Demonstration of silver-stained nucleolar organizer region associated proteins (AgNORs) after wet autoclave pretreatment in breast carcinoma: Correlation to tumor stage and long-term survival

Argyrophilic nucleolar organizer region associated proteins (AgNORs) are known to reflect cellular and nucleolar activity. Due to a novel staining procedure, which substantially improves visualisation of AgNORs on formalin-fixed and paraffin-embedded material, AgNORs can be reliably demonstrated as true substructures of the nucleoli. The aim of the present study was to apply a standardized morphometric AgNOR quantification on a large series of breast carcinomas with regard to its prognostic relevance. AgNOR quantity was evaluated on archival tumor tissues of 115 adenocarcinomas of the breast treated with the wet autoclave method prior to standardized silver-staining and morphometric analysis. AgNOR parameters were correlated to prognostic features (steroid hormonal receptor status, tumor type, tumor size, histological grading, pTNM, and UICC stage) carrying out both univariate and multivariate survival analyses. AgNOR number and area were proven to be statistically significantly related (Pearson correlation coefficient: 0.67, Bonferroni adjusted P = 0.0001). Almost all AgNOR parameters, in particular CV (coefficient of variation) of corrected area (-area) and CV of number, were statistically significantly correlated to estrogen and progesterone receptor status as well as histological grading of tumors. Increased AgNOR parameters were statistically significantly associated with early tumor relapse and cancer related death. Univariate and multivariate analysis by means of Cox regression revealed independent prognostic significance for CV of -area and number of AgNORs. Various AgNOR parameters (CV of number, CV of -area, CV of area, mean -area, and mean area of AgNORs per nucleus) determined on wet autoclave pre-treated formalin-fixed and paraffin-embedded breast cancer tissues are statistically highly significantly associated with the prognostic outcome, independently predicting tumor-free and overall survival.     

Comparative Immunohistochemical Studies of p53 and Proliferating Cell Nuclear Antigen Expression and Argyrophilic Nucleolar Organizer Regions in Pancreatic Duct Cell Carcinomas

Thirty-eight cases of pancreatic duct cell carcinoma were examined for p53 expression and proliferating cell nuclear antigen (PCNA) by enhanced immunohistochemistry, as well as for changes in numbers of argyrophilic nucleolar organizer regions (AgNORs). Fifteen cases (39.5%) showed p53 overexpression, which tended to increase in proportion to the histopathological grading of malignancy. However, tumor stage and lymph node status were not correlated to p53 overexpression. PCNA labeling index (LI) increased with both histologically malignant grading and pathological stage, but was not correlated with lymph node status. The expressions of p53 and PCNA thus did not necessarily reflect the degree of malignant development. In contrast, AgNOR number showed statistically significant correlations with these three indicators of malignancy. A comparative analysis of p53, PCNA LI and AgNOR number showed overexpression of p53 to be correlated to PCNA LI and essentially unrelated to AgNOR number. The present results thus indicate a close relation between p53 and PCNA, while AgNORs appear to be regulated separately from either of them.     

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.     

Silver-stained nucleolar organiser region counts are of no prognostic value in primary breast cancer.

The assessment of nucleolar organising regions have been reported to be of prognostic value both in a number of haematological and solid tumours. We have examined the relationship between the number of nucleolar organising regions (NORs) present in 75 primary breast cancers and various clinical and pathological features known to be associated with prognosis in patients with breast cancer. Formalin-fixed, paraffin-embedded tumour tissue was sectioned and stained by a one-stage argyrophil (AgNOR) method. Using light microscopy the mean number of AgNORs per cell was calculated. No correlation was observed between AgNOR counts and any of the prognostic variables studied, including oestrogen receptor (ER) status, histological grade of malignancy, lymph node stage or site of initial metastatic disease. Similarly there was no correlation between AgNOR counts and disease-free interval or survival. AgNOR counts do not appear to be a prognostic factor in primary breast cancer.     

Prognostic significance of argyrophilic nucleolar organizer staining in soft-tissue sarcomas.

The utility of argyrophilic stain for nucleolar organizer region (AgNOR) for estimating proliferative activity and prognosis of soft-tissue sarcomas (STS) was examined. Formalin-fixed and paraffin-embedded sections of 38 cases with STS were used; the reaction product of AgNOR stain was observed as dots mainly in the nucleoli. The mean number of AgNOR dots per nucleus of tumor cells was calculated in 200 cells (AgNOR count). The AgNOR count, ranging from 1.4 to 16.1 (mean, 7.5), showed a good correlation with cellularity (r = 0.483, p less than 0.003) and histologic grade (r = 0.626, p less than 0.00005), but less shown with mitotic counts (r = 0.350, p less than 0.04). The prognosis of cases with AgNOR low-count group (5-year survival rate was 74.6%) was much better than those in high count group (33.3%) (p less than 0.0005). The AgNOR count correlated well with reactivity of tumor cells for Ki-67 staining, which was available only in freshly prepared sections. These findings suggested that the AgNOR staining is a simple and useful method for estimating tumor-cell proliferation and prognosis of patients with STS.     

