Nucleolar Organizer Region Staining Research Articles

The proliferative activity in oral epithelial dysplasia analyzed by proliferating cell nuclear antigen immunostaining and argyrophilic nucleolar organizer region staining

The proliferative activity of leukoplakia without dysplastic change (LP), epithelial dysplasia (ED), and squamous cell carcinoma (SCC) in the oral mucosa was examined by means of proliferating cell nuclear antigen (PCNA) immunostaining, silver-binding argyrophilic nucleolar organizer region (AgNOR) staining, and the frequency of mitotic figures. Significant differences in the labeling index of PCNA immunostaining (PI) and mitotic index (MI) were noted between LP and ED and between ED and SCC. The mean numbers of AgNORs (AI) significantly differed between ED and SCC. There was a significant positive correlation between PI and MI in samples of ED. However, there was no significant correlation between AI and other indexes. The number and the distribution of PCNA-positive cells in ED varied among samples. Five samples with higher PI and MI indexes than the mean values were selected from those of dysplasia based on the correlation between PI and MI. Their histological features symptomatic of oral ED as defined by the World Health Organization (WHO) Collaborating Centre in 1978, were investigated and compared with 10 samples with lower indexes. Histological findings, such as “loss of polarity of the basal cells,” “an increased nuclearcytoplasmic ratio,” “cellular pleomorphism,” and “enlarged nucleoli,” were significant histological features of these five samples. This study showed that the four histological components described previously and the increased number of mitotic figures used as the index of proliferating activity were the main histological components related to severe ED of oral mucosa. They will provide a useful means of deciding the histopathological grade of oral ED.

Comparison between nucleolar organizer region staining and MIB-1 staining in dysplasia in inflammatory bowel disease (IBD): [formula omitted], M.R. Fadaie∗, V. Gross, J. Schölmerich, J. Rüschoff∗, Depts. of Internal Medicine I and ∗Pathology, University of Regensburg, 93042 Regensburg, Germany

Comparison between nucleolar organizer region staining and MIB-1 staining in dysplasia in inflammatory bowel disease (IBD): [formula omitted], M.R. Fadaie∗, V. Gross, J. Schölmerich, J. Rüschoff∗, Depts. of Internal Medicine I and ∗Pathology, University of Regensburg, 93042 Regensburg, Germany

Evaluation of three different AgNOR counting methods in advanced carcinoma of the prostate

We applied the silver staining of nucleolar organizer regions (AgNOR) technique to the pretreatment biopsies of 50 cases of advanced prostate cancer. Three different counting methods were utilized in the enumeration of AgNORs. All methods yielded statistically significant differences of mean AgNOR counts of groups defined by high and low WHO, and by Gleason grades. However, there was overlap among groups, and further analysis of counts by grouping of patients according to their stage, response to treatment, and prognosis was not conclusive. Replicate counts were performed in 10 cases. While intraobserver reproducibility was high by all methods, only the second counting method yielded nonsignificant interobserver variability. There was a significant intratumoral heterogeneity of AgNOR scores. Lack of technical standardization, low reproducibility, and lack of correlation with prognosis limit the use of AgNOR counts in advanced carcinoma of the prostate.

PCNA immunostaining combined with AgNOR staining in esophageal squamous cell carcinoma to identify patients with a poor prognosis

Immunostaining of the proliferating cell nuclear antigen (PCNA) provides important information about cell kinetics and is easily performed on routinely obtained formalin-fixed, paraffin-embedded materials. We report herein the results of a retrospective study on PCNA staining in esophageal cancer undertaken to determine its significance. As this study indicated that immunoreactivity was preserved in specimens fixed within 24h, only 31 specimens from surgical patients were available for this investigation. The mean PCNA index of the patients without invasion to the adventitia (35.7 +/- 17.9) was significantly lower than that of those with invasion to the adventitia or neighboring structures (49.7 +/- 14.5), while the PCNA index did not correlate with other clinicopathologic parameters such as histologic type, lymph node metastases, or prognosis. However, when an analysis of PCNA staining was combined with an analysis of argyrophilic nucleolar organizer region (AgNOR) staining, a correlation with prognosis was found. In fact, seven patients with a high PCNA index (> or = 44) and AgNOR count (> or = 6) had a significantly poorer prognosis than the remaining 22 (P = 0.0014), and six of these seven patients died within 2 years. These results indicate that this combined evaluation may be useful for the identification of patients with a poor prognosis among those undergoing surgery for esophageal squamous cell carcinoma.

