The value of nucleolar organizer region staining in the differential diagnosis of borderline melanocytic lesions

The technique of silver (Ag) staining of nucleolar organizer region-associated proteins (AgNORs) has been shown to be of value in differentiating between benign and malignant cells. We have studied 33 borderline melanocytic lesions, in which a diagnosis of melanoma had been seriously considered, in order to assess the value of this technique in a commonly encountered diagnostic situation. We found that benign naevus cells possessed single compact or granular AgNORs, whereas some malignant melanocytes possessed large, often loosely arranged groups of AgNORs. However, the pattern of AgNORs observed in melanocytes of some atypical but benign lesions was also seen in some melanomas. The differential diagnosis of borderline melanocytic lesions is not clarified by use of the AgNOR technique.     

Silver staining of nucleolar organizer regions in prostatic lesions

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

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.     

Silver staining method for nucleolar organizer regions in cervical smears

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

Silver-stained nucleoli and nucleolar organizer region counts are of no prognostic value in thick cutaneous malignant melanoma

Nucleolar organizer regions (NORs) can be stained by a simple one-step silver technique; the black dots formed are termed AgNORs. Often AgNORs are tightly clustered, appearing as one silver-stained nucleolus (AgNu). We have assessed this technique as a possible prognostic indicator for thick (greater than 3.0 mm) primary cutaneous malignant melanoma (CMM). Three groups were studied: (A) seven thick CMM that had not metastasized 8-20 years after excision; (B) three thick CMM that developed metastases 6-9 years after excision; and (C) twelve CMM that presented with metastases or developed them within 4 years of excision. Two methods of counting silver-stained black dots in nuclei were employed: one method counted easily discernible black dots consisting of AgNus and dispersed AgNORs; the other attempted to count actual AgNORs both dispersed and clustered within AgNus. Scores per nucleus by the first method were 1.5-6.7 in group A, 1.1-2.6 in group B, and 1.4-5.4 in group C. AgNOR counts by the second method were 6.2-13.0 in group A, 5.4-8.9 in group B, and 5.3-10.5 in group C. No significant difference was present between groups for scores by either method. Due to the subjectively, technical difficulty, non-reproducibility, and tedium associated with the second method of attempting to count individuals AgNORs, the first method is recommended. It is concluded that this technique is of no value in predicting prognosis for CMM.     

Correlation between DNA flow cytometric and nucleolar organizer region data in non-Hodgkin's lymphomas

The argyrophilic staining (AgNOR) technique, novel in histopathology, was applied to a series of 20 non-Hodgkin's lymphomas (NHL) of established Kiel subtype. The method demonstrates nucleolar organizer regions (NORs) by virtue of sulphydryl groups on their associated proteins and the enumeration of AgNOR foci has been previously shown to discriminate between NHL of low- and high-grade histological types. This finding was confirmed and the results were compared with those obtained by means of DNA flow cytometry performed on paraffin wax-embedded tissue from the same lymphomas. There was a very good linear correlation between the mean numbers of AgNOR sites per nucleus and the percentage of S-phase cells for each case, both values being high in high-grade NHL and low in low-grade lesions. Conversely there was no significant correlation between the DNA index, representing DNA aneuploidy, and AgNOR counts. It is suggested that the numbers of AgNORs in a lymphoma may be related to the dividing fraction of cells rather than, as might be expected, to ploidy alone. It is also proposed that the AgNOR technique, which is rapid, simple, and inexpensive, may provide, at least, an adjunct to DNA flow cytometry in the assessment of neoplasm in histopathology.     

Assessment of the argyrophilic nucleolar organizer region area/nucleus ratio in ovarian serous epithelial adenomas,borderline tumors and cancers

Introduction

There is a need to assess the value of the novel potentially useful biomarkers in ovarian tumors. The aim of study was to assess the value of sAgNOR analysis in ovarian serous epithelial tumors.

Material and methods

The analysis was performed in ovaries from 113 patients treated operatively due to serous ovarian tumors (30 adenomas, 14 borderline tumors and 69 cancers). After silver staining of paraffin specimens from surgery, sAgNOR in tumor cells was analyzed. Additionally, the value of the argyrophilic nucleolar organizer region area/nucleus ratio (sAgNOR) in the prediction of disease-free survival (DFS) and overall survival (OS) in 52 patients with serous ovarian cancer with complete follow-ups in November 2009 was evaluated. Age, grading, radicality of surgery and FIGO staging were analyzed as additional factors.

