The distinction of mesothelioma from adenocarcinoma in malignant effusions by electron microscopy

To determine the usefulness of the electron microscopic (EM) differential diagnosis between malignant mesothelioma and metastatic adenocarcinoma in cytologic specimens of serous fluids, we undertook a prospective study of 17 pleural and peritoneal effusions from 14 patients. In the nine effusions identified as malignant by routine cytologic examination, EM correctly diagnosed three mesotheliomas and six adenocarcinomas. EM resolved the differential diagnosis of mesothelioma versus adenocarcinoma in three cases in which routine cytologic examination could not. As with tissue specimens, EM cannot be used to diagnose the malignancy of cytologic specimens; it can, however, reliably identify the origin of cells diagnosed as malignant by routine cytologic examination. We conclude that, when EM is used to evaluate cytologically malignant effusions, it can accurately distinguish mesothelioma from adenocarcinoma. This technique will be diagnostically useful in selected cases and may be helpful in avoiding more invasive procedures as well as delays in diagnosis and therapy.     

Qualitative and quantitative analysis of peritoneal fluids from women with gynecologic diseases. Comparison of cytology and flow cytometry for the detection of malignancy in lavage and ascitic fluids

A prospective study was undertaken to compare flow cytometric (FCM) analysis to conventional cytologic evaluation for the detection of malignant cells in peritoneal fluids (peritoneal lavages and ascitic fluids) from women with gynecologic diseases. The 94 peritoneal fluids analyzed came from 63 cancer patients (with epithelial ovarian carcinomas) and 31 control patients (with benign gynecologic diseases). The FCM DNA histograms were generated using propidium iodide as a DNA fluorochrome. Samples for cytologic analysis were stained with the standard May-Grünwald-Giemsa or Papanicolaou stains. Of the 94 samples, 90 were evaluable cytologically while 70 were suitable for FCM analysis. The sensitivities were 55% for FCM DNA analysis and 80% for cytologic analysis. FCM DNA analysis had a 30% false-positive rate; cytologic analysis produced no false-positive results. These results indicate that there is no advantage in employing FCM analysis instead of conventional cytologic evaluation for the detection of malignant cells in peritoneal fluids from gynecologic cases.     

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.     

Flow cytometric analysis and cytopathology of body cavity fluids

A total of 75 samples of body cavity fluids from 71 patients were analyzed by both flow cytometry (FCM), to detect cells with an abnormal DNA content (aneuploidy), and by conventional cytopathology. Samples included 27 pleural fluids, 35 peritoneal fluids, 11 peritoneal washings and 2 pericardial fluids. For cytologic examination, the samples were prepared using standard techniques. Samples for FCM analysis were centrifuged and exposed to a hypotonic solution containing detergent and propidium iodide, a DNA intercalating fluorescent stain. Aneuploidy as well as cytologic malignancy were found in 17 samples. Forty-seven samples had normal DNA histograms by FCM and were also cytologically negative. Four samples suspicious by cytology but normal by FCM were from patients with renal-cell carcinoma (two samples from the same patient), endometrial adenocarcinoma without metastasis and chronic lymphocytic leukemia. Three samples abnormal by FCM but negative by cytology were from patients with ovarian cystadenoma, cirrhosis and uterine leiomyoma. FCM showed aneuploidy in four cytologically negative samples from patients with histologically proven malignancy (lymphoma, colonic adenocarcinoma, cervical squamous cell carcinoma, and endometrial adenosquamous carcinoma). Based on these results, FCM analysis combined with conventional cytopathology yielded 100% sensitivity, 100% predictive value of a negative result and 94% specificity. This rapid and quantitative FCM analysis of body cavity fluids can be a very useful adjunct to conventional diagnostic cytopathology.     

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.     

Oncofetal antigens as tumor markers in the cytologic diagnosis of effusions

To test the value of oncofetal antigens in the cytologic diagnosis of effusions, immunoperoxidase staining with antisera to carcinoembryonic antigen (CEA), alpha fetoprotein (AFP), pregnancy-specific beta 1-glycoprotein (SP1) and placental alkaline phosphatase (PLAP) was carried out on pleural and peritoneal fluids from 72 cases. Sections of formalin-fixed, paraffin-embedded cell blocks were used in most cases; cytocentrifuge preparations were used in some. Reactions were negative with all antisera in 23 of 24 nonmalignant effusions as well as in all 7 cases of malignant mesothelioma and 4 cases of malignant lymphoma. In 24 of 36 confirmed carcinomatous effusions, staining was positive with one or more antisera, including anti-CEA positivity in 23 of the 24 cases. In 5 of the 24 cases with positive staining, a confident diagnosis of malignancy had not been made on routine cytologic preparations. Immunoperoxidase staining for CEA appears to be of supportive value in the cytologic diagnosis of malignancy in effusions.     

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.     

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.     

