Usefulness of argyrophilic nucleolar organizer staining for histologic grading of soft-tissue sarcomas

Accurate histologic grading is essential for making a proper therapy decision in soft-tissue sarcomas (STS). The usefulness of the argyrophilic stain for nucleolar organizer region (AgNOR) in assessing the histologic grade of STS has been examined. One hundred and forty-two patients with STS confined to the extremity and trunk were selected. Tumors were classified based on the criteria of Enzinger and Weiss [“Soft-Tissue Tumors.” St. Louis: C.V. Mosby, 1983]. In addition, non-specific classification was made based on the shape of proliferating cells occupying more than 50% of the field in the sections such as pleomorphic cell, small round cell, spindle cell, epithelioid cell, myxoid, and unclassified tumors. The mean number of AgNOR dots per nucleus of tumor cells was calculated in 200 cells (AgNOR count). Each category of non-specific classification was divided into a high-count group (<8 AgNOR count) and a low-count group (>8 NOR). The low-count group showed a significantly better prognosis than the high-count group in small round cell and spindle cell tumors (P< 0.007 and P< 0.0005, respectively). Similar results were obtained in pleomorphic cell tumors, though they were statistically not significant because of the relatively small number of examined cases. Most patients with epithelioid cell and myxoid tumors were in the low-count group. These findings suggest that the assessment of histologic grading of STS could be made effectively by the non-specific classification and the aid of AgNOR Staining. © 1993 Wiley-Liss, Inc.     

Prognostic significance of Ki-67 reactivity in soft tissue sarcomas

Proliferative activity of soft tissue sarcomas (STS) in 34 cases was estimated by immunohistochemical procedures (avidin-biotin complex [ABC] method) with monoclonal antibody Ki-67 which reacts with a nuclear antigen expressed in all phases of cell cycle except G0. In 20 of 34 cases (59%), varying numbers of Ki-67-positive tumor cells were detected with a range from 5 to 382 per 10 high power fields (HPF) (mean 57.2/10 HPF). Ki-67 index (the number of Ki-67-positive tumor cells/10 HPF) positively correlated with mitotic count (r = 0.428, P less than 0.02), cellularity (r = 0.447, P less than 0.01), and histologic grade (r = 0.473, P less than 0.01). The Ki-67 low index group (less than 50/10 HPF) showed more favorable prognosis than the high index group (more than 50/10 HPF) (P less than 0.005). Three cases with low mitotic count and unfavorable prognosis were proved to be the Ki-67 high index group (142-382/10 HPF). These results indicated that reactivity of tumor cells for Ki-67 is a useful prognostic marker in the patients with STS, and might be used as one of the histologic factors for the grading of STS.     

Prognosis for gastric-cancer invading the serosa evaluated by argyrophilic nucleolar organizer region staining

Eighty-five patients with gastric cancer invading the serosa following curative resection were analyzed with respect to clinicopathological features and prognosis by staining the argyrophilic nucleolar organizer region (AgNOR). The AgNOR was counted in 200 cancer cells per tissue and the mean number per cell was identified as the AgNOR score. The AgNOR count varied from 1.79 to 5.71, while the mean value was 2.92 +/- 0.74 and the median score was 2.95. The patients were divided into two groups: the AgNOR high group (AgNOR count greater-than-or-equal-to 2.95, n=44) and the AgNOR low group (AgNOR count < 2.95, n=41). There were no differences between the two groups with respect to sex, age, tumor size, location of the tumor, macroscopic appearance, histological differentiation, growth pattern and vascular involvement. The lymphatic advancement of tumor cells was prominent and the rate of lymph node metastasis was higher (p<0.01) in patients in the AgNOR high group. The AgNOR high group had a higher rate of recurrence and also had a variety of recurrence styles. The prognosis of the AgNOR high group was poorer, with statistical significance (p<0.01), while the 10-year survival rate was 35.3% for the AgNOR high group and 77.2% for the AgNOR low group. Therefore, the AgNOR count was found to be closely related to tumor advancement and thus is considered to have a predictive value for the prognosis of patients with gastric cancer invading the serosa after curative resection.     

Correlation between nucleolar organizer regions visualized by silver staining and the growth rate in lung adenocarcinoma.

