Nucleolar organizer regions in human esophageal disorders: comparison with proliferating cell nuclear antigen by immunostaining.

A silver colloid technique to demonstrate nucleolar organizer region-associated proteins (AgNORs) was performed on sections of 15 samples of human esophageal tissue, including five nonpathological esophageal epithelium, two esophageal dysplasia of the squamous epithelium, and eight esophageal squamous cell carcinomas. Initially we examined various protocols for AgNOR staining. Staining performed on 4% paraformaldehyde-fixed paraffin-embedded specimens with an incubation time of 30 min yielded the most satisfactory results. In nonpathological esophageal epithelium, the mean number of AgNOR counts per nucleus in the four layers of esophageal epithelium was greatest in the parabasal layer and was statistically significant. No significant differences were observed among the mean number of AgNOR counts per nucleus in the nonpathological parabasal layer, dysplasia, and carcinoma. Positive correlation was observed between the PCNA labeling index of esophageal disorders and the mean number of AgNOR particles per nucleus. Therefore, in esophageal disorders, the AgNOR staining per nucleus appears to correlate with proliferative activity but is of little practical value in discerning malignancy and/or aggressive biological behaviors.     

Nucleolar organizer regions in human adrenocortical disorders

A silver colloid technique to demonstrate nucleolar organizer region-associated proteins (AgNORs) was performed on sections of 39 human adrenal glands, including normal adrenal cortex, bilateral adrenocortical hyperplasia, adenoma, and carcinoma. In mineralocorticoid and glucocorticoid-producing adrenocortical cells, the mean number of AgNORs per nucleus was lowest in the normal adrenal gland and highest in adenoma, with statistically significant differences. The mean number of AgNORs in bilateral adrenocortical hyperplasia was placed between that of normal adrenal and adenoma. AgNOR numbers in morphologically and clinically verified adrenocortical carcinoma (12 cases) were not different from those in adenoma (17 cases). In adrenal cortex, AgNOR numbers may be correlated with increased steroid hormone production but appear to be of little value in discerning malignancy in adrenocortical neoplasms.     

Nucleolar organizer regions in myogenic stromal tumours of the stomach

Summary Silver staining of nucleolar organizer regions (AgNOR) was studied in 26 primary benign and malignant gastric stromal tumours of myogenic origin. The absolute number of AgNOR per nucleus and the size of AgNOR were compared with histomorphologic features of the tumours.The total number of AgNOR per nucleus in epithelioid and spindle cell leiomyosarcomas significantly (p<0.001) exceeded that in leiomyomas, cellular leiomyomas and epithelioid leiomyoblastomas. The mean number or the size of AgNOR did not correlate with the number of mitoses or the tumour size. In addition, large and bizarre AgNOR were seen predominantly in histologically malignant tumours. Only one exceptional epithelioid leiomyoblastoma recurred despite a lack of conventional characteristics of malignancy and a low AgNOR count.Therefore, quantitative determination of the number of AgNOR is a new independent variable in myogenic gastric tumours. It provides additional information for the histopathological evaluation of this heterogenous group of mesenchymal tumours.     

鼻咽泡状核细胞癌中癌细胞群体的AgNORs定量分析

目的：通过癌细胞核仁组成区（NORs)蛋白的图像定量分析，评价鼻咽泡状核细胞癌的癌细胞群体增生能力。方法：对18例鼻咽泡状核细胞癌石蜡切片进行银染（AgNORs)，应用CAS200图像分析仪分别测定泡状核癌细胞群体和梭形癌细胞群体的AgNORs参数值，并作比较分析。结果：与梭形癌细胞群体相比，泡状核癌细胞群体具有较高的每核AgNOR计数、每核AgNOR面积和平均AgNOR面积／粒，差异均有显著性，而且与泡状核癌细胞群体的增殖细胞核抗原（PCNA）阳性率明显高于梭形癌细胞群体的前研究结果相一致。结论：泡状核癌细胞群体的增生能力明显高于梭形癌细胞群体，增生能力各异的癌细胞群体存在于同一组织类型鼻咽癌中，反映了鼻咽癌细胞群体增殖能力的异质性，其构成比例和增生差异程度将是影响患者对放射线治疗敏感性及其预后的重要因素之一。     

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     

Cytopathology and AgNOR counts in fine-needle aspiration cytology smears of thyroid lesions