Comparison of cytologic and acridine-orange flow-cytometric detection of malignant cells in human body cavity fluids

Flow cytometrically (FCM) derived DNA and RNA profiles were studied in acridine orange (AO)-stained body cavity fluid (BCF) specimens obtained from 78 patients with various solid tissue and hematologic malignancies. The ploidy (DNA index), RNA content (RNA index), proliferative activity (% S + G2M) and DNA and RNA scattergram patterns were tested "double-blind" against the cytologic scoring of specimens as malignant, benign or reactive. It was determined that expression of an "abnormal" RNA index (greater than or equal to 2.8) and an elevated proliferative activity (% S + G2M greater than or equal to 7.4) was dependent on the presence of malignancy; 21 of 22 specimens having those abnormal indices had DNA aneuploidy and were cytologically scored as positive. The AO FCM sensitivity and specificity for detecting malignant cells (when measured against cytology scoring) were 61% and 90%, respectively, using the "abnormal" RNA index and % S + G2M cut-offs together with the cellular DNA aneuploidy marker. By supplementing the cytologic scoring with AO FCM DNA and RNA features, the sensitivity for detecting malignant cells was 94%, as compared to 72% for cytology alone. Two specimens gave false-positive FCM results: a tuberculous effusion with a tetraploid subpopulation and a reactive mesothelial proliferation that was diploid and negative cytologically. Scoring for malignancy based on the visual pattern of the DNA and RNA FCM scattergrams, while showing good correlation for aneuploid specimens, in some cases failed to identify diploid disease. The results demonstrate the usefulness of FCM DNA and RNA analysis for supplementing cytologic examination of BCF specimens for the purpose of detecting malignant cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

An immunoperoxidase study of S-100 protein in neoplastic cells in serous effusions. Use as a marker for melanoma

S-100 protein has been demonstrated on histologic sections in a number of neural and nonneural tissues, including a variety of neoplasms. Since pleural or peritoneal effusions are frequently the initial presentation of cancer, a study was undertaken to determine if S-100 protein in exfoliated cancer cells could be used as a marker for melanoma. Cells in 36 serous fluids obtained from 32 patients were retrospectively examined for S-100 protein by the peroxidase-antiperoxidase technique. All samples had been previously studied as Papanicolaou-stained cytology specimens, and 25 samples had been studied by transmission electron microscopy. All benign effusions were negative for S-100 protein. Malignant effusions were negative except for some that contained malignant melanoma cells: two of five pigmented melanomas and both cases of amelanotic melanoma. This study indicates that S-100 protein in malignant cells is a useful marker for malignant melanomas, especially the amelanotic type.     

Significance of pericellular lacunae in cell blocks of effusions.

A retrospective study was conducted to assess the usefulness of pericellular lacunae in cell block sections of serous effusions in diagnosis. From January to December 1988, 286 cell blocks were prepared in our laboratory from pleural, pericardial and peritoneal fluids; 62 of them were excluded from this study because of inadequate cellularity, diagnostic uncertainty or lack of a proteinaceous background. The remaining consisted of 148 benign effusions from 128 patients and 76 malignant effusions from 56 patients. A single specimen from each patient was selected and reviewed to assess the presence and number of pericellular lacunae and to determine the relationship of this feature to cell arrangement (single cells versus cell clusters). Pericellular lacunae were found in 42 (75%) of the malignant effusions as compared to 41 (32%) of benign specimens. In the majority of malignant cases with lacunae, this feature was associated with greater than two-thirds of the cells, whereas in benign cases, when present, it was seen in less than one-third of the cells. Neoplasms characterized by large cell clusters more frequently had lacunae than did those with small groups or single cells. Lacunae were not evident in cases of malignant melanoma and lymphoma. We conclude that although pericellular lacunae are more often associated with malignant cells, their presence in itself cannot be used as a reliable indicator of malignancy in body cavity fluids.     

Diagnostic interest of the monoclonal antibody CA1 in malignant pleural effusion

The Ca1 antibody was used in an immunohistochemical procedure on smears of cells from 40 patients with malignant pleural effusion. The control group consisted of 25 benign pleural effusions with a high percentage of reactive mesothelial cells. The Ca1 Mc Ab was positive in 19 (79%) of the 24 pleural effusions with positive malignant cytology. In all the benign cases the Ca1 Mc Ab was negative (100% specificity). The Ca1 Mc Ab detected malignant mesothelial cells in two cases and was negative with reactive mesothelial cells and other nucleated cells present in the pleural effusion. We conclude that the Ca1 antibody offers a useful diagnostic method for malignant pleural effusions, when the morphological interpretation is doubtful.     

Bronchial cytology in pleural mesothelioma. A report of 3 positive cases, including 1 diagnosed initially on bronchial brushings

OBJECTIVE: To evaluate retrospectively the value of bronchial aspiration cytology in patients with histologically proven pleural mesothelioma, reappraising positive smears in light of conventional microscopic features and, when feasible, immunocytochemical investigations. STUDY DESIGN: In 3 cases of mesothelioma with bronchial brushings positive for malignant cells, the cytologic features were correlated with the histologic findings. RESULTS: Salient microscopic features included scant to moderate cellularity arranged in micropapillary clusters, morular aggregates with scalloped borders and isolated malignant cells. Intercellular clear spaces or windows suggesting a brush border on cell membranes were also noted. In cases with available material, immunocytochemistry was positive for keratins, epithelial membrane antigen and calretinin and negative for carcinoembryonic antigen. All the cases were histologically confirmed epithelial mesotheliomas. CONCLUSION: In rare cases, pleural mesothelioma cells are shed within the airway lumina and can be detected in bronchoscopic cytology specimens. Cytologic features seem comparable to their analogues in pleural effusions. Although no single criterion appears diagnostic, their combined documentation could ensure correct interpretation, especially if supported by a limited immunocytochemical panel.     

