Epithelial membrane antigen in the cytodiagnosis of effusions and aspirates: immunocytochemical and ultrastructural localization in benign and malignant cells

Immunoreactivity for epithelial membrane antigen (EMA) was evaluated in exfoliated benign and malignant cells using immunoperoxidase and immunogold techniques. In addition, protein A-colloidal gold was used for ultrastructural localization of EMA. Our results suggest that EMA is useful in distinguishing adenocarcinoma cells (strongly positive) from reactive mesothelial cells (negative or weakly positive), lymphoid cells (negative), and a variety of nonepithelial neoplasms (negative) with which they may be confused. Exfoliated cells from two mesotheliomas were also strongly positive for EMA. Ultrastructurally, EMA was distributed in a dense, even, linear pattern along the cell membrane and microvillous surface processes of adenocarcinoma cells. A similar but sparse distribution pattern was observed in reactive mesothelial cells. These observations reflect the increased sensitivity and higher resolution of the immunogold technique.     

Value of immunocytochemistry in the study of malignant effusions

Recognition of malignant effusion relies heavily on cytologic examination despite the difficulty of distinguishing atypical mesothelial hyperplasia from metastatic carcinoma. The combination of CEA, EMA, vimentin, keratin, high-molecular-weight cytokeratin (HMWK), low-molecular-weight cytokeratin (LMWK), and Alcian blue was tested in 51 cytologic specimens of pleural, peritoneal, and pericardial effusions. These showed metastatic carcinoma in 38 cases (ovary, 14; lung, 8; breast, 7; GI, 4; endometrium, 4; bladder, 1) and mesothelial processes in 13 (hyperplasia, 9; mesothelioma, 4). Strong positivity for EMA (92%), CEA (90%), and Alcian blue (71%) was noted in metastatic carcinoma but not in the mesothelial processes. Keratin was positive in all cases of mesothelioma but occurred also in mesothelial hyperplasias (44%) and metastatic carcinomas (47%). In mesothelial cells, HMWK was consistently stronger than LMWK, whereas in adenocarcinoma the reverse was true. There was no difference in the degree or distribution of positivity of any of the markers among the various primary sites of the neoplasms. Our findings are consistent with the view that immunocytochemistry with a battery of antibodies is useful in the recognition of malignant effusions but cannot, as yet, determine the site of origin of metastatic neoplasms.     

Differential expression of CD44s and CD44v3-10 in adenocarcinoma cells and reactive mesothelial cells in effusions

The detection of malignant cells in serous effusions obtained from patients diagnosed with cancer marks the presence of metastatic disease and is associated with a poor outcome. The purpose of this study was to evaluate the role of CD44s and CD44v isoforms in the distinction between mesothelial cells and malignant epithelial cells in effusions. Fifty-nine fresh pleural and peritoneal effusions were studied. These consisted of 41 specimens from patients with known gynecological neoplasms, 9 from patients diagnosed with breast adenocarcinoma, and 9 effusions from patients with various nongynecological malignancies or tumors of unknown origin. Forty-three effusions contained malignant/atypical epithelial cells, and 16 effusions were diagnosed as reactive. Three effusions contained exclusively malignant cells. Specimens were stained with anti-CD44s, v3, v5, v6, v7 and v3-10. The presence of staining in cancer cells, benign mesothelial cells and lymphocytes was evaluated. CD44s immunoreactivity was seen in 10 of 43 (23%) cases in malignant/atypical epithelial cells and in 53 of 56 (94%) cases in benign cells. In contrast, CD44v3-10 was seen in 23 of 43 (55%) cases in malignant/atypical epithelial cells and in 3 of 56 (6%) cases in benign cells. We advocate the use of CD44s and CD44v3-10 immunostaining in diagnostic evaluation of difficult serous effusions. Received: 30 August 1999 / Accepted: 8 November 1999     

Silver staining of interphase nucleolar organizer regions in cytologic smears previously stained by the Papanicolaou and May-Grünwald-Giemsa techniques.

