Autoantibodies against p53 are not increased in human ascites and pleural effusions

 Mutated human p53 may give rise to the formation of autoantibodies and may be a marker for a worse prognosis. We speculated that ascites or pleural effusions may enhance the formation of such autoantibodies in cancer patients and, therefore, we measured the presence of autoantibodies in the ascites or pleural effusion of 40 patients with advanced malignancies. As controls, p53 autoantibodies were measured in 15 patients with effusions who did not have a malignancy. Using a specific enzyme-linked immunosorbent assay, p53 autoantibodies could only be detected in the effusions of 5/40 patients (12.5%) with known malignancies. The formation of autoantibodies did not correlate with the presence or absence of tumor cells in the effusion. The effusions of the patients without tumor were all negative for p53 autoantibodies. Our study shows that malignant or reactive effusions do not stimulate the local or systemic production of autoantibodies against p53. Received: 14 November 1995 / Accepted: 8 February 1996     

Heparanase expression: a potential ancillary diagnostic tool for distinguishing between malignant cells and reactive mesothelium in body cavity effusions

OBJECTIVE: Heparanase, an endoglycosidase that cleaves heparan sulphate, is frequently expressed in carcinomas and was suggested to play a role in cell invasion and metastasis. We investigated whether heparanase expression may serve as a reliable marker to discriminate benign mesothelial cells from malignant cells shed into body cavities. METHODS AND RESULTS: Cytological smears of effusions from 51 hospitalized patients were immunostained for heparanase. Strong immunoreactivity was noted in 35 of 40 (88%) carcinoma samples and in all three malignant mesothelioma cases. Only rare (<3%) reactive mesothelial cells were noted showing a faint negligible staining. Specificity was 100%, sensitivity 88%, and positive and negative predictive values were 100% and 89% respectively. CONCLUSIONS: Our results suggest that heparanase may be of value as a complementary component in a diagnostic panel of markers, contributing to its reliability and accuracy.     

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.     

C-ErbB-2 Oncoprotein Immunostaining In Serous Effusions

The product of c-erbB-2 gene is detected in a proportion of carcinomas from various sites and is generally associated with a high degree of malignancy. A series of 58 effusions containing malignant cells and 16 cytologically negative serous effusions was assessed by immunocyto-chemical methods for c-erbB-2 expression using the monoclonal antibody NCL-B11, which recognizes the internal domain of the c-erbB-2 oncoprotein. Both alcohol-fixed smears and cell blocks from formalin-fixed specimens were used. A crisp, clear cut membrane-associated positive staining was evident in 51 % (30/58) malignant effusions and was restricted to metastatic adenocarcinomas. Breast and ovarian cancers showed the highest incidence of positivity. Mesotheliomas as well as non-neoplastic effusions were consistently negative. Paraffin blocks from formalin-fixed cells displayed a weak immunoreactivity when compared with their alcohol-fixed counterparts. the study shows that the c-erbB-2 oncoprotein can be easily identified in standard cytological smears: it can be of assistance in differentiating adenocarcinomas from mesotheliomas, and in selected cases it can provide a further prognostic indicator, replacing tissue immunohistochemistry.     

PINCH‐2 expression in cancers involving serosal effusions using quantitative PCR

Y. Yuan, H. P. Dong, D. A. Nymoen, J. M. Nesland, C. Wu and B. Davidson
PINCH‐2 expression in cancers involving serosal effusions using quantitative PCR Objective: The PINCH‐2 gene was previously shown to be overexpressed in malignant mesothelioma compared with ovarian/peritoneal serous carcinoma in Affymetrix array analysis. The objective of the present study was to validate this finding at the mRNA and protein level. Methods: Effusions (n = 91; 71 ovarian and 10 breast carcinomas, 10 malignant mesotheliomas) were assayed for PINCH‐2 mRNA expression using quantitative PCR. PINCH‐2 protein expression was analysed in 37 effusions using flow cytometry. Results: Quantitative PCR analysis showed significantly higher PINCH‐2 mRNA levels in mesotheliomas compared with carcinomas (P = 0.004). Values of <10 copies were found exclusively in carcinoma effusions (25.4% of ovarian and 50% of breast carcinomas). However, PINCH‐2 protein expression by flow cytometry did not differ significantly between the three cancer types. No association was observed between PINCH‐2 levels and patient survival or expression of previously‐studied molecules related to adhesion, metastasis and apoptosis inhibition in ovarian carcinoma. Conclusions: Our data suggest that PINCH‐2 mRNA is overexpressed in malignant mesothelioma compared with carcinomas involving serosal cavities, and that low levels of this gene argue against the diagnosis of mesothelioma. The frequent PINCH‐2 protein expression in all three studied cancers suggests a role for this molecule in cancer cell biology in effusions and merits further research.     