Nucleolar and argyrophilic nucleolar organizer region counts in urothelial carcinomas with special emphasis on grade II tumors.

Prognostic assessment of bladder carcinomas of intermediate differentiation is difficult. This study therefore investigated the prognostic values of nucleolar status and silver staining of argyrophilic nucleolar organizer regions (AgNORs) in grade II bladder carcinomas. In biopsies from 34 grade II transitional cell carcinomas of the urinary bladder the number of nuclei with nucleoli, the location of nucleoli within the nucleus and the number of AgNORs were determined in 1000 or 200 nuclei per section respectively. Ten biopsies showing normal urothelium, 18 cases with mild to severe atypia, 27 grade I, 34 grade II and 12 grade III transitional cell carcinomas were also studied. Significantly differing nucleolar and AgNOR values were found comparing normal urothelium/grade I carcinomas with severe urothelial atypia/grade III carcinomas. Grade II carcinomas, however, were inhomogeneous. One subgroup had nucleolar and AgNOR values resembling grade I carcinomas while the second group had values similar to those of grade III carcinomas. This subdivision of grade II carcinomas correlates with results reported for DNA-cytometry. The results suggest a subdivision of patients with grade II transitional cell carcinomas into a low risk and high risk group.

Nucleolar organizer regions in lining epithelium adjacent to squamous cell carcinoma of human oral mucosa

The value of silver staining of nucleolar organizer regions (AgNOR) counts as a diagnostic aid has been reported for several neoplastic entities. Previous studies have proved the value of the morphometric evaluation of AgNOR in the detection of incipient cellular alterations. A morphometric analysis of AgNORs was performed in oral mucosa epithelium adjacent to squamous cell carcinoma compared with normal mucosa epithelium and the carcinomatous parenchyma. Highly statistically significant differences in all 5 AgNOR-related parameters assessed were found between normal mucosa and mucosa adjacent to cancer. Conversely, the corresponding nuclear parameters failed to exhibit significant differences. The parameter AgNOR contour index plotted for individual cases affords a cutoff value that could prove useful in identifying epithelia at early stages of transformation. AgNOR evidenced significant variations in epithelium adjacent to oral squamous cell carcinoma, which did not exhibit morphologic signs of atypia. Based on this study, AgNOR would be a quantitative, discriminative aid, easy to monitor in a pathology laboratory, in detecting incipient cellular alterations. These findings contribute to the issue of early diagnosis and to the knowledge of tumoral growth.

Nucleolar organizer regions in small cell carcinoma of lung.

Agyrophilic staining of nucleolar organizer regions (NOR) has been used to differentiate between cells of small cell carcinoma and lymphocytes in tissue specimens. We used cytologic smears which were previously Papanicolaou-destained to study the one-step agyrophilic staining technique for nucleolar organizer regions (AgNOR) in cell of small cell carcinoma and lymphocyte. The purposes of this study were to assess the feasibility and usefulness of AgNOR staining in diagnostic cytology and to try to set up a procedure that could be used on prestained smears for retrospective study. While the NOR of each lymphocyte appeared to be one round dot after AgNOR staining, the NOR of cell of small cell carcinoma showed dots, slightly variable in size and shape. The mean number of NOR was significantly higher (p < 0.01) in cells of small cell carcinoma (4.7 +/- 0.7) than in lymphocytes (1.4 +/- 0.4). In conclusion, AgNOR staining was demonstrated to be a useful method to differentiate between cells of small cell carcinoma and lymphocyte in Papanicolaou-destained smears.

