AgNOR Method Research Articles

Assessment of proliferative activity of thyroid HĂźrthle cell tumors using PCNA, Ki-67 and AgNOR methods.

Assessment of proliferative activity of thyroid HĂźrthle cell tumors using PCNA, Ki-67 and AgNOR methods.

Mastocitoma cutâneo canino: estudo de 45 casos

Quarenta e cinco mastocitomas cutâneos caninos foram graduados histologicamente com o uso de hematoxilina-eosina. Foram empregados os métodos azul de toluidina e região organizadora nucleolar argirofílica (AgNOR) para, respectivamente, evidenciar os grânulos citoplasmáticos e avaliar o índice de proliferação celular. Diversas características histológicas foram observadas, como distribuição das células na pele, tamanho, forma, aspecto de citoplasma e núcleo, quantidade de estroma, presença de eosinófilos e alterações associadas. Com base nessas caracteríscas, 37,8% dos mastocitomas foram classificados como grau I, 51,1% como grau II e 11,1% como grau III. A média geral de AgNOR nos mastocitomas foi de 1,9 (1,2 a 4,3) e as médias para os graus I, II e III foram, respectivamente, de 1,5, 1,85 e 3,25. A técnica de AgNOR mostrou ser de fácil execução, custo acessível e confiável como meio auxiliar para estimar um prognóstico mais objetivo para os mastocitomas.

Extracellular matrix of reinke's space in some pathological conditions

ObjectiveReinke's space (RS) is a highly specific structure in which the majority of vocal fold pathology occurs. The vibratory equivalents of structurally normal RS are regular vocal fold vibrations and normal glottic mucosal waves. Recently, RS has been considered to be composed not only of fibres but also of extracellular matrix (ECM), which is an extremely important component of a normal mucosal wave. As Reinke's oedema (RO) is characterized by excessive ECM in RS, we investigated the cell activity in RO tissue which is possibly responsible for excessive ECM production. Material and MethodsTissue samples were obtained from 20 RO patients during microlaryngeal surgery. Tissue was analysed histochemically using the AgNOR method and the proliferation activity was assessed immunohistochemically using Ki67 monoclonal antibodies. ResultsAgNOR activity was detected in prominent stromal cells in 16 patients. Also, more pronounced activity, compared to the surrounding control tissue, was demonstrated in the epithelial cells of 17 patients using a Ki67 proliferation marker. Electron microscopy showed thickening and decomposition of the basement membrane in all RO tissue samples. ConclusionsVocal fold mucosa that has been damaged by smoking and phonotrauma may react by producing excessive ECM, resulting in RO. Both epithelial and stromal cells are in a state of higher metabolic activity, indicating their role in this production.

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.

AgNOR staining and quantification

Nucleolar organiser regions (NORs) are defined as nucleolar components containing a set of argyrophilic proteins, which are selectively stained by silver methods. After silver-staining, the NORs can be easily identified as black dots exclusively localised throughout the nucleolar area, and are called “AgNORs”. The NORs' argyrophilia is due to a group of nucleolar proteins, which have a high affinity for silver (AgNOR proteins). A number of studies carried out in different tumour types demonstrated that malignant cells frequently present a greater AgNOR protein amount than corresponding non-malignant cells. Moreover, in cancer tissues AgNOR protein expression was found to be strictly related to the cell duplication rate. Over the past 12 years, the “AgNOR method” has been applied in tumour pathology for both diagnostic and prognostic purposes. However, the lack of a standardised silver-staining protocol has led to much misinterpretation of actual structures evaluated in individual studies. Indeed, the absolute AgNOR scores reported by different authors for the same types of tumour are scarcely comparable and the results produced by these investigations sometimes seem to be conflicting. In order to achieve definitive standardisation of the AgNOR method and produce comparable data in all laboratories, the “International Committee on AgNOR Quantitation” was founded, and during the first Workshop “AgNORs in Oncology” held in Berlin in 1993 guidelines for AgNOR protein evaluation were first defined. The present paper discusses the main technical aspects of NOR silver-staining, and critically evaluates the methods commonly employed for AgNOR protein quantification in routine cyto-histopathology.

In situ standardised AgNOR analysis: a simplified method for routine use to determine prognosis and chemotherapy efficiency in colorectal adenocarcinoma

Standardised AgNOR analysis as recommended by the Committee on AgNOR Quantitation of the European Society of Pathology is now regarded as the gold standard, particularly when retrospective studies on archival, formalin-fixed, and paraffin-embedded material are performed. This article deals with the implementation of the standardised AgNOR method in colorectal carcinoma and its consequent impact on clinical decision making. First, the clinical relevance of AgNOR analysis as a major and independent prognostic factor in exclusively surgically treated adenocarcinomas of the colon and rectum is elucidated. Additionally, due to the fact that in last 10 years 5-Fluorouracil based chemotherapy and radiotherapy have become standard treatment in a variety of tumour stages, the outstanding predictive power of AgNOR analysis with regard to chemotherapy efficiency is emphasised.