BACKGROUND. The value of nucleolar organizer regions (NOR) visualized by silver staining (AgNOR) for the histologic differentiation, pathologic staging, and estimation of growth rate was assessed by the investigation of paraffin sections from 58 lung adenocarcinomas. AgNOR consist of NOR-associated proteins, and the number of AgNOR might be related to proliferative activity. METHODS. In lung adenocarcinoma, the growth rate can be measured by means of chest radiographs. Using this technique, the authors studied the correlation between the mean number of AgNOR and growth rate. RESULTS. The mean number of AgNOR ranged from 1.8 to 6.3 (mean +/- standard deviation, 4.0 +/- 0.8). Neither the degree of histologic differentiation nor the pathologic staging was related to the AgNOR count. The tumor growth rate was estimated on the basis of the doubling time in the chest radiographs of 13 patients. The doubling time ranged from 80 to 760 days. There was a high inverse correlation between the AgNOR count and the doubling time (r = -0.910; P less than 0.001). CONCLUSIONS. Thus, it appears to be possible to use the mean number of AgNOR as an index of proliferative activity.     

Argyrophilic nucleolar organizer regions in breast carcinoma. Correlation with DNA flow cytometry, histopathology, and lymph node status.

Argyrophilic nucleolar organizer regions (AgNOR) have been correlated with proliferative activity of neoplasms. Increased AgNOR may reflect increased proliferative activity of cells or ploidy. To explore this hypothesis, 41 breast carcinomas were processed for AgNOR silver staining and DNA flow cytometry. AgNOR counts were expressed as mean AgNOR/nucleus and percentage of tumor cells with more than five AgNOR/nucleus. The first count was designated mean AgNOR or mAgNOR, and the second count was designated AgNOR proliferative index or pAgNOR. Using Mantel-Haensel statistical analysis, carcinomas that exhibited mAgNOR of 2.4 or more had a high likelihood of aneuploidy (P less than 0.0001), an S-phase fraction of more than 5.8% (P less than 0.003), or a diameter greater than 2 cm (P less than 0.007). In addition, tumors with pAgNOR of 8% or more showed a statistically significant correlation with aneuploidy (P less than 0.004), tumor grade (P less than 0.04), and a more significant one with high S-phase fraction (P less than 0.0001). No significant correlation was obtained between pAgNOR and tumor size or lymph node status. These data indicate that AgNOR quantitation reflects changes in DNA ploidy and cell proliferation. They also suggest that the mean AgNOR counts correlate best with the DNA mass or ploidy and that the frequency of cells with higher AgNOR count best reflects proliferative activity or S-phase fraction.     

Prognostic value of AgNOR counting

The prognostic significance of the argyrophilic nucleolar organizer regions (AgNORs) in tumour pathology is still a matter of debate. A prospective study was performed in a series of renal cell carcinomas to clarify the prognostic value of AgNOR counting. Sections from 21 renal cell carcinomas were stained in 1990 with the method of Ploton. Black dots within the nucleus from 200 tumour cells were counted: the mean AgNOR count for the whole series was 6.13, the median 5.94 and the SD 1.78. Patients were then followed up for at least 6 years or to death: at the time of the survival analysis (June 1996), 13 patients were alive without evidence of recurrence or metastasis, 6 had died of the disease and 2 of myocardial infarction. All the patients with 5.94 AgNORs per cell or fewer were alive at 6-year follow-up, while only 60% of patients with more than 5.94 AgNORs per cell survived (p=0.01). In the multivariate analysis, only AgNOR count (p=0.015) retained an independent prognostic significance. With the limitation due to the small number of cases, this prospective study clearly indicates that AgNOR count has a significant prognostic role, at least in renal cell carcinoma.     

Could Argyrophilic Nucleolar Organizer Regions count mirror DNA Ploidy in Malignant Salivary Gland Tumors?

Objective: Nucleolar organizer regions (NORs) are DNA coils that transcribe to ribosomal RNA. The NOR-associated protein, termed argyrophilic NOR (AgNOR), was visible within the nucleus by staining with silver nitrate examination via the light microscope. AgNOR counting is a proliferation marker and may help in the diagnosis and prognosis of various neoplastic lesions. Aneuploidy (abnormal DNA content) can predict the progression, survival and prognosis of the tumors. The aim of this study was to evaluate the role of AgNORs, DNA ploidy status, and total S-phase fraction (TSPF) as prognostic parameters in malignant salivary gland tumors (MSGTs). Methods: The current study is a retrospective study on a cohort of MSGTs (N=47), to assess AgNORs using Silver Nitrate stain, DNA index (DI), and TSPF using flow cytometry (FCM). Data including tumor size and site, lymphovascular invasion (LVI), lymph node metastasis (LNM) were collected. Results: The AgNORs count was statistically significant with MSGT type. DI was found to have a significant association with tumor site, tumor size and MSGT type. In addition, TSPF was found to be significantly associated with LVI. A moderate positive correlation was noted between AgNORs count and TSPF. LNM, tumor site, high AgNORs and low DI were all associated with short disease-free survival (DFS) and poor overall survival (OS). Conclusion: The present study revealed that high AgNORs count, DNA aneuploidy and TSPF had a poor influence on MSGTs prognosis.     

