29, and in carcinoma 7.16 (P less than 0.001). Adenomas with diameters greater than 3 cm had significantly higher Ag-NOR counts than smaller adenomas. For normal cortex, the mean Ag-NOR value was 2.05 and in hyperplasia, 3.62. The results indicate that the Ag-NOR technique can help in differential diagnosis between benign and malignant adrenocortical lesions and thus may have a prognostic value.

"Nucleolar organizer regions" as a factor for the prognosis of laryngeal cancer?

The prognostic significance of silver-binding nucleolar organizer regions (AgNORs) was assessed in tissue sections of primary tumours taken from 30 patients with squamous cell carcinomas of the larynx. By means of semiautomated image analysis the AgNORs of 100 tumour cell nuclei were measured. Their number as well as their area were evaluated together with morphometrical features (obtained by automated image analysis) and histopathological and clinical data. Considering the mean number of AgNORs the multivariate discriminant analysis revealed significant differences between carcinomas of patients, who were alive of at least 3 years without local or distant recurrence and those, who had died of laryngeal cancer (p less than 0.03%). There was, however, no correlation between the AgNOR number and the DNA features. But, on the other hand, the DNA features, e.g. the 5c exceeding rate according to Böcking and the DNA index according to Atkin were found to be correlated with the tumour size (p less than 0.08%). These preliminary results indicate that the AgNOR number as well as the DNA features could be independent prognostic parameter in laryngeal squamous cell carcinoma, complementary to the staging categories.