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.     

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.     

Lectin microarrays differentiate carcinoma cells from reactive mesothelial cells in pleural effusions

The aim of this study was to evaluate the diagnostic utility of lectin microarrays in pleural effusions of patients with lung cancer. A lectin microarray, LTL, PSA, LCA, UEA-1, AAL, MAL-I, MAL-II, SNA, WGA, ECL, DSA, STL, SWGA, HPA, ConA, GNA, HHL, BPL, EEL, Jacalin, WFA, ACL, MPL, DBA, SBA, was used to determine the glycoprotein profile of cells in pleural effusions from patients with lung cancer (54 cases), and with benign lung disease (54 cases). The A549 cell line, used as an experimental control, was positive for AAL, MAL-I, WGA, STL, Jacalin and ACL binding. Adenocarcinoma cells in pleural effusions were positive for ECL, DSA, AAL, MAL-I, WGA, STL, Jacalin, and ACL binding. AAL, WGA, and ACL positive binding was the most common, found in 54, 48, and 38 samples, respectively. ECL and DSA binding was positive in only 4 samples. In comparison, reactive mesothelial cells displayed positive binding for all markers in the microarray panel. SNA and AAL positive binding was detected in the majority of samples; 50/54 and 48/54 samples, respectively. Positive binding of DBA, MAL-II and EEL was present in only 2, 4 and 4 samples, respectively. SNA binding had the highest sensitivity (92.6 %), specificity (100 %), and accuracy (96.3 %). SNA may be used as a biomarker to distinguish reactive mesothelial cells from adenocarcinoma cells. The lectin microarrays proved able to distinguish carcinoma cells from reactive mesothelial cells in pleural effusions.     

Immunocytochemical panel for distinguishing carcinoma cells from reactive mesothelial cells in pleural effusions

Objective:  The aim of this study was to evaluate the individual and combined diagnostic utility of carcinoembryonic antigen (CEA), cytokeratin 19 fragments (CK19) and HBME-1 in pleural effusions of patients with lung cancer.
Study design:  CEA, CK19 and HBME-1 were detected by immunocytochemistry in pleural effusions from patients with lung cancer (86 cases) and without lung cancer (40 cases).
Results:  CEA and CK19 expression were significantly higher in the carcinoma cell group and in three subgrouped as adenocarcinoma (AC), squamous cell carcinoma (SCC) and small cell lung cancer than in the mesothelial cell group, whereas HBME-1 expression was lower in the former group ( P <  0.01). In the subgrouped tumours, CEA expression was higher in AC than in SCC ( P <  0.05), whereas HBME-1 expression was higher in SCC than in AC ( P <  0.01). Used alone, CK19 had the highest sensitivity (95.3%) and accuracy (93.7%), whereas CEA had the highest specificity (97.5%). When combinations of antibodies were evaluated together and membrane staining with HBME-1 taken as a negative outcome, CK19 and HBME-1 gave a high diagnostic performance: sensitivity of 100.0% and accuracy of 95.2% respectively.
Conclusion:  A panel of CEA, CK19 and HBME-1 monoclonal antibodies proved to be suitable for distinguishing carcinoma cells from reactive mesothelial cells in pleural effusions.     

Distinction between carcinoma cells and mesothelial cells in serous effusions. Usefulness of immunohistochemistry

The usefulness of an immunoperoxidase battery to distinguish carcinomatous from benign effusions was examined. Cell block sections from 90 previously diagnosed effusions were stained with antibodies to Leu-M1, B72.3, epithelial membrane antigen (EMA), carcinoembryonic antigen (CEA) and vimentin. The 90 cases comprised 69 carcinomas (23 mammary, 16 ovarian, 10 pulmonary, 7 gastrointestinal [GI] and 13 others), 2 malignant mesotheliomas and 19 cases with reactive mesothelial cells only. EMA and vimentin were the most useful markers for distinguishing carcinoma cells from reactive mesothelial cells. EMA reacted with 86% of the carcinomas while vimentin reacted with 90% of the reactive cases. Leu-M1, B72.3 and CEA, although generally less sensitive than EMA, were also helpful in this regard. Additionally, the use of Leu-M1 and CEA together may help to distinguish pulmonary from GI carcinomas.     