Malignant cells are known to display a distinctive argyrophilic nucleolar organizer region (Ag-NOR) protein distribution compared with distribution in their benign counterparts. In this study, Ag-NOR staining was applied to smears from reactive and malignant serous effusions. The smears were either unstained or had been previously stained with the Papanicolaou or May-Grünwald-Giemsa (MGG) technique. The purpose of this study was to assess the feasibility and usefulness of Ag-NOR staining in diagnostic cytopathology and to set up a procedure that could be used on prestained smears in retrospective studies. The one-step silver staining method was applied to prestained smears after a weak destaining step in trichloracetic acid and to unstained smears after a postfixation step in Carnoy's fluid. Positive results were observed on both destained and unstained preparations, MGG-stained smears showing a better visualization of the silver deposits than Papanicolaou-stained smears. Major differences in size, shape, and distribution patterns of the Ag-NOR-positive granules were observed between neoplastic and reactive cells. Furthermore, the mean number of silver-stained dots per cell was significantly higher in malignant cells (33.32 +/- 2.23) than in reactive mesothelial cells (9.71 +/- 0.66). These data indicate that this Ag-NOR technique can be profitably applied to prestained cytologic smears to assist in the diagnosis of malignancy and that the technique has the advantage that cellular morphology and silver staining can be evaluated on the same slide.     

Cytopathology of extramedullary hemopoiesis in effusions and peritoneal washings: A report of three cases with immunohistochemical study

The clinical, cytopathologic, and immunohistochemical features of three cases of extramedullary hemopoiesis are presented. Differentiation of megakaryocytes from malignant cells can be difficult in cytologic material; we believe familiarity with cytologic features of megakaryocytes, along with factor VIII-related antigen immunoreactivity, is helpful in providing an accurate diagnosis. Diagn Cytopathol 1986;2:326-329.     

Ultrastructural studies of malignant cells in fluids

An ultrastructural study was performed on 104 sequential fluids in which more than eight malignant cells per ten high-power fields were found by routine light microscopy. The study included fluids associated with mesotheliomas, melanomas, lymphomas, squamous-cell carcinomas, small-cell anaplastic (oat-cell) carcinomas, and adenocarcinomas. Electron microscopic examination reliably separated lymphoid from epithelial malignancies and benign from reactive and malignant mesothelial cell proliferations. It also suggested or identified a primary site for the adenocarcinomas. Ultrastructural examination of fluids can be a valuable adjunct to routine light microscopy of cytology specimens. No false-positive diagnoses were encountered. Sampling was the most significant limitation for this technique.     

Immunocytochemical typification of mesothelial cells in effusions: In vivo and in vitro models

We have performed immunocytochemical, immunoelectron microscopy. Western blot, and culture techniques using monoclonal antibodies against cytokeratin, vimentin, and desmin on 17 benign and 20 malignant effusions of pleural and ascitic origin. Triple coexpression of these three antigens was observed in benign reactive mesothelial cells as well as in one case of mesothelioma. All metastatic adenocarcinoma cells were consistently negative to desmin and positive to cytokeratin and vimentin. Present results were helpful to distinguish reactive and malignant mesothelioma from metastatic carcinoma cells in effusions. © 1994 Wiley-Liss, Inc.     

Morphometric analysis of hyperthermia-induced nuclear and nucleolar size modifications in a human melanoma cell line

Although no prominent morphologic alterations were observed by light microscopy in a human melanoma cell line (M-14) exposed at 42 degrees C for 4 hr, the computerized image analysis demonstrated a significant enlargement of both nuclei and nucleoli. The mean nuclear and nucleolar areas were found to be 32% and 94% increased, respectively, in comparison with normal controls. The gaussian distribution of absolute frequency of the measures demonstrated that this nuclear and nucleolar enlargement was distributed among the whole cell population, taking place in clonogenic cells and in "sterilized" cells, ultimately doomed to die. This finding, not previously described in the context of the heat shock response, may be regarded as morphological evidence of an increase in the protein content of the nuclei of heat-treated cells, as determined by biochemical methods.     

