CEA, ZGM and EMA localization in cells of pleural and peritoneal effusion: a preliminary study

Carcinoembryonic antigen (CEA), the zinc glycinate marker (ZGM) and epithelial membrane antigen (EMA) have been described as epithelial or tumour markers of varying specificity. These antigens were studied by immunoperoxidase localization in selected cell blocks of 62 pleural or peritoneal effusions and compared to cytological findings and review of the clinical records. By cytological criteria, 25 of the cell blocks were positive for malignancy, 30 negative, and 7 inconclusive. CEA, ZGM, and EMA by immunoperoxidase staining were localized on the cell surface and often in the cytoplasm of malignant cells, in 11/25 (44 per cent), 17/25 (68 per cent) and 22/25 (88 per cent) of the positive cell blocks respectively. Ten (40 per cent) of these cases were positive for all three antigens, 7 (28 per cent) for two, and 6 (24 per cent) for one. Of the 7 cases which were inconclusive on routine cytological reporting, 5 were positive for at least one marker. In 3 of the 5 a diagnosis of malignancy was confirmed, and in the other two was strongly suspected as malignant on clinical grounds. Macrophages were sometimes positive for one or more markers (but showed cytoplasmic staining only) and mesothelial cells in some cases stained positively for EMA but were always negative for CEA and ZGM. Localization of the 3 antigens in cells of malignant effusions was compared with their localization in the primary tumours in 9 cases. Localization corresponded for CEA in 7 of 9 cases, for EMA in 8 of 8 an for ZGM in only 2 of 9. Effusion fluid levels for CEA were compared with the cytological and immunocytochemical findings in 30 cases. Mucin stains performed on the cell blocks were also compared with the immunoperoxidase findings.     

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.     

Immunoperoxidase staining for the detection of herpes simplex virus antigen in cervicovaginal smears

Destained cervicovaginal smears from eight patients with herpes simplex virus (HSV) infections were stained by means of the peroxidase-antiperoxidase (PAP) technique to demonstrate the presence of the HSV type 2 (HSV-2) antigen. Positive results were obtained in six of the eight cases, with intense staining for the HSV-2-specific antigen throughout the cytoplasm and nuclei of cells having a ground-glass nuclear appearance as well as in multinucleated giant cells. Virus isolation was successfully performed for the HSV-2-positive case that also had a histologically confirmed squamous-cell carcinoma of the cervix. The combined use of cytology and the PAP staining technique was of great value in the demonstration of cervical HSV infections.     

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.     

Immunocytochemical Staining of Serous Effusions With the Monoclonal Antibody Ber-Ep4

Cytospin preparations were made from 102 serous effusions for immunocytochemical staining using a panel of monoclonal antibodies including a new monoclonal antibody Ber-EP4. On cytological examination, 32 fluids were reported to contain tumour cells consistent with metastatic adenocarcinoma; 66 contained benign cells only and three were reported to contain cells suspicious of malignancy. One effusion contained tumour cells consistent with malignant mesothelioma. Positive staining of the tumour cells with Ber-EP4 was observed in the 32 effusions (100%) which contained adenocarcinoma cells. No staining of the mesothelial cells in these 32 specimens was observed. Carcinoembryonic antigen, epithelial membrane antigen Ca2 and CD15 staining of tumour cells was noted in 53%, 50%, 50% and 9% of these cases, respectively. None of the mesothelial cells in the benign effusions stained with Ber-EP4. Nor did the malignant mesothelial cells in the only case of malignant mesothelioma. These findings suggest that Ber-EP4 is a valuable addition to antibodies available for the differential diagnosis of mesothelial cells and adenocarcinoma cells in serous effusions.     