Prognostic significance of argyrophilic nucleolar organizer regions in esophageal carcinoma

The argyrophilic nucleolar organizer region (AgNOR) of 100 cancer cells from biopsy specimens of esophageal squamous cell carcinomas in 98 surgically treated cases was examined, using a silver colloid staining technique on biopsy specimens. The number of AgNOR per nucleus (AgNOR number) was higher in the more advanced groups with regard to the length of the tumor (P less than 0.01), the depth of penetration (P less than 0.05), and lymph node metastasis (P less than 0.01). The survival of the patients with a high AgNOR number (greater than or equal to 6) was significantly poorer than those with either a medium range AgNOR number (4 less than or equal to-less than 6) (P less than 0.05) or a low AgNOR number (less than 4) (P less than 0.01). In the multivariate analysis including conventional clinicopathological factors, the AgNOR number was found to be one of the independent and significant variables (P less than 0.01). Because the AgNOR method is simple and can be applied to paraffin-embedded sections, the AgNOR number may provide potential benefit in the pretherapeutic assessment of malignant potentiality in esophageal carcinoma.     

食管鳞癌组织AgNOR计数与DNA倍体的关系

本文应用ＡｇＮＯＲ技术对２０例食管鳞癌手术切除标本中癌细胞核内ＡｇＮＯＲ进行计数，同时运用流式细胞分析技术测定其ＤＮＡ指数（ＤＩ）及Ｓ期细胞百分率，并与ＡｇＮＯＲ计数进行比较。结果显示：食管鳞癌细胞核内平均ＡｇＮＯＲ数目与Ｓ期细胞百分率呈明显正相关（ｒ＝０．７３）。而ＤＩ与ＡｇＮＯＲ计数无明显相关关系（ｒ＝０．３０）。     

Nucleolar organizer region count and subjective AgNOR pattern assessment (SAPA) score in skin tumors.

OBJECTIVE: To study the argyrophilic nucleolar organizer region (AgNOR) count and subjective AgNOR pattern assessment (SAPA) score in cytologic and histologic specimens of various skin tumors. STUDY DESIGN: The study group consisted of 37 patients (14 benign and 23 malignant) of various skin tumors. In all cases, cytology by fine needle aspiration cytology (FNAC), and histological specimens were studied by conventional staining and silver staining for AgNOR. RESULTS: The mean count in benign tumors in cytologic specimens was 2.08 +/- 0.01, compared with 5.50 +/- 1.12 in malignant tumors (P<0.001). In histologic specimens, mean count was 2.13 +/- 0.51 in benign, compared with 5.38 +/- 1.10 in malignant tumors (P<0.001). The SAPA score in benign tumors (P<0.001) in cytologic specimens, was 6.07 +/- 0.83, compared with 10.65 +/- 1.27 in malignant tumors, and in histology, it was 6.07 +/- 0.87 in benign, compared with 10.83 +/- 1.15 in malignant tumors (P < 0.001). Melanoma showed the higher AgNOR count compared with squamous cell carcinoma and basal cell carcinoma. The parameters were statistically significant between the grade of tumor in squamous cell carcinoma and the positivity of lymph nodes as demonstrated by SAPA score. No correlation was found between the clinical stage and Clark level of melanoma. Although, AgNOR count and SAPA score showed similar results, the indicators of validity were higher in SAPA than AgNOR count. CONCLUSION: Although, AgNOR count and SAPA score gave similar results, but the indicators of validity were higher in SAPA score than AgNOR count.     

Prognostic Significance of Two Dimensional AgNOR Evaluation in Local Advanced Rectal Cancer Treated with Chemoradiotherapy