Cytology of pericardial effusions in aids patients

Pericardial effusions in patients with the acquired immunodeficiency syndrome (AIDS) can be due to a variety of causes and are often large enough to be sampled for cytologic examination. Over a period of 46 months, 15 cytologic specimens from 14 patients with AIDS were examined. Thirteen patients were male, one was female; the age range was 26 to 43 years. All male patients were homosexual or intravenous drug abusers, and the female patient was the spouse of an intravenous drug abuser. In general, the cytology specimens were moderately cellular with inflammatory cells seen in all cases. Atypical or reactive mesothelial cells were found in 12 cases (80%), and the atypia in one of these 12 was so marked that carcinoma was suspected; cells suspicious for malignant lymphoma were found in 2 cases (13%); degenerated mesothelial cells were present in one case. No infections were identified in this series. Ten patients (66%) had subsequent pericardial biopsies. Marked cellularity and nuclear pleomorphism in lymphoid cells with an altered nuclear cytoplasmic ratio were the dominant findings in the two suspected lymphoma cases. Both patients had known lymphoma elsewhere; in one, involvement by lymphoma was also found on pericardial biopsy. Mesothelial proliferations showing papillary formations with psammoma bodies were seen in three cases; in one of these, histoplasmosis was later diagnosed by pericardial biopsy. To our knowledge this is the first series to describe cytologically the marked mesothelial atypia seen in pericardial fluid in AIDS patients. We contrast this atypia with that seen in malignant effusions and caution against overinterpretation of pericardial fluids from AIDS patients.     

Utility of cytokeratin 20 in identifying the origin of metastatic carcinomas in effusions

Using a commercially available monoclonal antibody (K_s20.1) and the avidin-biotin peroxidase method on cytospins and cell blocks, we analyzed cytokeratin (CK) 20 expression in 169 serous effusions. Cytoplasmic staining was observed in 44/151 malignant fluids. Colon, gastric, and pancreatic adenocarcinomas and mucinous ovarian tumors were most frequently positive. Single cases of transitional-cell and squamous cell carcinomas were reactive as well. Lung and breast cancers were mostly negative. Nonmucinous ovarian tumors were invariably unlabeled as were mesotheliomas and normal mesothelial cells. the study shows that CK 20 is valuable in distinguishing tumor cell origin in effusions. in particular, it identifies a set of carcinomas with the majority arising from the gastrointestinal tract, and represents a highly characteristic marker for colorectal cancer. © 1995 Wiley- Liss, Inc.     

Malignant melanoma mimicking giant cell variant of glioblastoma multiforme: a case report and review of literature

We present a case of metastatic malignant melanoma in a patient initially diagnosed with glioblastoma multiforme, giant cell variant. A forty year old female presented to our institution for a re-resection of a recurrent right parietal lobe mass, presumed to be recurrent glioblastoma multiforme. PET scan during preoperative evaluation revealed a 3 cm left lower lobe lung mass. Metastatic glioblastoma to lung was considered in the differential diagnosis. Resection of the brain mass revealed a highly pleomorphic giant and spindle cell lesion with an immunophenotype strongly supportive of melanoma. Immunostains for melanocytic markers were subsequently performed on the lung biopsy specimen, and demonstrated diffuse staining of the atypical cells, supporting the diagnosis of malignant melanoma in the lung. This case demonstrates the importance of considering melanoma in the differential in any tumor with high grade features.     

