Quantitative ultrastructural study of nuclei from exfoliated benign and malignant mesothelial cells and metastatic adenocarcinoma cells

Various approaches, including morphometric image analysis, are currently being used to improve the distinction between diffuse mesothelioma and metastatic adenocarcinoma of the serous membranes. Since exfoliated cells of malignant mesotheliomas were thought to have nuclear profile contours with greater irregularity than the similar profiles in metastatic adenocarcinoma cells in pleural effusions, this and other nuclear parameters were measured in ultrastructurally examined preparations from three cases of reactive mesothelial hyperplasia, seven examples of diffuse mesothelioma and three cases of metastatic adenocarcinoma (with primaries in the ovary, esophagus and prostate). Contrary to the subjective impression, the nuclei in metastatic adenocarcinomas actually had a mean nuclear contour index greater than that found in diffuse mesotheliomas; statistically, the difference was not significant. Likewise, such other nuclear parameters as nuclear area, condensed chromatin area and contour index, percentage of condensed chromatin and number of condensed chromatin clumps per nuclear profile did not discriminate between malignant mesotheliomas and adenocarcinomas metastatic to pleural surfaces. These morphometric results quantitate the similarities in nuclear size, nuclear shape and condensed chromatin arrangement in these two types of tumor and explain why the cytopathologist has such great difficulty in distinguishing between exfoliated mesothelioma and adenocarcinoma cells in most cases.     

Cytophotometric determination of DNA in mesotheliomas and reactive mesothelial cells

Cytophotometry was used to study the nuclear DNA content of cells in Feulgen-stained effusion specimens from 18 patients with mesothelioma and 14 patients with reactive mesothelial proliferations. The mean DNA content (MDNA) of mesothelioma cells was significantly higher than that of reactive mesothelial cells (P less than .001). Other parameters reflecting the DNA content also differed significantly between the two kinds of cells, including (1) the ratio of mean mesothelial DNA to mean lymphocyte DNA, (2) the percentage of mesothelial cells with DNA content exceeding three times the lymphocyte MDNA and (3) the coefficient of variation of the DNA content. Since these parameters were highly correlated, only one was accepted in a stepwise linear discriminant model for distinguishing reactive from mesotheliomatous effusions. The model correctly classified all of the reactive effusions studied and 89% of the mesotheliomatous effusions. These results indicate that DNA analysis, using the Feulgen stain and cytophotometry, yields criteria that may be useful in distinguishing benign reactive mesothelial cells from malignant mesothelioma in effusions when used in conjunction with other traditional parameters.     

Appearance of mesothelioma cells in sputum. A case report.

A case in which mesothelioma cells appeared in sputum is reported. There has been only one earlier reported case of the exfoliation of mesothelioma cells into sputum. A postmortem examination in our case disclosed a lepidic growth of mesothelioma cells along the alveolar wall of the lung, suggesting that this mode of spread in the lung was probably responsible for the shedding of tumor cells in sputum. Cytopathologists should be aware of this possibility in differentiating this entity from adenocarcinoma involving the pleura.     

Ultrastructure of pleural mesothelioma and pulmonary adenocarcinoma in malignant effusions as compared with reactive mesothelial cells.

OBJECTIVE: To determine the ultrastructural features of diffuse malignant pleural mesothelioma cells in cytologic specimens from pleural effusions. STUDY DESIGN: We retrospectively studied 35 pleural effusions: 12 diffuse malignant pleural mesotheliomas (8 epithelial type, 4 biphasic type), 12 pulmonary adenocarcinomas and 11 cases of reactive mesothelial cells. RESULTS: In the cytoplasm, reactive and malignant mesothelial cells had more-abundant intermediate filaments (P < .05, P < .01) and fewer free ribosomes (P < .001, P < .001) than adenocarcinoma cells. Reactive mesothelial cells had fewer mitochondria than mesothelioma cells (P < .05). Mesothelioma cells had longer, thinner microvilli on the cell surfaces (P < .001); length/diameter ratios of microvilli were 19.1 +/- 7.0 (mesothelioma) vs. 9.1 +/- 2.2 (adenocarcinoma) and 9.2 +/- 2.4 (mesothelial cells). Giant intercellular junctions (desmosomes or desmosomelike structures > 1 micron in length) were found in eight cases of mesothelioma. Core filaments or rootlets in microvilli were present in two cases of adenocarcinoma. CONCLUSION: Because cytologic specimens from pleural effusions were easy to obtain, we think ultrastructural cytology is useful in distinguishing mesothelioma from adenocarcinoma and benign effusions.     

Distribution of silver-stained interphase nucleolar organizer regions as a parameter to distinguish neoplastic from nonneoplastic reactive cells in human effusions

The distribution of interphasic nucleolar organizer regions (NORs) was studied in cytologic preparations of human serous effusions in order to differentiate malignant cells from nonmalignant reactive cells. The study was carried out on 80 cases of metastatic adenocarcinoma, 10 cases of mesothelioma, 10 reactive pleural effusions and 5 peritoneal washings. Visualization of NORs at the light microscopic level was obtained using a silver-staining technique for acidic proteins selectively associated with NORs. The morphologic data were also statistically evaluated by means of an automated image analyzer. The quantity of silver-stained NORs was higher in cancer cells (both mesothelioma and adenocarcinoma) than in reactive mesothelial cells. Moreover, NORs were more irregularly distributed within the nucleoli and were more variably sized in cancer cells than in reactive mesothelial cells.     