Ribonucleotide reductase immunoreactivity in adenocarcinoma cells and malignant or reactive mesothelial cells in serous effusions

OBJECTIVE: Ribonucleotide reductase (RNR) is a cytoplasmic enzyme that is essential for DNA synthesis. Its activity is strongly associated with cell proliferation. We assessed the value of immunostaining for RNR in distinguishing between reactive mesothelia (RM), malignant mesotheliomas (MM) and adenocarcinomas (AC) in serous effusions. STUDY DESIGN: Cytocentrifuged cell smears of serous effusions from 38 RM, 10 MM and 36 AC were immunostained with the monoclonal antibody KM1054 raised against the R2 subunit of RNR (RNR-R2) using the labeled streptavidin-biotin method. Quantitative RNR-R2 values were determined by counting the percentages of immunoreactive cells. RESULTS: RNR-R2 immunostaining was confined to the cytoplasm. The median RNR-R2 value was 1.4% (range, 0-7.9%) for RM, 11.2% (4.1-15.3%) for MM and 12.1% (2.0-40.6%) for AC. Significant differences in RNR-R2 values were found for both AC versus RM (P < .001) and MM versus RM (P = .009). There was no difference between AC and MM (P = .26). An RNR-R2 value > or = 7% was found in 30 of 36 AC, 8 of 10 MM and 2 of 38 RM. CONCLUSION: RNR-R2 immunostaining can be useful as an adjunct for differentiating AC or MM from RM in serous effusions.     

Distribution pattern of concanavalin A on carcinoma cells, histiocytes and mesothelial cells from effusions

Fixed and unfixed cancer cells, mesothelial cells and histiocytes were exposed to fluorescein-labeled concanavalin A (ConA-FITC). Cancer cells, whether fixed or unfixed, showed a similar pattern of fluorescence, as a continuous layer over the whole periphery of the cell. This pattern of ConA-FITC distribution was also obtained on fixed mesothelial cells and fixed histiocytes. Redistribution of ConA-FITC in form of "caps" and "patches" was recorded on unfixed mesothelial cells and unfixed histiocytes. Of the three cell types studied, only the histiocytes were lysed by the incubation with ConA-FITC.     

Light and electron microscopic characteristics of signet-ring adenocarcinoma cells in serous effusions and their distinction from mesothelial cells

Signet-ring adenocarcinoma cells in serous fluids have been classically described as possessing vacuolated cytoplasm and eccentrically placed, crescent-shaped nuclei. We studied serous fluids from six patients that contained signet-ring adenocarcinoma cells by light microscopy; one case was also studied by transmission electron microscopy. We found that the adenocarcinoma cells were more often present in a non-signet-ring configuration. The typical crescent-shaped nucleus was rarely displayed in smears and may be seen only in the cell-block preparation. Special stains (PAS, mucicarmine and Diff-Quick) showed globular cytoplasmic positivity in signet-ring adenocarcinoma cells but not in mesothelial cells. Significant characteristic electron microscopic findings in the signet-ring adenocarcinoma cells included (1) cytoplasmic lumens or invaginations or both, (2) cytoplasmic protrusions and (3) mucin granules of various sizes and densities. Singly or in combination, all of the above features were located on one side of the nucleus, which suggests that signet-ring adenocarcinoma cells retain some degree of cellular polarity.     

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.     

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.     

Surface configuration of mesothelial cells in effusions. A comparative light microscopic and scanning electron microscopic study.

Surface configuration of mesothelial cells identified by light microscopy (LM) has been studied by scanning electron microscopy (SEM). It has been shown that mesothelial cells may have a variable SEM appearance. The surfaces of a small proportion of mesothelial cells are covered by regular microvilli (MV) and show openings of the pinocytotic vesicles. The surfaces of the majority of these cells are covered by vesicles or blebs. An intermediate population of mesothelial cells, i.e., cells displaying side-by-side blebs and MV, has also been observed. The latter cells no longer display pinocytotic vesicles. Occasional mesothelial cells have smooth surfaces. It has been shown by LM and transmission electron microscopy that cells with blebs are viable and capable of mitotic activity. It is concluded that mesothelial cells, detached from their epithelial setting, lose microvilli and pinocytotic vesicles and acquire surface blebs. The possible relationship between mesothelial cells and macrophages based on surface features has been discussed.     

