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A quantitative analysis combined with Ag staining was carried out to study the size variation of the main nucleolar organizer region (NOR) bearing chromosome pair 11 of Salmo trutta. A standardized NOR size measurement was developed by comparing the length of the short arm (NOR-bearing region) to the total chromosome length. Statistical procedures support arguments for the existence of a large and structural polymorphism within this species for this chromosome region. A minimum of five different chromosome classes were identified, which account for the total variation found. Size variation among classes was due both to changes in the number of NOR clusters as well as to the amount of rDNA genes within each cluster. NOR size values were normally distributed in the sample analyzed.Key words: nucleolar organizer region, size polymorphism, brown trout.

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 (P0.007 and P0.0005, 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.

Summary The number of silver-stained nucleolar organizer regions (Ag-NORs) was counted in stromal myofibroblasts in 125 cases of breast cancer. The mean (S.E.) of Ag-NORs in stromal myofibroblasts was 4.4 (0.1). The number of Ag-NORs in myofibroblasts was related to histological grade (p = 0.028), histological type (p = 0.013), tumour necrosis (p = 0.026), mitotic frequency (p = 0.091) and S-phase fraction (p = 0.032). The number of Ag-NORs in stromal myofibroblasts predicted survival with a borderline significance (x2 = 3.4, p = 0.052) and also in axillary lymph node negative tumours (X2 = 2.8, p = 0.095). The recurrence-free survival in the entire cohort (X2 = 7.1, p = 0.0075) and in axillary lymph node negative tumours (X2 = 7.1, p = 0.0078) could be predicted on the basis of the number of Ag-NORs in stromal myofibroblasts. In axillary lymph node positive tumours the number of Ag-NORs in stromal myofibroblasts had no prognostic value. In multivariate analysis the number of Ag-NORs in stromal myofibroblasts had no independent prognostic value. The results suggest a close interaction between stromal myofibroblasts and epithelial cancer cells in breast cancer, and stromal changes are clearly a subject for further analyses.

We present a case of desmoplastic cerebral astrocytoma of infancy (DCAI) in a 7 1/2-month-old girl and include immunohistochemical, ultrastructural and proliferative activity studies. The dural-based cystic tumor showed a biphasic pattern consisting of glial fibrillary acidic protein (GFAP)-positive astrocytes embedded in a desmoplastic stroma. The astrocytic processes were lined with basal lamina at their surface contacting the collagen. Scattered islands of undifferentiated small cells were seen acquiring GFAP positivity at their peripheral zone facing the collagen. Studies with silver nucleolar organizer region and proliferating cell nuclear antigen disclosed a high proliferative activity. Flow cytometric study showed an elevated S phase and 15% hypertetraploid cell population. These findings contrast the favorable prognosis of the tumor at 26 months follow-up. Probably, extracellular-matrix-induced maturation of the undifferentiated cells with the formation of basal lamina may account for this unique disparity.

Proliferating cell nuclear antigen (PCNA) expression in renal cell carcinoma (RCC) was determined by immunohistochemical staining using the PC10 clone. PCNA indices ranged from 2.4 to 53.1 per cent with mean indices of 12.6, 19.0, and 31.6 per cent for grades 1 to 3 RCC and 31.9 per cent for sarcomatoid RCC. There was a significant difference between the indices of grades 1 and 3 and grades 2 and 3 tumours and between grades 1 and 2 RCC and sarcomatoid RCC. AgNOR scores and mitotic indices were determined for each tumour and comparison of PCNA indices with mean AgNOR scores and mitotic indices showed only a weak correlation (PCNA/AgNOR r = 0.406, PCNA/mitotic index r = 0.315). Tumours were divided according to PCNA index (or = 18 per cent and18 per cent) and there was a significant difference in survival between the two groups, for all cases, and for each of the Robson stages. Multivariate analysis using Cox's proportional hazard model showed PCNA index, tumour stage, and mean AgNOR score to be independent predictors of survival, while tumour grade and mitotic index were found to be dependent variables.

Flow cytometric analysis (FCA) and silver colloidal nucleolar organizer region-associated protein staining (AgNOR) have been used individually in assessing the histopathologic nature of various human tumors. However, few researchers have investigated the relationship between the two techniques in a single series. In a retrospective study, we examined 36 premalignant lesions of the oral cavity by FCA and AgNOR on formalin-fixed, paraffin-embedded tissue submitted to the University of Tennessee, Memphis, oral pathology laboratory. Three categories of epithelial dysplasia were represented (9 mild. 9 moderate, 6 severe), as well as four epithelial hyperplasias without dysplasia, three squamous cell carcinomas, and five fibrous nodules as controls. Parameters recorded for each case included age. race, gender, site, light microscopic diagnosis (LMD). DNA index (DI), total proliferative index (TPI), S-phase (S), range of nucleolar organizer regions (RNOR), and mean number of nucleolar organizer regions (MNOR). The average maximum nucleolar organizer region count (AMXNOR) for each LMD category was also calculated. The objective of the study was to determine if FCA or AgNOR aided in the subjective LMD of oral premalignant lesions and if the parameters recorded for the specimens exhibited any positive correlation. The FCA results indicated an abnormal DI in 6 of the 24 dysplastic lesions. A positive partial correlation was seen between DI and MNOR (r = 0.434: P

Transitional cell carcinomas of the urinary bladder vary in their biologic potential, which may be correlated with the grade and stage of the tumor. Cellular proliferation may prove to be another measure of predicting tumor biologic potential. We have compared five different methods to assess proliferation in 26 tumors and correlated these results with tumor grade and stage. A portion of each tumor was incubated in vitro with bromodeoxyuridine (BrdUrd). For each tumor this was compared with at least three of the following four other markers of proliferation: mitotic count, silver-stained nucleolar organizer regions, immunohistochemical staining with Ki67, and proliferating cell nuclear antigen. Statistical correlations were seen between tumor grade and stage and these markers. There were strong correlations between the BrdUrd labeling index (LI) and both the Ki67 LI and proliferating cell nuclear antigen LI. The correlation between the BrdUrd LI and mitotic count was more tenuous; no significant correlation was found between BrdUrd LI and silver-stained nucleolar organizer region count. The correlation between these measurements of proliferation and tumor grade and stage was less strong. Our data suggest that cellular proliferation of transitional cell carcinomas can be reliably assessed with several different markers and that most of these markers can be correlated with tumor grade but not with stage.