Results

sAgNOR in adenomas, borderline tumors and cancers was in the following ranges: (0.73 ±0.23) × 10⁶, (0.81 ±0.18) × 10⁶ and (0.96 ±0.33) × 10⁶ [AgNOR/cm²] respectively. In cancers from G1 to G3 sAgNOR was (1.02 ±0.32) × 10⁶ (G1), (0.98 ±0.37) × 10⁶ (G2) and (0.82 ±0.24) × 10⁶ (G3) [AgNOR/cm²] respectively. In univariate analysis, but not in multivariate analysis, staging negatively correlated with better DFS and OS. sAgNOR, age of patients, grading and radicality of surgery were not associated with DFS or OS in either univariate or multivariate analysis.

Conclusions

sAgNOR analysis is not sufficient to precisely characterize cellular kinetics in serous ovarian tumors, and the analysis of sAgNOR, mAgNOR and pAgNOR should be performed commonly. The prognostic significance of sAgNOR in patients with serous ovarian cancer was not proven.     

Nucleolar organizer regions and prognosis in renal cell carcinoma

The prognostic significance of nucleolar organizer regions (NORs) in renal cell carcinoma (RCC) was evaluated. NORs were quantified in a series of 182 cases of RCC using the silver-colloid method. The cases were staged according to Robson's method (48 stage I, 26 stage II, 33 stage III, 75 stage IV) and mean NOR numbers for each tumour were correlated with survival over a 5-year period. Localized tumours (stages I and II) with low NOR numbers had an almost 100 per cent 5-year survival. Those patients with clinical evidence of metastases at presentation showed a high mortality, although those with low numbers of NORs had a significantly increased disease-free interval. Statistical analysis using the log rank test indicated NORs to be a significant predictor of survival over the whole series (P = 0.0001) and within each of Robson's stages (P = 0.0008 stage I, P = 0.0154 stage II, P = 0.0009 stage III, P = 0.0001 stage IV). Analysis of data using Cox's proportional hazard model showed mean NOR numbers to be independent of stage as a predictor of survival.     

The utility of Ki67 immunostaining, nuclear organizer region counting, and morphology in the assessment of follicular lymphomas

Three methods, the enumeration of large non-cleaved cells (LNCC), the enumeration of nucleolar organizer regions (AgNORs), and the estimation of the percentage of Ki67-positive cells, have been compared on a series of 36 follicular lymphomas for their utility in subclassification. All three methods produced similar subdivisions to those obtained using the histological classification of the Working Formulation for clinical usage. Twenty cases have been followed clinically to assess any relationship between these parameters and short-term (less than 4 years) survival. Each technique showed a trend towards identifying patients with a poor short-term prognosis with no one method being superior to any other. Unless larger series with longer follow-up suggest otherwise, there is no indication to use either the AgNOR or Ki67 methods which offer no advantage over the simple and inexpensive Working Formulation or Berard's LNCC counting.     

Growth fractions in human prostatic carcinoma determined by Ki-67 immunostaining

Until recently, [3H]-thymidine incorporation, DNA analysis by flow cytometry, and cell doubling times have been the main methods of studying tumour cell kinetics. All these techniques are laborious, expensive, and difficult to perform in a routine diagnostic laboratory. This study examined fresh frozen sections from 31 prostatic biopsy specimens with the hybridoma antibody Ki-67, a marker of proliferating cells, using a modified avidin-biotin-peroxidase complex technique. The percentage of glandular cells decorated by this antibody, representing the growth fraction, was determined for both benign and malignant samples. Benign prostatic glands showed an average Ki-67 score of 4 per cent, significantly less than the 16.3 per cent mean growth fraction found in prostatic carcinomas. There was a significant correlation between the tumour growth fraction as assessed by Ki-67 staining, and the histological grade. A positive correlation was also found between the Ki-67 score and the intensity of staining, and a definite trend was noted between the Ki-67 score and the tumour clinical stage. Ki-67 promises to be a useful marker in determining the prognosis of prostatic cancer.     

Nucleolar organizer regions in melanocytic dysplasia and melanoma

Using silver (Ag) staining to demonstrate nucleolar organizer region-associated proteins (AgNORs), pigmented naevi exhibiting features of melanocytic dysplasia have been examined and compared with benign intradermal and compound naevi and with malignant melanomas. A highly significant difference was found between the numbers of AgNORs demonstrated in benign naevus cells and atypical melanocytes and in malignant melanocytes, suggesting that this technique may have a role in differentiating between difficult melanocytic lesions.     