Correlation between the papanicolaou stain and the Wright-Giemsa stain in body fluids: a quality assurance study

OBJECTIVE: To compare the use of Papanicolaou and Wright-Giemsa stains for the evaluation of body fluids in cytology and hematology laboratories and determine whether other factors account for discrepancies in diagnosis. STUDY DESIGN: We retrospectively reviewed cytopathology reports of peritoneal, pleural, and cerebrospinal fluids received by hematology and cytology laboratories for 1 year. Cases were divided into 3 categories-benign, atypical, and malignant--and slides of discrepant diagnoses were reviewed. RESULTS: During this period, 198 of 3212 (0.61%) cases received by the hematology laboratory and 252 of 4402 (0.57%) cases received by the cytology laboratory were diagnosed as malignant or atypical. Of 3212 cases simultaneously received by the cytology and hematology laboratories, 17 diagnosed as malignant by hematology were diagnosed benign by cytology (sensitivity 96%). Sixteen cases diagnosed as malignant by cytology were diagnosed as benign by hematology (sensitivity 97%). No benign cases were diagnosed as malignant (specificity 100%). Review of the glass slides of the discrepant cases revealed 8 cases undercalled by hematology and 7 cases undercalled by cytology. CONCLUSION: Papanicolaou stain is superior for carcinoma and Wright-Giemsa stain for hematopoietic disorders, but used together they may reduce false negative results. Delays in processing, staining technique, and interobserver variability contribute to discrepancy.     

DNA analysis of malignant effusions. Comparison with cytologic diagnosis and carcinoembryonic antigen content.

Twenty-three consecutive malignant effusions from 19 patients submitted for cytologic examination were analyzed for carcinoembryonic antigen (CEA) content and for DNA analysis by flow cytometry. The study was undertaken to determine if the addition of DNA analysis would improve the sensitivity of cytologic diagnosis and CEA assay. CEA examination was performed on Papanicolaou-stained smears and hematoxylin-and-eosin-stained cell blocks. Final diagnoses were correlated with histologic examination (four patients), clinical and radiologic studies, and follow-up. The malignant effusions in 19 patients were secondary to carcinoma of the breast (5), lung (5), ovary (1), endometrium (1), mucinous carcinoma of the colon (1), unknown primary (1), extraovarian papillary carcinoma (1), mesothelioma (2) and large cell lymphoma (2). The sensitivity of cytologic diagnosis was 100% and specificity 100%. DNA aneuploidy, defined as the presence of two separate peaks in the histogram, was present in 7 of 23 fluids (sensitivity, 30%). Four fluids had insufficient cells for analysis, and one histogram showed debris (following chemotherapy). DNA aneuploidy was detected in effusions secondary to carcinoma of the breast (4), lung (1) and lymphoma (2). Using 5 ng/mL as the cutoff, the sensitivity of CEA was 68%. DNA analysis of cells in malignant effusions is less sensitive than cytologic diagnosis, and CEA assay and is not recommended for routine use in the diagnosis of malignant effusions.     

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.     

Early diagnosis of mesothelioma in serous effusions using AgNOR analysis

OBJECTIVE: To compare the results of conventional cytology, DNA image cytometry, immunocytochemistry and argyrophilic nucleolar organizer region (AgNOR) analysis for the diagnosis of malignant cells in serous effusions. STUDY DESIGN: One hundred twenty effusions, 40 with carcinoses, 40 with malignant mesotheliomas and 40 without tumor cells on follow-up were studied by conventional cytology and three adjunctive methods. RESULTS: Unequivocal tumor cells were detected in 92.5% of effusions due to carcinoses and in 45% due to mesotheliomas. Applying immunocytochemistry with BerEP-4 positivity and DNA image cytometry with aneuploidy as a marker revealed 100% of carcinoses and 71.7% of mesotheliomas. Applying the experimentally found thresholds of 2.5 AgNORs as "satellites" and 4.5 AgNORs as "satellites and clusters" together as mean values per nucleus resulted in a 95% correct rate of mesothelioma and 100% rate of carcinoma cell identification without false positive diagnoses. CONCLUSION: AgNOR analysis may be a useful adjunct to other methods in the routine diagnosis of malignant serous effusions. It seems to be the most sensitive method in early cytologic diagnosis of mesotheliomas in effusions. Seventy-three percent of malignant mesotheliomas were diagnosed cytologically at first on effusions. Forty-seven percent of patients with malignant mesotheliomas were identified at the early tumor stage T1 N0 M0.     

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.