Immunocytochemical staining of effusions; an external quality control study in The Netherlands

Immunocytochemical staining of effusions; an external quality control study in The Netherlands
In The Netherlands an external quality control study of immunocytochemical (IC) staining of effusions was initiated, consisting of three test rounds. The 12 participating laboratories received samples of malignant effusions (runs 1, 2 and 3), and five unstained control specimens prepared from the same material in runs 2 and 3. The laboratories used their own protocols to prepare and stain the samples ('in‐house' specimens). Two persons viewed and scored the slides following preset criteria concerning number and morphology of diagnostic cells, background staining and staining specificity. Better scoring results were found for control specimens, compared with 'in‐house' specimens, primarily caused by cell loss in the latter. This finding underlines the view that high quality IC needs well organized processing and staining procedures, and warrants external quality control systems.     

Cytological Aspects of Pleural, Peritoneal and Pericardial Fluids From Patients With Systemic Lupus Erythematosus

Serous effusions of nine of 33 patients with systemic lupus erythematosus contained lupus erythematosus (LE) cells, identifiable in Papanicolaou-stained smears, wet films stained with toluidine blue, and cell blocks stained with haematoxylin and eosin. Specimens in which LE cells were found contained at least a moderate number of polymorphonuclear neutrophilic leucocytes. Most specimens containing LE cells also contained cells that resembled LE cells (tart cells), which appeared to be small macrophages that had phagocytosed a non-homogenized nucleus of a cell that had undergone degeneration. In 34 years of cytologic practice we have recognized LE cells in serous effusions only from patients who were already diagnosed as having systemic lupus erythematosus.     

Expression of Insulin-Like Growth Factor-I Receptor and Transferrin Receptor By Breast Cancer Cells In Pleural Effusion Smears

Smear preparations were made from cells harvested from pleural fluid from 90 patients with breast cancer and stained for transferrin receptor (TRFr) and insulin-like growth factor-I receptor (IGF-Ir) using an immunocytochemical technique. the results were correlated with those from 36 benign effusion smears. In malignant smears from the breast cancer cases TRFr was demonstrated in 84.4% of the cellular deposits and IGF-Ir in 91.1%. TRFr was demonstrated in two (11%) of the tuberculous effusion smears and in six (100%) effusions from patients with collagen disease. IGF-Ir was not demonstrated in any of the smears from patients with benign disease. the sensitivity and specificity of TRFr staining were 84.4% and 77.7%, respectively, and for IGF-Ir staining were 91.1% and 100%, respectively. the underlying metabolic changes in the tumour cells which give rise to positive staining with these markers are discussed. Les préparations cytologiques ont été obtenues à partir de cellules recueillies dans le liquide pleural chez 90 patientes ayant un cancer du sein puis ont fait I'objet de techniques immunocytochimiques pour mettre en évidence les récepteurs de la Transferrine (TRF-r) et du facteur de croissance Insulin Like-I (IGF-Ir). Les résultats ont été corrélés avec ceux obtenus sur 36 épanchements bénins. Dans les étalements provenant de patientes traitées pour cancer du sein, TRF-r est positif dans 84,4% des groupements cellulaires et I'IGF-Ir dans 91,1%. Une activité pour le TRF-r est observée dans deux cas (11%) d'epanchements tuberculeux et dans les 6 cas (100Y0) d'épanchement survenant chez des patients atteints de collagénose. Aucune activité IGF-Ir n'est présente dans les cellules des épanchements des patients atteints d'affection bénigne. La sensibilité et la spécificité de I'activité TRF-r sont de 84,4% et de 77% respectivement, celles de I'activité IGF-Ir étant de 91,1% et de 100% respectivement. Les modifications métaboloques sous-jacentes á la positivité en immunocytologie des cellules tumorales avec ces marqueurs sont discutiés. Ausstriche von Pleuraergüssen von 90 Patientinnen mit Mammakarzinom wurden hinsichtlich Tranferrinrezeptoren (TRF-r) und Insulinwachstumsrezeptor (IGF-r) untersucht. 36 benigne Ergüsse dienten als Vergleich. In tumorösen Ergüssen waren TFGr in 84,4% und IGFr in 91,1% nachweisbar. TRFr war ausserdem in 2 (11Y0) der tuberkulösen und 6 (100%) der rheumatischen Ergüsse positiv währen IGFr in keinem der benignen Fälle positiv ausfiel. Sensitivität und Spezifität waren für TRFr 84,4% bzw. 77,7% und IGFr 91,1y0 bzw. 100%. Die metabolischen Veränderungen der Tumorzellen werden diskutiert.     

