Value of computer‐assisted quantitative nuclear morphometry for differentiation of reactive renal tubular cells from low‐grade urothelial carcinoma

H. Ohsaki, E. Hirakawa, K. Kagawa, M. Nakamura, H. Kiyomoto and R. Haba Value of computer‐assisted quantitative nuclear morphometry for differentiation of reactive renal tubular cells from low‐grade urothelial carcinoma Objective: To assess whether computer‐assisted quantitative morphological parameters can be an effective tool for objectively distinguishing reactive renal tubular cells from low‐grade urothelial carcinoma cells (LG‐UCs) in voided urine. Methods: Nuclear morphometry was performed by a computer‐assisted image analyser system on Papanicolaou‐stained cytological specimens. The circumference of reactive renal tubular cells (n = 40) or LG‐UC (n = 20) nuclei were manually traced, and the following nuclear morphometric parameters were analysed: (i) area, (ii) perimeter, (iii) roundness factor, (iv) maximum length, and (v) linear factor. For each nuclear measurement, we calculated the maximum, minimum, mean and standard deviation. Results: The mean nuclear area and nuclear perimeter were higher in reactive renal tubular cells compared to the LG‐UCs. The mean of roundness and linear factors (reflecting a tendency for the nuclear outline to be regular and oval, respectively) were higher in LG‐UCs compared with reactive renal tubular cells. Among nuclear areas, the nuclear perimeter, roundness factors and maximum length did not show any significant differences between reactive renal tubular cells and LG‐UCs. On the other hand, the linear factor showed a mean higher value among LG‐UCs than reactive renal tubular cells (P = 0.023). Conclusions: Of five quantitative nuclear morphological parameters, only linear factor was statistically significant in differentiating reactive renal tubular cells in renal disease from LG‐UCs.     

Density distribution, cytomorphologic features and immunologic characteristics of ovarian and endometrial clear cell carcinomas

Three subpopulations of cancer cells with different morphologic features were separated by density gradient centrifugation of two ascitic fluids and one cystic fluid from one patient with ovarian clear cell carcinoma and one with endometrial clear cell carcinoma. Immunophenotypic analyses of isolated fractions using polyclonal and monoclonal antibodies against ovarian carcinoma-associated antigens revealed significant immunologic heterogeneity among the tumor cells. The identical histopathologic structures of the ovarian and endometrial clear cell carcinomas and the similar distribution and immunologic reactivity of the cell types isolated from the ascitic and cystic fluids confirmed the common histogenesis of both cancers. These findings suggest that the conventional cytologic diagnosis of clear cell carcinomas could be supplemented by immunofluorescent staining. Density gradient centrifugation appeared to be a useful method for the separation of mesothelial cells.     

Expression of matrix metalloproteinases-2 and -9 by cells isolated from the peritoneal fluid of women with ovarian carcinoma

OBJECTIVE: To investigate the level of expression of matrix metalloproteinases (MMP)-2 and -9 by cells isolated from the peritoneal fluid of women with ovarian carcinoma. STUDY DESIGN: Tumor tissue specimens and cells isolated from peritoneal fluid from 20 patients with epithelial ovarian carcinoma were examined for MMP-2 and -9 expression using immunostaining. Six benign peritoneal effusions containing mesothelial cells were also included in the study. RESULTS: Expression of both MMP-2 and -9 was noted in cancer cells in peritoneal fluid of all cases studied. Peritoneal fluid cancer cells showed increased expression of both MMP-2 and -9 relative to mesothelial cell expression of these MMPs. Positive immunoreactivity of these MMPs in primary tumor tissues was confirmed by immunohistochemistry. CONCLUSION: Our findings suggest that both MMP-2 and -9 are frequently overexpressed in ovarian cancer cells disseminated in the peritoneal cavity and that determination of cellular MMP-2 and -9 expression could be useful in distinguishing cancer cells from mesothelial cells in peritoneal fluid cytologic specimens from women with ovarian epithelial carcinoma.     

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.     