Chromosome Banding Techniques

Chromosome banding techniques produce a series of consistent landmarks along the length of metaphase chromosomes that allow for both recognition of individual chromosomes within a genome and identification of specific segments of individual chromosomes. These landmarks facilitate assessment of chromosome normalcy, identification of sites of chromosome breaks and alterations, and location of specific genes. This unit covers these basic banding techniques (Q-banding, G-banding, and R-banding), which produce virtually identical patterns of bands along the length of human chromosomes, although the bands and polymorphic regions highlighted may differ with each technique. These techniques highlight reproducible landmarks along the length of the chromosome and specialized staining techniques can be used to highlight particular regions of chromosomes, such as heterochromatic and repeated-sequence segments. These specialized techniques, nucleolar organizer region (NOR) staining, centromeric heterochromatin staining (C-banding), methylated satellite DNA staining (distamycin-DAPI banding), and replication banding are also presented in this unit.

The proliferative activity in dysplasia and carcinoma in situ of the uterine cervix analyzed by proliferating cell nuclear antigen immunostaining and silver-binding argyrophilic nucleolar organizer region staining

The proliferative activity of dysplasia and carcinoma in situ (CIS) in the uterine cervix was examined using proliferating cell nuclear antigen (PCNA) immunostaining and silver-binding argyrophilic nucleolar organizer region (AgNOR) staining. A significant difference in the labeling index of PCNA immunostaining obtained from dysplastic cells in each histopathologic category was demonstrated between severe dysplasia and CIS. There was no significant difference among each category of dysplasia. The frequency of mitotic figures was highest in CIS, followed in decreasing order by severe, moderate, and mild dysplasia, and was closely correlated with the histopathologic classification. There was an intimate correlation between the PCNA and mitotic indexes in severe dysplasia and CIS. However, the mean numbers of AgNORs in each category of dysplasia and CIS were not significantly different, and there was no apparent relationship with the histologic classification. The PCNA index seemed to be a useful means of evaluating proliferative activity in dysplasia and CIS, and especially in distinguishing CIS from severe dysplasia. In addition, the present PCNA index suggests that a considerable alteration of the biologic behavior involving genetic changes occurs during the progression of carcinogenesis from severe dysplasia to CIS. However, AgNOR staining did not appear to reliably represent the proliferative activity in these lesions.

Histochemical characterization of endometrial proliferation using AgNOR (nucleolus organizer region)

Nucleolar organizer regions (NORs) are loops of DNA that transcribe to ribosomal RNA. We used histochemical staining of NORs for 78 sections of endometrial tissue, obtained either from curettage or from hysterectomy specimens (normal endometrium 14, simple hyperplasia 10, complex hyperplasia 18, atypical hyperplasia 8 and adenocarcinoma 28). There was a significant positive correlation between mean AgNOR counts and the degree of hyperplasia. In adenocarcinoma, no significant difference was found between AgNOR counts in tissues of different histologic grades. Our study suggests that AgNOR counts are reliable markers of endometrial proliferation and allow a clear distinction between benign, premalignant and malignant epithelial changes.

Computerized nuclear morphometry and survival in renal cell carcinoma: comparison with other prognostic indicators

Morphometric nuclear parameters were compared with patient survival for a series of 174 renal cell carcinomas (RCC) collected over a 30 yr period. Stepdown regression showed long diameter, average feret diameter, form factor and the ratio of average feret diameter to equivalent diameter to be significantly associated with survival. Nuclear area, nuclear perimeter, equivalent diameter, ratio of long diameter to average feret diameter and coefficients of variation of nuclear area and nuclear perimeter were not significantly correlated with survival. All parameters were correlated with a 3 division nuclear grading classification using analysis of variance. Multivariate analysis showed nuclear form factor, tumor stage, silver staining nucleolar organizer region numbers and proliferating cell nuclear antigen expression to be independently associated with survival. The results of this study indicate that form factor is the most discriminate morphometric parameter for RCC, providing survival data additional to that derived from tumor staging and from markers of tumor proliferation.

