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 Hepatocarcinogenesis Induced by N-2-Fluorenylacetamide in Rats: Comparison with Bromodeoxyuridine Immunohistochemistry

The number of silver-stained nucleolar proteins (AgNOR) was counted in preneoplastic and neoplastic rat liver lesions induced by N-2-fluorenylacetamide (FAA) and was compared with that of bromodeoxyuridine (BrdU)-incorporating cells detected immunohistochemically using monoclonal antibody against BrdU. Male ACI/N rats were given diet containing 200 ppm FAA for 12, 16 or 20 weeks to induce hepatocellular foci and tumors. The mean numbers of AgNOR stained by a one-step silver colloid method and BrdU-labeling indices in various liver cell lesions were as follows: nontreated liver (n = 20), 1.20 and 0.08; nonlesional areas (n=20), 1.33 and 0.13; altered liver cell foci (n = 80), 2.04 and 4.05 [eosinophilic cell type (n = 20), 1.78 and 1.82; clear cell type (n=20), 1.45 and 1.77; basophilic cell type (n=20), 1.99 and 4.58; hyperbasophilic cell type (n=20), 2.94 and 8.02]; neoplastic nodules (n = 10), 3.11 and 2.99; hepatocellular carcinomas (n = 10), 7.22 and 8.29. Thus, the mean number of AgNOR and the value of BrdU-labeling index were well correlated and both values showed a stepwise increase from normal liver cells to liver cell carcinoma, although some scatter was present. These data suggest that mean number of AgNOR may reflect the cellular kinetics in rat hepatocarcinogenesis, and the one-step silver colloid method for demonstration of AgNOR may therefore be a simple and useful staining to examine the proliferative nature of cells.     

Nucleolar organizer regions in hepatocarcinogenesis induced by N-2-fluorenylacetamide in rats: comparison with bromodeoxyuridine immunohistochemistry

The number of silver-stained nucleolar proteins (AgNOR) was counted in preneoplastic and neoplastic rat liver lesions induced by N-2-fluorenylacetamide (FAA) and was compared with that of bromodeoxyuridine (BrdU)-incorporating cells detected immunohistochemically using monoclonal antibody against BrdU. Male ACI/N rats were given diet containing 200 ppm FAA for 12, 16 or 20 weeks to induce hepatocellular foci and tumors. The mean numbers of AgNOR stained by a one-step silver colloid method and BrdU-labeling indices in various liver cell lesions were as follows: nontreated liver (n = 20), 1.20 and 0.08; nonlesional areas (n = 20), 1.33 and 0.13; altered liver cell foci (n = 80), 2.04 and 4.05 [eosinophilic cell type (n = 20), 1.78 and 1.82; clear cell type (n = 20), 1.45 and 1.77; basophilic cell type (n = 20), 1.99 and 4.58; hyperbasophilic cell type (n = 20), 2.94 and 8.02]; neoplastic nodules (n = 10), 3.11 and 2.99; hepatocellular carcinomas (n = 10), 7.22 and 8.29. Thus, the mean number of AgNOR and the value of BrdU-labeling index were well correlated and both values showed a stepwise increase from normal liver cells to liver cell carcinoma, although some scatter was present. These data suggest that mean number of AgNOR may reflect the cellular kinetics in rat hepatocarcinogenesis, and the one-step silver colloid method for demonstration of AgNOR may therefore be a simple and useful staining to examine the proliferative nature of cells.     

The estimation of proliferative activity by pcna and AgNORs in leukoplakia and squamous-cell carcinoma of the oral cavity

The proliferating activity of leukoplakia and squamous cell carcinoma (SCC) was estimated using proliferating cell nuclear antigen (PCNA) immunostaining and silver-binding argyrophilic nucleolar organizer region (AgNOR) staining. Twenty-eight leukoplakias and 15 SCCs of the oral cavity were used in this study. The mean+/-S.D. of PCNA LI and AgNOR counts were 7.7+/-6.0% and 2.43+/-0.68/nucleus in leukoplakias, and 22.3+/-11.6% and 4.77+/-1.49/nucleus in SCCs. Both of PCNA LI and AgNOR counts were significantly higher in SCCs than in leukoplakias. There was a significant linear correlation between PCNA LI and AgNOR counts (p=0.0022) in leukoplakias, but not in SCCs. In the series of leukoplakias, PCNA LI apparently increased in leukoplakias with dysplasia and malignant transformed cases. Our data suggest that PCNA LI and AgNOR counts are useful markers of proliferating activity, and PCNA LI might be a prognostic factor of leukoplakias.     