Nuclear chromatin texture and sensitivity to DNase I in human leukaemic CEM cells incubated with nanomolar okadaic acid

It is now known that the analysis of chromatin texture can be used in oncology as a sensitive detection method, either to define diagnostic classifications or to locate a lesion along a defined trend curve. However, the functional significance of these variations in textural features remains sometimes unclear. Several drugs have been shown to be able to modulate chromatin structure. Among them, the phosphatase inhibitor okadaic acid at low concentration can increase accessibility to DNA in chromatin of carcinoma cells. This paper demonstrates that short exposures (0–3h) to a 10-nM dose of okadaic acid induced an increased sensitivity to DNase I digestion in human CEM leukaemic cell nuclei and that this sensitization was associated to variations of nuclear texture characteristics, as evaluated by image cytometry. CEM cells treated with okadaic acid for 0–3h displayed changes in chromatin supraorganization with a more homogeneous and fine chromatin texture, as compared to control cells. This suggests that the appearance of an open configuration of chromatin structure as evaluated by biochemical methods corresponds to a more decondensed texture of nuclei measured by image cytometry. Longer exposures (6–24h) of CEM cells to 10nM okadaic acid lead to apoptosis. As reported previously for camptothecin-treated HL60 cells, okadaic acid-treated CEM cells display biphasic nuclear chromatin texture changes, i.e. a decondensation phase followed by the appearance of typical apoptotic cells with a smaller nuclear area and a highly condensed chromatin. Finally, using the multidrug-resistant CEM-VLB cell line, it was confirmed that these multidrug-resistant cells also display cross-resistance to okadaic acid, as this compound was unable to induce either increased DNase I sensitivity, apoptosis, or altered nuclear texture in this particular cell line.     

Computerized morphometric quantitation of cellular features in splenocytes from normal and nutrient-restricted mice

The morphometric analysis of splenocytes reveals quantitative changes in the chromatin and cytoplasm that may be used to distinguish between cells from normally fed animals and cells from subjects fed restricted diets. Analysis of Feulgen-stained and Papanicolaou-stained cells from mice fed normal diets and mice fed either calorie-restricted diets or isocaloric but protein-restricted diets showed an approximately 10% reduction in the nuclear area and in the total optical density (TOD) of stained chromatin in cells from diet-restricted mice. Some changes in chromatin texture features were also observed. Utilization of nuclear area, TOD and one textural feature in a linear discriminant analysis produced a distinct separation of the cells from dietary-restricted mice and the cells from normally fed subjects; this was observed with both Feulgen-stained and Papanicolaou-stained cells. The cellular effect of dietary restriction was more noticeable in the cytoplasm than in the nucleoplasm; Papanicolaou-stained cells from diet-restricted animals showed a 23% reduction in the cytoplasmic TOD and a 10% reduction in the nuclear TOD. This study shows that computerized morphometric analysis may be used in place of or in conjunction with other measurement procedures and chemical tests to quantitate and differentiate cells subjected to different types and levels of nutritional stress.     

Application of morphometric methods to the cytologic study of intradermal nevi.

Twenty-one intradermal nevi were studied by morphometric methods in an attempt to morphologically characterize the two types of nevus cell--epithelioids, type A, and fusiforms, type C--and to quantify the differences between them. Morphometric parameters of the intradermal nevi were compared with similar parameters of melanocytes and melanoma cells so that the maturation rates of the nevi cells could be established and to see if the parameters might indicate the degree of malignancy. Superficial nevus cells were differentiated from deep cells by their larger size and larger nuclear area. Nuclear area appeared to have potential for differentiating benign from malignant tumors. Decrease in cellular area appeared to indicate maturation rather than atrophy. Melanoma cells were differentiated by their larger size. Cell nuclear perimeter appeared to have confirmatory value, while cell perimeter was inconclusive.     

Quantitative and qualitative differences between benign and malignant mesothelial cells in pleural fluid

Quantitative as well as qualitative cellular parameters were investigated in 20 cases of reactive mesothelial proliferations and 40 cases of primary pleural mesotheliomas. The two groups showed statistically significant differences in the nuclear areas, cytoplasmic areas and standard deviations. In the mesotheliomas, the mean nuclear area and the mean cytoplasmic area were larger than in the reactive proliferations. Nine cases could not be properly classified with these quantitative parameters alone. Qualitative analysis revealed highly characteristic features in the mesotheliomas. Morula formation as well as irregular and coarse reticular chromatin patterns were strongly indicative of mesothelioma. The location and shape of the nucleus and the type and amount of cytoplasmic vacuolization gave additional information for distinguishing between the two groups.     