We assessed the usefulness of silver staining of nucleolar organizer regions in the diagnosis of pigmented conjunctival tumors. Fifty-one biopsy specimens were silver stained to identify the nucleolar organizer regions. Nineteen nevi without atypia, three nevi with atypia, eight primary acquired melanosis lesions, and 14 melanomas were studied. In each specimen, silver staining of the nucleolar organizer regions was counted in 100 cells to yield an average of the silver staining of the nucleolar organizer region count. The mean silver staining of the nucleolar organizer region counts per cell was correlated with the degree of malignancy of pigmented conjunctival lesions as follows: nevi, 3.0; primary acquired melanosis, 3.2; nevi with atypia, 3.9; primary acquired melanosis with atypia, 5.0; and melanoma, 5.7 (Spearman correlation [rS] = .83, P = .0001; analysis of variance [ANOVA] F test = 20.9, P = .0001). A cutoff value of 4.0 (mean silver staining of nucleolar organizer regions per cell) will differentiate melanoma and primary acquired melanosis with atypia from other lesions (sensitivity, 100%; specificity, 96%). The silver staining of nucleolar organizer regions is a useful adjunct in determining the malignancy of pigmented conjunctival tumors.

Proliferative potential of four cases of central neurocytoma were assessed by a silver colloid staining technique for argyrophilic protein associated with nucleolar organizer regions (AgNORs). All but one tumor had an Ag-NOR score of less than 2 and were regarded as a benign entity. However, one tumor tissue from a patient suffering from a recurrent central neurocytoma after 13 years from the first operation possessed a 2.06 AgNOR score, which is not significantly different from others. It is considered that central neurocytoma is an essentially benign neoplasm, even though the tumor is recurrent. The present study revealed the usefulness of AgNOR staining for a retrospective assessment of the proliferative potential of central neurocytomas.

Using cytogenetic analysis following Giemsa staining, nucleolar organizer region (NOR) staining, and C-banding, three distinct karyotypes in three species of curimatids belonging to the fish genus Potamorhina were identified: 2n = 54/44 M + 10 SM (P. pristigaster), 2n = 56/52 M + 2 SM + 2 ST (P. latior), and 2n = 102/2 M + 2 SM + 98 A (P. altamazonica). A 2n = 54 was considered to be the ancestral diploid number and the different karyotypes were probably the result of centric fissions. Both the NOR pattern and constitutive heterochromatin pattern are species specific.Key words: centric fissions, karyotypic evolution, Amazon fish, Curimatidae.

The three-dimensional (3-D) organization of rDNA-containing chromatin and the set of protein markers of active ribosomal genes, the Ag-NOR proteins, were investigated by confocal laser scanning microscopy (CLSM). The rDNA genes of marsupial cells (PtK1) were mapped using biotinylated DNA probes for 45S rDNA sequences and the Ag-NOR protein distribution was revealed by specific Ag-NOR staining. We used PtK1 cells because each nucleolus possesses only one nucleolar organizer region (NOR). In metaphase chromosomes, nonisotopic in situ hybridization demonstrated the presence of rDNA in the secondary constriction of the X chromosomes with an axial distribution and also lateral expansions. 3-D reconstruction of the Ag-NOR protein signals revealed the presence of these proteins in the secondary constriction where they formed a crescent-shaped structure around the axial chromatin pedicule. The organization of the secondary constriction in PtK1 chromosomes is discussed. During interphase, nonisotopic in situ hybridization in intact cell monolayers and isolated nuclei showed the rDNA genes distributed as intense fluorescent spots linked by weak signals in the inner regions of the nucleoli. We conclude that the rDNA is not homogeneously distributed in the internal regions of the nucleoli. In the same nucleolar regions, the Ag-NOR proteins were revealed as granules linked by thin filaments. These images indicate similar 3-D distributions for rDNA probes and Ag-NOR proteins. The beaded organization of the transcriptional regions in the nucleoli is discussed.

Bromodeoxyuridine (BrdU) is a thymidine analog that is incorporated into cellular DNA in the synthetic phase. The BrdU-labeling index (BLI) thus reflects the S-phase fraction. The argyrophilic nucleolar organizer regions (AgNORs) are silver-stained granules that have been correlated with ploidy and/or S-phase fraction. Two AgNOR counting methods have been proposed to distinguish between ploidy and S-phase fraction: the mean AgNOR (mAgNOR) count (the mean number of AgNOR granules in 100 cells), which is believed to reflect ploidy, and the AgNOR proliferative index (pAgNOR) (the percentage of cells exhibiting five or more AgNOR granules per nucleus), which is believed to reflect S-phase fraction. To evaluate the latter hypothesis we studied 19 tumors metastatic to the brain in patients who had received preoperative low-dose BrdU injections. Formalin-fixed, paraffin-embedded sections of the resected tumors were stained using the indirect immunoperoxidase technique and a monoclonal antibody to BrdU. The BLI was determined by counting 1,000 tumor cells in each case; AgNOR silver staining was performed on sections of the same blocks studied for BLI. The correlation between pAgNOR counts and BLI was statistically significant (r = .649, P = .003). No statistically significant correlation could be obtained between mAgNOR counts and BLI (r = .421, P = .17). We conclude that the pAgNOR count can reliably reflect the proliferative activity of cells. The method can prove beneficial in situations in which cell kinetic analysis is needed and in which invasive procedures, such as BrdU injection, are not feasible or are contraindicated.

