Nuclear morphometry and AgNOR quantification: computerized image analysis on ovarian mucinous tumor imprints

OBJECTIVE: To determine the morphometric characteristics of nuclei and silver-stained nucleolar organizer regions (AgNORs) on cytologic imprints and their value in differential cytodiagnosis of benign, atypical proliferative (borderline) and malignant ovarian mucinous tumors. STUDY DESIGN: Forty-six mucinous ovarian tumor imprints (16 benign, 15 borderline, 15 malignant), were analyzed. Nuclear area, outline, "shape factor" and "form factor" were measured on Papanicolaou-stained smears. AgNOR quantification included 7 variables related to the number and area of single, cluster, total and relative AgNOR content per nucleus and the size distribution of AgNORs. RESULTS: Nuclear area and shape factor allowed distinguishing borderline and malignant tumors. The nuclear area in benign tumors was larger than that in borderline tumors; malignant tumors had the highest values. Single and cluster AgNORs were statistically significantly different in borderline tumors compared with malignant tumors, except for the cluster AgNOR area. The total AgNOR area, number and relative area increased from benign through malignant tumors, with statistically significant differences among all groups. By AgNOR size distribution, small AgNORs discriminate malignant from borderline and benign tumors. CONCLUSION: Combining nuclear morphometry and AgNOR analysis on cytologic imprints could be a diagnostically useful method in the assessment of mucinous ovarian tumors.     

Morphometry and digital AgNOR analysis in cytological imprints of benign, borderline and malignant serous ovarian tumours

AIM: The aim of the study was to determine values of a quantitative morphometry analysis of nuclear characteristics and argyrophilic nucleolar organizer regions (AgNORs) in differential cytodiagnosis of benign, atypically proliferating (borderline) and malignant serous ovarian tumours. METHODS: Cytological imprints of benign (n = 20), borderline (n = 19) and malignant (n = 20) ovarian serous tumours were analysed. A computerized, digital analysis was used to determine morphometric nuclear features, the number and characteristics of single AgNORs, cluster AgNORs, total AgNOR and AgNOR area/nucleus (relative area) ratio. According to their size AgNORs were classified in three categories. A one-way variance analysis and post hoc test (Scheffé) were used for statistical analysis. RESULTS: The morphometric nuclear analysis showed that benign, borderline and malignant serous ovarian tumours are statistically different (P < 0.001) according to the area and outline, the values being highest in malignant tumours and lowest in the borderline group. Digital analysis of AgNORs in benign, borderline and malignant groups showed that the total AgNOR number increases with progression of the lesion (meaning tumour malignancy) significantly (P < 0.001) between benign and malignant as well as between borderline and malignant serous ovarian tumours (P < 0.001). The progression of the lesion malignancy was accompanied by a significant (P < 0.001) progressive increase of the total and relative AgNOR area per nucleus. The AgNOR size increases from benign to malignant tumours and a statistically significant difference (P < 0.001) was observed in all three groups regarding small and large AgNORs. CONCLUSION: Combining different markers of morphometric nuclear characteristics and AgNOR values could improve differential cytodiagnosis of benign, borderline and malignant serous ovarian tumours.     

MicroRNA expression in ovarian carcinoma and its correlation with clinicopathological features