The object of the present work is to study the cytomorphological features and the value of silver colloidal staining method in distinguishing the non-neoplastic, benign, and malignant neoplasm in fine-needle aspiration cytology (FNAC) smears of thyroid nodules. One hundred forty histologically confirmed aspirated samples of thyroid lesions were studied and classified cytologically. These smears were stained for AgNOR counts. The number and location of AgNOR dots as well as clusters in nuclei were studied by two different observers independently. Lower AgNOR counts were recorded in cases of thyroiditis (1.375 ± 0.414), whereas follicular carcinoma had a higher number of AgNOR counts (5.04 ± 0.52). The clusters of AgNOR dots were centrally located in colloid goitre, but no cluster arrangement was observed in cases of carcinomas. Increased nuclear size, nucleoli, and chromocentres were helpful in the diagnosis of follicular carcinoma. AgNOR counting cannot be reliably used on an individual case basis to differentiate adenoma from carcinoma. Therefore, AgNOR study in thyroid lesions can be used as an additional diagnostic method with cytomorphological features to differentiate benign and malignant follicular neoplasms. Diagn. Cytopathol. 1998;19:238–243. © 1998 Wiley-Liss, Inc.     

Nucleolar organiser regions in normal, cirrhotic, and carcinomatous livers.

A series of 54 liver biopsy specimens was studied by means of the argyrophil (AgNOR) technique for nucleolar organiser region (NOR)-associated proteins. These included normal livers and livers affected by chronic active hepatitis, cirrhosis, hepatocellular carcinoma and adenoma. Four of the cases of cirrhosis showed liver cell dysplasia. The mean numbers of NOR sites in normal, cirrhotic, and carcinomatous livers were significantly different: adenoma had similar mean counts to those in chronic active hepatitis (CAH). There was no overlap between the ranges of NOR counts in normal, cirrhotic, and malignant liver specimens. Where cirrhosis and hepatocellular carcinoma were present in the same specimen, the AgNOR counts were higher in the carcinomatous than cirrhotic areas. To investigate the prospective value of the method a further seven biopsy specimens were studied; in these it had not been possible to decide on a diagnosis between normality and cirrhosis or cirrhosis and hepatocellular carcinoma. In all seven specimens a repeat biopsy or necropsy gave results as predicted by AgNOR staining. It is therefore proposed that quantitation of staining for NOR-associated proteins is a diagnostically useful method in liver disease.     

p53 Protein,PCNA staining,and DNA content in follicular neoplasms of the thyroid gland

Forty-nine follicular adenomas and 11 follicular carcinomas of the thyroid were investigated by immuno-histochemistry for the expression of p53 protein and proliferating cell nuclear antigen (PCNA). The DNA ploidy and the S-phase fraction (SPF) of the neoplasms were analysed by flow cytometry. Twelve adenomas (24 per cent) and six carcinomas (55 per cent) were DNA non-diploid (P=0·07). The carcinomas had a higher proliferation rate than the adenomas when assessed either by SPF size (median 9·9 per cent vs. 2·9 per cent, P=0·0003) or by PCNA staining intensity (P<0·0001). Some scattered nuclei in two (4 per cent) adenomas and in three (27 per cent) carcinomas stained positively for p53 (P=0·04). The two adenomas with positive staining for p53 were subserially sectioned, but no signs of invasion were found; both patients are alive and well 6 and 7 years after surgery. One of the two adenomas showing positive p53 nuclear staining was DNA aneuploid, and both were positive in PCNA staining, but their SPFs were low (2·1 and 3·3 per cent). We conclude that p53 protein expression is not confined to follicular carcinomas; scattered p53-positive cells may also be present in histologically and clinically benign follicular adenomas. Because both follicular adenomas and carcinomas may be DNA aneuploid and their SPF and PCNA staining distributions overlap, the distinction between follicular adenoma and carcinoma should still be based on histological criteria.     

Correlation of two argyrophilic nucleolar organizer region counting methods with the Ki-67 labeling index in uterine smooth muscle cell tumors

The argyrophilic nucleolar organizer regions (AgNOR) are silver stained granules that are thought to correlate with cell proliferation activity. Two AgNOR counting methods: the mean AgNOR count (mAgNOR, the mean number of AgNOR granules in 100 cells) and the AgNOR proliferative index (pAgNOR, the percentage of cells exhibiting five or more AgNOR granules per nuclei) have been proposed. In this study, the two counting methods were applied to 58 cases of normal uterine corpus and uterine corpus tumors and were compared with the Ki-67 labeling index (Ki-67 LI) using MIB-1 monoclonal antibody and other histopathological criteria. Notable differences in the number of AgNOR and the Ki-67 LI were observed between benign and malignant smooth muscle tissue. Histopathologic features are well correlated to the proliferative activity of tumors. Although the most reliable method of predicting malignant potential cannot be determined, the methods outlined by this study are thought to be highly useful in assessing proliferative activities.     

