Nucleolar organizer regions in normal skeletal muscle and benign and malignant rhabdomyoblastic tumors

The silver staining of interphase nucleolar organizer regions (NORs) has been shown to have an important application in diagnostic histopathology for distinguishing some benign from malignant conditions. In this study, normal fetal and adult skeletal muscles and tissue from fetal and adult rhabdomyomas as well as rhabdomyosarcomas were stained with the silver method for NORs. The morphologic distribution of NORs in rhabdomyosarcomas was found to be very different from that in normal skeletal muscles. In addition, cases of rhabdomyoma were easily differentiated from rhabdomyosarcomas. Statistical analysis of data, from all cases, regarding the diameter of NORs and number per nucleus confirmed these observations.     

Nucleolar organizer regions in soft tissue sarcomas

Argyrophilic proteins of nucleolar organizer regions (AgNORs) were studied in a series of 65 sarcomas of the soft tissues and 2 cases of fibrohistiocytic tumors of intermediate malignancy. The numbers of AgNORs per nucleus and the size of AgNORs were determined and compared with pathomorphologic parameters such as grading of malignancy, cellularity and tumor diameter. The main finding of this study was that AgNOR counts showed significant differences between low-grade malignant (G 1) and high-grade malignant (G 3) tumors predominantly based on a correlation with the frequency of mitosis. All the other criteria of tumor grading (nuclear pleomorphism, differentiation, amount of necrosis), tumor diameter and cellularity showed no differences with regard to AgNOR counts. Though it was observed that tumor giant cells possessed predominantly coarse granular AgNORs, no correlation was found between AgNOR size and any of the parameters investigated. In spite of a clear statistically significant result gained by an evaluation of all cases, a heterogeneity of AgNORs in relation to mitotic activity within a few histological tumor types could be observed, making it difficult to suggest the determination of AgNORs as a parameter with general validity for all soft tissue sarcomas.     

NUCLEOLAR ORGANIZER REGIONS IN CUTANEOUS T CELL LYMPHOMAS

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

Nucleolar organizer regions in precancerous and cancerous lesions of the bronchus

Using a silver staining technique, nucleolar organizer region-associated proteins (Ag-NOR) were studied in paraffin sections of five specimens of normal bronchial epithelium, eight of atypical squamous metaplasia, five of carcinoma in situ, and seven of microinvasive squamous cell carcinoma. The mean number of Ag-NOR in the nucleus were normal epithelium 1.2 +/- 0.1 (mean +/- SD), atypical squamous metaplasia (borderline lesion) 2.2 +/- 0.5, carcinoma in situ 3.8 +/- 0.6, and microinvasive squamous cell carcinoma 4.8 +/- 1.1. There was a highly significant difference between the Ag-NOR numbers in the atypical squamous metaplasia and those in the carcinoma in situ (P less than 0.01). The Ag-NOR staining is a useful technique for the differential diagnosis of difficult borderline lesions in the bronchial epithelium.     

Nucleolar organizing regions in primary intracranial malignant lymphomas

Twenty-two non-immunocompromised patients with primary intracranialmalignant lymphomas were examined on surgical material by using anargyrophilic method for the demonstration of nucleolar organizer regions asAg-NORs. The histopathological classifications of 22 patients included 3small lymphocytic, 7 small cleaved, 9 large cell, 1 mixed large and small,and 2 small non-cleaved type. The numbers of Ag-NOR of malignant lymphomapatients varied from 1.36 to 5.02 (mean 3.46 ± 0.25). The mean Ag-NORnumbers in the histopathological subtypes were small lymphocytic 1.63, smallcleaved 3.18, large 4.21, mixed large and small 3.47, and small non-cleaved3.78. The number of Ag-NORs in small lymphocytic type was significantly lessthan small cleaved or large cell type (p < 0.05). The small cleaved typealso had a smaller Ag-NOR number than the large cell type (p < 0.05).Except for two patients who had postoperative deterioration, 20 patientsreceived postoperative irradiation ranging from 36 to 54 Gy (median, 46 Gy).Sixteen patients had complete response to radiotherapy, and 4 had goodpartial response. Ten patients had tumor recurrence within the remissionperiod of 3 months to 7 years and 10 months (median 4.8 months). Threepatients with intracranial relapse at a remote site had a significantlylonger remission period (mean 57.3 months) than 7 with local relapse (mean6.29 months), p < 0.05. The mean Ag–NOR number of the short andlong remission period were 3.13 ± 0.34 and 3.81 ± 0.80,respectively. No significant difference was found between these two groups.The survival period was 3.2 months to 12 years (median 20 months). TheAg-NOR numbers of survival period less than or more than 20 months were 3.62± 0.40 and 3.27 ± 0.37, respectively. The Ag-NOR numbers didnot correlate with either the remission or the survival period. Theseresults indicate that Ag-NOR numbers may correlate with the histopathologictypes, but not with the prognosis of primary intracranial malignantlymphomas.     

