An unusual cytologic presentation of mesothelioma in serous effusions

Three cases of diffuse malignant mesothelioma in which samples of pleural fluid showed an unusual cytologic picture are presented. Instead of cells of an obvious mesothelial type forming organized clusters, the smears were dominated by foamy macrophage-like cells, with or without certain nuclear features suggesting malignancy. It is suggested that these cells were derived from neoplastic mesothelial cells by a process of differentiation.     

Immunocytochemical diagnosis of lymphoma in serous effusions

An immunoalkaline phosphatase technique was used to examine the lymphoid cells in serous effusions from five patients with malignant lymphoma. The results were interpreted along with the morphologic studies and retrospective assessments of the clinical conditions of the patients. Two patients had no involvement of the serous cavities, and two had proven involvement. The fifth patient was studied while his lymphoma was evolving from inapparent to disseminated disease. In the two patients without involvement of the serous cavities, the effusion lymphocytes were predominantly monoclonal T cells, comparable to those in six patients with diseases other than lymphoma. In those with involvement of the serous cavities, the effusion lymphocytes were predominantly monoclonal B cells. In the patient with lymphoma in evolution, immunocytochemical studies accurately reflected the progression of disease. We conclude that immunocytochemical studies of the lymphocytes in serous effusions help not only to differentiate reactive from neoplastic lymphoproliferation but also to assess the status of lymphomatous involvement of the serous cavities. The immunocytochemical studies are most effective when correlated with clinical and cytologic studies.     

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.     

Usefulness of ploidy, AgNOR and immunocytochemistry for differentiating benign and malignant cells in serous effusions

OBJECTIVE: The objective of this study was to establish the value of different markers in differentiating reactive mesothelial cells from metastatic adenocarcinomatous cells in serous effusions (SE). METHODS: Forty-five SE were processed for morphological examination (Papanicolaou stain), assessment of ploidy, AgNOR counting and immunocytochemical assay of carcinoembryonic antigen (CEA), epithelial membrane antigens (EMA), Ber-EP4 and Leu-M1. Ploidy was established in an image-analyser in smears stained by the Feulgen stain method. AgNOR dots were counted in the smears stained with the silver nitrate assay for non-histone proteins present in the nucleolar organizer region. CEA, EMA, Ber-EP4 and Leu-M1 were evaluated by immunocytochemistry using the streptavidin-biotin complex method. RESULTS: All the smears with positive cytology were aneuploid. Three false negatives by morphological studies were aneuploid, with AgNOR values in two of them corresponding to the neoplastic group. CEA and Leu-M1 showed a low specificity; EMA and Ber-EP4 showed moderate sensitivity. CONCLUSIONS: The assessment of ploidy and the study of AgNOR were better methods than immunocytochemistry for distinguishing between reactive mesothelial cells and adenocarcinomatous cells in serous fluid.     

Additional techniques in serous effusions.

Cytological examination is a valuable diagnostic tool in case of a serous effusion. The first manifestation of malignancy may be an effusion of the pleural, pericardial, or peritoneal cavity, especially in carcinoma of the ovary, or lung, and malignant mesothelioma. In other malignancies effusions may occur in the course of the disease. The contribution by Mother by et al. in this issue of ACP focuses on the contribution of image and flow cytometry to establish the presence or absence of malignancy in serous effusions. They point out that the sensitivity of DNA image cytometry in equivocal effusions may be as high as 87.5%, and that for the detection of malignancy, DNA image cytometry is superior to flow cytometry.     

Analysis of rare high-DNA cell populations in serous effusions using continuous-motion imaging

The rare high-DNA cell sub-populations in a series of serous effusion specimens were analysed to determine whether such measurements could provide a basis for the improved diagnosis of malignancy. Monolayer specimens stained with gallocyanin chrome-alum were scanned with the CERVIFIP continuous-motion image analyser to locate and measure the highest-DNA cells in the sample. Two types of features were obtained for the detected sub-populations; firstly, 'percentile ploidy' values which characterise the ploidy levels above which specified proportions of the total cells are found; and secondly 'percentage abnormal' values which characterise the proportion of the cells diagnosed as malignant during examination by a cytopathologist. The classification accuracy for one or both of these features was then obtained by comparison with the clinical outcome of each patient. The results gave a classification error of 9/44 (20%) using the 0.01% percentile ploidy alone, 6/44 (14%) using the 75% percentage abnormal feature alone, but only 2/44 (5%) from a box discriminant using both features. It was therefore concluded that the analysis of the high-DNA cell population could be of value in the diagnosis of malignancy in serous effusion specimens.     