Measurement of apoptosis in cytological specimens by flow cytometry: comparison of Annexin V,caspase cleavage and dUTP incorporation assays

H. P. Dong, A. Holth, M. G. Ruud, E. Emilsen, B. Risberg and B. Davidson Measurement of apoptosis in cytological specimens by flow cytometry: comparison of annexin V, caspase cleavage and dUTP incorporation assays Objective: To compare the performance of different assays for measuring apoptosis in cytological specimens. Methods: Apoptosis was assessed in 27 specimens (22 effusions, five fine needle aspirates; 20 malignant, seven reactive) using flow cytometry, applying assays for the measurement of annexin V expression, caspase‐3 and ‐8 cleavage and deoxynucleotidyl transferase deoxyuridine triphosphates (dUTP) incorporation. Results were studied for differences between reactive and malignant specimens, as well as performance across assays. Results: Wide variation in the degree of apoptosis was observed in both benign and malignant specimens using all assays. However, the percentage of annexin V‐positive cells was higher compared with those showing caspase cleavage or dUTP incorporation in the majority of cases, irrespective of specimen type. Comparative analysis of benign and malignant specimens showed no significant differences in expression of any of the studied parameters. However, tumour cells and reactive mesothelial cells in pleural effusions had a significantly lower level of dUTP incorporation compared with their counterparts in peritoneal specimens (P = 0.001). Conclusions: The present data are in agreement with our previous observation in ovarian carcinoma effusions, that measurement of apoptosis by the annexin V assay provides higher expression values than those obtained by other assays, suggesting that this assay does not accurately reflect the degree of apoptosis in benign or malignant cells in effusions.     

Flow cytometric measurement of cellular FLICE-inhibitory protein (c-FLIP) in ovarian carcinoma effusions

H. P. Dong, A. K. Ree Rosnes, A. J. Bock, A. Holth, V. A. Flørenes, C. G. Trope’, B. Risberg and B. Davidson Flow cytometric measurement of cellular FLICE‐inhibitory protein (c‐FLIP) in ovarian carcinoma effusions Objective: The objective of this study was to establish a flow cytometry assay for measuring c‐FLIP in serous effusions. In addition, we studied the clinical relevance in ovarian carcinoma effusions of this inhibitor protein in the death receptor signalling pathway of apoptosis. Methods: Two c‐FLIP antibodies were tested using Western blotting and the best performing one was used for titration of c‐FLIP expression in a panel of five cell lines, consisting of ovarian carcinoma, breast carcinoma and malignant mesothelioma. The concentration that provided the best signal‐to‐noise ratio was used for comparison of the performance of three fixation and permeabilization protocols. The best performing protocol was chosen for analysis of 69 ovarian carcinoma effusions. c‐FLIP expression was analysed for association with clinicopathological parameters and survival. Results: Rabbit polyclonal c‐FLIP by Abcam and the IntraStain kit by Dako performed best. c‐FLIP expression was detected in tumour cells in all 69 effusions (expression range 21–100%, median = 80%). No association was found between c‐FLIP expression and clinicopathological parameters, including chemoresponse and survival. However, an inverse correlation was found between c‐FLIP levels and expression of the previously studied apoptosis marker cleaved caspase‐3 (P = 0.029). Conclusions: An assay for measuring c‐FLIP in cytology specimens is presented. c‐FLIP is frequently expressed in ovarian carcinoma effusions, but its expression appears to be unrelated to disease aggressiveness.