Increased cell proliferation and transforming growth factor-α (TGFα) in the gall-bladder epithelium of patients with pancreaticobiliary maljunction

Pancreaticobiliary maljunctlon is associated frequently with gall-bladder carcinoma. Although increased turnover of the gall-bladder epithelium in patients with pancreaticobiliary maljunction is thought to predispose to carcinogenesis, there is little data to confirm this hypothesis. In addition, no previously published study has addressed the process underlying cell proliferation. In this study, cell kinetics were first evaluated using two methods, proliferating cell nuclear antigen (PCNA) Immunohistochemical staining and argyrophilic nucleolar organizer region (AgNOR) staining. Second, immu-nohistochemistry was used to investigate the expression of transforming growth factor-α (TGFα), a potential regulator of cell proliferation in the gall-bladder. The gall-bladders of 11 patients with pancreaticobiliary maljunction were studied, and 11 gall-bladders removed from patients during other surgery were used as controls. The number of PCNA-positive cells and the number of AgNOR per nucleus were significantly greater in the gall-bladders of patients with pancreaticobiliary maljunction than in the control gall-bladders. The expression of TGFα was also significantly greater in the gallbladders of patients with pancreaticobiliary maljunction than in the control gall-bladders. In conclusion, these results suggest that the increased TGFα expression induced by pancreaticobiliary maljunction promotes proliferation of the gall-bladder epithelium, which may lead to carcinogenesis.     

Prognostic significance of nucleolar organizing regions (NORs) in carcinomas of the sigmoid colon and rectum.

The prognostic significance of silver-binding nucleolar organizing regions (AgNORs) has been evaluated in tissue sections of biopsies taken from 70 primary adenocarcinomas of the colon sigmoid (n = 25) and rectum (n = 45) prior to their curative resection. A significant correlation between five-year survival rate and the mean AgNOR number per tumour cell (p less than 0.001) and the mean size of silver stained dots (p less than 0.05) was found according to the univariate Kaplan-Meier survival analysis. There was no significant relationship between AgNOR content and grade of malignancy, pT categories or pN categories. Multivariate survival analysis of covariates (Cox regression model) revealed a set of five variables that significantly influenced the patients' outcome: pN categories, AgNOR content, pT categories, maximum grade of malignancy and number of inflammatory cells. From the clinical and pathological parameters studied, pN and pT categories as well as the mean AgNOR number were the most important variables predicting death from colorectal carcinoma. Since the analysis of AgNORs can be performed on routinely processed paraffin-embedded tissue, this method may be of potential use in pretherapeutic assessment of the biologic aggressiveness of the disease.     

Growth fraction in lung tumours determined by Ki67 immunostaining and comparison with AgNOR scores

Estimation of the growth fraction with the use of monoclonal antibody Ki67, which recognizes a nuclear antigen in proliferating cells, was compared with the nucleolar organizer region staining in 95 lung tumours. There was nuclear staining in most tumours; 12 tumours were negative. Cytoplasmic staining was observed in another seven tumours. The small cell carcinoma group had the highest mean Ki67 index (23.75); squamous carcinomas had a mean value of 15.71, adenocarcinomas 10.99, and large cell anaplastic 20.76. Carcinoids had few stained cells. Nucleolar organizer regions were demonstrated by the argyrophilic method (AgNOR). No correlation was found between Ki67 indices and AgNOR scores. Kinetic data obtained by the AgNOR technique were less discriminating in view of the overlap between scores of various groups including carcinoids. We conclude that use of the monoclonal antibody Ki67 is a more reliable method of assessing proliferative activity in lung tumours. This antibody may be effective in identifying slowly proliferating tumours which are less sensitive to chemotherapeutic agents.     