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.     

前列腺癌AgNOR计数与DNA含量测定的意义

作者应用银染色技术对３０例前列腺癌、２０例前列腺增生症组织中的核仁组成区相关嗜银蛋白（ＡｇＮＯＲ）进行检测，并采用图像分析技术测定其ＤＮＡ含量。结果显示，在前列腺癌组织中，ＡｇＮＯＲ计数与ＤＮＡ含量均明显高于前列腺增生症组织（Ｐ＜０．００１）；肿瘤分化越差，ＡｇＮＯＲ计数和ＤＮＡ含量越高，并且与患者预后相关（Ｐ＜０．００１）；ＡｇＮＯＲ计数与ＤＮＡ含量呈明显的正相关（Ｐ＜０．０１）。结果提示ＡｇＮＯＲ计数与ＤＮＡ含量可作为反映前列腺癌生物学行为的指标。     

用形态测量方法研究卵巢上皮性肿瘤的诊断及预后

背景与目的:形态测量学在肿瘤的病理诊断及预后评估中日渐重要。本文探讨图像分析法在卵巢上皮性肿瘤的诊断及预后中的意义。方法:联合应用图像分析、AgNOR计量分析及DNA含量测定3种形态测量学方法,对110例原发性卵巢上皮性肿瘤的诊断及预后进行研究,选用的参数为核面积(nucleararea,NA)、核周长(nuclearperimeter,NP)和核形状因子(nuclearformfactor,NFF)、DNA含量(DNAcontent,DC)、DNA指数(DNAindex,DI)、G0/G1期细胞百分率(percentageofG0/G1phases,P2c)、高倍体细胞百分率(percentageofDNAmultiploid,P>4c),并于光镜下进行AgNOR计数。结果:(1)良性、交界性及恶性3组卵巢上皮性肿瘤相比较,NA、NP、NFF、DC、DI及AgNOR计数在各组间差异有极显著性意义(P<0.01或P<0.001)。(2)形态测量学结果与预后关系研究表明,卵巢上皮性癌患者生存≥5年及<5年两组病例中,NA、NP、NFF参数两组间差异有显著性或极显著性意义(P<0.05或P<0.01),不同的图像分析结果提示其预后不同。AgNOR计数与上皮性卵巢癌患者的生存时间呈负相关(r=-0.73,P<0.001)。DNA高倍体含量与上皮性卵巢癌患者生存时间亦呈负相关(r=-0.75,P<0.001)。结论:形态测量学可为不同性质的卵巢上皮性肿瘤的诊断和预后提供可靠的客观依据。     

Cell adhesion molecule E-cadherin and cellular proliferation marker AgNOR in bladder lesions--usefulness and limitations.

The objective of this study on bladder lesions was to assess the relationship of (a) the proliferative activity measured by AgNOR counts, and (b) the loss of cell adhesion leading to acquisition of invasive properties as assessed by E-Cadherin expression. Paraffin embedded tissue biopsies from normal urothelium and malignant urothelial lesions were randomly selected from our surgical pathology files. AgNORs were analysed by the silver staining method and E_cadherin expression by immunohistochemistry. An increase in M&P AgNOR counts was observed from normal through benign to malignant tumors. A corresponding decrease in E Cadherin expression was noticed from normal through different grades of transitional cell carcinoma (TCC). A correlation was found between a high P AgNOR and decreased E_cadherin expression with stromal and muscle invasive tumors. It is deduced that when low grade tumors show a high P AgNOR and loss of E_cadherin expression, it is a significant indicator of the possible aggressive behavior in terms of stromal or muscle invasion and progression to a higher grade in future.     