Comparison of Monoclonal Antibodies Aua1 and Ber Ep4 With Anti-Cea For Detecting Carcinoma Cells In Serous Effusions and Distinguishing Them From Mesothelial Cells

Commercially available monoclonal antibodies AUA1, BER EP4 and carcinoembryonic antigen (CEA) were applied to cell blocks from 95 serous effusions. AUA1 and BER EP4 were reactive with 89% of effusions known to contain carcinoma cells, and anti-CEA with 71%. They also reacted with cells in two effusions from patients with malignant disease which were regarded as negative on conventional cytological examination of Papanicolaou-stained smears. They were negative in all but one of the benign effusions. Using all three antibodies, 95% of effusions containing carcinoma cells were detected. Use of these antibodies could improve the cytological diagnosis of serous effusions.     

Relation between ultrastructural presence of CEA using peroxidase-antiperoxidase (PAP) method and tissue and serum levels of CEA in patients with breast cancer.

Ultrastructural studies were performed to detect the presence of carcinoembryonic antigen (CEA) in breast cancer by peroxidase-antiperoxidase (PAP) method. The evidenced presence of CEA was compared with the serum and tissue concentrations. A correlation between the presence of CEA at the ultrastructural level and tissue concentration was observed but not with serum levels. These studies revealed positive immunocytochemical staining for CEA when antigen concentration was 700 ng/g tissue and the reaction was strongly positive when the concentration was greater than or equal to 2000 ng/g tissue.     

Density distribution and immunological reactivity of tumor cells from peritoneal effusions of patients with ovarian neoplasms.

Ascitic samples from 19 patients with primary ovarian non-mucinous carcinomas, three with Krukenberg tumors and eight with noncancerous peritoneal effusions were studied by conventional cytology and immunocytochemical staining. Density gradient centrifugation was applied to fractionate ascitic fluid cells. The enrichment of cell types by this method facilitated their cytomorphological characterization and identification of neoplastic cell subpopulations existing in peritoneal effusions. Immunophenotypic studies of cells were made using monoclonal antibodies (mAbs) against ovarian carcinoma-associated antigens (OC 125, 10B, 8C) and carcino-embryonic antigen (CEA). Non-specific cross-reacting antigen (NCA) was applied as a marker for granulocytes which often accompany peritoneal effusions. Our results indicated that immunofluorescence (IF) staining contributed to the distinction between the primary and secondary ovarian carcinomas. Density gradient centrifugation appeared to be a useful method for separation of mesothelial cells.     

Detection of benign or malignant origin of ascites with combined indirect immunoperoxidase assays of carcinoembryonic antigen and lysozyme

The indirect immunoperoxidase method was used to study the presence of the intracellular carcinoembryonic antigen (CEA) and lysozyme (LZ) in alcohol-fixed cytologic smears of peritoneal fluids from 2 patients with chronic active hepatitis, 31 patients with liver cirrhosis and 7 patients with malignant liver disease. In the two patients with hepatitis, LZ was positive in both CEA was positive in one and negative in the other. Of the 31 patients with liver cirrhosis, 21 (67.5%) were LZ positive, 27 (87%) were CEA negative and only 4 (13%) were CEA positive. Of the seven patients with malignant disease, six were CEA positive and six were LZ negative. It is of interest that 23 of 24 (96%) LZ-positive results and 28 of 29 (97%) CEA-negative results corresponded to negative cytologic diagnoses for malignancy. Cytologic diagnosis of "reactive mesothelial cells" seemed to correlate better (71%) with CEA-negative and LZ-positive results. The data suggest that the investigation of CEA and LZ in the cells of peritoneal fluids appears to have promise as an adjunct to cytology in differentiating benign from malignant origins of the fluid.     