Significance of immunocytochemical expression of E-cadherin, N-cadherin and CD44 in serous effusions using liquid-based cytology

OBJECTIVE: To assess the diagnostic utility of E-cadherin (E-cad), N-cadherin (N-cad) and CD44 to discriminate adenocarcinoma cells from benign and malignant mesothelial cells in body cavity fluids and to clarify the origin of cancer cells. STUDY DESIGN: A total of 120 ThinPrep (Cytyc Corp., Boxborough, Massachusetts, U.S.A.) cytologic specimens of serous effusions, which included 22 cases of reactive mesothelium, 6 cases of malignant mesothelioma and 92 cases of metastatic adenocarcinoma from various sites, were immunostained for E-cad, N-cad and CD44. RESULTS: Eighty-three of 92 metastatic adenocarcinomas (90.21%) expressed E-cad, while 1 of 6 malignant mesotheliomas and 1 of 22 cases of reactive mesothelium were positive for E-cad. All 6 cases of mesothelioma expressed N-cad, whereas most cases of metastatic adenocarcinomas were negative. CD44 immunoreactivity was seen in 18 of 22 (81.81%) benign effusions and in 21 of 92 (22.82%) metastatic adenocarcinomas. CONCLUSION: The combination of E-cad, N-cad and CD44 appears to be a useful panel for distinguishing metastatic adenocarcinoma, mesothelioma and reactive mesothelium and also for clarifying the exact histogenetic origin of cancer cells. This is of great importance in a few otherwise-insoluble cases because of differences in tumor treatment and prognosis.     

Study of chromatin structure in ataxia-telangiectasia cells

Micrococcal nuclease was used as a probe to study chromatin structure in control and ataxia-telangiectasia cells. The rate and extent of release of acid-soluble nucleotide was similar in both cell types. Production of mono- and oligonucleosomes by micrococcal nuclease as determined by gel electrophoresis also failed to reveal differences in chromatin structure between control and ataxia-telangiectasia cells. Radiation exposure did not significantly alter the kinetics of digestion. These results indicate that there are no gross alterations in chromatin structure in ataxia-telangiectasia cells.     

Computer-aided image analysis for the differentiation of mononuclear cells in peripheral blood smears from leukemic patients

Different leukemias originating from the lymphatic system and from the myeloid tissue of the bone marrow were selected to develop and test algorithms to cytophotometrically define leukemic cells. Two of these cases were of low-grade, one of intermediate-grade and one of high-grade malignancy. The malignant cell types of each leukemic form could be identified and characterized by assessing the chromatin texture of the nucleus in the leukemic cells. With this method, normal peripheral white blood cells could be differentiated from leukemic ones. The results correlate directly with established hematologic parameters as they are described in the literature. The investigation demonstrated the usefulness of computer methods in the differentiation of closely related cell structures.     

Discrimination of osteoarthritic and rheumatoid human synovial cells in culture by nuclear image analysis.

Rheumatoid arthritic (RA) and osteoarthritic (OA) synovial cells in culture differ in their metabolic and proliferative behaviour. To assess links between these properties and nuclear changes, we used image analysis to study chromatin texture, together with nuclear morphometry and densitometry of OA and RA cells in primary culture. Chromatin pattern at the third day (D3) was heterogeneous and granular with chromatin clumps whereas at the final stage (D11) of culture a homogeneous and finely granular chromatin texture was observed. This evolution indicates global chromatin decondensation. These characteristics were more marked for RA than for OA nuclei. At each culture time, RA nuclei could be discriminated with high confidence from OA ones from parameters evaluating the organization of the chromatine texture. Nuclear image analysis is thus a useful tool for investigating synovial cell biology.     

Cytoskeletal proteins as tissue-specific markers in cytopathology

Antibodies to intermediate filament proteins react in a tissue-specific manner and can be used to characterize tumor cells present in thin-needle aspirates from solid tumors, from palpable lymph nodes and cells present in samples from peritoneal and pleural effusions. From our studies so far the following conclusions can be drawn: Polyclonal antisera to cytokeratins can identify carcinoma metastases in thin-needle aspirates from palpable lymph nodes and distinguish them from malignant lymphomas and nonmalignant lesions such as chronic lymphadenitis, which show only vimentin-positive cells. Monoclonal antibodies to specific cytokeratin polypeptides are able to distinguish between different types of epithelial tumor metastases, i.e. metastases from adenocarcinomas and metastases from squamous cell carcinomas. Cells present in peritoneal and pleural effusions can be partly characterized using intermediate filament antisera. We have found that metastatic adenocarcinoma cells from breast, ovary, endometrium, cervix, colon and stomach, as well as squamous cell carcinomas and malignant mesothelioma stain specifically with antibodies to cytokeratin while mesenchymally derived tumors such as malignant lymphomas, malignant melanomas, and fibrosarcomas, are positive for vimentin only. Metastatic tumor cells of epithelial origin present in aspirates from human serous body cavity fluids may coexpress vimentin next to their original cytokeratin intermediate filaments. Benign mesothelial cells present in body cavity fluids frequently coexpress cytokeratins and vimentin. Tumor cells present in thin-needle aspirates from solid tumors such as pleomorphic adenomas of the parotid gland can be identified as such because of their typical patterns of intermediate filament (co-)expression.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

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.