Nucleolar organizer regions and glycoprotein-hormone α-chain reaction as markers of malignancy in endocrine tumours of the pancreas

The value of silver staining of nucleolar organizer regions (AgNORs) and human chorionic gonadotropin α-chain reaction (HCG-α) as markers of malignancy was investigated in 60 primary pancreatic endocrine tumours, 37 of which had metastasized at the time of surgery, and in one of which metastases developed 4 years after surgery. Assessment of AgNORs by digital image analysis revealed few but large AgNORs (mean number 2.5 ± 1.1; mean area ± in the 22 benign tumours and many but small AgNORs (mean number 5.1 ± 1.9, P <0.05; mean ± 9 μm². P <0.01) in the malignant tumours. Quantification of the number of AgNORs per tumour cell AgNOR distribution score) showed that 96% (26/27) of tumours exhibiting at least 5% of cells with more six AgNORs per nucleolus showed metastases either at the time of diagnosis or up to 4 years after surgery. HCG-α immunoreactive cells were present in 25/38 (66%) malignant tumours and in 4/22 (18%) benign tumours. Combined evaluation of AgNOR distribution and HCG-α scores showed a high positive predictive value of 96% in cases with a raised AgNOR distribution score irrespective of the HCG-α status. A good negative predictive value (81%) was, however, only obtained if both parameters, AgNOR distribution and HCG-α scores, were negative. Thus, investigation of AgNORs HCG-α is helpful in predicting malignancy in a high percentage of pancreatic endocrine tumours.     

Prognostic value of argyrophilic nucleolar organizer regions (AgNOR) count in adenoid cystic carcinoma of salivary glands

A retrospective study was performed on 34 patients with adenoid cystic carcinoma (ACC) to see if quantification of argyrophilic nucleolar organizer regions (AgNOR) was correlated with their prognosis. Mean follow-up period was 113 months. According to detailed clinical data that included tumor recurrences, metastases and patient survival conditions, two groups were classified. Group 1 consisted of 20 live patients without metastases, of whom 16 were free of disease, while group 2 comprised 14 patients with metastases among whom 12 died of tumor. The silver staining technique was applied to paraffin embedded specimens. In each case, 300 nuclei were randomly examined and the mean AgNOR per nucleus was calculated. All patients from group 2 had mean AgNOR counts greater than 4, whereas 65% of patients from group 1 had mean AgNOR counts less than 4. In addition, statistical analysis showed that the pooled mean AgNOR count in group 2 was significantly higher than in group 1 (P < 0.01). The present results suggest that silver staining technique promises to be a useful supplementary method for prognostic evaluation of salivary ACC, and may be performed before planning the treatment.     

Salivary gland tumours studied by means of the AgNOR technique

Difficulty is sometimes encountered in distinguishing between pleomorphic adenoma, adenoid cystic carcinoma and adenocarcinoma, especially in small biopsies from salivary glands. The argyrophil (AgNOR) staining technique for nucleolar organizer regions (NORs) has been applied to a series of benign and malignant salivary gland tumours. We studied 35 salivary gland tumours, 13 benign and 22 malignant. In all specimens clearly defined silver-stained intranuclear AgNOR dots were visible. The differences between the numbers of AgNORs in the benign and malignant groups, notably pleomorphic adenomas, adenoid cystic carcinoma and adenocarcinoma, were highly significant. In view of this difference we propose that the AgNOR staining technique is of diagnostic help in distinguishing between these salivary gland tumours.     

The prognostic importance of the nucleolar organizer region (AgNOR), Ki-67 and proliferating cell nuclear antigen (PCNA) in primary nonurachal bladder adenocarcinoma

The aim of this study was to investigate the role of tumor proliferation in patients with nonurachal bladder adenocarcinoma. Samples were obtained from 16 patients (12 men and 4 women, mean age 62 years) with primary nonrurachal bladder adenocarcinoma. The 16 formalin-fixed specimens were stained immunohistochemically for Ki-67 antigen and PCNA using MIB-1 and PC-10 antibodies. In addition, the AgNOR quantity was assessed using the colloid silver nitrate staining technique in all cases. The Ki-67, PCNA and AgNOR proliferation indices were found to be significantly higher in high-grade and invasive tumors. The higher the grade (p<0.01) and stage (p<0.01), the higher were the proliferation indices. Patients whose tumor samples had a high Ki-67, PCNA and AgNOR proliferation index showed a higher incidence of local recurrence (p<0.01) and distant metastasis (p<0.01). In conclusion, our results suggest that Ki-67, PCNA and AgNOR proliferation scores may be important prognostic indices in nonurachal bladder adenocarcinomas.     

PCNA in the ovary of zebrafish (Brachydanio rerio, Ham.-Buch.)