Immunofluorescent staining of metastatic carcinoma cells in serous fluid with carcinoembryonic antibody, epithelial membrane antibody, AUA-1 and BER-EP4

Using an indirect immunofluorescence technique, we assessed the accuracy and clinical usefulness of a panel of monoclonal and polyclonal antibodies. the panel consisted of carcinoembryonic antibody (CEA) and epithelial membrane antibody (EMA), AUA-1, and Ber-EP4 conjugated with fluorescein isothiocyanate. Twenty-six specimens from pleural, peritoneal or pericardial effusions known to contain carcinoma cells (adenocarcinoma or large cell anaplastic carcinoma) and 16 specimens without carcinoma were first examined. the sensitivity and specificity for each of the antibodies were as follows: CEA, 71% and 75%; EMA, 96% and 81%; AUA-1, 80% and 100%; and Ber-EP4, 85% and 100%, respectively. the panel of antibodies was then applied to a group of 14 'problematic' fluids. These had been identified as causing dilemmas in interpretation, either because the cells in the fluids were of equivocal appearance on light microscopy, or the cytological diagnosis was different from that expected in the light of the clinical condition of the patient. Insufficient cellular material was present in one specimen. In five (39%) of the cases the immunochemical staining supported the light microscopic diagnosis. In four (30%) cases, however, the results indicated that the original light microscopic report was incorrect. Two of these were examples of large cell carcinoma of the lung, in which false negative reports had been issued on pleural fluids. the other two were cases of benign ovarian tumours in which a false positive report had been issued. the immunostaining also clarified the final diagnosis in the three patients (23%) on whom 'suspicious' cytological reports had previously been issued. the remaining case, fluid from a patient with a high grade mixed Mullerian tumour of the ovary, was unresolved. We conclude that immunofluorescent staining by AUA-1, EMA and Ber-EP4 is an aid in the cytological interpretation of serous fluids. CEA is much less helpful.     

Diagnostic role of p16/INK4A protein in Human Papillomavirus (HPV) induced cervical dysplasia

The p16/INK4A protein is a cellular regulatory polypeptide over-expressed in the presence of high levels of the Human Papillomavirus (HPV) coded E7 protein. This review outlines the use of p16 antigen staining in cervical biopsies as well as in PAP smears summarizing the corresponding literature and commenting the authors’ own experience. The p16 antigen is a reliable marker for dysplastic cells in CINII/CINIII (HSIL) lesions as viewed in cervical biopsies. When PAP smears were examined at large scale screening for p16 antigenreactive and atypical cells, considerable variations could be found especially in ASCUS graded lesions. Therefore, the presence of p16-reactive atypical cells in PAP smears should be interpreted together with the cytological signs of dysplasia, such as the altered N/C ratio. In addition, women revealing p16-positive ASCUS/LSIL specimens should be examined for the presence of HPV DNA. Detection of HPV DNA alone, i.e. in the absence of cytological screening has a low predictive value, since the clearance of HPV may occur even in the absence of morphological alterations. Combined cytological as well as molecular follow up contributes to the efficiency of diagnostic and increases the probability of correct interpretation of the pre-cancerous lesions by non-invasive techniques.     

A highly sensitive method for the demonstration of carcinoembryonic antigen in normal and neoplastic colonic tissue

Summary A highly sensitive method for the immuno-histochemical localisation of carcinoembryonic antigen (CEA) is described. This method is based on the binding of a peroxidase-antiperoxidase complex (PAP) to anti-CEA antibodies by means of an anti-gamma-globulin which reacts with both the anti-CEA and the antiperoxidase antibodies. Using the technique described here, CEA was localised in conventionally processed normal and cancerous colonic tissue. In normal as well as in neoplastic tissues, a CEA-specific staining of cell membranes and cytoplasm was demonstrated. In frozen sections of normal colonic tissue CEA was found even at high dilutions of the first antibody; this indicates the high sensitivity of the method. The applicability of the method to conventionally processed and thereby well preserved tissue specimens opens the possibility to identify CEA by light microscopy even at very low concentrations.     