A silver-staining technique to identify nucleolar organizer region-associated proteins (Ag-NOR) was applied to 55 gastric myogenic tumors. The mean numbers of Ag-NOR in the nucleus were: leiomyoma (30 cases), 2.0 +/- 0.6 (mean +/- SD); low-grade leiomyosarcoma (11 cases), 3.0 +/- 0.7; and high-grade leiomyosarcoma (14 cases), 3.9 +/- 0.7. The Ag-NOR counts were compared with DNA ploidy as determined by flow cytometry in 46 tumors. The Ag-NOR counts were significantly different in the aneuploid leiomyosarcomas (4.1 +/- 0.6: mean +/- SD) and diploid leiomyosarcomas (2.9 +/- 0.6: mean +/- SD). Thus, this parameter may serve as an objective histological discriminant for malignancy.

The histological features of adenoid cystic carcinoma are varied. In general, the tumors are classified into 4 histological patterns: tubular, cribriform, trabecular and solid. Numerous previous reports have indicated that the tubular pattern usually represents a favorable prognosis, the solid pattern a poor prognosis and the cribriform pattern an intermediate prognosis. Therefore, the present study was undertaken to determine precisely the proliferative potential of each histological pattern of adenoid cystic carcinomas. A silver colloid technique to identify nucleolar organizer region associated protein (AgNORs) was applied to paraffin sections in a total of 16 adenoid cystic carcinomas. A morphometric analysis of highly magnified photographic images of AgNORs in light microscopic preparations was performed. Of the 16 tumors, 8 showed a mixture of different histological patterns in the same section. In comparing the AgNOR number among different histological patterns in the same section, the value was highest for the solid pattern, lowest for the cribriform pattern and intermediate for the trabecular pattern. Moreover, the mean AgNOR number also showed a stepwise increase from the cribriform pattern (2.3 +/- 0.3) through the trabecular pattern (2.9 +/- 0.2) to the solid pattern (3.3 +/- 0.6). There was a significant difference in AgNOR numbers between cribriform and trabecular and between cribriform and solid patterns. Our results indicate that the proliferative potential of histological patterns of adenoid cystic carcinoma is lowest in the cribriform, highest in the solid, and intermediate in the trabecular pattern area. The AgNOR staining technique appears to be of value in estimating the proliferative activity of adenoid cystic carcinomas.

Histochemical analyses with Ki-67 and anti-DNA polymerase alpha monoclonal antibodies were done, and argyrophilic nucleolar organizer regions (Ag-NOR) were counted to estimate the proliferative potential of 200 brain tumors. The findings were compared with the bromodeoxyuridine labeling index (BrdU LI), or S-phase fraction. The proliferating cell indexes (PCI), determined by Ki-67 and anti-DNA polymerase alpha monoclonal antibodies were higher and Ag-NOR more numerous in medulloblastomas, glioblastomas, and metastatic carcinomas than in astrocytomas or meningiomas. The Ki-67 and DNA polymerase alpha PCI correlated with the BrdU LI (r = 0.87 and r = 0.84, respectively) and with each other (r = 0.94). The number of Ag-NOR correlated less strongly with these indexes in some tumors. These findings suggest that Ki-67 and anti-DNA polymerase alpha monoclonal antibodies may be useful for estimating the proliferative potential of individual tumors in routine clinical practice. The number of Ag-NOR, however, does not always reflect the growth potential of each tumor. Cancer 1993; 71:199-206.

Five specimens of Rhamdia quelen collected from the Lindóia Stream, PR, Brazil, were cytogenetically analyzed. The diploid chromosome number found was 58, including 30 metacentric, 16 submetacentric, 10 subtelocentric, and 2 acrocentric chromosomes. Supernumerary or B chromosomes, frequently observed in this fish group, were not detected. One of the individuals was triploid, with 3n = 87. A silver-stained nucleolar organizer region was found on a pair of submetacentric chromosomes of the diploid specimens, and on three chromosomes of the triploid individual, confirming triploidy. Treatment with fluorochrome chromomycin A(3) revealed fluorescent bands coincident with those of the silver-stained nucleolar organizer region, in both diploid and triploid individuals, showing that this is a GC-rich region. Heterochromatin distribution was visualized by the C-banding technique, mainly in the terminal chromosome regions of the individuals and was also observed in the pericentromeric regions of some chromosomes and at both telomeres.