Clinical characteristics of malignant melanomas developing in persons with dysplastic nevi

A total of 452 patients with dysplastic nevi (DN) were followed prospectively by repetitive, complete cutaneous examinations in order to determine the clinical features of early malignant melanomas (MM) arising in them. Sixteen patients (3.5%) developed 18 newly diagnosed MM during an average follow-up period of 27 months. Twelve of the 18 MM were in situ and all of the primary invasive MM diagnosed prospectively in this follow-up were less than 0.89 mm in Breslow thickness, implying an excellent prognosis. The principal clinical clue to the diagnosis of MM was change in a preexisting pigmented lesion. Total-body photographs were very useful in helping to identify the early MM in these patients.     

Nucleolar organizer regions in aggressive and nonaggressive basal cell carcinoma of the skin.

Nucleolar organizer regions (NOR) were investigated on routine paraffin-embedded histologic sections of 11 aggressive basal cell carcinomas that recurred and/or metastasized (BCC2) and 11 nonaggressive basal cell carcinomas (BCC1). The absolute number of NOR per nucleus was higher in BCC2 than in BCC1, and their distribution pattern was also different. In fact, the means of argyrophilic staining of NOR (AgNOR) counts in the two groups of tumors by two observers were 6.56 with a SD of 1.98 for the nonaggressive and 9.48 with a SD of 2.12 for the aggressive basal cell carcinomas. A statistical analysis of these data using the Student's t test confirmed these observations (t = 64.49). Problems in the evaluation of NOR and possible comparison with other experiences are also discussed. The authors conclude that a quantitative assay of AgNOR and perhaps their distribution pattern may provide information useful to recognize BCC2 and hence may be of help in their prognostic prediction.     

Morphometric analysis of the free stromal cells of nevi and malignant melanomas

A morphologic study of 114 cases of pigmented skin tumors was carried out. The cases were divided into 5 groups on the basis of the degree of nevus proliferation and depth of melanoma invasion. The percentage and correlation between different types of free stromal cells were obtained for different skin zones in 6 cases of each group. Correlation analysis established the set character of cell cooperation at different stages of tumor growth. Diagrams illustrating correlations between free stromal cells and pigmented formations patterns are discussed. The results of the study may be helpful in running diagnostic tests of nevi for malignancy as well as identifying the depth of melanoma invasion whenever visual morphologic examination is insufficient.     

Nucleolar organizer regions as a prognostic indicator for stage I non-small cell lung cancer.

When the number of silver-stained nucleolar organizer regions (Ag-NORs) was counted in 274 patients with non-small cell lung cancer, the mean number per nucleus in patients overall was 5.07 +/- 1.92 (SD). With the use of the tumor (T)-nodes (N)-metastasis (M) classification, the mean Ag-NOR count for patients with T1 and T2 disease was statistically lower than that for those with T3 and T4 disease (P less than 0.01). The mean Ag-NOR counts were lower in patients with N0 disease than in those with N1 and N2 disease (P less than 0.01); lower in patients with stage I disease than in those with stage II, IIIA, IIIB, or IV disease (P less than 0.01); and lower in patients with adenocarcinoma than in those with squamous cell carcinoma (P less than 0.01) or large-cell carcinoma (P less than 0.05). In 131 patients with stage I disease, the mean Ag-NOR count was 3.80 +/- 1.32, and the 5-year survival rates of patients with Ag-NOR counts of less than 3.80 and greater than or equal to 3.80 were 78 and 44%, respectively, including 78% and 25% for adenocarcinoma, respectively (P less than 0.01). However, there was no statistically significant difference for those in stage II, IIIA, IIIB, or IV, and in stage I (without an adenocarcinoma). Because patients with stage I non-small cell lung cancer and a high number of Ag-NORs had a poor prognosis, Ag-NORs can serve as a pertinent marker of an early recurrence.     

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.     

Immunohistochemical expression of VEGF, HIF1-a, and PlGF in malignant melanomas and dysplastic nevi