A comparison of the avidin-biotin-peroxidase complex (ABC) and peroxidase-anti-peroxidase (PAP) immunocytochemical techniques for demonstrating Sendai virus infection in fixed tissue specimens

Mice were infected with Sendai virus and killed 8 days later. Lungs were removed and perfused with ethanol, 10% neutral formalin, Bouin's, B-5, or Zenker's fixatives. Tissues were dehydrated, embedded in paraffin, sectioned and stained for the presence of Sendai virus using the avidin-biotin-peroxidase-complex (ABC) and peroxidase antiperoxidase (PAP) immunocytochemical techniques. Results of these techniques were compared. The ABC technique was more sensitive than the PAP. Sendai antigen was demonstrated by the ABC technique in lung tissue fixed with any fixative, whereas antigen could be demonstrated with consistency only in ethanol-fixed lung by the PAP technique. Trypsin treatment of lung prior to immunoperoxidase treatment failed to enhance staining with either technique and actually caused a decrease in staining in ethanol, B-5 and Zenker's-fixed specimens.     

Characterization of cell types in human breast tumor primary cultures

Primary cultures of cells from breast carcinomas were attempted in 74 cases. Growth was observed in 46 cases. Using immunochemical demonstration of keratin proteins (KER), epithelial membrane antigen (EMA) and carcinoembryonic antigen (CEA), three morphologically distinct cell populations were characterized and described. Two cell types (E- and E'-cells) were identified as epithelial by their positive staining for KER and EMA. The third type (F cells) displayed a negative staining for these two epithelial markers; they were considered as stromal cells (fibroblasts). More than 50% of the cultures consisted of pure epithelial cells. Positive CEA staining was observed only in KER- and EMA-positive cells. Out of the 30 cultures, 15 displayed positive staining for CEA. In 7 of these, 30-50% of cells displayed positive, diffuse staining for CEA. The other 8 cultures consisted of more than 50% CEA-positive cells. Strong and homogeneous positivity was restricted to the E-cells, while in the same cultures, E'-cells displayed heterogeneous and diffuse staining. Efficiency and value of this cell culture system are discussed, in comparison with other human breast tumor cell (HBTC) culture techniques. Identification of growing cells and cellular composition of primary cultures are emphasized.     

The value of epithelial membrane antigen in the diagnosis of ovarian tumors.

The value of epithelial membrane antigen (EMA) in the diagnosis of ovarian tumors was investigated using an indirect immunoperoxidase staining technique on 91 histologic sections (88 tumors and 3 normal tissues) and 39 ascitic fluid smears (28 from patients with epithelial ovarian tumors and 11 from cases of myoma uteri). The rate of positive EMA staining was highest in malignant tumors (89.2%), second highest in tumors of low malignant potential (33.3%) and lowest in benign tumors (25.0%); normal ovarian tissues were negative for EMA. Of the malignant tumors, all 48 serous cystadenocarcinomas (100%) and 18 of 26 mucinous cystadenocarcinomas (69.2%) stained positively for EMA. In serous cystadenocarcinomas, the EMA staining was mainly localized on the luminal membrane of cells in well-differentiated tumors, but appeared on the entire cell surface and cytoplasm of cells in poorly differentiated tumors. The results of EMA staining on ascitic fluid smears were almost the same as the results for the histologic sections. The intensity and the localization of EMA staining were related to the grade of malignancy in these ovarian tumors. In comparison with staining for other antigens (carcinoembryonic antigen, CA-125 and human keratin protein), EMA was found to be one of the most sensitive markers for the diagnosis of ovarian cancer.     

The value of the immunoperoxidase slide assay in the diagnosis of malignant pleural effusions in breast cancer

Whether immunocytochemical studies of malignant pleural effusions due to breast cancer would increase the diagnostic yield as compared with conventional effusion cytology was examined in 30 cases with biopsy-proven metastatic spread to the pleura. Conventional cytology was performed on air-dried smears as well as on cytocentrifuge preparations stained with the May-Grünwald-Giemsa stain. Immunocytochemistry was performed with monoclonal antibodies against carcinoembryonic antigen (CEA), epithelial membrane antigen (EMA) and human leukocyte antigen (HLA) and the peroxidase-antiperoxidase technique on glass slides after Ficoll-Hypaque centrifugation. By conventional cytology, 13 cases (43%) were positive for malignant cells, 6 cases (20%) were suspicious, and 11 cases (37%) were negative. In marked contrast, all 30 cases were immunocytologically positive for malignancy. Tumor cells in all cases demonstrated a positive reaction for EMA. Some mesothelial cells were also positive for EMA, but their reaction pattern was clearly distinguishable from that of the tumor cells. Twenty-one cases (70%) also showed CEA-positive tumor cells; mesothelial cells never reacted with CEA. Some tumor cells showed a loss of HLA expression. In conclusion, this immunocytologic method can be recommended as a routine procedure for greatly increasing the diagnostic yield of cytology in pleural effusions due to breast cancer.     