Made available in DSpace on 2013-08-28T14:11:32Z (GMT). No. of bitstreams: 1 WOS000279672400001.pdf: 785188 bytes, checksum: 0c5db4db1c8ff870d918d8baa4e1397f (MD5) Made available in DSpace on 2013-09-30T18:46:58Z (GMT). No. of bitstreams: 2 WOS000279672400001.pdf: 785188 bytes, checksum: 0c5db4db1c8ff870d918d8baa4e1397f (MD5) WOS000279672400001.pdf.txt: 38740 bytes, checksum: 3a795b7b63bb8e9676c7c00041c6cb52 (MD5) Previous issue date: 2010-07-02 Submitted by Vitor Silverio Rodrigues (vitorsrodrigues@reitoria.unesp.br) on 2014-05-20T13:55:04Z No. of bitstreams: 2 WOS000279672400001.pdf: 785188 bytes, checksum: 0c5db4db1c8ff870d918d8baa4e1397f (MD5) WOS000279672400001.pdf.txt: 38740 bytes, checksum: 3a795b7b63bb8e9676c7c00041c6cb52 (MD5) Made available in DSpace on 2014-05-20T13:55:04Z (GMT). No. of bitstreams: 2 WOS000279672400001.pdf: 785188 bytes, checksum: 0c5db4db1c8ff870d918d8baa4e1397f (MD5) WOS000279672400001.pdf.txt: 38740 bytes, checksum: 3a795b7b63bb8e9676c7c00041c6cb52 (MD5) Previous issue date: 2010-07-02 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Fundação para o Desenvolvimento da UNESP (FUNDUNESP) Within the Orthopteran species, those of the suborder Ensifera have been rarely studied from the cytogenetic point of view, mainly due to the difficulties for taxonomic identification of its species. The Gryllidae is the second largest family of this suborder and possesses some genera, such as Anurogryllus, that occur only on the American continents. The aim of this work was to determine the karyotype characteristics, the meiotic chromosome behaviour, and the nucleolar organizer region (NOR) pattern of Anurogryllus sp (Orthoptera: Gryllidae). In the analyzed sample, high levels of numerical, morphological, and NORs polymorphisms were detected. Within five distinct karyotypes that were found, the basic karyotype of Anurogryllus sp. showed 2n(male) = 22 + X0 with acrocentric autosomes and a metacentric X sex chromosome; furthermore, a conspicuous secondary constriction related to the NOR was present along the entire short arm on pair 5. The other four types of karyotypes arose from centric fusions between elements of pairs 1/3, 2/6, 4/7 and a NOR partial translocation from pair 5 onto the long arm terminal region of one element of the fused pair 2/6. Such intraspecific variability and the consequences of high levels of polymorphism are discussed, leading to conjectures about the mechanisms that led to these chromosome rearrangements. Univ Fed São Paulo, Dept Ciencias Biol, São Paulo, Brazil Univ Fed Vicosa, Dept Biol Geral, Vicosa, MG, Brazil Univ Estadual Paulista, Dept Biol, Inst Biociencias, São Paulo, Brazil Univ Estadual Paulista, Dept Biol, Inst Biociencias, São Paulo, Brazil FAPESP: 06/53275-3

Karyotype and chromosomal characteristics, i.e. number and location of ribosomal DNA (rDNA) clusters, and sequence variation of the ribosomal internal transcribed spacer 2 (ITS2) were studied in a monozoic (unsegmented) tapeworm, Caryophyllaeides fennica (Caryophyllidea), using conventional and Ag-staining, fluorescent in situ hybridization (FISH) with 18S rDNA probe, and PCR amplification, cloning and sequencing of the complete ribosomal ITS2 spacer. The karyotype of this species was composed of ten pairs of metacentric (m) chromosomes (2n = 20). All chromosomes except the pair No. 2 displayed DAPI-positive heterochromatin in centromeric regions. In addition, two distinct interstitial DAPI-positive bands were identified on chromosome pair No. 7. FISH with 18S rDNA probe revealed four clusters of major ribosomal genes situated in the pericentromeric region of the short arms in two pairs of metacentric chromosomes Nos. 8 and 9. Hybridization signals were stronger in the pair No. 8, indicating a higher amount of rDNA repeats at this nucleolar organizer region (NOR). Analysis of 15 ITS2 rDNA sequences (five recombinant clones from each of three individuals) showed 13 structurally different ribotypes, distinguished by 26 nucleotide substitutions and variable numbers and combinations of short repetitive motifs that allowed sorting the sequences into four ITS2 variants. These results contribute to recently published evidence for the intraindividual ribosomal ITS sequence variability in basal tapeworms with multiple rDNA loci and imply that both phenomena may be mutually linked.

International audience; The organization of chromosomes is important for various biological processes and is involved in the formation of rearrangements often observed in cancer. In mammals, chromosomes are organized in territories that are radially positioned in the nucleus. However, it remains unclear whether chromosomes are organized relative to each other. Here, we examine the nuclear arrangement of 10 chromosomes in human epithelial cancer cells by three-dimensional FISH analysis. We show that their radial position correlates with the ratio of their gene density to chromosome size. We also observe that inter-homologue distances are generally larger than inter-heterologue distances. Using numerical simulations taking radial position constraints into account, we demonstrate that, for some chromosomes, radial position is enough to justify the inter-homologue distance, whereas for others additional constraints are involved. Among these constraints, we propose that nucleolar organizer regions participate in the internal positioning of the acrocentric chromosome HSA21, possibly through interactions with nucleoli. Maintaining distance between homologous chromosomes in human cells could participate in regulating genome stability and gene expression, both mechanisms that are key players in tumorigenesis.

Karyotype analysis of selected species from arachnid orders Amblypygi and Uropygi Whip spiders (Amblypygi) and whip scorpions (Uropygi) represent relict arachnid orders which has been found already at Upper Carboniferous strata. Although cytogenetic data from amblypygids and uropygids might be important to reconstruct karyotype evolution of arachnids, cytogenetics of these orders is almost unknown. Presented study is aimed in analysis of karyotype and meiosis in 16 species of Amblypygi and 4 species of Uropygi. Both groups are characterized by considerable range of diploid chromosome numbers (2n = 24 - 86 in Amblypygi and 36 - 66 in Uropygi). Analysed species does not exhibit morfologically differentiated sex chromosomes. Differentiation of sex chromosomes on molecular level was revealed in amblypygid Paraphrynus mexicanus by comparative genome hybridization. Obtained data indicate XY/XX sex chromosome system in this species. Comparison of karyotype data indicates reduction of chromosome numbers during evolution of both orders. In Amblypygi, this reduction was accompanied by increase of number of biarmed chromosomes. This trend is not apparent in Uropygi. Karyotypes of most analysed amblypygids and uropygids are also characterized by low amount of heterochromatin. Most studied species exhibit two pairs...