Properties of a potassium-selective ion channel in human melanoma cells

Currents through ion channels were measured from cells of a human melanin-producing melanoma cell line (IRG 1) with the patch clamp technique. In these cells the most frequently observed channel is a potassium channel. The channel activates slowly at depolarizing voltage steps but does not inactivate. Single channel potassium currents can be measured in cell-attached patches at the resting potential of melanoma cells. The channel has a conductance of approximately 10 pS. As measured from the reversal potentials of single channel currents, the permeability ratio for sodium and potassium, P _Na/P _K, is between 0.03 and 0.04. Open probability is increased at positive potentials. Mean open times are prolonged at voltage steps to more positive potentials. Closed time histograms are fitted by two exponentials. The slow shut time is decreased at positive potentials. In whole cell measurements, cell conductance measured between –20 and +70 mV was reduced by 10 mM tetraethylammonium chloride from 6.4±1.2 nS (n=4) to 0.8±0.3 nS (n=3). Application of isoproterenol decreases the probability of the channel being open without any change in the single channel conductance. A possible role of the 10 pS potassium channel in the growth of melanoma cells is discussed.     

Expression of the chromatin remodeling factor Rsf-1 is down-regulated in breast carcinoma effusions

We recently identified Rsf-1, a chromatin-remodeling gene, as a potential oncogene that is frequently amplified and overexpressed in ovarian serous carcinoma, and demonstrated that its expression in carcinoma cells in effusions is associated with poor prognosis. In the present study, we assessed the clinical significance of Rsf-1 overexpression in breast carcinoma effusions. Formalin-fixed paraffin-embedded sections from 47 effusions were analyzed for Rsf-1 expression by immunohistochemistry. Matched primary tumors (n = 30) and solid metastases (n = 26) from 30 patients were additionally studied. Rsf-1 expression in tumor cells in effusions was analyzed for association with clinicopathologic parameters and survival. Rsf-1 protein expression was found in carcinoma cells in 34 (72%) of 47 effusions, 24 (80%) of 30 primary carcinomas, and 24 (92%) of 26 metastases. Rsf-1 immunoreactivity in effusions showed no association with HER-2 or hormone receptor status. Rsf-1 expression level was significantly lower in effusions compared with primary tumors (P = .026 and P = .011 for extent and intensity, respectively) and lymph node metastases (P = .023 and P = .013 for extent and intensity, respectively). Staining extent and intensity were both significantly lower in breast compared with ovarian carcinoma effusions (P = .001 for extent, P < .001 for intensity). Rsf-1 expression showed no association with survival. In conclusion, in contrast to ovarian carcinoma, Rsf-1 expression is down-regulated in breast carcinoma cells in effusions compared with the solid counterparts and has no prognostic role at this anatomic site.     

Cytology of pleural effusions in rheumatoid arthritis

Rheumatoid pleural effusions are relatively uncommon. The cytologic examination of such effusions can be diagnostic of the underlying disease; this is of great clinical significance when the rheumatoid condition has not been diagnosed prior to the pleural involvement. The diagnostic cytologic abnormalities include large elongated and multinucleated giant cells and macrophages in a background of granular and necrotic debris. The cytologic characteristics parallel the histologic features of pleural rheumatoid nodules.     

Preoperative cytodiagnosis of primitive carcinoid tumor of the wirsung duct: A case report with immunocytochemical study

A case of primitive carcinoid tumor of the Wirsung duct detected by fiberoptic guided brushing cytology is reported. The authors describe the main cytomorphologic characteristics and underline the role of immunocytochemistry in helping to reach a reliable preoperative diagnosis.     