增殖细胞核抗原与AgNOR检测在前列腺癌临床中的意义

目的研究前列腺癌中增殖细胞核抗原（ＰＣＮＡ）表达及ＡｇＮＯＲ计数的意义。方法应用免疫组织化学方法和银染色技术检测前列腺癌和良性前列腺增生组织中ＰＣＮＡ表达和ＡｇＮＯＲ计数。结果ＰＣＮＡ增殖指数与ＡｇＮＯＲ计数在癌组织中均明显高于良性前列腺增生组织（Ｐ均＜０．００１）,且两者均与肿瘤组织学分级和预后有密切关系（Ｐ均＜０．０１和Ｐ均＜０．００１）;前列腺癌ＰＣＮＡ增殖指数与ＡｇＮＯＲ计数间存在非常显著的正相关（Ｐ＜０．０１）。结论ＰＣＮＡ增殖指数和ＡｇＮＯＲ计数结合分析,在鉴别前列腺良恶性病变和判断前列腺癌的恶性程度及预测患者预后方面具有十分重要的意义。     

Quantitative-analysis of silver staining nucleolar organizer regions (AgNORs) in human tongue carcinoma cell-lines

Six morphometric parameters related to nuclear size, number of AgNORs, and area of AgNORs in two tongue carcinoma cell lines were analyzed by an image analyzing system. It was discovered that AgNOR numbers, the area of all AgNORs in the nucleus, the ratio of mean area and size, and nuclear size are the parameters demonstrating the most consistent pattern of change against passage numbers in T1 and T2 human tongue carcinoma cells. These changes may be related to 'dedifferentiation' of tumor cells in prolonged culture.     

AgNORs and their relationship to cell size,histological grade,lymph node involvement,metastases, and survival pattern in carcinoma of the breast: A study from south India

An AgNOR count using the Smith and Crocker [Histopathology 12:113–125, 1988] method of staining was performed on 200 cases of carcinoma of the breast. A count of coarse AgNORs per nucleus was made on 50 random cells and the mean of their number per nucleus calculated. The relationship of a single variable “AgNOR count” to other variables such as cell size, histological grade, number of positive ipsilateral axillary lymph nodes, and presence of metastasis in regions other than the ipsilateral axillary lymph nodes was found using a univariate method of analysis. Also, the effect of different independent variables, e.g., number of AgNORs, cell size, histological grade, number of positive axillary lymph nodes, and metastasis on a single variable, i.e., 4-year period of survival, was also assessed by a univariate method of statistical analysis. It was found that the AgNOR count was significantly related to the cell size, histological grade, and presence of metastasis. Large cells, grade III tumors, and neoplasms with evidence of metastasis showed larger numbers of AgNORs in their nuclei. It was observed that the number of AgNORs significantly affected the 4-year survival of patients. The higher the AgNOR counts, the poorer were the chances of surviving for 4 years. The other factors that influenced survival in the present study were the number of positive axillary lymph nodes and metastasis to sites other than axillary lymph nodes. © 1996 Wiley-Liss, Inc.     

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）。     

Soft-tissue sarcomas of adults; study of pathological prognostic variables and definition of a histopathological grading system

The pathological features of 155 adult patients with soft-tissue sarcomas were studied retrospectively, in an attempt to set up a grading system for these tumors. As the first step, seven histological criteria (tumor differentiation, cellularity, importance of nuclear atypia, presence of malignant giant cells, mitosis count, pattern of tumor necrosis and presence of vascular emboli) were evaluated in a monofactorial analysis. Five of these (tumor differentiation, cellularity, mitosis count, tumor necrosis, and vascular emboli) were correlated with the advent of metastases and with survival. A multivariate analysis, using a Cox model, selected a minimal set of three factors (tumor differentiation, mitosis count, and tumor necrosis) the combination of which was necessary and sufficient to retain all the prognostic information. A grading system was elaborated, which turned out to be correlated with the advent of metastasis and with patients' survival. A second multivariate analysis introducing clinical prognostic features showed that the histological grade was the most important prognostic factor for soft-tissue sarcomas. Thus, this grading system appears to be highly interesting because of its prognostic value and the facility of its elaboration. However, its reproducibility should be tested.     

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.     

Evaluation of argyrophilic nucleolar organizer regions in tongue squamous cell carcinomas

Using a silver staining technique, argyrophilic nucleolar organizer regions (AgNORs) were studied on the biopsy specimens taken from 67 tongue squamous cell carcinomas (SCCs), and the relations of the results of AgNORs to the clinicopathological findings, to the labelling indices of proliferating cell nuclear antigen (PCNA LI), and to the outcomes of the disease were investigated. The mean numbers of AgNORs per cell were 1.67 +/- 0.19 (n = 27) in the control squamous epithelia and 3.58 +/- 1.15 (n = 67) in the tongue SCCs, respectively, and a significant difference was found between the two groups (P < 0.0001). Concerning the clinicopathological findings, the mean numbers of AgNORs in the progressive cases (T3, T4), in the factor N-positive groups (N1, N2), and in the advanced stages (stages III, IV) were statistically higher than those in T1, T2, N0, and earlier stage (stages I, II) tumours, respectively. Similarly, a higher value of the AgNOR count was present in the histological grade III or diffuse invasive tumour. There was also a directly significant correlation between the AgNOR counts and the labelling indices of PCNA (r = 0.53, P < 0.0001). Concerning the outcome of the disease, the mean numbers of AgNORs were higher in the group with local recurrence or with lymph-node metastasis. A lower rate of 5 years' survival was found in the high value group (63.5%) of AgNORs compared with that of the low value group (86.0%), with a significant difference between the two groups (P < 0.05). The results suggest that AgNORs may reflect the degree of malignancy and cellular proliferation in tongue SCCs.     