The histologic distinction between craniopharyngioma and epidermoid is equivocal. Eight craniopharyngiomas and ten epidermoids (including three dermoids) as well as nine rat (Wistar) abdominal skins that acted as control were investigated by a silver colloid method for nucleolar organizer regions (Ag-NORs). The mean number of Ag-NORs in at least 200 cells of both basal (columnar) epithelial cells and suprabasal (prickle) cells was evaluated. It was found that the mean number of Ag-NORs in basal cells of epidermoid and rat skin was significantly greater than that in suprabasal cells (P

The prognostic significance of argyrophilic nucleolar organizer regions (AgNORs) has been evaluated in bone marrow trephine biopsies from 64 patients with multiple myeloma (MM) prior to therapy. The univariate Kaplan-Meyer survival analysis showed a significant correlation between survivals and AgNOR counts (median of survival 51.3 months for cases with 4.62 AgNORs per plasma cell (PC) versus 16 months for cases with >4.62 AgNORs per PC; P=0.0000) or AgNOR distribution in PC nucleus (AgNOR configuration) (median of survival 71.67 months for cases with tightly grouped AgNORs. 16.26 for partially grouped and 11.74 for dispersed AgNORs; P=0.001). Significant prognostic correlations were also found for monoclonal immunoglobulin type (P=0.008), platelet counts (P=0.0078). serum creatinine level (P=0.0001), Durie's clinical stage (P=0.02). percentage of plasma cells in bone marrow biopsies (BMPCX) (P= 0.005), pattern of medullary involvement (P=0.003) and PC atypia (P= 0.009). Borderline result was detected for the percentage of PCs in aspirates (P=0.06). No significant correlation was found between prognosis and patients age, sex, haemoglobin level, serum albumin or calcium level, marrow cellularity and excess of haemosiderin. Multivariate survival analysis showed that only two variables were significantly correlated with prognosis: AgNOR counts (P=0.003) and AgNOR configuration (P

Silver-stained nucleolar organizer regions (AgNORs) in human breast carcinoma were studied using a computer-assisted system of image analysis. Standardized, automatic measurements of 7 morphometric parameters (area, perimeter, shape factor, bend energy, angle, and small and large diameters) performed on paraffin sections and cell imprint were compared and correlated with nuclear morphometry, histopathological grading, tumor growth fraction, (monoclonal Ki67-immunostaining), DNA nuclear content (stoechiometric Feulgen staining) and axillary lymph node invasion. The major findings were as follows: (i) variations in AgNORs and nuclear parameters were correlated, (ii) the ratio of AgNOR area/nuclear area was significantly different in low and high grade tumors, (iii) mean AgNOR parameter values increased significantly with the tumor growth fraction and tumor hyperploidy and were significantly higher in patients with axillary lymph node metastases and (iv) AgNOR evaluation was more accurate for cell preparations than for tissue sections.

To study megakaryocyte activation, the argyrophilic staining method of nucleolar organizer regions (AgNOR) has been applied to decalcified bone marrow biopsies of 16 individuals with no haematopoietic disorders and 59 patients with chronic myeloproliferative disease. Of the 59 patients, 18 had chronic myeloid leukaemia (CML), 21 chronic megakaryocytic granulocytic myelosis (CMGM), 13 polycythaemia vera (PV) and 7 essential thrombocythaemia (ET). The AgNOR number of megakaryocytes in CML was significantly lower, and in CMGM, PV and ET significantly higher than in healthy individuals. The high number and the clusters of fine-grained AgNORs of megakaryocytes in CMGM, PV and ET are suggestive of active, proliferating cells. The AgNOR number of megakaryocytes and the platelet counts of the patients did not show a convincing correlation. In CMGM, PV and ET the pyknotic, heterochromatinized megakaryocytes with narrow rims of cytoplasm called bare (nude) nuclei, possessed few, large AgNOR granules. The AgNOR staining of bare nuclei and the roughly identical number of granules found in CMGM, PV and ET indicate a common, active mechanism of apoptosis.

In this study, surgically excised mammary tumors from 98 bitches were graded histologically, and the grade was compared with the mean nucleolar organizer region (NOR) count in silver-stained paraffin-embedded sections. Histologically benign tumors, papillary adenocarcinomas, and intraductal carcinomas showed relatively little variation; the mean count for each category was between three and four NOR per nucleus. There was, however, a significant increase in the NOR counts in tubular and solid carcinomas. This increase was most pronounced for tumors that showed evidence of infiltration into the surrounding connective tissues. The mean NOR count for noninfiltrative carcinomas was 5.1, and that for invasive carcinomas was 7.3 ( P < 0.03). The mean NOR count for individual carcinomas ranged from 2.0 to 12.3, and a significant correlation was found between an increased NOR count and tumor-related death during the first post-surgical year. The 39 bitches in which the tumor had an NOR count less than 8.0 had a generally favorable prognosis; only six (15%) died as a result of the original neoplasm. In contrast, 18/21 dogs (85%) with a carcinoma having an NOR count greater than 8.0 died from the tumor during the first post-surgical year. A similar, although less pronounced result was obtained specifically for invasive carcinomas, in which 3/12 (25%) tumors with an NOR count less than 6.0 resulted in the death of the host, compared with 17/20 (85%) that had an NOR count greater than 6. By using this technique, it is possible to identify a subgroup of bitches with invasive mammary carcinomas that have a very poor prognosis following apparently adequate surgical ablation of the primary tumor.

The “conventional” histochemical and enzyme techniques that can be applied to the diagnosis of malignant lymphomas, including Hodgkin's disease, are reviewed. Their historical perspectives are outlined and their value for today is assessed. Many of these conventional methodologies are still useful diagnostic tools, although some, especially enzyme histochemistry, have been replaced by immunohistochemical and gene rearrangement techniques.The recently popularized argyrophil nucleolar organizer region (AgNOR) staining method and its potential usefulness to lymphoma cell kinetics and prognostication are also described, although there is still much work to be done to increase our understanding of this reaction and its place in lymphoma diagnosis and management. (The J Histotechnol 15:185-197, 1992)

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.