ABSTRACT: BACKGROUND: MicroRNA (miRNA) expression is known to be deregulated in ovarian carcinomas. However, limited data is available about the miRNA expression pattern for the benign or borderline ovarian tumors as well as differential miRNA expression pattern associated with histological types, grades or clinical stages in ovarian carcinomas. We defined patterns of microRNA expression in tissues from normal, benign, borderline, and malignant ovarian tumors and explored the relationship between frequently deregulated miRNAs and clinicopathologic findings, response to therapy, survival, and association with Her-2/neu status in ovarian carcinomas. METHODS: We measured the expression of nine miRNAs (miR-181d, miR-30a-3p, miR-30c, miR-30d, miR-30e-3p, miR-368, miR-370, miR-493-5p, miR-532-5p) in 171 formalin-fixed, paraffin-embedded ovarian tissue blocks as well as six normal human ovarian surface epithelial (HOSE) cell lines using Taqman-based real-time PCR assays. Her-2/neu overexpression was assessed in ovarian carcinomas (n = 109 cases) by immunohistochemistry analysis. RESULTS: Expression of four miRNAs (miR-30c, miR-30d, miR-30e-3p, miR-370) was significantly different between carcinomas and benign ovarian tissues as well as between carcinoma and borderline tissues. An additional three miRNAs (miR-181d, miR-30a-3p, miR-532-5p) were significantly different between borderline and carcinoma tissues. Expression of miR-532-5p was significantly lower in borderline than in benign tissues. Among ovarian carcinomas, expression of four miRNAs (miR-30a-3p, miR-30c, miR-30d, miR-30e-3p) was lowest in mucinous and highest in clear cell samples. Expression of miR-30a-3p was higher in well-differentiated compared to poorly differentiated tumors (P = 0.02), and expression of miR-370 was higher in stage I/II compared to stage III/IV samples (P = 0.03). In multivariate analyses, higher expression of miR-181d, miR-30c, miR-30d, and miR-30e-3p was associated with significantly better disease-free or overall survival. Finally, lower expression of miR-30c, miR-30d, miR-30e-3p and miR-532-5p was significantly associated with overexpression of Her-2/neu. CONCLUSIONS: Aberrant expression of miRNAs is common in ovarian tumor suggesting involvement of miRNA in ovarian tumorigenesis. They are associated with histology, clinical stage, survival and oncogene expression in ovarian carcinoma.     

Lipocalin2 Expressions Correlate Significantly With Tumor Differentiation in Epithelial Ovarian Cancer

We recently identified lipocalin2 (LCN2) as being upregulated in ovarian cancer cell lines. The purpose of this study was to validate LCN2 upregulation in ovarian cancers and to investigate its potential as a serum biomarker. We assayed LCN2 expression in ovarian cancers using real-time PCR and IHC. To evaluate the potential of LCN2 as a biomarker, we measured serum LCN2 levels in 54 ovarian cancers, 15 borderline and 53 benign ovarian tumors, and 90 healthy controls. SYBR green PCR and IHC showed LCN2 overexpression in ovarian cancers. LCN2 immunoreactivity was significantly associated with tumor differentiation (p=0.009), as well-differentiated tumors showed the highest LCN2 expression. Serum LCN2 level in ovarian cancer was significantly higher than in the other study groups (p<0.001), and in accordance with IHC results, it also correlated with tumor differentiation, with well-differentiated tumors having the highest value. The sensitivity and specificity of LCN2 in detecting ovarian cancer was 72.2% and 50.4%, respectively. By Cox univariate analysis, LCN2 positivity was an independent prognostic factor for overall survival (hazard ratio = 1.47, p=0.012). In conclusion, LCN2 expressions are upregulated and related to tumor differentiation in ovarian cancers and should be included in future research assessing potential biomarkers for ovarian cancer. (J Histochem Cytochem 57:513–521, 2009)     

Clinicopathological study of metallothionein immunohistochemical expression, in benign, borderline and malignant ovarian epithelial tumors

Metallothioneins (MTs) are a family of cystein-rich metal-binding proteins, which are expressed in normal cells during fetal and postnatal life but also in a variety of human neoplasms. MT expression in human tumors has been linked to resistance to anticancer drugs and differentiation and progression in some types of tumors. This study examined the immunohistochemical expression of MTs in benign, borderline and malignant tumors of ovarian surface epithelium and the possible correlations with clinicopathological parameters and survival. A total of 87 cases with diagnosis of ovarian surface epithelial tumors were included. Specifically, 21 cases of benign cystadenomas (11 serous and 10 mucinous), 14 borderline (low malignant potential tumors, 8 mucinous and 6 serous) and 52 cases of ovarian cancer were analysed. Immunohistochemical expression of MT (cut-off level > 10% of tumor cells) was clearly associated with malignancy. A statistically significant correlation was found between the expression of MT in cancer cases and benign tumors (p < 0.0001) and cancer cases and borderline tumors p = 0.003. In cancer cases a difference was observed between grade I and III (p = 0.002). There was no correlation of MT overexpression with survival in the small number of ovarian carcinoma patients where it was analysed. MT constitutes a marker that characterizes aggressiveness and a high malignant potential in ovarian epithelial tumors. In diagnostic problems MT may help distinguish between benign, borderline and malignant tumors.     