Increased expression of melanoma stem cell marker CD271 in metastatic melanoma to the brain

Human melanoma contains multipotent stem cells that express the neural crest stem cell marker CD271. CD271-expressing melanoma cells in murine xenografts give rise to metastatic tumor. However, a comprehensive clinical investigation of its role in different stages of melanomagenesis has not been well studied. We studied CD271 expression with immunohistochemistry in 11 cases of banal melanocytic nevus, 9 cases of primary cutaneous melanoma, 10 cases of primary mucosal melanoma, 5 cases of metastatic melanoma in regional lymph nodes, and 11 cases of metastatic melanoma in the brain. In addition, 9 cases of metastatic, high-grade adenocarcinomas from breast and lung to the brain were studied as controls. The staining was scored based on the number of positive cells and analyzed by student t-test. All banal melanocytic nevi showed negative to equivocal staining. Primary cutaneous melanomas showed variable patterns, mucosal melanomas were mostly negative, and metastases to lymph nodes ranged from negative to moderate positivity. In contrast, all 11 cases of metastatic melanoma to the brain showed moderate (4 cases) to strong positivity (7 cases). Metastases from lung and breast origin were used as controls and showed negative to weakly positive staining in all but one case. Statistically, CD271 has significantly increased expression in metastatic melanoma to the brain when compared to the other groups studied (P < 0.05). The findings suggest that CD271 expression is specifically increased in metastatic melanoma to the brain. Further prospective study for the role of CD271 in prediction of melanoma brain metastasis as well as prognosis assessment will be of great clinical significance.     

Malignant melanoma in goats: a clinico-pathological study

The clinicopathological features of 62 cases of malignant melanoma in goats in Sudan are described. The tumours occurred most frequently in grey or brown goats. The predilection site was the perineum. The tumour invaded locally and metastasized through lymphatics and blood vessels. Surgical excision of the tumour was followed or preceded by lymphadenectomy in some cases. The tumour was highly malignant and carried a poor prognosis. Possible aetiological factors are discussed.     

Detection of cancer cells in effusions from patients diagnosed with gynaecological malignancies

The detection of malignant cells in pleural, peritoneal, and pericardial fluids of cancer patients marks the presence of metastatic disease and is associated with a grave prognosis. We evaluated five epithelial markers for the detection of cancer cells in 94 fresh pleural, peritoneal and pericardial effusions. Eighty-four of the samples were regarded as adequate for analysis after evaluation of cytological smears, including 61 samples from patients known to have gynaecological neoplasms. The other 23 samples were from patients with various non-gynaecological malignancies or tumours of unknown origin. Our control cases were 10 fallopian tubes not affected by any malignancy and 12 malignant mesotheliomas. Cell blocks from all cases were stained for CA-125, BerEP4, carcinoembryonic antigen (CEA), BG8 (Lewis Y blood antigen), and B72.3 (TAG-72). Fifty-one of 84 cases were diagnosed as malignant or suggestive of malignancy in cytological smears and/or cell block sections. However, staining for epithelial markers highlighted the presence of malignant cells in 7 additional cases. When membrane staining was evaluated, the sensitivity of the markers studied in detecting malignant cells was as follows: CA-125: 88%, BerEP4: 78%, CEA: 26%, BG8: 86%, B72.3: 79%. Membrane positivity for CEA, B72.3 and BerEP4 was not detected in reactive mesothelial cells. However, membranous staining in mesothelial cells was evident in 13% and 31% of cases with the use of BG8 and CA-125, respectively. Weak cytoplasmic staining for CEA was observed in mesothelial cells in 2 cases. When Ber-EP4, B72.3, and BG8 staining results in cancer cells were combined, the following sensitivity levels were observed: BG8+B72.3: 91%; BG8+Ber-EP4: 90%; B72.3+Ber-EP4: 93%; BG8+ Ber-EP4+B72.3: 95%. The detection of malignant cells in effusions is facilitated by the use of immunocytochemistry using a wide panel of antibodies. BerEP4 and B72.3 appear to be the best markers when both sensitivity and specificity are considered, followed by BG8, while CEA and CA-125 have a limited role in the detection of metastases from gynaecological tumours owing to the low sensitivity of the former and the low specificity of the latter. Analysis of all staining results should be based on a thorough morphological examination. Received: 14 December 1998 / Accepted: 23 February 1999