Polymorphism of the nucleolus organizer regions (NORs) in Physalaemus petersi (Amphibia, Anura, Leptodactylidae) detected by silver staining and fluorescence in situ hybridization.

The nucleolus organizer regions (NORs) of both karyotypes I and II of Physalaemus petersi (Jiménez de la Espada, 1872) from the Brazilian Amazon were studied by Giemsa staining, and by the Ag-NOR method. Karyological group I specimens were also studied by the fluorescence in situ hybridization (FISH) technique. Multiple NOR-bearing chromosomes were detected in both karyotypes. The coincident results of the Ag-NOR and FISH methods rule out the occurrence of silent NORs in this anuran. There was no intraindividual NOR variability in either group, but interindividual variability of NORs was high in group I. Seven different patterns of active NOR distribution were definitely recognized among fifteen specimens. This was considered to be a NOR site polymorphism. These results, combined with the C-band polymorphism previously reported for P. petersi, demonstrate a high rate of chromosome evolution in this group.

1-33-06 High proliferation rate in dystrophinopaties: A histochemical study by AgNOR method

1-33-06 High proliferation rate in dystrophinopaties: A histochemical study by AgNOR method

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.

Characterization of the proliferation state in canine mammary tumors by the standardized AgNOR method with postfixation and immunohistologic detection of Ki-67 and PCNA.

Paraffin-embedded tissue sections of 96 surgically removed mammary tumors from female dogs were analyzed for their proliferation state using three different methods. The AgNOR method, originally developed by Ploton and coworkers in 1986, modified and standardized by the AgNOR committee, is an easy, inexpensive silver-staining procedure used to determine cell proliferation and prognosis of various tumors. Due to the standardized staining protocol of the AgNOR method and a postfixation step, results obtained were of excellent quality for image-analysis processing. The growth fraction was evaluated by counting of immunohistologically positive-stained cells for Ki-67 or proliferating-cell nuclear antigen (PCNA). The values determined were, in general, lower with Ki-67 (MIB1) than with PCNA (PC10). Nevertheless, the labeling indices of these antigens correlated significantly (P < 0.001). Though the differences of the means between the tumor groups according to the classification system of the World Health Organization were significant for all three investigated methods (P < 0.001), there was a considerable overlap between the tumor groups concerning all investigated parameters. An exploratory data analysis (multivariate analysis) as performed to evaluate the prognostic relevance of the three methods including further anamnestic, clinical, gross, and histopathologic variables. Besides the histopathologic diagnosis (survival P < 0.001; survival time P < 0.05; reappearance of tumor growth P < 0.05), only the PCNA-labeling index (time until reappearance of tumor growth P < 0.001) was of prognostic significance.

The AgNOR count in ulcerative colitis with and without dysplasia

The numbers of specific intranuclear AgNOR silver precipitates (AgNORs) and their relation to the Inflammatory Bowel Disease-Dysplasia Morphology Study Group's classification were studied in 134 colonscopic biopsy specimens of ulcerative colitis (UC) with and without dysplastic findings from 73 patients with active and inactive disease. In addition, 40 cases of colonic adenocarcinomas and 22 normal colonic mucosae were also assessed for comparison with the one-step silver staining AgNOR technique. Statistical comparison of the mean AgNOR number revealed a highly significant inter-class difference (P < 0.001) between UC without dysplasia and UC with dysplasia as well as between active and inactive UC. On the contrary, the AgNORs count did not separate colonic adenocarcinomas from UC with dysplasia either high or low grade nor these three classes from the group of UC with indefinite dysplasia. The quantitative evaluation of epithelial changes occurring in UC by the AgNOR method may help to establish the clinical management of colitis patients, in addition to qualitative morphological criteria.

Karyotype differentiation between two species of carangid fishes, genusTrachurus (Perciformes: Carangidae)

A karyological study ofTrachurus trachurus andT. mediterraneus (Perciformes: Carangidae) was conducted by standard, fluorochrome staining (CMA3, mithramycin, quinacrine mustard, DAPI), C-, Ag-NOR, and Alu-I banding methods. The karyotypes of both species consisted of 2n = 48 chromosomes, but of different FN:T. trachurus possessed a chromosome complement of 2 metacentric and 46 acrocentric elements, fundamental number (FN) = 50 andT. mediterraneus, a chromosome complement of 4 metacentric, 4 submetacentric, 14 subtelocentric and 26 acrocentric chromosomes, FN = 70. In neither of the two taxa investigated were heteromorphic sex chromosomes observed. The nucleolar organizer region was interstitially located on the long arm of the Ist pair of chromosomes in both species, intermediate inT. mediterraneus and subterminal inT. trachurus. Constitutive heterochromatin was found in nearly all centromeric and telomeric regions inT. trachurus; inT. mediterraneus it formed less intense telomeric and centromeric bands and thin interstitial bands on eight chromosome pairs. In addition, the C-positive material reacted differently to the digestion with endonuclease Alu-l in the two species. The results are discussed and compared with karyological data known for other species of Carangidae.