Diagnostic utility of sialosyl-Tn in discriminating carcinomatous cells from benign mesothelium in body cavity effusions

OBJECTIVE: Detecting malignant cells in the setting of reactive mesothelium can be difficult. Several techniques have been tried but without widespread acceptance. Sialosyl-Tn (STn) is an aberrantly glycosylated precursor of the MN blood group antigen frequently expressed in carcinomas and dysplastic epithelium. We investigated the STn monoclonal antibody for its clinical utility as an isolated stain to discriminate benign mesothelium from malignant cells. STUDY DESIGN: Cell block material from 72 cases of body cavity fluids were immunostained for STn using the avidin-biotin complex method without antigen retrieval. Slides were incubated overnight at 4 degrees C in a humidified chamber. RESULTS: Strong immunoreactivity was noted in 31/40 (77%) carcinomatous cases. Only moderate staining was noted in 1 of 28 (4%) benign effusions and weak staining in 5 (18%) additional benign cases. Specificity was 100%, sensitivity 78%, positive predictive value 100% and negative predictive value 76%. No staining was noted in four noncarcinomatous malignant effusions. CONCLUSION: STn may have diagnostic value in this cytologic setting as part of a diagnostic panel but not as an isolated stain.     

Hyperdiploidy and apparent aneusomy in mesothelial cells from non-malignant effusions as detected by fluorescence in situ hybridization (FISH)

Interphase cytogenetics by fluorescence in situ hybridization (FISH) can be used to detect malignant cells characterized by chromosomal aneuploidy. However, apparent aneusomy in normal "control" tissues has to be considered when using FISH as diagnostic tool. In effusions as model tissue exposed to metastasis, the definition of cut-off levels for background aneusomy by FISH was aimed in this study. Using centromeric probes representing chromosomes 7, 8, 11, 12, 17 and 18, extensive chromosome copy number enumeration by single-color FISH analysis was performed in pleural and ascitic effusions derived from 15 patients with various, non-malignant diseases. In all effusions, cells with gain of hybridization signals for several or all chromosomes tested were found (in up to 1.94% of cells). A consistent finding was high grade hyperdiploidy (>4 centromeric signals). Mesothelial elements mainly contributed to hyperdiploidy in effusions, as demonstrated by a combined analysis of FISH and immunocytochemistry with staining for cytokeratin. Dual-color FISH analysis showed that hyperdiploidy was predominantly corresponding to polyploidization; however, there were always minor cell populations classified as aneuploid by dual-color FISH. In conclusion, stringent criteria have to be applied to distinguish malignancy-related aneuploidy from background aneusomy by FISH.     

Immunohistochemical differentiation of malignant mesothelioma, mesothelial hyperplasia and metastatic adenocarcinoma in serous effusions, utilizing staining for carcinoembryonic antigen, keratin and vimentin

The cytologic diagnosis of malignant mesothelioma and its distinction from mesothelial hyperplasia and metastatic adenocarcinoma is consistently difficult; tissue studies utilizing the immunohistochemical profiles of carcinoembryonic antigen (CEA) and keratin have demonstrated a reproducible distinction between these tumors. Mesothelium contains vimentin in addition to keratin, but its characterization is hindered by its poor preservation in formalin fixatives; alcohol fixation is far superior. Alcohol-fixed, Papanicolaou-stained smears of serous fluids from five cases of reactive mesothelium, five metastatic adenocarcinomas and five malignant mesotheliomas were stained with polyclonal CEA, antikeratin monoclonals AE1 and AE3 (combined) and monoclonal vimentin utilizing the peroxidase-antiperoxidase method. The study revealed the excellent preservation of mesothelial vimentin staining in all three groups. The reactive mesothelium and mesothelioma groups were strongly positive for vimentin and keratin whereas the metastatic adenocarcinoma group was only positive for keratin and CEA (except one case). These findings support the results of previous tissue studies, disclosing CEA staining in the metastatic adenocarcinomas, but not in the mesotheliomas, and the inability of keratin staining to distinguish between the two. The findings also emphasize that positive vimentin staining will usually exclude a metastatic adenocarcinoma, but will not distinguish between neoplastic and reactive mesothelial states.