The prognostic significance of AgNOR proteins in stage II-III rectal cancers treated with chemoradiotherapy was evaluated. Silver staining was applied to the 3μm sections of parafin blocked tissues from 30 rectal cancer patients who received 5-FU based chemoradiotherapy from May 2003 to June 2006. The microscopic displays of the cells were transferred into the computer via a video camera. AgNOR area (nucleolus organizer region area) and nucleus area values were determined as a nucleolus organizer regions area/total nucleus area (NORa/ TNa). The mean NORa/TNa value was found to be 9.02±3.68. The overall survival and disease free survival in the high NORa/TNa (>9.02) patients were 52.2 months and 39.4 months respectively, as compared to 100.7 months and 98.4 months in the low NORa/TNa (<9.02) cases. (p<0.001 and p<0.001 respectively). In addition, the prognosis in the high NORa/TNa patients was worse than low NORa/TNa patients (p<0.05). In terms of overall survival and disease-free survival, a statistically significant negative correlation was found with the value of NORa/TNa in the correlations tests. Cox regression analyses demostrated that overall survival and disease-free survival were associated with lymph node status (negative or positive) and the NORa/TNa value. We suggest that two-dimensional AgNOR evaluation may be a safe and usable parameter for prognosis and an indicator of cell proliferation instead of AgNOR dots.     

Predictive value of Ki-67 and argyrophilic nucleolar organizer region staining for lymph node metastasis in gastric cancer

Proliferative activities in 91 primary gastric carcinomas and 36 corresponding metastatic perigastric lymph nodes were investigated using Ki-67 labeling percentage and an argyrophilic nucleolar organizer region (AgNOR) count. Tumors with a high proliferative activity often metastasized to lymph nodes, and the proliferative activities of the primary lesion and the perigastric lymph node metastases were similar. A significant correlation was recognized between the Ki-67 labeling percentage and the AgNOR count (r = 0.744; P less than 0.001). The Ki-67 labeling percentage and AgNOR count proved to be useful predictors of nodal metastasis regardless of tumor size, depth of invasion, and histological type. Even when tumors are smaller (less than 7 cm) or the stage of the disease is early (pT1, 2), the formation of metastasis increased with an increased Ki-67 labeling percentage or AgNOR count. The combination analysis of depth of invasion with Ki-67 labeling percentage or AgNOR count gives a more precise prediction of nodal metastasis, compared with histological analysis alone.     

Histologic grading in soft-tissue sarcomas. An analysis of 194 cases including agnor count and mast-cell count

In order to establish a new histologic grading system for STS, we evaluated histologic prognostic factors. For this purpose, we selected 194 patients with STS: 31 in the upper extremities, 63 in the trunk, and 100 in lower extremities. All the patients were treated by surgery, followed by chemotherapy in 74 cases, radiotherapy in 11, chemotherapy and radiotherapy in 30, or no adjuvant treatment in 79. Histologic factors evaluated were mitotic count, extent of necrosis, cellularity, cellular pleomorphism, extent of myxoid change, sclerosis, non-specific histologic diagnosis, counting of reaction product in silver stain for nucleolar organizer regions (AgNOR) and mast-cell counts. Univariate analysis revealed mitotic count, necrosis, cellularity, cellular pleomorphism, non-specific histologic classification, AgNOR count and mast-cell count to be significantly related to prognosis. Multivariate analysis revealed that AgNOR count, cellularity and necrosis were independent prognostic factors. A new grading system was introduced: low-grade, intermediate-grade and high-grade. The survival between each group were significantly different; the 5-year-survival rate in patients of the low-, intermediate- and high-grade groups was 87%, 74% and 35% respectively. Our findings suggest that this histologic grading system may be useful for making therapeutic decisions.     

Bax expression has prognostic significance that is enhanced when combined with AgNOR counts in glottic carcinomas.

Using nucleolar organizer regions (NORs) as a proliferative marker and Bax expression as a marker for apoptosis, we have studied the individual and combined prognostic significance of these markers. Successive sections of diagnostic, formalin-fixed and paraffin-embedded specimens from 69 patients with T1-4 tumours were stained with a rabbit anti-human Bax polyclonal antibody and silver nitrate for visualization of NORs (AgNORs). After classification for staining intensity and the percentage of Bax expression, a final score resulting in four classes of increasing Bax expression was obtained. AgNOR counts were expressed as mean counts (mAgNOR) and the percentage of tumour nuclei with more than one AgNOR (pAgNOR>1). Both AgNOR parameters were grouped in three classes with increasing values. Low Bax scores correlated significantly with poor prognosis (P = 0.0106). For mAgNOR and pAgNOR>1, high values correlated with poor prognosis (P = 0.0185 and P = 0.0003 respectively). A combined parameter, for which the Bax score was subtracted from the AgNOR scores, appeared to be statistically stronger than the individual parameters (P < 0.0001). Both Bax expression and AgNOR scores, and in particular the combination of these parameters, appear to be strong prognostic markers in glottic squamous cell carcinomas.     