AgNOR quantity in needle biopsy specimens of prostatic adenocarcinomas: correlation with proliferation state, Gleason score, clinical stage, and DNA content

Aims-To define the relation between the quantity of silver stained nucleolar organiser regions (AgNORs) and histological grade, clinical stage, DNA content, and MIB-1 immunostaining in needle biopsy specimens of prostatic adenocarcinomas.Methods-Histological grade was determined according to the Gleason system. AgNOR quantity, DNA content and MIB-1 immunostaining were evaluated by image cytometry on routine histological sections stained with silver, Feulgen reaction and MIB-1 antibody, respectively.Results-The mean AgNOR area increased with increasing Gleason score. A significant difference was found in the AgNOR values between low, intermediate and high grade tumours. Patients with clinically localised tumour (stages A and B) had lower AgNOR values than patients with advanced disease (stages C and D), but the difference in the mean AgNOR values between the two groups was not statistically significant. Non-diploid tumours had a significantly higher mean (SD) AgNOR area than diploid tumours (3.68 (1.04) mum(2)v 2.73 (0.60) mum(2), respectively), while no significant difference was observed in the mean AgNOR values between aneuploid and tetraploid tumours (3.68 (1.04) mum(2)v 3.70 (1.05) mum(2)). When AgNOR and MIB-1-PI values were compared using linear regression analysis, a highly significant correlation was found.Conclusions-These data demonstrate that AgNOR quantity reflects the proliferative potential of prostatic adenocarcinomas, and is significantly related to histological grade and DNA content. The ease of application on routine sections, maintaining the morphological integrity of the tissue, the ability to evaluate selected histological areas of limited size and objective quantification by image cytometry make the AgNOR method particularly suitable for cell kinetic analysis in prostatic needle biopsy specimens.

A sensitive staining method for NORs.

A sensitive staining method for nucleolar organizer regions (NORs) is described, using blue toning of AgNORs. NORs are loops of DNA which are transcribed into ribosomal RNA. NORs can be demonstrated by staining with silver nitrate, since NOR-associated proteins are argyrophilic, producing structures termed AgNORs. Normal blood lymphocytes were stained with both methods. The number and resolution of NORs increased 2-3 times by blue toning (30 mmol/l FeCl3, 11 mmol/l potassium hexacyanoferrate(III), and 33 mmol/l oxalic acid) compared with silver staining. A significant difference in the number of NORs was noticed between silver-stained and blue-toned cells (P < 0.001). The blue toning technique thus appears to be more sensitive in detecting NORs than the AgNOR method and may prove a useful alternative for applications in histopathology.

Determination of epidermal proliferative activity in experimental mouse tail test by AgNOR analysis

Nucleolar organizer regions are segments of DNA coding ribosomal genes, which can be histologically detected by silver technique as so-called AgNORs. The estimation of AgNOR number and AgNOR size are currently under investigation as markers of cellular proliferation activity. We therefore examined the epidermal AgNOR expression after topical application of different antiproliferative compounds using the mouse tail test. The epidermis of 0.025% fluocinolone acetonide-treated mouse tails had the lowest AgNOR expression. Pretreatment with 0.2% Anthralin, 1% propyl gallate and 2% 3,4-hexaalkylbenzoylacrylic acid, an experimental phospholipase-A2 inhibitor, also revealed significant inhibition effects of epidermal AgNOR expression. Native and petrolatum-treated epidermis as control showed the highest AgNOR expression. The AgNOR results in basal cells proved to be more informative than these in the stratum spinosum, the best parameter was the AgNOR number. The obtained results were closely related to the values of the corresponding studies of PCNA expression. The AgNOR method seems to be useful for estimation of antiproliferative efficiency of pharmacological substances. This technique is simple in handling and can be applied using paraffin-embedded tissue sections.

PCNA expression and nucleolar organizer regions in malignant melanoma and nevus cell nevus.