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。     

Correlation between nucleolar organizer region score and bromodeoxyuridine labeling index in C6 glioma cell line

Correlation between the mean number of nucleolar organizer region-associated argyrophilic proteins (AgNORs) per cell and BrdU labeling index (LI), the percentage of S-phase cells, was investigated in vitro using rat C6 glioma cells under several different conditions such as increased concentration of fetal calf serum (FCS) in a stepwise manner in medium. In addition, the effect of dibutyryladenosine cyclic monophosphate (d-cAMP) on the mean number of AgNORs and the BrdU LI was examined. C6 glioma cells cultured with no FCS showed 5.1% BrdU LI and 3.46 AgNOR score. BrdU LI and AgNOR score of the cells cultured with 1% FCS were 26.5% and 6.25, respectively. Those with 5% FCS were 44.7% and 8.41, with 10% FCS; 49.8% and 10.85, and with 20% FCS; 48.4% and 10.64. Treatment of d-cAMP with 10% FCS induced morphological differentiation and reduced BrdU LI (9.4%) and AgNORs score (4.90), compared with C6 cells cultured in medium containing 10% FCS without d-cAMP. Thus, the BrdU LI and AgNOR score of the C6 cells cultured under several different concentration of FCS were shown to be well linearly related (r = 0.97) and both values were decreased with morphological alteration of C6 glioma cells in the presence of 1 mM d-cAMP.     

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）。     

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.     

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。     

Prognostic utility of silver stained nucleolar organiser region counting in non Hodgkin's lymphoma

Silver stained Nucleolar Organiser Region (AgNOR) counting is a well established method of estimating the proliferative activity of a tumour. Their utility in prognostication in Non Hodgkin's Lymphoma (NHL) has not been fully investigated. We analysed hundred cases of NHL, all of which had a minimum of two and a half years of follow up, using histology by International Working Formulation (IWF) and their AgNOR scores. We found the IWF grading to have a good correlation with the outcome. The two and a half year survival for low grade NHL was 90 percent, for intermediate grade NHL was 47 percent and for high grade NHL was 30 percent. AgNOR scores correlated well with IWF grading and classification, with average AgNOR score of 1.82 (SD 0.34) in low grade NHL; 4.47 (SD 2.25) in intermediate grade NHL and 7.07 (SD 4.40) in high grade NHL. Over histology, AgNOR counting yielded additional prognostic information only in the diffuse small cleaved cell NHL (DSC), where finely dispersed NORs were seen. Of the 22 case of DSC included, 11 cases with AgNOR scores less than three had a much better outcome with 75 percent survival at two and a half years. Cases of DSC with AgNOR scores greater than three had a significantly worse outcome with only 25 percent survival at two and a half years (p=0.0103). Such sub-stratification could not be done in any other catergory of NHL using AgNOR scores. Thus, counting of AgNORs in lymphomas with finely dispersed NORs as in DSC is of prognostic value.     

肾上腺皮质肿瘤增殖活性检测及预后意义

 目的：探讨细胞增殖活性检测对肾上腺皮质肿瘤的诊断价值和预后。方法：采用DNA含量测定、AgNOR和PCNA染色对正常肾上腺皮质、皮质增生、腺瘤和癌进行细胞增殖活性检测。结果：正常肾上腺与皮质增生DNA含量、AgNOR计数、PCNA指数均值相接近(P>0.05),DNA倍体主要呈二倍体。腺瘤较增生组相比三种指标均值相差显著(P<0.01)。结论：提示细胞增殖活性检测对肾上腺皮质肿瘤诊断和预后判断有较大参考价值。     

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.     

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.     

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.     