Interactions of human ovarian tumor cells with human mesothelial cells grown on extracellular matrix : An in vitro model system for studying tumor cell adhesion and invasion

Human ovarian tumors metastasize by direct extension into the peritoneal cavity leading to tumor cell implantation onto peritoneal surfaces. Successful formation of peritoneal implants is dependent on the ability of ascitic tumor cells to infiltrate the mesothelium, and become firmly adherent to the underlying extracellular matrix (ECM). In order to investigate this process in more detail, an in vitro model system was developed employing human mesothelial cells grown on ECM-coated culture dishes. The ability of human ovarian carcinoma cells derived from ascitic fluid to attach to the mesothelial cell monolayer grown on ECM, ECM alone or plastic was quantitated with the use of 51Cr radio-labelled tumor cells. Tumor cells exhibited a more rapid and firmer attachment to ECM than to the mesothelial cells or to plastic. Using agitation to stimulate peritoneal fluid dynamics and shear forces in vivo, tumor cell arrest was found to be limited to the ECM, but it occurred at a slower rate than it did without agitation. Tumor cell attachment was also restricted to areas of exposed ECM in wounded mesothelium as assessed by phase-contrast microscopy. Morphologic alterations of the mesothelium induced by tumor cells were observed with the use of scanning electron microscopy (SEM) and immunohistochemical staining which included disruption of intercellular junctions leading to retraction of mesothelial cells, exposure of underlying ECM, subsequent attachment and proliferation on ECM. This model system would appear to be useful for elucidating mechanisms of ovarian tumor cell adhesion and proliferation, and for assessing various therapeutic modalities for their ability to block tumor cell implantation, invasion and growth on peritoneal surfaces.     

Pseudomyxoma peritonei associated with ovarian mucinous tumors. Cytologic appearance in five cases.

The cytologic findings in five cases of pseudomyxoma peritonei associated with ovarian mucinous tumors are reported. The evidence suggests that if mucinous or gelatinous ascitic fluid is received in the laboratory and is shown to contain a dual cell population of round cells of mesothelial origin and spindle-shaped cells of fibroblastic origin together with lakes of fibrillar mucin, a cytologic diagnosis of pseudomyxoma peritonei can be made with confidence. It will usually indicate mucinous neoplasia of the appendix or ovary. In the case of ovarian neoplasia, the tumor is most likely to be a low-malignant-potential mucinous tumor. The patient's prognosis, however, will be that of mucinous carcinoma rather than of usual borderline tumors of the ovary.     

Choroid plexus carcinoma. Cytologic identification of malignant cells in ascitic fluid

Abdominal deposits of a choroid plexus carcinoma in a patient with a ventriculoperitoneal shunt were cytologically diagnosed by examination of ascitic fluid after regression of the primary tumor. The morphology of the malignant cells in ascitic fluid was more similar to that of mesothelial cells than to the appearance of cells from this lesion in cerebrospinal fluid.     

Manual and automated AgNOR count in differentiating reactive mesothelial from metastatic malignant cells in serous effusions

OBJECTIVE: To distinguish reactive mesothelial cells from malignant cells in serous effusions using manual and automated methods of enumeration of argyrophilic nucleolar organizer regions (AgNORs). STUDY DESIGN: In this prospective study, 38 samples of benign (19 cases) and malignant (19 cases) serous effusions were included. AgNOR stain was used in each case along with routine Papanicolaou stain. The smears were examined under an oil immersion objective, and AgNOR dots were counted by direct observation independently by 2 observers. Automated AgNOR counting and morphometry were performed with a Quantimet 600 image cytometer (Leica, Cambridge, England). At least 100 cells were counted in each case. The number of AgNOR dots in individual cells, AgNOR area, nuclear area, AgNOR vs. nuclear area and nuclear perimeter were measured. Data on benign and malignant cells were compared. RESULTS: The AgNOR dots were discrete and smaller in benign effusion cases as compared to coarse and aggregated in malignant effusion cases. In benign reactive effusion cases the mean number of AgNOR dots per nucleus was 2.33 +/- 0.71 and 2.83 +/- 1.15 by the manual and automated method, respectively, whereas that for malignant effusion cases was 7.48 +/- 2.51 and 8.09 +/- 1.69 by the manual and automated method, respectively. Mean total AgNOR areas in benign and malignant groups were 4.77 +/- 2.66 microns 2 and 38.22 +/- 13.71 microns 2, respectively. Mean nuclear area, nuclear perimeter and ratio of AgNORs vs. nuclear area were 48.72 +/- 19.30 microns 2, 24.68 +/- 10.25 microns and .098 in benign effusion cases as compared to 174.25 +/- 82.36 microns 2, 69.03 +/- 27.23 microns and 0.22 in malignant effusion samples. All these values were significantly higher (P < .001, Student's t test) in malignant cells as compared to benign reactive cells. CONCLUSION: AgNOR dot enumeration, AgNOR area and ratio of AgNORs to nuclear area are valuable adjuncts to cytomorphology in differentiating reactive mesothelial cells from malignant cells in serous effusions. Automated AgNOR counting is rapid and less cumbersome.     