Diagnostic Value of AgNOR Staining in Follicular Cell Neoplasms of the Thyroid

The diagnostic value of argyrophil staining of nucleolar organizer regions (AgNOR) was studied in 95 nonneoplastic and neoplastic follicular lesions of the thyroid. Different AgNOR parameters such as number, size, and distribution pattern were determined using digital image analysis. In addition, nuclear and nucleolar size as well as the percentage of nucleoli touching the nuclear membrane (nucleolar margination) were assessed. A stepwise increase in nuclear size and AgNOR counts from normal thyroid tissue to follicular adenoma as well as from differentiated follicular to anaplastic carcinoma was found (mean nuclear area [micron2]/mean AgNOR number per cell: 21.5/1.6 vs. 34.4/3.5 and 45.3/5.0 vs. 66.5/10.8, p < 0.01/p < 0.001). There was, however, no clear separation between these diagnostic groups. In contrast, an almost total discrimination between follicular adenoma and carcinoma was achieved by quantification of AgNORs per tumor cell nucleolus (AgNOR distribution score). In benign adenomas, 3.3% (range, 0-8.8%) of the cells showed nucleoli with at least five AgNOR dots within one focal plane, whereas in follicular carcinomas, the corresponding value was 34.1% (range, 12-75%). Two of four cases of so-called atypical adenomas showed values in the range of benign adenomas, and two were in the range of follicular carcinomas. In comparison with other nuclear and nucleolar parameters, the AgNOR distribution score proved the most valuable diagnostic criterion for the cytomorphological differentiation between follicular adenoma and carcinoma of the thyroid.

Proliferative activity in the malignant cellular blue nevus

The proliferative activity of four malignant cellular blue nevi (MCBN) was assessed in routinely fixed, paraffin-embedded material using staining for the argyrophilic nucleolar organizer regions (AgNORs), immunohistochemical staining for proliferating cell nuclear antigen (PCNA [PC10]), and DNA flow cytometry. The objective was to determine whether the evaluation of proliferative activity could represent a useful diagnostic parameter. Four cellular blue nevi (CBN), 10 melanocytic nevi (MN), four common blue nevi (BN), and 10 conventional malignant melanomas (MMs) were selected as controls. In the MCBN the mean AgNOR number, evaluated on the basis of 100 tumor cells, was 8.33 ± 0.83; NORs were small and dispersed throughout the nucleus; the mean PCNA score was 31.93% ± 4.4; and two of the cases were aneuploid and two diploid. In the CBN the AgNOR count was 3.69 ± 0.56; NORs were large and mainly grouped in a central cluster; the mean PCNA score was 3.53% ± 1.28; and three of the cases were diploid and one aneuploid. The AgNOR counts in the MCBN were significantly different from those in the CBN ( P = .0002), MN (3.04; P = .00001), and BN (2.93; P = .00006), whereas they were not significantly different from those in the conventional MMs (7.64; P = .58). The PCNA (PC10) scores in the MCBN were significantly different from those in the CBN ( P = .00003), MN (2.05%; P = .00001), and BN (5.06%; P = .00002), whereas they were not significantly different from those in the conventional MMs (28.9%; P = .49). In all the cases a linear relationship between AgNOR counts and PCNA scores was observed ( r = .94, P = .00001). Our results indicate that AgNOR analysis and PCNA immunostaining can be regarded as useful additional parameters for the diagnosis of MCBN.