High-performance capillary electrophoretic analysis of hyaluronan in effusions from human malignant mesothelioma

A procedure to quantify hyaluronan in effusions from human malignant mesothelioma using a highly sensitive and reproducible high-performance capillary electrophoresis (HPCE) method is presented. Following ethanol precipitation, hyaluronan and galactosaminoglycans were degraded to Δ^4.5-dissacharides with a mixture of chondroitinases ABC and AC. Heparan sulphate and proteins/glycoproteins were separated by ultrafiltration on a Centricon 3 membrane, and hyaluronan-derived disaccharides were analysed by direct injection of the filtrate into a HPCE system. Determination of hyaluronan in effusions from five healthy individuals and three patients with mesothelioma gave values comparable to those found using the HPLC method. One of the advantages of the HPCE method as compared to HPLC is the low solvent consumption. The much lower detection limit (attomole level) of the HPCE method may also allow the analysis of hyaluronan content in serum. The contribution of HPCE in diagnosis of a neoplasm, such as human malignant mesothelioma, illustrates the great potential of this technique in the field of life sciences.     

Image analysis of mesothelioma. II. Discrimination of mesothelioma from metastatic serous ovarian adenocarcinoma

OBJECTIVE: To examine the utility of karyometric measurements in the differentiation of mesothelioma from metastatic serous ovarian adenocarcinoma. STUDY DESIGN: High-resolution images of 1,631 nuclei from 32 cases of mesothelioma and 742 nuclei from 15 cases of ovarian adenocarcinoma were recorded. A stepwise discriminant analysis and nonparametric classifier were applied. RESULTS: Nuclei from these two diagnostic categories appear very similar and occupy feature space with significant overlap. A nonparametric classification procedure provided acceptable correct classification. CONCLUSION: For certain regions in feature space, cases could be unequivocally classified.     

Monoclonal antibodies in the cytodiagnosis of serous effusions

In order to assess the value of immunocytochemical staining as a method of discriminating between benign reactive mesothelial cells and malignant epithelial cells in serous effusions, we have studied the reactions of a panel of commercially available antibodies on cells harvested from 83 pleural and peritoneal fluids and compared the results with the clinical and cytological diagnoses. The antibodies used were raised against cytokeratin (PKK1), epithelial membrane antigen (EMA), carcino-embryonic antigen (CEA), pregnancy specific B1-glycoprotein (SP1) and leucocyte common antigen (LCA). Anti-CEA was positive in 16 of 39 effusions (41%) containing carcinoma cells. Pregnancy specific B1-glycoprotein (SP1) was positive in 33% of the same samples. Mesothelial cells did not stain with these antibodies. Thus anti-CEA and SP1 can be used to discriminate between benign mesothelial and malignant epithelial cells in effusions. Anti-PKK1 stained both benign reactive mesothelial cells and malignant epithelial cells and cannot be used to discriminate between these two cell types. Strong positive staining of malignant cells was noted with anti-EMA. However, as occasional weak staining of mesothelial cells was also noted, strong staining with this antibody may be regarded as suspicious but not conclusive of malignancy.     

Utility of HBME-1 immunostaining in serous effusions

Utility of HBME-1 immunostaining in serous effusions
HBME-1 is an anti-mesothelial cell monoclonal antibody derived from human mesothelioma cells. We investigated 227 body cavity effusions to test its utility in differentiating mesothelioma from adenocarcinoma. HBME-1 outlined cell membranes in non-neoplastic mesothelial cells. Thick surface staining was observed on all mesotheliomas. HBME-1 reactivity was also detected in 24% of metastatic carcinomatous effusions. Most ovarian carcinomas (83%) reacted with this antibody, showing surface staining. Cytoplasmic HBME-1 immunoreactivity was observed in a small proportion of non-ovarian adenocarcinomas (14%). Despite its limited specificity, HBME-1 might be added to the battery of other markers of epithelial and/or mesothelial differentiation to be used in cases of suspected mesothelioma. Evaluation of suspicious cells should include careful study of the pattern of immunostaining.     