Ascitic fluid cytologic features of a malignant mixed mesodermal tumor of the ovary

A case of malignant mixed mesodermal tumor of the ovary in a postmenopausal patient presenting with abdominal distension is reported. Cytologic examination of smears of the ascitic fluid showed the presence of adenocarcinomatous and sarcomatous cells (with some of the latter being giant cells) plus numerous unidentifiable cells that bore some resemblance to either mesothelial cells or macrophages. Electron microscopic studies showed a clear differentiation of the adenocarcinomatous and sarcomatous cells from positively identified mesothelial cells and macrophages also present in the ascitic specimen, indicating that the unidentified cells in fact originated in the adenocarcinoma (endometrioid carcinoma), chondrosarcoma and unclassified sarcoma found in the surgical specimen. The differential diagnostic cytomorphologic and electron microscopic features are described in detail.     

Fractalkine receptor CX(3)CR1 is expressed in epithelial ovarian carcinoma cells and required for motility and adhesion to peritoneal mesothelial cells

Epithelial ovarian carcinoma (EOC) is a deadly disease, and little is known about the mechanisms underlying its metastatic progression. Using human specimens and established cell lines, we determined that the G-protein-coupled seven-transmembrane fractalkine receptor (CX(3)CR1) is expressed in primary and metastatic ovarian carcinoma cells. Ovarian carcinoma cells robustly migrated toward CX(3)CL1, a specific ligand of CX(3)CR1, in a CX(3)CR1-dependent manner. Silencing of CX(3)CR1 reduced migration toward human ovarian carcinoma ascites fluid by approximately 70%. Importantly, adhesion of ovarian carcinoma cells to human peritoneal mesothelial cells was dependent on CX(3)CL1/CX(3)CR1 signaling. In addition, CX(3)CL1 was able to induce cellular proliferation. Together, our data suggest that the fractalkine network may function as a major contributor to the progression of EOC, and further attention to its role in the metastasis of this deadly malignancy is warranted.     

The value of calretinin and cytokeratin 5/6 as markers for mesothelioma in cell block preparations of serous effusions

Objective: To determine the value of calretinin and cytokeratin (CK) 5/6 in discriminating mesothelioma from adenocarcinoma in serous effusion specimens. Methods: A total of 101 recent, histologically or clinically confirmed malignant effusions with immunostained cell block preparations were reviewed. The cases consisted of 34 mesotheliomas and 67 adenocarcinomas. This included 17 ascitic fluid and 84 pleural fluid samples. The adenocarcinomas included metastatic carcinomas from the breast (12), lung (19), stomach (3), colon (1), pancreas (2), ovary (6) endometrium (1) and 23 histologically confirmed metastases from unknown primary sites. The cases were assessed as negative or positive (>5% of cells stained). The staining pattern was recorded as cytoplasmic, cell membrane, nuclear or cytoplasmic and nuclear staining. Results: Calretinin staining was present in 97% (33/34) of the mesothelioma cases with a majority of them showing both cytoplasmic and nuclear staining (29/33). Only 3% (2/67) of adenocarcinomas were positive for calretinin, one being a lung adenocarcinoma and the other an adenocarcinoma of unknown primary site in an ascitic fluid. Cytokeratin 5/6 staining was also present in 33/34 (97%) of mesothelioma cases. Six (9%) adenocarcinomas were positive, including metastases from the lung (1), breast (1), ovary (2) and unknown primary site (2). Four of the six adenocarcinoma cases positive for CK5/6 were in ascitic fluids. No cases of mesothelioma were negative for both calretinin and CK5/6. Only one adenocarcinoma case, (which was from unknown primary site in an ascitic fluid sample), was positive for both markers. Conclusions: The results confirm that calretinin and CK 5/6 are useful markers for mesothelioma in effusion specimens. CK5/6 staining may be less useful for peritoneal fluid specimens where metastatic adenocarcinomas may be more likely to express the antigen. Further study of ascitic/peritoneal specimens is warranted. However, positive staining, particularly for both antigens, is highly indicative of a mesothelial origin for cells. The two markers make a useful addition to EMA and the panel of adenocarcinoma markers routinely applied to effusion specimens.     