Nucleolar Organizer Regions (NORs) Staining and Proliferating Cell Nuclear Antigen (PCNA) Immunostaining in Mucosa-Associated Lymphoid Tissue (MALT) Gastric Lymphomas

Nucleolar organizer region-associated proteins (AgNOR) and proliferating cell nuclear antigen (PCNA) have been studied by means of a silver staining technique and immunohistochemistry, in paraffin-embedded, gastrectomy specimens of 12 low-grade and 13 high-grade gastric MALT lymphomas respectively. A significant difference was found between the AgNOR count and PCNA index of low-grade lymphomas (mean AgNOR count 2.5 and mean PCNA index 8.33%) and high-grade lymphomas (mean AgNOR count 8.67 and mean PCNA index 49.7%). It is suggested that both methods are useful adjuncts to histopathology for the distinction between low and high grade gastric MALT lymphomas. We also found heterogeneity in AgNOR counts and PCNA index among individual cases of either low or high grade MALT gastric lymphomas. This suggests that the AgNOR count and PCNA index is helpful in the individual approach of the proliferation rate of each tumour, a parameter of potential importance for predicting the biological behaviour of the tumour and the prognosis of the disease.

Leuprolide acetate depot decreases the number of nucleolar organizer regions in uterine leiomyomata

Women with symptomatic uterine myomas were randomized to receive LA depot or placebo for 12 weeks before myomectomy. Silver staining nucleolar organizer regions (AgNORs) per nuclei were assessed in the tissue obtained at the time of myomectomy. Myoma tissue from women treated with LA depot before myomectomy had significantly fewer AgNORs per nuclei than myoma tissue obtained from women treated with placebo. Leuprolide acetate depot may reduce the number of proliferating cells in myomas.

Nucleolar Organizer Regions in Determining Malignancy of Pigmented Conjunctival Lesions

We assessed the usefulness of silver staining of nucleolar organizer regions in the diagnosis of pigmented conjunctival tumors. Fifty-one biopsy specimens were silver stained to identify the nucleolar organizer regions. Nineteen nevi without atypia, three nevi with atypia, eight primary acquired melanosis lesions, and 14 melanomas were studied. In each specimen, silver staining of the nucleolar organizer regions was counted in 100 cells to yield an average of the silver staining of the nucleolar organizer region count. The mean silver staining of the nucleolar organizer region counts per cell was correlated with the degree of malignancy of pigmented conjunctival lesions as follows: nevi, 3.0; primary acquired melanosis, 3.2; nevi with atypia, 3.9; primary acquired melanosis with atypia, 5.0; and melanoma, 5.7 (Spearman correlation [rS] = .83, P = .0001; analysis of variance [ANOVA] F test = 20.9, P = .0001). A cutoff value of 4.0 (mean silver staining of nucleolar organizer regions per cell) will differentiate melanoma and primary acquired melanosis with atypia from other lesions (sensitivity, 100%; specificity, 96%). The silver staining of nucleolar organizer regions is a useful adjunct in determining the malignancy of pigmented conjunctival tumors.

Myxopapillary ependymoma. Results of nucleolar organizing region staining.

Although myxopapillary ependymomas are generally benign with a tendency for slow growth and local recurrence, they are capable of spread within the nervous system and of extraneural metastasis. Histologic features have not been helpful in determining which patients are at risk for recurrence or dissemination, making management decisions difficult. A retrospective review was conducted of 14 cases of myxopapillary ependymoma. The nucleolar organizing region (NOR) staining method was used to determine if this technique was useful in the management of these tumors. Five patients had total resections of encapsulated lesions, four had total resections of adherent tumors, and four had subtotal resections. Twelve received postoperative radiation therapy. With a mean follow-up of 80 months, 12 patients are well and disease-free. Two patients have had recurrences after surgery and irradiation, leading to death in one and disability in the other. The mean number of NOR per cell in eight specimens ranged from 0.4-1.64. The patient who died with intracranial spread had the highest number of NOR per cell. Based on these data and a review of the literature, it is recommended that radiation be delayed until recurrence in tumors that have been totally resected. Local radiation therapy may be indicated in subtotally resected tumors. NOR staining shows promise in predicting the likelihood of spread of tumor. Patients with myxopapillary ependymomas should be followed indefinitely because of the potential for late recurrence, even after aggressive therapy.