The argyrophil method for nucleolar organizer regions (AgNOR) known as the AgNor technique and mucin histochemical stain were applied to investigate the dysplasia and cancaration in ulcerative colitis including 58 cases of biopsy specimens and three cases operative specimens. The numbers of AgNOR have been gradully increased with the grades of dasplasis. Similarly the percentage of sialomucin having vicinal diols on side chain of sialic acid also gradully increased with the grades of dysplasia. The AgNOR reflects rDNA transcriptional activity responsible for degrees of differentiation of cell. Epithelial cells secreting a heterogeneous mucin, could be taken as a signal of abnormal cellular differentiation. AgNOR and mucin chages might be assumed as a criteria in representing malignant transformation.

Summary The number and the size of silver-stained intranuclear granules, which correspond to the nucleolus and nucleolar organizer regions, have been determined by means of quantitative methods in cultured primary human mesotbelial cells obtained from normal, reactive and malignant mesotbelium. The mean values per nucleus of the number, the total area, the average area, and the relative area of the silver-stained granules and the mean nuclear area were determined for each of the three conditions. Normal, reactive and malignant mesotbelial cells differed significantly in all the features. These findings at the optical level reflect the differing rate of the nucleolar biosynthetic activity related to the different biological properties of the three cell types, and the features can be useful morpbometric descriptors in the diagnostic pathology of the mesothellum.

The possible contribution of the silver colloid technique for staining nucleolar organizer regions in the distinction between benign and malignant adrenocortical neoplasms was investigated. Nine cases of adenoma, eight cases of carcinoma, nine cases of hyperplasia and four normal adrenal cortex specimens were examined. The mean silver binding nucleolar organizer region (Ag-NOR) value in adenoma was 4.29, and in carcinoma 7.16 (P less than 0.001). Adenomas with diameters greater than 3 cm had significantly higher Ag-NOR counts than smaller adenomas. For normal cortex, the mean Ag-NOR value was 2.05 and in hyperplasia, 3.62. The results indicate that the Ag-NOR technique can help in differential diagnosis between benign and malignant adrenocortical lesions and thus may have a prognostic value.

Whether borderline serous tumors of the ovary can be differentiated from invasive serous cystadenocarcinomas by morphometric analysis of nuclear features of the neoplastic epithelium was examined. Multiple descriptors extracted from nuclear tracings and intranuclear gray-level analysis of argyrophilic nucleolar organizer regions were evaluated in 11 borderline and 18 invasive tumors using computerized interactive morphometric analysis. These descriptors included: nuclear area and perimeter; number, area, and perimeter of argyrophilic nucleolar organizer regions; standard deviations of these findings; and a size distribution of nuclear areas in discrete classes. Multivariate statistical analysis showed the internal consistency of the two groups in terms of physical descriptors and the discriminating value of the parameters of nuclear size and pleomorphism (independently of nucleolar organizer region parameters). The results indicated that interactive morphometric analysis of nuclear features, combined with appropriate statistical methods, could be used to distinguish between these tumors.

Gastrointestinal stromal tumours are lesions in which it is difficult to predict clinical outcome from the histological appearances. Sixty cases were studied using three different methods of assessing aspects of cellular proliferation; these were (i) immunostaining for proliferating cell nuclear antigen (PCNA), (ii) interphase nucleolar organizer region staining (AgNORs), and (iii) a histological grading system based on mitotic counts. Both PCNA immunostaining and AgNOR counts were found to correlate well with histological grading and all three methods independently showed good correlations with survival. This suggests that these proliferation-associated markers may be used as additional features to support histological grading in this relatively uncommon group of tumours.

The frequency of nucleolar organizer regions (NORs) in each glioma tissue and the relation between the expression of glial fibrillary acidic protein (GFAP) and the frequency of NORs was investigated. The number of Ag-NORs per cell for glioblastoma multiforme was significantly higher than that for anaplastic astrocytoma (P less than 0.05) and that for astrocytoma (P less than 0.01). The number of Ag-NORs per cell for GFAP-positive cells was significantly lower than that for GFAP-negative cells in each histopathological grade (P less than 0.01). Moreover, the linear relationship was demonstrated between the Ag-NORs numbers of GFAP-negative cells and bromodeoxyuridine (BUdR) labeling indices. From these results, it is concluded that many GFAP-positive glioma cells may have low growth potential in glioma tissue and GFAP-negative cells may have a close relation to cell proliferation. The combination of immunohistochemical and silver colloid staining is a useful method for investigating the biological characteristics of brain tumors.

Summary Nonhistone nucleoproteins associated with the nucleolar organizer region (NOR) can be visualized by a silver-staining technique on paraffin-embedded tissues. The number of black dots (Ag NORs) appearing on the nuclei are thought to reflect cell differentiation of certain tumors and can be used as an adjunct in predicting their evolution. We applied this method to determine if Ag NORs counts could be used as a diagnostic aid in borderline tumors of the ovary. Thirty-two cases of adenocarcinomas, 25 cases of borderline tumors, and 14 cases of adenomas were selected from Bouin-fixed archival material after histological examination. Both mean values of Ag NORs counts demonstrated a progressive increase from adenomas to borderline tumors and to carcinomas; the differences were statistically significant. Using discriminant analysis, all cases of benign and malignant tumors except one adenoma and one carcinoma were discriminated as belonging to an individualized group. No difference was found between borderline tumor with peritoneal implants and those without peritoneal implants. The results indicate that the Ag NORs counting procedure may be useful in distinguishing borderline tumors from carcinomas and adenomas. Ag NORs counts cannot, however, predict the clinical behavior of borderline tumors of the ovary.