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.     

Automated image morphometry of lobular breast carcinoma

OBJECTIVE: To analyze the role of automated image morphometry (AIM) in distinguishing infiltrating lobular carcinoma (ILC) of the breast from benign, borderline and infiltrating ductal carcinoma (IDC). STUDY DESIGN: Only histopathologically proven lobular carcinoma, ductal carcinoma, borderline lesions and benign breast lesions were selected for the study. There were 19 cases of ILC and 30 cases of IDC, 20 cases of benign lesions (fibroadenoma, 18; fibrocystic disease, 1; and fibroadenosis, 1); 10 cases were borderline lesions (mild epithelial hyperplasia, 3; moderate epithelial hyperplasia, 2; florid epithelial hyperplasia 4; intraductal papillary carcinoma, 1). In all cases hematoxylin and eosin-stained slides were used for AIM. At least 100 cells from each case were subjected to analysis randomly with an image cytometer with Leica Quantimet 600 software (Cambridge, England). Nuclear area, diameter, perimeter, convex perimeter, convex area and roundness were measured in each case with random, unbiased selection of cells and 40 x objectives (one pixel = 0.46 microm). AIM data on the cases were analyzed in relation to final cytologic diagnosis. RESULTS: All the nuclear morphometric features of ILC were much lower than those of IDC and borderline lesions, whereas nuclear morphometric data on ILC were only marginally more than those on benign cases. ANOVA showed that mophometric data were significant (P < .05) in all the variables between ILC and IDC. However, there was no significant difference between ILC, and borderline and benign cases. CONCLUSION: Image morphometry may be useful in distinguishing ILC from IDC on cytologic smears. However, morphometric data may not be helpful in distinguishing benign and borderline lesions from ILC.     

Morphometric image analysis of follicular lesions of the thyroid

OBJECTIVE: To determine the role of image morphometry in distinguishing various follicular lesions of the thyroid in cytologic smears. STUDY DESIGN: Archival fine needle aspiration smears of 10 cases each of follicular hyperplasia, follicular adenoma, follicular carcinoma and follicular variant of papillary carcinoma were used for the study. All cases were histopathologically proven. At least 100 random nuclei from each case were subjected to analysis with an image cytometer. Area, convex area, length, width, perimeter, convex perimeter and roundness of nuclei were measured using a 40 x objective (1 pixel = 0.446 micron). RESULTS: ANOVA showed that all the nuclear variables studied were significantly different (P < .05) in follicular hyperplasia as compared to follicular carcinoma and papillary carcinoma. All nuclear variables except roundness were also significantly different (P < .05) between follicular hyperplasia and follicular adenoma. However, between follicular adenoma, follicular carcinoma and papillary carcinoma there was considerable overlap of nuclear morphometric parameters. CONCLUSION: Image morphometry may help to distinguish nonneoplastic follicular lesions (hyperplasia) from neoplastic lesions (adenomas and carcinomas). However, to distinguish benign from malignant follicular lesions, image morphometry might not improve the accuracy of standard cytologic examination.     