We evaluated the role of vascular endothelial growth factor (VEGF), placental growth factor (PlGF), and hypoxia-inducible factor 1-a (HIF1-a) in melanoma angiogenesis and investigated their expression in dysplastic nevi, as potential melanoma precursors. In addition, we examined a possible correlation of VEGF expression with PlGF and HIF1-a. These factors were detected immunohistochemically in 95 melanomas of all types and stages and in 28 dysplastic nevi. We used 10 intradermal melanocytic nevi as controls. HIF1-a was expressed in 93 out of 95 (97.89%) melanomas and in none of the dysplastic or control nevi. HIF1-a expression was more intense in melanocytes around necrotic areas but did not correlate with melanoma type, the Clark staging or the Breslow thickness. A strong positive association was detected between HIF1-a and VEGF expression in all cases. VEGF was detected in 82 out of 95 (86.31%) melanomas and in 21 out of 28 (75%) dysplastic nevi, whereas it was expressed weakly in neoplastic cells of the controls. Its expression was more intense in melanomas, especially in nodular melanomas of elevated stage and thickness. PIGF was detected in 46 out of 95 (48.42%) melanomas and in none of the nevi. Expression did not correlate with melanoma staging nor thickness; however, it was more intense in superficial spreading melanomas, where a weak positive association between VEGF and PlGF was also detected. There was no association between HIF1-a and PlGF in any melanoma type. Hypoxia, through the expression of HIF1-a, plays a key role in melanoma progression; it activates VEGF secretion, which induces angiogenesis and metastasis. The role of PlGF seems to be limited.     

Extracutaneous malignant melanomas

Extracutaneous malignant melanomas are rare tumors with vexing clinical presentation and grim prognosis. Only 4%-5% of all primary melanomas do not arise from the skin. These tumors are almost uniformly fatal, even in 2006. Although a fairly good number of these lesions were reported in the literature, the lack of a side-by-side analysis of these studies has resulted in tentative conclusions that merely offer a first glimpse at the clinicopathologic diversity of these lesions. To remedy this issue, this article took an aim at presenting a literature review concerning extracutaneous malignant melanomas. It also reports several cases of extracutaneous melanomas, which I came across in my 15 years of surgical and molecular pathology practice. The study raises several notions. Extracutaneous malignant melanomas are rare but extremely aggressive lesions with a grim outcome. They include ocular, metastatic, anorectal, mucosal, nail beds, conjunctival, vaginal, urogenital, orbital, esophageal, and leptomeningial malignant melanomas. The development of these lesions lacks an association with sun damage, family history, or precursor nevi. These lesions cause considerable diagnostic consternation and their distinction from other types of tumors (such as undifferentiated carcinomas, high-grade sarcomas, and lymphomas) is critical both from a diagnostic and prognostic point of view. In the proper clinical, histological, and cytological context, immunopositivity for S100 protein, HMB45, and vimentin allows the distinction of these malignant melanomas from other histologically similar malignancies. To conclude, extracutaneous melanoma should be considered while undifferentiated neoplasms, especially those displaying prominent eosinophilic nucleoli, and the coexistence of epithelioid and spindle cells. Special staining and immunohistochemistry should be resorted to establish the diagnosis.     

Cytogenetic analysis of melanocytes from premalignant nevi and melanomas

We karyotypically analyzed cultured melanocytes from a variety of lesions, including congenital and dysplastic nevi, primary melanoma, and metastatic melanoma. The cells derived from congenital nevi had normal karyotypes, as did 22 of the 26 cultures derived from dysplastic nevi. The karyotypes of melanocytes from primary and metastatic melanomas were all abnormal. The only chromosome change in common between the nevi with abnormal karyotypes and the melanomas was the loss of one copy of chromosome 9 (two of four nevi and four of 11 melanomas, including three from the same patient) or the loss of the short arm of chromosome 9, especially of region 9pter-p22 (three of 11 melanomas). We suggest that deletion of a gene or genes on 9p, possibly interferon genes, is an initial step in the malignant transformation of melanocytes.     

Immunohistochemical analysis of malignant melanomas and nevocellular nevi with monoclonal antibodies to distinct monomorphic determinants of HLA antigens

Using an indirect immunoperoxidase technique, 20 nevocellular nevi, 5 dysplastic nevi, 14 primary cutaneous melanomas, and 24 metastatic melanomas were tested with a panel of monoclonal antibodies to monomorphic determinants of Class I (HLA-A,B,C) and Class II (la-like) major histocompatibility complex antigens. Class I HLA and beta 2-microglobulins were not detected on the majority of nevus cells but were expressed by 3 of 5 dysplastic nevi, by the majority of tumor cells in 12 of 14 primary cutaneous melanomas, and in 13 of 24 metastases. The different expression of Class I HLA and beta 2-microglobulins in primary and metastatic lesions suggests that loss of these antigens may be associated with progression of malignancy. Class II HLA were not detected in common nevi but were locally present in 1 of 5 dysplastic nevi, 7 of 14 cases of primary cutaneous melanoma, and all 24 cases of metastatic lesions tested. These findings suggest that increase in Class II HLA expression may be associated with progression of malignancy. The staining patterns obtained with monoclonal antibodies to distinct determinants of Class I HLA and Class II HLA were superimposable within each type of antigen. Therefore, the discrepancies in the literature about the expression of histocompatibility antigens by lesions of melanocytic origin are not likely to reflect the different specificity of the antibodies used by the various investigators.