We present a patient with early bile duct carcinoma who underwent pancreatoduodenectomy for a mass forming type of chronic pancreatitis and describe the histologic and immunohistochemical findings of

The expression of two cell proliferation indices, the proliferating cell nuclear antigen (PCNA), using the monoclonal antibody PC-10 in the immunoperoxidase method, and the nucleolar organizer regions (NORs), using the colloid silver nitrate staining technique, was assessed in formalin-fixed paraffin-embedded material of 50 transitional cell urinary bladder carcinomas. A relationship was found between the histologic grade and each of the two indices used. A significant difference was observed, particularly between carcinomas of grade II and III (p < 0.001). A relationship was also demonstrated between each of PC-10 and AgNOR scores and the clinical stage, but it was attributed mostly to the close correlation of the latter with the histologic grade of these tumors. The linear correlation coefficient between PC-10 and AgNOR scores was 0.757 (p < 0.001). Our results suggest that PC-10 and AgNOR scores may be important prognostic indices in urinary bladder cancer.

A review of 78 renal tumors in patients under 6 months of age revealed 35 congenital mesoblastic nephromas (CMNs). Based on cellular criteria, 14 were classified as classical, 4 as partly cellular, and 17 as cellular CMN. The mean ages were 24, 11, and 70 days, respectively. There were 13 intrarenal tumors (stage I) but 9 classical, 3 partly cellular, and 5 cellular CMNs extended to the perirenal fat (stage II) and 5 cellular tumors ruptured (stage III). In order to assess cellular proliferative activity, silver staining of nucleolar organizer region (Ag-NOR) proteins was performed on 19 CMNs. The number of Ag-NOR dots per cell was significantly lower in classical and partly cellular CMN than in cellular CMN, whatever the stage (P.01). Within the cellular CMNs, the mean number of Ag-NOR dots was statistically higher in the single case that recurred with fatal outcome (P.02). Counting of Ag-NOR dots appears to be a reproducible method for evaluating the biologic potential of CMNs. The number of Ag-NOR dots, DNA content measurements, the histological subclassification, and the presence or absence of tumor at the surgical margins may be useful features in selecting those patients who will benefit from further treatment after nephrectomy.

Twenty-eight cases of prostatic adenocarcinomas have been selected and the mean argyrophil nucleolar organizer region (AgNOR) count for each case was evaluated. Nine of these tumors were of high, 13 of median and 6 of low differentiation. It was found that as the tumors became less differentiated, the AgNOR count increased (p less than 0.001). Stage of the disease, serum prostate-specific antigen (PSA) value, and DNA ploidy of all these cases have been examined as well but no statistically significant correlation among these three parameters and the AgNOR count was found.

Distinctions between benign and malignant tumors are less evident in the central nervous system than in other tissues. Since the level of cell proliferation is an important feature in tumor grading, we applied AgNOR in 50 cases of brain tumors with different grades and histological origins in order to check its efficiency in discriminating between benign and malignant cases. We found significant differences between the means of grade I (1.76) and grade IV (2.46) tumors. No significant differences were observed considering the same grading with distinct histological types or age of patients, reinforcing the efficiency of AgNOR.

J Oral Pathol Med (2010) 39: 223–229 Background: The aim of this study was to assess the expression, distribution and comparison of tenascin, a glycoprotein of the extracellular matrix in ameloblastoma and ameloblastic fibroma, both odontogenic neoplasms with diverse biological behavior and to understand the proliferative activity by using the morphometric analysis. Methods: Paraffin embedded tissue from 25 cases of odontogenic tumors i.e., ameloblastoma (n = 15) and ameloblastic fibroma (n = 10) were used. The expression of tenascin was evaluated using immunohistochemistry. Morphometric analysis of nucleolar organizer regions (NORs) from ameloblastoma and ameloblastic fibroma was carried out by silver staining. Results: A heterogeneous expression of tenascin was found in ameloblastoma which was mainly localized at the epithelial–mesenchymal interface and a patchy distribution was observed in the stroma (80%), while strong positivity was observed in the stroma and at the basement membrane zone of ameloblastic fibroma (100%). argyrophilic nucleolar organizer regions (AgNORs) revealed higher mean counts in ameloblastoma (3.093 ± 0.902) when compared with those of ameloblastic fibroma (1.553 ± 0.250). Ameloblastoma presented more than two NORs (two to five) per nucleus in majority of the cells, while ameloblastic fibroma exhibited only one NORs per nucleus. Conclusions: Expression of tenascin in these neoplasms suggest that it could play a role in epithelial- mesenchymal interaction, while AgNORs reveal that ameloblastomas are more aggressive when compared with ameloblastic fibromas.

Chromosomes of Corbicula fluminalis were characterized by karyotype analysis and nucleolar organizer region (NORs) localization. The triploid chromosome number was confirmed as 54; the karyotype is composed of 3 metacentric, 15 submetacentric and 36 subtelo-acrocentic chromosomes. Silver staining revealed nucleolar organizers on the telomeric regions of three subtelo-acrocentic chromosomes. This is the first study on chromosomes of C. fluminalis. The results are discussed with regards to Corbicula species as well as its relationships to other mollusc species based on cytogenetic characters and morphometric of the shells.

Hypertrophic scarring remains a major problem for patients who have suffered from surgeries or burns. Vascularization plays an important role in the early phase of hypertrophic scarring. Therefore, the inhibition of angiogenesis might be used as a preventive strategy. In this study, we assessed the effect of anti-angiogenesis resulting from adenovirus-mediated METH1 (metalloprotease and thrombospondin1) gene expression on the hypertrophic scar formation in a rabbit ear model of hypertrophic scarring. We first investigated the number of microvessel and microcirculatory perfusion in untreated scars on days 10, 30, 60, and 90 after epithelialization. Then, we examined the effect of anti-angiogenesis by adenovirus-mediated METH1 expression on hypertrophic scar formation by calculating the scar elevation index, counting the microvessel and argyrophilic nucleolar organizer region particle, and detecting the amount of collagen on days 30 and 60 after treatment. We found that untreated scar tissues at the proliferative phase (days 10-60 after epithelialization) had a significantly higher density of microvessel and microcirculatory perfusion than those at the mature phase (day 90 after epithelization) (both p