Argyrophilic nucleolar organiser region counts and prognosis in pharyngeal carcinoma.

The prognostic significance of argyrophilic nucleolar organiser regions (AgNORs) has been evaluated in biopsy specimens from 61 primary squamous and undifferentiated carcinomas of the pharynx prior to therapy. The univariate Kaplan-Meyer survival analysis showed a significant correlation between 3- and 5-year survival rates and the mean AgNOR number per tumour cell (P less than 0.001). No significant correlation was found between prognosis and patients age and sex, tumour location, clinical stage, histologic grade, extent of lymphocytic infiltration, HMFG-2 positivity of tumour cells and UCHL1, LN2, MB2 positivity of infiltrating lymphocytes. There was no significant association between AgNOR counts and tumour histologic grade or clinical stage. Multivariate survival analysis showed that only two variables were significantly correlated with prognosis: AgNOR counts (P less than 0.001) and the extent of lymphocytic infiltration (P less than 0.027). Our results indicate the prognostic value of AgNOR counts and suggest the use of this method as a significant parameter in the pretherapeutic assessment of the aggressiveness of pharyngeal carcinomas.     

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 factors in soft tissue sarcomas. A multivariate analysis of 109 cases

Prognostic factors were evaluated in 109 soft tissue sarcomas of the extremities, walls of the trunk, head, and neck. All lesions were graded according to the systems proposed by the National Cancer Institute (NCI) and the French Federation of Cancer Centers (FNCLCC), and a correlation was found between tumor grade and prognosis. Univariate analysis selected the following variables as unfavorable prognostic factors: invasive tumor margins, extra-compartmental status, deep tumors, tumor diameters over 5 cm, inadequate excision, presence of necrosis, high mitotic count, histologically undifferentiated tumors, and blood vessel invasion. These variables were found to be interdependent. Multivariate analysis selected quality of surgery as the most important variable for predicting local recurrences. The factors selected with regard to overall and metastasis-free survival were tumor size, tumor margins, necrosis, and adequacy of excision. These results permitted classification of patients into four prognostic groups: two with good and two with bad prognosis. Five-year survival for the four groups was 100%, 83%, 53%, and 0%; 5-year metastatic rates were 0%, 12%, 67%, and 100%. Similar groups were obtained when the variables of tumor margins and size were combined with an adaptation of the NCI grading (low-grade tumors/high-grade tumors without necrosis/high-grade tumors with necrosis). Comparative analysis showed that patients with tumors of the same histologic grade or type were not necessarily classed in the same prognostic groups. A better clinicopathologic correlation was obtained using a combination of prognostic factors than with histologic grading or typing alone.     

Prognostic significance of histologic parameters of soft tissue sarcomas.

BACKGROUND AND METHODS. A univariate and multivariate analysis for the correlation between histomorphologic factors and prognosis was made using data from 1116 patients with soft tissue sarcoma, including 1005 cases available with complete histologic and follow-up data. RESULTS. The overall 5-year survival rate was 43.6%. The univariate analysis using Kaplan-Meier survival curves showed that tumor differentiation, cellularity, nuclear atypia, cellular pleomorphism, mitotic activity, amount of fibrous stroma, extent of myxoid areas, extent of tumor necrosis, and histologic grading (determined by the estimated range of malignancy for each type of sarcoma using a reported guideline) were all significant prognostic factors in the overall soft tissue sarcoma group. By a multivariate analysis using a procedure based on the Weibull model to failure data, the histologic grading and extent of tumor necrosis were proved to be prognostically significant in the overall sarcoma group. However, after additional analysis on each histologic type of sarcoma, it was determined that both tumor necrosis and histologic grading were applicable in only two types: malignant fibrous histiocytoma and leiomyosarcoma. Liposarcoma, synovial sarcoma, and malignant schwannoma each also showed some determinable factor of prognostic significance. CONCLUSIONS. It was concluded that there seemed to be no single universal prognostic parameter that could be applied to each soft tissue sarcoma type. These results suggest that the predictive significance of the various histologic parameters should be based on each specific type of sarcoma.