Positive effusion cytology as the initial presentation of malignancy

During a period of four years (1981 to 1984), 641 ascitic, 860 pleural and 47 pericardial fluid specimens were examined cytologically. Of these, 154 ascitic samples, 174 pleural specimens and 10 pericardial effusions, obtained, respectively, from 108, 133 and 7 patients, were found to contain malignant cells. In 7 patients, ascites, and in 18 cases, pleural effusions were the first indication of cancer. None of the positive pericardial fluids was the initial presentation of malignancy. The cytologic findings and follow-up data on these 25 patients are the subject of this study. The most common type of neoplasm in these effusions was adenocarcinoma (86% of the ascitic and 78% of the pleural fluids). Most of the malignant neoplasms in ascitic fluids were derived from ovarian tumors (5 of 7) while those in pleural effusions came mainly from lung tumors (12 of 18). Mammary carcinoma, which was the most common malignant tumor found in cases of pleural effusions, did not present initially with an effusion in any of our cases. The cytologic diagnosis was confirmed in all cases by either biopsy or strong clinical evidence. The prognosis in patients who initially presented with an effusion was poor. All of the patients with an adequate follow-up died within 29 months in cases of ascites and within 19 months in cases of pleural effusions.     

Ovarian carcinoma and serous effusions. Changing views regarding tumor progression and review of current literature.

Carcinoma of the ovary is the leading cause of death from gynecological cancer in western countries. Ovarian carcinoma is commonly associated with the accumulation of fluid containing malignant cells in the peritoneal, and not infrequently in the pleural cavity. The differentiation of these cells from reactive mesothelial cells is at times difficult. In addition, tumor progression in ovarian carcinoma and the biological characteristics of carcinoma cells in effusions compared to their counterparts in solid tumors are poorly understood. This review details the current knowledge regarding diagnostic and biologic aspects of effusion cytology, with emphasis on ovarian carcinoma. Results from our first studies of effusions are subsequently presented. These attempt to address several issues. First, to improve the diagnostic ability to detect cancer cells in effusions using antibodies designed for the differentiation of epithelial cells from mesothelial cells. Secondly, to study genotypic and phenotypic differences between ovarian carcinoma cells in effusions, solid primary tumors and metastatic lesions, as well as to compare malignant cells in peritoneal and pleural effusions. These studies of carbohydrate antigens, E-cadherin complex and matrix metalloproteinases (MMP) attempted to evaluate whether ovarian carcinoma cells in effusions possess true metastatic properties, or are similar to the cells in primary tumors, thereby merely representing the result of a shedding process. Finally, the prognostic role of these molecules was studied in solid tumors from a patient cohort consisting of long- and short-term survivors, followed for up to 20 years. Figure 1 on http://www.esacp.org/acp/2001/23-3,4/davidson.htm.     

Specific tumor markers in diagnostic cytology. Immunoperoxidase studies of carcinoembryonic antigen, lysozyme and other tissue antigens in effusions, washes and aspirates

The immunoperoxidase technique was used to identify specific tumor markers in exfoliated cells in fine needle aspirates and body fluids. Carcinoembryonic antigen (CEA) and lysozyme staining was evaluated in cytocentrifuge preparations from 42 malignant effusions and aspirates and 16 benign effusions. Reactive mesothelial cells were negative for CEA and lysozyme or showed faint peripheral cytoplasmic staining. Malignant cells from 50% of the adenocarcinomas studied were positive for CEA. All tumors studied were negative for lysozyme. These staining patterns are helpful in the differential diagnosis of reactive mesothelial and adenocarcinoma cells, a frequent diagnostic dilemma. Moreover, demonstration of specific tumor antigens (e.g., prostatic acid phosphatase, calcitonin and immunoglobulin) helped define the origin of metastatic malignancy in selected cases. Estrogen receptor activity was also identified in tumor cells using this technique. Immunoperoxidase was helpful in the evaluation of malignant cytologic specimens from patients with more than one tumor. Interpretation of staining patterns is discussed, with reference to the limitations of the technique. Immunoperoxidase methods maintain cytologic detail, are readily adaptable to diagnostic cytology and increase the specificity of cytologic diagnosis.     

Clear cell carcinoma of the ovary diagnosed in ascitic fluid. A case report

BACKGROUND: Clear cell carcinoma of the ovary (CCC) is a rare variety of ovarian cancer. CASE: A case of CCC in a 49-year-old woman was diagnosed in asciticfluid on thin-layer preparations. Peritoneal fluid cytology revealed papillary clusters of cells with clear cytoplasm and extracellular hyaline material generally without neoplastic cells. The tumor was excised, and the histologic sections confirmed the cytologic diagnosis. CONCLUSION: CCC has a distinctive cytomorphologic appearance, and the entity may be diagnosed on ascitic fluid cytology.