Malignant ascites of serous papillary ovarian adenocarcinoma. An immunocytochemical study of the tumor cells

In 17 malignant peritoneal effusions due to papillary serous adenocarcinoma of the ovary, the reaction patterns of the tumor cells to monoclonal antibodies (MAbs) against surface antigens were studied and compared with the reaction patterns of mesothelial cells in the same effusions. The following surface markers were used with the adhesive slide method: epithelial membrane antigen (EMA), human epithelium-specific cell surface antigen (HEA-125), human endothelial antigen (BMA-120), carcinoembryonic antigen (CEA 3-13), an antibody against natural killer cells and cytotoxic cells (BMA-070), granulocyte antigen (Leu M1) and leukocyte antigen of class I (HLA-1). In all cases, from 30% to 95% of the tumor cells reacted with EMA and HEA-125. Tumor cells showed a positive staining with CEA 3-13 in only five cases. In all cases, from 75% to 95% of the tumor cells reacted positively with BMA-120. The reactivity of a few mesothelial cells with EMA and of all mesothelial cells with BMA-120 did not interfere with the identification of positive tumor cells since the reaction patterns were different. Interestingly, our study demonstrated that BMA-070, an MAb identifying natural killer cells and cytotoxic cells, is also a most useful tumor marker. The same was found to be true for Leu M1, an MAb originally thought to react only with granulocytes. The tumor cells showed a partial or total loss of the expression of HLA-1 reactivity. Since all cases were immunocytochemically positive for tumor cells while conventional cytology was positive in only 13 of the cases, the immunocytochemical analysis of malignant peritoneal effusions due to papillary serous adenocarcinoma of the ovary seems able to improve the cytologic diagnosis of the fluids.     

Tumor marker studies of cervical smears. Potential for automation

The distribution of three tumor markers, epithelial membrane antigen (EMA), carcinoembryonic antigen (CEA) and prekeratin (PK) was studied in exfoliated epithelial cells in cervical smears using an immunoalkaline phosphatase staining technique to demonstrate the antigens. EMA was expressed by abnormal cells in a consistent and reproducible fashion whereas the other two markers were variably expressed by both normal and abnormal cells. Our results suggest that immunocytochemical staining for EMA could be of value not only for the diagnosis of cervical intraepithelial neoplasia but also for the automated screening of cervical smears.     

Fine needle aspiration cytodiagnosis of secretory carcinoma of the breast

Secretory carcinoma (SC) of the breast is a rare variant of breast malignancy and its cytological features in fine needle aspirates have only recently been described. In this communication, our experience with four cases of SC of the breast is presented in which the diagnosis was established on fine needle aspiration cytology (FNAC). In all cases, the samples were cellular and featured diffuse, prominent, intracytoplasmic vacuoles and secretion in malignant cells and occasional signet-ring like forms. The cytodiagnosis of SC in all the cases correlated with subsequent examination of cell blocks of the aspirate and tissue. Cytochemical stains showed diffuse positivity for mucin by alcian blue stain in the vacuolated cells which was periodic acid-Schiff positive and resistant to diastase digestion. Oil-red O staining was negative. Immunopositivity to carcinoembryonic antigen, cytokeratin (CAM 5.2), B72.3 and epithelial membrane antigen was found in malignant cells. The cytodiagnostic criteria for SC of the breast, characteristic cytological features which are useful in a correct FNAC diagnosis and differentiation from other pertinent breast carcinomas, are discussed.     

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.