NUCLEOLAR ORGANIZER REGIONS IN CUTANEOUS T CELL LYMPHOMAS

The present work studied the application of AgNOR count to differential diagnosis between cutaneous T cell lymphoma (CTCL) and cutaneous pseudolymphoma (CPL). Paraffin sections from 50 mycosis fungoides (22 MFI-Premycotic stage, 24 MF Ⅰ infiltrative stage and 4 MF Ⅲ - tumor stage), 2 nonepidermotropic cutaneous T cell lymphoma (NECTCL) and 9 CPL were investigated. In each case, 200 cells randomly selected were examined using a × 100 oil immersion lens. The mean number, standard deviation and standard error of the mean of AgNOR counts were as follows: MFⅠ 1.17±0.09, SEM = 0.01; MⅡ 1.17±0.01, SEM = 0.01; MF Ⅲ. 3.55±0.87, SEM = 0.43; NECTCL 4.5±0.28, SEM -0.199; CPL 1.17±0.1, SEM ± 0.03. The results revealed a highly significant difference between CTCL (MFⅢ NECTCL) and CPL (t = 4.75, P<0.001), tumor stage (MF Ⅲ) and pretumor stage (MFI, MF Ⅱ) of mycosis fungoides (t = 4.75, P<0.001). Thus. AgNOR count is valuable in differential diagnosis.     

Proliferating cell nuclear antigen and argyrophilic nucleolar organizer regions in patients with thymic disease

We examined argyrophil nuclear organizer regions (AgNOR) and proliferating cell nuclear antigen (PCNA) in 41 patients with surgically-treated thymic disease. AgNOR count and PCNA labeling index (LI) in thymic carcinoma were significantly higher than those in thymoma and thymic hyperplasia. A positive correlation was observed between the PCNA LI and argyrophilic nucleolar organizer region (AgNOR) counts within all thymic disease (r=0.31, P=0.002). The PCNA LI in invasive thymoma was lower than that in non-invasive thymoma. In survival analysis, the cut-off values for the PCNA LI and AgNOR count were chosen to produce two categories with equal numbers of 26 thymoma patients. There were no significant difference in the survival rate between the lower and higher group patients in relation with AgNOR count and PCNA LI. We conclude that combining AgNOR and PCNA may discriminate the biological activity of thymic disease. These staining methods can be performed with ease and, applied in a clinical laboratory on a routine basis to help predict cytological malignancy of thymic disease.     

INVESTIGATION OF RELATIONSHIPS BETWEEN KI－67SCORE，DNA INDEX，AND HISTOLOGIC GRADEIN SOFT TISSUE SARCOMAS

ＩＮＶＥＳＴＩＧＡＴＩＯＮＯＦＲＥＬＡＴＩＯＮＳＨＩＰＳＢＥＴＷＥＥＮＫＩ－６７ＳＣＯＲＥ，ＤＮＡＩＮＤＥＸ，ＡＮＤＨＩＳＴＯＬＯＧＩＣＧＲＡＤＥＩＮＳＯＦＴＴＩＳＳＵＥ　ＳＡＲＣＯＭＡＳＷａｎｇｙａｎｏｎｇ王亚农；ＳｈｉＤａｒｅｎ施达仁；ＳｈｅｎＺ...     

Agyrophilic nucleolar organizer region counts as related to bromodeoxyuridine incorporation scores in ovarian clear cell carcinoma cells]

The value of agyrophilic nucleolar organizer region (AgNOR) stain as potential technique for the estimation of cell kinetics was assessed in this study. The daily AgNOR counts and the scores of the proliferation-associated marker bromodeoxyuridine (BrdU) in a human ovarian carcinoma cell line-OCC1 for 10 days after subculture were determined. The correlation between AgNOR counts and BrdU incorporation scores was highly significant (r = 0.86, P less than 0.01). Therefore AgNOR counts offer a potential tool to provide cytokinetic information.     

Proliferative activity as a prognostic factor in Borrmann type 4 gastric carcinoma.

Proliferative activities in 181 primary Borrmann type 4 gastric carcinomas were investigated using percentage labelling of proliferating cell nuclear antigen (PCNA) and an argyrophilic nucleolar organiser region (AgNOR) count. Tumours with a high proliferative activity often metastasised to lymph nodes (P < 0.01), and these patients had a lower survival rate (P < 0.05). A significant correlation was recognised between the PCNA labelling percentage and AgNOR count (r = 0.452, P < 0.001). Cox''s regression analysis showed that PCNA labelling percentage is an independent prognostic factor. These results indicate that estimating proliferative activity may be useful in predicting lymph node metastasis and patients'' prognosis in cases of Borrmann type 4 gastric carcinoma.