Background Cytogenetic studies were carried out on samples of Eigenmannia virescens (Sternopygidae, Gymnotiformes) obtained from four river systems of the Eastern Amazon region (Para, Brazil). Results All four populations had 2n = 38, with ZZ/ZW sex chromosomes (Z, acrocentric; W, submetacentric). Constitutive heterochromatin (CH) was found at the centromeric regions of all chromosomes. The W chromosome had a heterochromatic block in the proximal region of the short arm; this CH was positive for DAPI staining, indicating that it is rich in A-T base pairs. The nucleolar organizer region (NOR) was localized to the short arm of chromosome pair 15; this result was confirmed by fluorescent in situ hybridization (FISH) with human 45S rDNA, and CMA3 staining indicated that the region is G-C rich. FISH with telomeric probes did not show any evidence of interstitial telomeric sequences (ITS). Conclusion Previous studies have shown that the species Eigenmannia sp. 2 and E. virescens have differentiated sex chromosomes, and diverse sex chromosome systems have been described for E. virescens specimens obtained from different Brazilian rivers. A comparative analysis of the present data and prior reports suggests that the sex chromosomes of Eigenmannia may have arisen independently in the different populations.

The human papillomavirus (HPV)-associated effect can mimic invasive squamous cell carcinoma. Measurements of the argyrophilic nucleolar organizer region (AgNOR) area of cells from smears with HPV infection of the cervix with marked atypia were carried out to differentiate this pathology from keratinizing carcinomas. After destaining, the smears were incubated in the dark for 25 min with a mixture of silver nitrate and gelatin in formic acid. After washing with deionized water and sodium thiosulfate, the slides were dehydrated and mounted with Canada balsam. The average AgNOR area was determined by image cytometry using the immersion oil objective and selecting 100 cells in each smear. Twenty-three patients with a mean AgNOR area of 1.32 microm(2) among their samples showed normal colposcopies and cervical smears after 18 months. In four patients, whose samples were diagnosed as atypical squamous cells of undetermined significance with viral atypia, the average AgNOR area was 7.70 microm(2); biopsies showed keratinizing squamous carcinomas in three of these cases and moderately differentiated squamous carcinoma in one of them. We propose a cutoff of 2.2 microm(2) for the AgNOR area of cells from smears with HPV infection of the cervix with marked atypia to differentiate this group from keratinizing carcinomas.

Objective: To introduce new parameters of diploid histogram of image DNA cytometry, and new types of the nucleolar organizer region (AgNOR) and to validate resulting proliferative kinetic index (PKI) in a prognostic study in patients with the chronic leukemic lymphoproliferative disorders (CLLPD). Study Design. A total of 413 smears of in various tumor mass compartments: bone marrow (BM), peripheral blood (PB) and lymph node (LN) were analyzed in CLLPD as a whole and in the B-chronic lymphocytic leukemia with variants (B-CLL+V) in separate. The analysis of diploid histogram included percentage of cells in peak of DNA histogram, and percentage of cells with lower and higher contents of DNA than cells in peak. The newly types of AgNOR were described: homogeneous, inhomogeneous and annular. Results. The newly introduced parameters of DNA and AgNOR are significant predictors of survival. Based on the most representative AgNOR and DNA characteristics related to survival, the PKI score was calculated. The CLLPD (p=0, 00118) and B-CLL+V (p=0, 03589) patients had a statistically significantly better prognosis when PKI was less than 4. Conclusion. PKI have confirmed the hypothesis that different prognostically subgroups could be identified within the homogeneous groups of neoplasms with relatively low malignancy (CLLPD and B-CLL+V).

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.

A silver colloid technique to identify nucleolar organizer region associated protein (AGNORs) has been applied to paraffin sections in a total of 43 endometrial hyperplasias (24 adenomatous and 19 adenocystic) 26 endometrial carcinomas and 22 normal endometria (11 of proliferative and 11 of secretory phase). A morphometric analysis of highly magnified photographic images of AGNORs in light microscopic preparations was performed. Malignant tumor cells showed significantly higher AGNOR numbers, maximum diameter and mean area compared with normal and hyperplastic endometrium, with the exception of adenocystic hyperplasia whose Dmax and mean area were significantly larger. Regarding the distribution pattern of AGNOR dots in the cases studied, it was found that normal and hyperplastic endometrium had a mainly clustered distribution while endometrial adenocarcinomas revealed a scattered one. The significant differences observed in the number of AGNORs, their size and mean area between benign and malignant endometrial epithelia suggest that the AG-NOR staining technique is of diagnostic importance in distinguishing between these two groups.

Chromosomal nucleolar organizer region (NOR) phenotypes are documented for all four extant species in the North American cyprinid fish genus Pimephales. All four species (P. notatus, P. promelas, P. tenellus, and P. vigilax) possess 2n = 50 chromosomes and a pair of NOR-bearing chromosomes with the NOR situated terminally on the short arm of a medium-sized to large submetacentric chromosome (NOR phenotype C). Trypsin G-banding demonstrated that the C NOR chromosome in all four species is homologous. Two of the species (P. tenellus and P. promelas) also possess a C′ NOR chromosome, which is defined as an NOR situated terminally on the short arm of a large submetacentric chromosome that is also the largest chromosome in the complement. The C′ NOR chromosome occurs infrequently in P. promelas, being found in only 8% or so of all metaphases examined. Trypsin G-banding demonstrated that the C′ NOR chromosomes in the two Pimephales species are homologous to one another and to the C′ NOR chromosomes found in the cyprinid genus Cyprinella. A presumed derivative of the C′ NOR chromosome occurs in the monotypic cyprinid genus Opsopoeodus. The NOR chromosomal data support monophyly of the four extant species of Pimephales, and further suggest that the genus Pimephales belongs in a monophyletic assemblage with, among others, the cyprinid genera Cyprinella and Opsopoeodus. The data do not support the previous hypothesis that Pimephales is a basal clade outside of a larger assemblage of "Notropis"-like, shiners.