Immunocytochemical study of malignant lymphoma in serous effusions

OBJECTIVE: To cytologically evaluate a large series of serous effusions associated with malignant lymphoma (ML), identify the immunoreaction patterns of the cells from selected positive cases and to investigate the correlation of cytomorphology with tissue section diagnosis. STUDY DESIGN: From 1966 to 1990, a review of the files of the Department of Anatomic Pathology, A. C. Camargo Hospital, disclosed 4,297 cases of serous effusions, 256 of which were associated with ML. Cytopathologically positive cases were selected for immunocytochemical study. All paraffin sections stained with hematoxylin and eosin were reviewed to confirm the malignancy of the cases. Immunostaining was performed on both cytocentrifuge slides previously stained with Papanicolaou stain and new sections of the biopsy samples using the immunoperoxidase method and avidin-biotin complex with monoclonal mouse antihuman B-cell marker L-26 and monoclonal (mouse) antihuman T-cell marker UCHL-1. RESULTS: Immunocytochemical reactions were performed in 54 cases: 22 were pan-B positive and 10 pan-T positive; 24 cases showed no reactivity for either monoclonal antibody. Immunohistochemical reactions were performed in 24 available cases: 15 were pan-B positive and pan-T positive; 3 cases showed no reactivity for either monoclonal antibody. Cytohistoimmunoreactions were similar in 11 pan-B positive cases and 2 pan-T positive cases. Three cases were negative for both markers, 4 cases were pan-T positive in tissue samples and negative in cytocentrifuge smears, 3 cases were pan-B positive in tissue and negative on cytology and 3 cases were negative for both markers in both tissue and cytologic specimens. CONCLUSION: Cytology is an effective method of evaluating serous effusions associated with malignant lymphoma: no false positive diagnosis was observed in this series. Immunophenotyping of lymphoid cells is useful to classify and confirm the cytologic diagnosis.     

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.     

Rapid diagnosis of malignant pleural mesothelioma and its discrimination from lung cancer and benign exudative effusions using blood serum

Malignant pleural mesothelioma (MPM), an aggressive cancer associated with exposure to fibrous minerals, can only be diagnosed in the advanced stage because its early symptoms are also connected with other respiratory diseases. Hence, understanding the molecular mechanism and the discrimination of MPM from other lung diseases at an early stage is important to apply effective treatment strategies and for the increase in survival rate. This study aims to develop a new approach for characterization and diagnosis of MPM among lung diseases from serum by Fourier transform infrared spectroscopy (FTIR) coupled with multivariate analysis. The detailed spectral characterization studies indicated the changes in lipid biosynthesis and nucleic acids levels in the malignant serum samples. Furthermore, the results showed that healthy, benign exudative effusion, lung cancer, and MPM groups were successfully separated from each other by applying principal component analysis (PCA), support vector machine (SVM), and especially linear discriminant analysis (LDA) to infrared spectra.     

Cytologic presentation of malignant mesothelioma in pleural effusions

1,601 pleural effusions were found to be malignant between 1976 and 1987. Among these were 26 (1.6% of the malignant effusions) mesothelioma. Only 2 cases showed pronounced cytologic features that made a definite diagnosis possible on cytologic criteria alone. In 20 cases diagnosis of mesothelioma was strongly suggested by the patient's history and cytology of the effusion was compatible with mesothelioma. In the other 4 cases special examinations (histo- and immunohistochemistry, electron microscopy) led to the final diagnosis. The cytologic features of mesothelioma and other examination techniques, needed to resolve the differential diagnosis of mesothelioma versus other neoplasm in pleural effusions, are discussed.     

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.     

Diagnostic utility of BCA-225 in detecting adenocarcinoma in serous effusions

OBJECTIVE: To determine the diagnostic value of the BCA-225 antibody in discriminating adenocarcinoma from benign mesothelium in body cavity effusions. STUDY DESIGN: One hundred four cases of unequivocally benign (34 cases) and malignant (70 cases) serous effusions with cell block material were immunostained for BCA-225 using the ABC method without antigen retrieval. The percentage of positively staining cells in each case was estimated in a blind fashion. RESULTS: BCA-225 stained at least 10% of morphologically malignant cells in 28 of 32 (88%) breast carcinomas and 58 of 67 (87%) adenocarcinomas overall. Neuroendocrine carcinomas (two cases) and one mesothelioma were positive in < or = 5% of their respective tumor cells. Of 34 benign cases, 6 (18%) exhibited positive staining, albeit in rare, morphologically benign cells. CONCLUSION: BCA-225 is able to discriminate adenocarcinoma from reactive mesothelium in cell block preparations and may prove useful as part of an antibody panel.