Central neurocytoma: proliferative assessment by nucleolar organizer region staining

Proliferative potential of four cases of central neurocytoma were assessed by a silver colloid staining technique for argyrophilic protein associated with nucleolar organizer regions (AgNORs). All but one tumor had an AgNOR score of less than 2 and were regarded as a benign entity. However, one tumor tissue from a patient suffering from a recurrent central neurocytoma after 13 years from the first operation possessed a 2.06 AgNOR score, which is not significantly different from others. It is considered that central neurocytoma is an essentially benign neoplasm, even though the tumor is recurrent. The present study revealed the usefulness of AgNOR staining for a retrospective assessment of the proliferative potential of central neurocytomas

A comparative assessment of proliferating cell nuclear antigen, c- myc p62, and nucleolar organizer region staining in non-Hodgkin's lymphomas: A histochemical and immunohistochemical study of 200 cases

Proliferating cell nuclear antigen (PCNA) and c- myc p62 oncoprotein are two nuclear proteins expressed in proliferating and transformed cells. They can be recognized immunohistochemically in paraffin sections by the monoclonal antibodies PC-10 and c- myc 1–9E10, respectively. On the other hand, nucleolar organizer regions (NORs) are loops of DNA that carry the r-RNA genes and can be visualized in paraffin sections as black dots (AgNORs) using a silver impregnation method. It has been suggested that the mean number of AgNORs may reflect the cellular kinetics of a tumor. We independently examined 200 cases of non-Hodgkin's lymphomas using the monoclonal antibodies PC-10 and c- myc 1–9E10, as well as the AgNOR method. Our study shows a very significant correlation between PCNA, c- myc expression, and AgNOR count on the one hand and histologic grade on the other ( P < .001), although a significant overlap among the three grades exists. PC-10, c- myc 1–9E10, and AgNOR scores are all shown to be linearly related, even though significant discrepancies were observed, and the correlation is stronger between PCNA and AgNORs (PCNA v c- myc p62, r = .551; PCNA v AgNORs, r = .746; c- myc p62 v AgNORs, r = .529; P < .001). A remarkable finding is that the intermediate group of lymphomas is heterogeneous as far as the proliferative rate is concerned: diffuse large cell cleaved/non-cleaved lymphomas (category G of the Working Formulation) are characterized by a significantly higher proliferative index, as evidenced by the elevated PCNA, c- myc p62, and AgNOR scores, in comparison with the other types of intermediate-grade lymphomas ( P < .001). However, the proliferative rate is lower than that of the high-grade lymphomas (PCNA, P < .05; c- myc p62, P < .001; AgNORs, P < .005). No significant difference exists between B-cell and T-cell lymphomas except for the higher expression of c- myc p62 in intermediate-grade B-cell lymphomas, obviously due to the higher proliferative rate of diffuse large cell lymphomas. Based on our findings, it appears that the combination of PCNA, c- myc p62, and AgNORs provides an accurate estimate of the proliferative rate of non-Hodgkin's lymphomas in paraffin sections. Clinical studies may show whether this information has prognostic value independent of histologic classification. In addition, our results suggest that category G (diffuse large cell) lymphomas may belong to a malignancy grade higher than the intermediate grade, a suggestion consistent with their more aggressive biologic behavior.

Karyotype evolution in Curimatidae (Teleostei, Characiformes) from the Amazon region. II. Centric fissions in the genus Potamorhina

Using cytogenetic analysis following Giemsa staining, nucleolar organizer region (NOR) staining, and C-banding, three distinct karyotypes in three species of curimatids belonging to the fish genus Potamorhina were identified: 2n = 54/44 M + 10 SM (P. pristigaster), 2n = 56/52 M + 2 SM + 2 ST (P. latior), and 2n = 102/2 M + 2 SM + 98 A (P. altamazonica). A 2n = 54 was considered to be the ancestral diploid number and the different karyotypes were probably the result of centric fissions. Both the NOR pattern and constitutive heterochromatin pattern are species specific.