An Outlook on Ovarian Cancer and Borderline Ovarian Tumors: Focus on Genomic and Proteomic Findings

Among the gynaecological malignancies, ovarian cancer is one of the neoplastic forms with the poorest prognosis and with the bad overall and disease-free survival rates than other gynaecological cancers. Ovarian tumors can be classified on the basis of the cells of origin in epithelial, stromal and germ cell tumors. Epithelial ovarian tumors display great histological heterogeneity and can be further subdivided into benign, intermediate or borderline, and invasive tumors. Several studies on ovarian tumors, have focused on the identification of both diagnostic and prognostic markers for applications in clinical practice. High-throughput technologies have accelerated the process of biomolecular study and genomic discovery; unfortunately, validity of these should be still demonstrated by extensive researches on sensibility and sensitivity of ovarian cancer novel biomarkers, determining whether gene profiling and proteomics could help differentiate between patients with metastatic ovarian cancer and primary ovarian carcinomas, and their potential impact on management. Therefore, considerable interest lies in identifying molecular and protein biomarkers and indicators to guide treatment decisions and clinical follow up. In this review, the current state of knowledge about the genoproteomic and potential clinical value of gene expression profiling in ovarian cancer and ovarian borderline tumors is discussed, focusing on three main areas: distinguishing normal ovarian tissue from ovarian cancers and borderline tumors, identifying different genotypes of ovarian tissue and identifying proteins linked to cancer or tumor development. By these targets, authors focus on the use of novel molecules, developed on the proteomics and genomics researches, as potential protein biomarkers in the management of ovarian cancer or borderline tumor, overlooking on current state of the art and on future perspectives of researches.Key Words: Ovarian cancer, borderline ovarian tumors, markers, genomics, proteomics, oncogenes.     

Volume-corrected mitotic index and p53 immunoreactivity in borderline and malignant epithelial ovarian tumors

OBJECTIVE: To determine if there are differences in p53 immunoreactivity and proliferation rate using the volume-corrected mitotic (M/V) index between borderline and malignant ovarian tumors. STUDY DESIGN: Thirteen borderline (7 serous and 6 mucinous) and 17 malignant (14 serous and 3 mucinous) ovarian neoplasms were included in this study. M/V index was calculated according to the formula of Haapasalo et al. Formalin-fixed, paraffin-embedded tissue sections were immunostained with a monoclonal antibody against p53 protein using an avidin-streptavidin method. RESULTS: Carcinomas had a considerably higher M/V index (17.7 +/- 9.8) than borderline tumors (1.5 +/- 1.7, P < .001). The usual mitotic index was also higher in carcinomas than in borderline tumors (24.9 +/- 16.6 versus 1.6 +/- 1.9, P < .001). p53 Immunoreactivity was found in 5/17 carcinomas (29.4%), while all borderline tumors were negative. CONCLUSION: Even though the number of our cases was too small to draw definitive conclusions, our results suggest that M/V index and p53 immunoreactivity may be of some help in the differentiation between borderline and malignant ovarian epithelial tumors.     