Relationship between AgNORs,MIB-1 and oncogene expression in male breast carcinoma and papillary superficial bladder neoplasm

The expression of p53, c-erbB-2, bcl-2 and c-myc proteins was compared to the quantity of the nucleolar organiser regions (AgNORs) and MIB-1 antigen to elucidate the relationship between oncogene expression and rapidity of cell proliferation and tumor growth fraction. Sections from 50 male breast carcinomas (MBC) and 62 superficial papillary bladder neoplasias were stained with the standardised AgNOR method and monoclonal antibodies MIB-1, DO7, CB11, bcl-2 124 and 9E11. p53 immunopositivity was associated with high AgNOR quantity and MIB-1 scores both in MBC and bladder neoplasm. c-erbB-2 expression was associated with high AgNOR quantity in bladder neoplasm. bcl-2 expression was associated with low AgNOR quantity in MBC. c-myc expression was associated with high AgNOR quantity in MBC. MBC patients with low AgNOR quantity, and p53, c-erbB-2 and c-myc immunonegativity had the longest overall survival. Patients with bladder neoplasia with low AgNOR quantity, negative p53 and positive c-erbB-2 immunostaining had the longest disease-free survival time. Our results indicate that p53 overexpression reflects both the rapidity of cell proliferation, as assessed by AgNOR quantity, and tumor growth fraction, as assessed by MIB-1 scores, while c-erbB-2, c-myc and bcl-2 expression mainly reflects the rapidity of cell proliferation. The combination of AgNOR quantity and oncogene expression may stratify patients into different risk groups.     

Nucleolar organizer regions in Spitz nevi and malignant melanomas

Nucleolar organizer regions (NOR) are loops of DNA that transcribe ribosomal RNA; they can be easily identified in formalin fixed paraffin embedded tissue using a silver (Ag) method. It has been suggested that the number of AgNOR per cell can differentiate between benign and malignant melanocytic lesions of skin. We have studied 29 Spitz nevi (SN) and 39 invasive malignant melanomas (MM) by the same silver method. SN showed between 1.0 and 1.6 AgNOR per cell with a mean of 1.2. MM counts ranged from 1.2 to 4.2 with a mean of 2.0. It is concluded that the AgNOR method cannot reliably differentiate SN from MM; however, a count of more than 2.0 AgNOR per cell would favor a diagnosis of MM rather than SN.     

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.     

Interphase nucleolar organizer region distribution in the tissues of oral leukoplakia, oral submucous fibrosis and oral cancer

The AgNOR technique, was applied to oral tissue sections of 185 oral cancer, 42 oral leukoplakia, 37 oral submucous fibrosis and 10 normal subjects to investigate whether any correlation held good in these different tissues. Compared to the AgNOR counts in normal oral epithelium, there was a gradation in increase in the mean AgNOR counts from oral leukoplakia to oral submucous fibrosis to oral carcinoma (P<0.01). This suggests that AgNOR count parallels with the degree of neoplastic transformation of oral epithelium. Three oral submucous fibrosis patients who showed very high AgNOR counts as that of oral cancer patients, later developed oral carcinoma. Among the oral cancer tissues, the moderately and poorly differentiated subtypes showed higher AgNOR counts and scattered distribution pattern than the well differentiated subtype which showed a clustered distribution pattern. These results suggest that AgNOR technique can be utilised as a diagnostic and prognostic indicator in premalignant and malignant oral tissues.     

Argyrophilic nuclear organiser region counts in locally advanced breast carcinoma treated by chemotherapy before surgery

The value of argyrophilic nuclear organiser region (AgNOR) counts in assessing histologically the effects of combination chemotherapy given to eleven patients with locally advanced breast cancer before mastectomy was studied. AgNOR counts were significantly reduced (P < 0.001) in the post-chemotherapy, surgically excised residual tumour specimens compared with the initial diagnostic biopsy specimens. AgNOR counts could be used to monitor the effects of chemotherapy on breast cancer.     

Prognostic value of histobiological factors (malignancy grading and AgNOR content) assessed at the invasive tumour front of oral squamous cell carcinomas.

Tumour cells at the invasive front of carcinomas have been found to differ substantially from the rest of tumour cells in a variety of human cancers. The present multivariate survival analysis of 94 oral squamous cell carcinomas (OSCCs) revealed that both the argyrophilic nucleolar organizer regions-associated protein (AgNOR) content of invading tumour cells and a multiparametric histopathological tumour front grade were significantly and independently associated with tumour-related death, irrespective of conventional Broders'' grade and clinical stage of the tumours. High tumour front scores and AgNOR content at the invasive OSCC front thus seem to reflect increased malignant potential. Proliferative activity, assessed by standardized AgNOR analysis, most probably represents one of the biological features underlying the usefulness of evaluating the invasive tumour front.