The histogenesis of malignant melanoma with particular reference to the role of melanocytic nevus is still controversial in oncological pathology. In order to differentiate between the proliferative activity of malignant melanomas and benign melanocytic tumors, immunohistochemical analysis was carried out. We used monoclonal antibody PC10 to identify the Proliferating Cell Nuclear Antigen (PCNA), a highly conserved 36 KD acidic nuclear protein which correlates with cell proliferation, and the AgNOR method to stain the Nucleolar Organizer Regions (NORs) whose number and configurations may reflect protein synthesis activity. 47 cases of primary melanoma, 63 metastatic melanoma lesions, and 23 cases of nevi are included in this study. Spitz nevi showed a higher index of cell proliferation compared with other common benign nevi but a much lower index compared with malignant melanoma cells. The metastatic lesions of malignant melanomas had a higher index of proliferation and activity of cells than the primary lesions. Skin-metastatic lesions had higher indexes of cell proliferation and NOR activity than lymph node-metastatic lesions. These results support the idea that metastatic melanoma cells are derived from a more advanced stage of tumor progression. These data suggest that the combination of PCNA and AgNOR is an useful marker for differentiating benign melanocytic tumors from malignant melanomas.

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.

Demonstration of nucleolar organizer regions in lung carcinoma by silver staining

Nucleolar organizer regions (NORs) were investigated in lung carcinomas by silver staining. This method was applied to 111 lung carcinoma specimens, including 40 with squamous cell carcinoma (SCC), 42 with adenocarcinoma (ADENO), 8 with adenosquamous carcinoma (ADESQ), 8 with small cell carcinoma (SMCC), 6 with large cell carcinoma (LGCC), and 7 with typical carcinoid tumors (CAOID). The mean AgNOR counts of ADENO, SCC, ADESQ, SMCC, and LGCC were significantly higher than those of the normal bronchial surface and those of the glandular or alveolar epithelium. The mean AgNOR count of CAOID was significantly higher than those of the normal glandular and alveolar epithelium but not that of the surface epithelium. The mean AgNOR count of SCC was significantly higher than that of bronchial squamous metaplasia, and the count of SMCC was significantly higher than that of CAOID. Within the same cancer category, the mean number of AgNORs increased in parallel with the histological tumor grades. These results indicate that the AgNOR method is useful for differentiating lung carcinoma from its normal counterparts and for evaluating histological tumor grades in the same lineage of lung carcinoma.

Morphometric analysis of nucleolar organizer regions in benign and malignant peritoneal effusions using backscattered electron microscopy.

Nucleolar organizer regions are loops of DNA associated with silver-stainable proteins (AgNORs). In general, malignant cells have more and larger AgNORs than benign cells. An inconsistent argyrophilic method and difficulties in objectively evaluating AgNORs account for some of the reluctance to utilize AgNOR staining as a diagnostic tool to differentiate benign and malignant lesions. Sections from paraffin-embedded cell blocks of 10 cases of benign and malignant peritoneal effusions were stained with a modified AgNOR method. Backscattered electron imaging in the scanning electron microscope, together with image analysis, was used to evaluate more objectively a number of AgNOR parameters and to determine which measurement was the most reliable discriminant of the two types of fluids. One hundred nuclei per case were identified and imaged. In contrast to benign nuclei, AgNORs in malignant nuclei were more numerous (P less than 0.0001) and larger (P less than 0.0001). A cut-off mean AgNOR area of 1.1 microns 2 (P less than 0.0001) correctly categorized all malignant (greater than 1.1 microns 2) and benign (less than or equal to 1.1 micron 2) cases. This system's objectivity and specificity could be used to enhance the cytological interpretation of effusions, where the separation of reactive mesothelial cells and malignant cells is extremely difficult.

AgNOR (Nucleolar Organizer Regions) Staining in Malignant Melanoma

Nucleolar organizer regions (NORs) are loops of ribosomal DNA seen in nuclei, which are demonstrable as black dots (AgNOR) in tissue sections by silver (Ag) colloid staining. The number of such AgNORs is correlated with cellular activity and is an indicator of the degree of malignancy. In this study, 76 melanocytic lesions were analyzed by AgNOR staining, and the clinical and histopathological characteristics of malignant melanoma and melanocytic nevi were considered. Although the AgNOR counts for melanocytic nevi were significantly different from those in malignant melanoma, an obvious overlap between them was detected. The number of AgNORs in melanocytic nevi per cell was usually 1 or 2. On the other hand, the number of AgNORs per malignant melanoma cell was variable. Morphologically, malignant melanoma cells often showed dispersal of AgNORs throughout the nucleus as well as multiple nucleoli containing clustered AgNORs, whereas melanocytic nevus cells tended to have a regular nucleolus with tightly clustered AgNORs. The correlation between AgNOR count and pathological staging was uncertain, but a slight correlation between AgNOR count and thickness of the primary lesion was obtained. However, the AgNOR count in malignant melanoma was not a prognostic factor for the disease. Therefore, the AgNOR method is difficult to use for differential diagnosis between benign pigmented lesions and malignant melanoma. Nonetheless, an AgNOR count of more than two per cell favors a diagnosis of malignant melanoma.