Digital image analysis of silver-stained nucleolar organizer region--associated proteins in endometrial cytologic samples

OBJECTIVE: Digital image analysis was applied to determine the number, area and size of silver-stained nucleolar organizer regions (AgNORs) in cytologic samples from curettage in normal, hyperplastic and malignant endometrium. STUDY DESIGN: Thirty-two archival cytologic smears from curettage (previously stained by the Papanicolaou method) with the histologic diagnosis (4 inactive endometrium, 5 secretion, 5 proliferation, 5 simple hyperplasia, 5 complex hyperplasia, 3 atypical hyperplasia, 5 adenocarcinoma, grade 1) were analyzed with the AgNOR technique. Count, area and size of AgNORs were analyzed in 50 cells per sample using a magnification of 1,000x. Quantitative analysis was performed on an SFORM digital imaging system. Data were analyzed with the SPSS/PC+ program. Mann-Whitney and chi 2 tests were performed. RESULTS: The average value of AgNOR count increased from normal to hyperplastic endometrium and well-differentiated adenocarcinoma. Differences were significant except between atypical hyperplasia and adenocarcinoma. Four, five and more AgNORs in 40% or more of the nuclei were found in complex and atypical hyperplasia and adenocarcinoma. Proliferation, and simple and atypical hyperplasia had similar mean values of AgNOR area. The mean total AgNOR area value increased from normal to hyperplastic had similar mean values of AgNOR area. The mean total AgNOR area value increased from normal to hyperplastic and well-differentiated adenocarcinoma. Differences were statistically significant. AgNOR size in well-differentiated adenocarcinoma was significantly different from that in normal endometrium and different grades of hyperplasia. CONCLUSION: Digital image analysis of AgNOR count, area and size enabled a distinction to be made between normal, hyperplastic and malignant endometrium.     

Flow and image cytometric DNA ploidy,including 5c exceeding cells,of serous borderline malignant ovarian tumors. Correlation with clinicopathologic characteristics

OBJECTIVE: To analyze DNA ploidy of serous borderline ovarian tumors by flow cytometry (FCM) and image cytometry (ICM), with 5c exceeding cells also analyzed, and to evaluate their correlation with clinicopathologic characteristics of patients and tumors. STUDY DESIGN: Cell suspensions were prepared according to a modified Hedley method from formalin-fixed, paraffin-embedded tissue blocks of 43 tumors. One part of the suspension was used for flow cytometric measurement; from the other part, filter slides were prepared for ICM. RESULTS: FCM and ICM found 2 aneuploid (peridiploid) serous borderline ovarian tumors, and FCM found 1. ICM found 3 tumors with 5c exceeding cells and 2 tumors with octaploid cells. There was no correlation between DNA aneuploidy and presence of 5c exceeding cells with tumor size, International Federation of Gynecology and Obstetrics stage or survival. CONCLUSION: The results confirm a good correlation between FCM and ICM DNA ploidy and the ability of ICM to detect 5c exceeding cells. The prognostic value of DNA ploidy and 5c exceeding cells in serous borderline malignant ovarian tumors warrant further evaluation.     

Prognostic value of GLUT-1 expression in ovarian surface epithelial tumors: a morphometric study

OBJECTIVE: To investigate the reported increase in the expression of the glucose transporter GLUT-1 in borderline and malignant ovarian epithelial tumors and its relationship to prognosis. STUDY DESIGN: In this study, areas in which immunohistochemical membranous staining with GLUT-1 were most evident were selected, and the proportions of GLUT-1 expression in 46 benign, 11 borderline and 42 malignant cases of ovarian epithelial tumors were determined quantitatively with a computer and Zeiss Vision KS 400 3.0 (G?ttingen, Germany) for Windows (Microsoft, Redmond, Washington, U.S.A.) image analysis. RESULTS: GLUT-1 expression was determined in all borderline tumors (11 of 11) and in 97.6% of malignant tumors (41 of 42). No GLUT-1 expression was observed in benign tumors. The intensity of GLUT-1 staining was lower in borderline tumors than in malignant cases. This was statistically significant (p = 0.005). As differentiation in malignant tumors increased, proportions of GLUT-1 expression showed a relative increase, but this difference was not statistically significant (p = 0.68). CONCLUSION: When GLUT-1 expression in borderline and malignant ovarian epithelial tumors was analyzed against prognosis, no statistically significant difference was identified. Assessment of GLUT-1 expression using the image analysis program was more reliable, with higher reproducibility than in previous studies.     