Progressive activity in latent prostate carcinoma defined by argyrophilic staining of the nucleolar organizer regions (AgNOR)

Argyrophilic staining of the nucleolar organizer regions (AgNOR) was studied in 30 cases of benign prostatic hyperplasias (BPH), 17 cases of latent prostate carcinomas, 50 cases of clinical carcinomas and seven cases of metastatic lesions from prostate carcinomas. The criteria for these comparisons were the number of positive-staining dots per nucleus, the area of the dots, and a relative score determined by multiplying the number of positive-staining dots in the nuclei by the areas of the dots. Overall, there were no significant differences in these three parameters between BPH and latent carcinomas. Among latent carcinomas, however, significantly higher AgNOR scores were observed for infiltrative lesions than for non-infiltrative lesions. AgNOR dot number, area and score increased as tumors became less differentiated, with no significant differences detected in metastatic versus non-metastatic carcinomas. These results suggest that some latent tumors are similar in biological behavior, such as cell proliferation, to clinical carcinoma.     

Argyrophilic nucleolar organizer region counts in multiple myeloma: A histopathological study on bone marrow trephine biopsies

Summary Argyrophilic nucleolar organizer region (Ag-NOR) analysis was performed on bone marrow biopsies from 90 patients with multiple myeloma (MM) at presentation. The pattern of AgNOR expression and its relationship to histological features were evaluated. The mean AgNOR number per plasma cell was directly correlated with the degree of MM differentiation (3.18 for G1, 4.36 for G2, 6.13 for G3;P<0.0001), with the per-centage of bone marrow plasma cells (BMPC%) (3.06 for BMPC%20, 4.28 for BMPC% 21–50, 5.14 for BMPC%>50;P<0.0001), with the pattern of medullary involvement (3.63 for interstitial, 4.44 for nodular, 5.17 for diffuse involvement;P<0.001) and with medullary fibrosis (5.23 for cases with fibrosis, 4.29 for cases without fibrosis;P<0.05). The plasma cells of G1 MM showed 2–3 large AgNORs, tightly grouped in a central nuclear cluster; those of G2 MM showed a central nuclear cluster composed of 4–5 medium-size dots and/or two clusters of 2–3 dots; the G3 MM plasma cells showed many small dots scattered in the nucleolus or dispersed in the nucleus. Our results indicate the diagnostic value of AgNOR analysis in MM and suggest the use of this method for identifying clones of atypical plasma cells with different proliferative activity in bone marrow biopsies. It allows simultaneous evaluation of the morphology and kinetics of MM cells in routinely fixed, decalcified, paraffin-embedded material.     

Relevance of Ki-67 expression in the classification of non-Hodgkin''s lymphomas: a morphometric and double-immunostaining study

The proliferation of reactive and neoplastic cells was retrospectively assessed in 92 cases of non-Hodgkin's lymphoma by morphometry using a double-immunoenzymatic technique including surface markers and the monoclonal antibody Ki-67. The findings were compared with the histological diagnosis. The overall Ki-67 positivity is not always a good measure of the corresponding corrected values and therefore we recommend that a correction should be made for the total number of complementary lymphocytes in the tumour. Taking the macrophages and the Ki-67 positivity of the reactive cells into account does not generally add any information. There was no difference in reactive cell content between follicular (counted within follicles) and diffuse lymphomas within the tumour areas. The value of the group mean for low-grade follicular (nodular) lymphomas was significantly higher than that of diffuse low-grade lymphomas, but not significantly different from that of intermediate-grade lymphomas. High-grade lymphomas exhibited significantly greater Ki-67 positivity than those of intermediate grade. In 76% of the cases there was significant agreement between malignancy grade (low/intermediate malignant versus high malignant) at 45% corrected Ki-67 counts.     

Comparative study of the prognostic value of nuclear grade and silver-binding nucleolar organizer region in renal cell carcinomas.

Silver-binding nucleolar organizer region (AgNOR) and nuclear grade were histologically assessed in 112 renal cell carcinomas. Nuclear grade was better capable of stratification than AgNOR in terms of survival prediction. It also had prognostic significance when total metastatic rates were evaluated. Although higher mean AgNOR numbers were constantly associated with higher nuclear grades, two arbitrary classifications of AgNOR (less than 1.5, 1.5-3.5, greater than 3.5; less than 2, 2-3, greater than 3) failed to resolve the survival difference.     

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.     

Nucleolar organizer regions in human maxillary sinus squamous cell carcinoma

A silver colloid technique for nucleolar organizer regions (AgNOR) was applied to paraffin sections of maxillary sinus squamous cell carcinomas (MSSCC) of 25 patients. The patients were divided into two groups, one with MSSCC recurring in the primary lesion after treatment with radiotherapy, chemotherapy and for surgery and one without recurrence. Notable differences between the numbers of NOR in neoplastic epithelia and the normal mucosa were observed ( P = 0.0001), but there were no differences between the numbers of NOR in the recurrent and non-recurrent carcinomas. This investigation found no prognostic importance in the number of AgNOR in MSSCC.     

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

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