PCNA, a nuclear protein intimately involved in DNA synthesis, has been localized immunohistochemically in ovaries of zebrafish (Brachydanio rerio, Ham.-Buch.). Positively stained cells were detected in the follicle epithelium of all stages of growing oocytes and degenerating oocytes. Furthermore, PCNA was found to be present in nuclei of oogonia and oocytes in meiotic prophase. Follicle epithelium showed an increasing number of PCNA-positive cells during egg cell development with a very low index of <0.5% at the beginning and a maximum of >7% in stage IV oocytes that are rapidly incorporating yolk protein. In this phase, follicle epithelium often showed groups of stained cells in limited zones of proliferation. In degenerating oocytes, over 60% of the former follicle cells were positive for PCNA. Nuclei of oogonia were always positive and the nucleus of stage III oocytes showed strong positivity. Stage III is the stage in which amplification of rRNA genes occurs and numerous nucleoli apear in the peripheral caryoplasm. It is concluded that PCNA expression is related to amplification of rDNA.     

Adenoid cystic carcinoma: A study of nucleolar organizer regions (AgNOR) counts and their relation to prognosis

We studied the AgNOR counts in 30 cases of adenoid cystic carcinoma from salivary glands (n = 18) and non-salivary sites (n=12) in an attempt to correlate them with the evolution of the disease. AgNOR counts per nucleus varied between 1·96 and 6·12 (mean value 4·2 ± 0·99) as compared with 1·21 ± 1·4 in normal salivary tissue. There was no significant difference between cases that had an unfavourable clinical outcome (recurrence, metastases, and/or died of the disease) and cases without disease complications (4·31 vs. 4·03 AgNORs per nucleus). No difference was found between tumours located at salivary and non-salivary sites or between major and minor salivary glands. AgNOR counts also did not correlate with the grade of differentiation of the neoplasms. In adenoid cystic carcinoma, AgNOR counts do not seem to be a prognostic indicator, in contrast to the usefulness of this method in other tumour types.     

Contribution of cell proliferative activity to malignancy potential in testicular seminoma

Testicular anaplastic seminoma, which has a high mitotic activtiy, is regarded aa more malignant than typical seminoma, although its prognosis is still unclear. To determine whether seminoma with relativety greater malignancy potential can be ldentified based on the cell proliferative activity, the mitotic rate (MR; mitotic count per high-power field), mitotic Index (MI; mitotic count per 1000 cells), Ki-67 labeling Index (K1–67 LI; the percentage of positive cells) and prolifereting cell nuclear antigen LI (PCNA LI; the percentage of positive cells) were histologically examined In 44 patients. The MI, Kl-67 LI and PCNA LI In patients with metastatlc disease were signmeantly higher than those in patients without metastatic disease, and the MI In patients with fatal disease was signmcantly higher than those in patients cured of the disease. However, these distributions of the MI, K1–67 LI and PCNA LI values overiapped for both pairs of groups. There were no significant differences in the YR. These results suggest that the cell proliferatbe activity makes a small contribution to the malignancy potential in testicular seminoma, with the activity being not necessarily indicative of metastasis and prognosis.     

Nucleolar organiser regions (AgNORS) in anal intraepithelial neoplasia and invasive anal squamous cell carcinoma.

AIM: To evaluate the usefulness of counting nucleolar organiser region associated proteins (AgNORs) in the management of anal squamous neoplasia. METHOD: Using a silver staining technique for NOR associated proteins, 32 routinely processed paraffin wax embedded sections of anal epithelium were assessed. These consisted of normal anal epithelium (n = 9), anal intraepithelial neoplasia (AIN) grades I (n = 5), and III (n = 13), and invasive squamous neoplasia of the anus (n = 5). RESULTS: The median AgNOR counts for every 100 cells are as follows: normal anal epithelium 2.15 (95% CI 1.89-3.94); AIN I 3.21 (95% CI 2.89-7.14); AIN III 4.32 (95% CI 4.00-8.10); and invasive squamous cell carcinoma of the anus 5.51 (95% CI 2.48-10.62). There were significant differences between AgNOR counts in anal cancer and normal epithelium (p < 0.05; Mann-Whitney U test)), AIN III and normal anal epithelium (p < 0.005), and AIN III and AIN I (p < 0.05). No significant differences were observed between AIN I and normal anal epithelium, anal cancer and AIN I, and anal cancer and AIN III. There was a considerable degree of overlap among the different groups. CONCLUSIONS: Despite the strong association between AgNOR values and degree of dysplasia, the variability within pathological grade may preclude the adoption of this technique on its own as a prognostic indicator. It may, however, be useful in conjunction with other markers of neoplastic growth such as c-myc oncogene amplification or overexpression as a marker of disease progression in AIN and invasive anal squamous cell cancer.