Fascin in ovarian epithelial tumors

Fascin contributes to the formation of actin-based protrusions involved in cell migration. Fascin has emerged as a prognostic marker in some carcinomas. We examined ovarian neoplasms to check any correlation between fascin expression and established clinicopathologic parameters. Fascin immunoreactivity was semiquantitavely scored in 100 ovarian tumors (62 carcinomas, 15 borderline tumors and 23 cystadenomas). Double staining for fascin and Ki-67 was performed in selected carcinomas. Western Blotting was done in frozen samples. Fascin immunoreactivity was highest in carcinomas, lowest in cystadenomas and intermediate in borderline tumors; these results were in accordance with those from Western blotting analysis. Fascin was statistically increased in carcinomas of advanced stage and in serous carcinomas. It was also increased in metastatic foci and in tumor foci with lower Ki-67 labeling. We conclude that in ovarian tumors fascin is associated with certain features of increased tumor aggressiveness. Future studies could determine if fascin may become a routinely helpful marker in gynecological pathology or clinical oncology.     

Nerve growth factor induces the expression of chaperone protein calreticulin in human epithelial ovarian cells

Epithelial ovarian cancer is highly angiogenic and high expression of Nerve Growth Factor (NGF), a proangiogenic protein. Calreticulin is a multifunctional protein with anti-angiogenic properties and its translocation to the tumor cell membrane promotes recognition and engulfment by dendritic cells. The aim of this work was to evaluate calreticulin expression in human normal ovaries, benign and borderline tumors, and epithelial ovarian cancer samples and to evaluate whether NGF regulates calreticulin expression in human ovarian surface epithelium and in epithelial ovarian cancer cell lines. Calreticulin mRNA and protein levels were analyzed using RT-PCR, Western blot and immunohistochemistry in 67 human ovarian samples obtained from our Institution. Calreticulin expression induced by NGF stimulation in cell lines was evaluated using RT-PCR, Western blot and immunocytochemistry. We found a significant increase of calreticulin mRNA levels in epithelial ovarian cancer samples as compared to normal ovaries, benign tumors, and borderline tumors. Calreticulin protein levels, evaluated by Western blot, were also increased in epithelial ovarian cancer with respect to benign and borderline tumors. When HOSE and A2780 cell lines were stimulated with Nerve Growth Factor, we found an increase in calreticulin protein levels compared to controls. This effect was reverted by GW441756, a TRKA specific inhibitor. These results suggest that NGF regulates calreticulin protein levels in epithelial ovarian cells through TRKA receptor activation.     

Expression of PTEN in ovarian epithelial tumors and its relation to tumor behavior and growth

OBJECTIVE: To evaluate the expression of tumor suppressor gene phosphatase and tensin homologue on chromosome 10 (PTEN) in ovarian epithelial tumors and its correlation with tumor growth and clinicopathologic features in ovarian adenocarcinomas. STUDY DESIGN: Immunohistochemical staining with anti-PTEN antibody was performed in 54 adenocarcinomas and 23 borderline tumors of the ovary. The apoptotic cells were visualized by terminal deoxynucleotidyl transferase mediated dUTP nick-end labeling, and proliferative cells were visualized by staining with Ki-67 antibody. RESULTS: Reduced PTEN expression was significantly higher among the adenocarcinomas than the borderline tumors (p < 0.001). Reduced PTEN expression in adenocarcinomas did not correlate with International Federation of Obstetrics and Gynecology (FIGO) stage. The Ki-67 index (KI) and apoptotic index were significantly higher in adenocarcinomas as compared with borderline tumors (p < 0.001). Tumors with reduced PTEN expression in ovarian adenocarcinomas had a significantly higher KI than those with normal PTEN expression (p < 0.01). By univariate analysis, FIGO stage and histologic type correlated with survival. However, FIGO stage was the only independent prognostic factor by multivariate analysis. CONCLUSION: Our results suggest that alteration of the PTEN gene may be associated with malignant transformation of ovarian epithelial tumors. The PTEN gene seems to be a negative regulator of cell proliferation in ovarian adenocarcinomas.     

Fine needle aspiration of borderline ovarian lesions. Is It useful?

Borderline ovarian tumors are a low grade form of epithelial ovarian carcinoma with a low rate of growth and a low potential to invade or metastasize. According to the new World Health Organization classification, these tumors are placed between clearly benign and obviously malignant tumors because they exhibit some, but not all, of the morphologic features of malignancy. For a distinction between borderline lesions and cystadenomas or carcinomas, 2 criteria are of the utmost importance: presence of nuclear atypia and absence of stromal invasion. The pathologic subtype ofperitoneal implants is probably one of the main prognosticfactors in patients with serous tumors of low malignant potential, while the prognostic value of micropapillary serous carcinoma in patients with noninvasive implants remains debatable. Although fine needle aspiration (FNA) is the most accurate diagnostic method in cytopathology, its value in the diagnosis of borderline lesions is limited, mainly because of its inability to establish the absence of stromal invasion. The diagnostic accuracy of RNA can be improved by supplementing cytologic examination with such diagnostic techniques as immunocytochemistry and cytometry.     

Copper content in ascitic fluid is associated with angiogenesis and progression in ovarian cancer

BackgroundAscites is associated with the poor prognosis of malignant tumors. The biological importance of the changes in the content of trace elements in the ascitic fluid is unknown. Herein, we analyzed trace elements in the ascitic fluid of patients with ovarian tumors and used cultured cells to determine the copper (Cu)-induced changes in gene expression in ovarian cancer.MethodsInductively coupled plasma mass spectrometry (ICP-MS) was used to compare ascitic fluid trace element levels in patients with benign ovarian tumors (n = 22) and borderline/malignant tumors (n = 5) for primary screening. Cu levels were validated using atomic absorption spectrometry (AAS) in 88 benign, 11 borderline, and 25 malignant ovarian tumor patients. To confirm Cu-induced gene expression changes, microarray analysis was performed for Cu-treated OVCAR3, A2780, and Met5A cells. The vascular endothelial growth factor (VEGF) concentration in the cell supernatant or ascitic fluid (ovarian cancer samples) was measured using ELISA.ResultsICP-MS showed that Co, Ni, Cu, Zn, As, Se, and Mo levels significantly increased in patients with malignant/borderline ovarian tumors compared to those in patients with benign ovarian tumors. AAS showed that malignant ovarian tumors were independently associated with elevated levels of Cu in ascites adjusted for age, body mass index, alcohol, smoking, and supplement use (p < 0.001). Microarray analysis of both Cu-treated ovarian cancer cell lines OVCAR3 and A2780 and the mesothelial cell line Met-5A revealed the upregulation of the angiogenesis biological process. Real-time polymerase chain reaction and ELISA demonstrated that an increased Cu content significantly enhanced VEGF mRNA expression and protein secretion in OVCAR3, A2780, and Met-5A cells. VEGF levels and clinical stages of the tumors correlated with the ascitic fluid Cu content in patients with malignant ovarian tumors (correlation coefficient 0.445, 95 % confidence interval [CI]: 0.069–0.710, p = 0.023 and correlation coefficient 0.406, 95 % CI: 0.022–0.686, p = 0.040, respectively).ConclusionCu levels significantly increased in patients with malignant ovarian cancer. Cu induced angiogenic effects in ovarian cancer and mesothelial cells, which affected ascites fluid production. This study clarifies the link between elevated Cu in ascites and malignant ovarian tumor progression. Strategies to decrease Cu levels in the ascitic fluid may help downregulate VEGF expression, thereby improving the prognosis of ovarian malignancies.     

卵巢上皮肿瘤淋巴转移与血管内皮生长因子C的表达

The aim of the present study was to explore the role of vascular endothelial growth factor-C (VEGF-C) in the process of angiogenesis, lymphangiogenesis and lymphatic metastasis in epithelial ovarian tumors. In situ hybridization and immunohistochemical staining for VEGF-C were performed in 30 epithelial ovarian carcinomas, 9 borderline tumors and 26 benign cystadenomas. Endothelial cells were immunostained with anti-VEGFR-3 pAb and anti-CD31 mAb, and VEGFR-3 positive vessels and microvessel density (MVD) were assessed by image analysis. VEGF-C mRNA and protein expression in ovarian epithelial carcinomas were significantly higher than that in borderline tumors and benign cystadenomas (p < 0.05 or p < 0.01). In ovarian epithelial carcinomas, VEGF-C protein expression, VEGFR-3 positive vessels and MVD were significantly higher in the cases of clinical stage III-IV and with lymphatic metastasis than those of clinical stage I-II and without lymphatic metastasis respectively (p < 0.05 or p < 0.01), VEGFR-3 positive vessels and MVD was significantly higher in the VEGF-C protein positive tumors than negative tumors (p < 0.05), VEGFR-3 positive vessels was significantly correlated with MVD(p < 0.01). These data suggest that VEGF-C might play a role in lymphatic metastasis via lymphangiogenesis and angiogenesis in epithelial ovarian carcinomas, and VEGF-C could be used as a biologic marker of metastasis in ovarian epithelial carcinomas.     

Nucleolar organizer region-associated proteins in cancer cells. Quantitative investigations on gliomas, meningiomas, urinary bladder carcinomas and pleural lesions.

Using a modified silver staining technique, we investigated nucleolar organizer region-associated proteins (AgNORs) in paraffin sections of 156 neoplastic tissues and other lesions, including gliomas (n = 41), meningiomas (n = 20), urinary bladder carcinomas (n = 58), and neoplastic and reactive lesions of the mesothelium of the pleural cavity (n = 37). We found significant differences in the mean number and area of AgNORs per nucleus between nonanaplastic and anaplastic astrocytomas. In meningiomas AgNOR analysis may be useful to distinguish between mostly benign tumors (grade 1 tumors) and atypical ones. Urinary bladder carcinomas exhibited a statistically significant increase in both AgNOR number and area as the grade of malignancy increased. Diagnostically useful differences in the AgNOR configuration between inflammatory and neoplastic processes were found for mesothelial lesions. In general, a higher grade of malignancy correlated with an increase in the AgNOR number. This was accompanied by an increase in the total AgNOR area per nucleus, irrespective of whether the size of the individual AgNORs had changed.     

Proliferative activity in endometrial hyperplasia and adenocarcinoma

Studies on the proliferative activity of cells in endometrial hyperplasia and adenocarcinoma were performed using techniques detecting Proliferating Cell Nuclear Antigen (PCNA) and Nucleolar Organizer Regions (NORs). PCNA expression was defined as the percentage of nuclei showing reactivity in 200 cells per sample. The mean AgNOR count per cell was calculated following the analysis of at least 100 nuclei per sample at a magnification of x 400. Student-t test was used for the statistical analysis. The results obtained indicate that the evaluation of cell proliferative activity expressed by AgNOR count and PCNA index can help in the distinction between atypical hyperplasia and well-differentiated adenocarcinoma, and thus can serve as a useful pathological criterion.     

Peritoneal washings in ovarian tumors. Potential sources of error in cytologic diagnosis

Peritoneal washings were performed on 48 patients with suspected or known ovarian carcinoma. The procedure was part of the initial surgical staging in 27 patients with presumed stage I and II ovarian cancer and was performed during second-look operations in 21 other cases with proven ovarian malignancy. This paper presents the microscopic features of the washings, with particular emphasis on the cytologic differentiation between benign and malignant findings outside of the ovary. Thirty-four cases showed benign or reactive mesothelial cells and no evidence of peritoneal disease. The washings of six patient showed malignant cells, which were confirmed histologically. Notable atypia that mimicked ovarian carcinoma was found in eight patients who had benign or borderline lesions. These findings included papillary and glandlike epithelial structures, with varying degrees of cellular atypia and psammoma bodies. The histologic counterparts of these atypicalities were Müllerian inclusions, mesothelial proliferations and borderline serous tumors. The differential diagnosis between these entities is essential because false-positive cytologic diagnoses may alter postoperative treatment in some patients.