Immunocytological staining reactions of anti-carcinoembryonic antigen, Ca, and anti-human milk fat globule monoclonal antibodies on benign and malignant exfoliated mesothelial cells.

A panel of three monoclonal antibodies were used in an immunoalkaline phosphatase staining method on a series of serous effusion samples from cases of mesothelioma, lung carcinoma, and benign disease. The antibodies used were anti-carcinoembryonic (CEA) antigen, Ca, and anti-human milk fat globule membrane antigen. Antibodies to the Ca antigen and human milk fat globule membrane antigen stained 75% and 83% of mesothelioma and 75% of cases of lung carcinoma, respectively. The anti-CEA antibody stained most cases of lung carcinoma strongly but was negative on 11 of 12 cases of mesothelioma and showed weak staining on one case. Benign cases were negative with all three antibodies. These three antibodies may be useful in distinguishing benign and malignant mesothelial cells and lung carcinoma in serous effusions.     

Immunohistological staining of reactive mesothelium, mesothelioma, and lung carcinoma with a panel of monoclonal antibodies.

A panel of seven monoclonal antiepithelial antibodies of different specificities, including anticytokeratin, human milk fat globule membrane, C, and carcinoembryonic antigen (CEA) were used with the alkaline phosphatase-antialkaline phosphatase (APAAP) immunostaining technique to determine their value in the differentiation between benign and malignant mesothelial cells and lung carcinoma in histological preparations. The anticytokeratin antibody reacted strongly with all cases of reactive mesothelium, mesothelioma, and lung carcinoma. Antibodies to human milk fat globule membrane and the Ca antigen stained mesothelioma and carcinoma and 43% of cases of reactive mesothelium. Staining for carcinoembryonic antigen was not detected in reactive mesothelium or mesothelioma, but was present in most of the lung carcinomas. CEA seemed to be the single most useful marker in distinguishing carcinoma from mesothelioma in that a positive reaction for CEA would indicate carcinoma rather than mesothelioma.     

Value of BCA-225 in the cytologic diagnosis of malignant effusions: an immunocytochemical study of 197 cases

An immunocytochemical study of routinely prepared paraffin embedded cell block material from 72 effusions containing adenocarcinoma and 125 benign effusions was performed using a commercially available monoclonal antibody to the breast carcinoma associated glycoprotein BCA-225. Positive staining was observed in cells in 77.8% of the malignant effusions but not in any of the benign effusions. We conclude that BCA-225 is a highly specific and very useful discriminator in the differential diagnosis of adenocarcinoma and reactive mesothelial cells. However, because this marker is not expressed in all adenocarcinomas, studies with a panel of antibodies will provide better sensitivity.     

Immunocytochemistry of malignant mesothelioma: OV632 as a marker of malignant mesothelioma.

In pleural or ascitic effusions the cytomorphological distinction of adenocarcinoma cells, reactive mesothelial cells, and malignant mesothelioma cells often causes a diagnostic dilemma. The value of immunocytochemistry was investigated on cytological smears of 24 well-established cases of malignant mesothelioma, a selected series of 31 metastatic adenocarcinomas, and 20 smears of patients without known malignancy. In these smears we scored the immunoreactivity with a panel of four monoclonal antibodies. In addition to antibodies for epithelial membrane antigen (EMA) and carcinoembryonic antigen (CEA), the monoclonal antibody MOC31 and the ovarian carcinoma specific antibody OV632 were incorporated in the panel. With none of these four antibodies was immunostaining of reactive mesothelial cells found. CEA- and MOC31-positive tumour cells were frequent in metastatic adenocarcinomas, but occurred rarely in malignant mesotheliomas. EMA-positive tumour cells were found in all metastatic adenocarcinomas (100 per cent) and in most malignant mesotheliomas (83 per cent). In addition to the expected reactivity of OV632 with ovarian carcinomas, 22 of 24 malignant mesotheliomas contained immunopositive tumour cells, while only a small proportion of non-ovarian adenocarcinomas reacted with this antibody. This selective staining of malignant mesothelioma cells, but not reactive mesothelial cells, with OV632 now permits the positive identification of malignant mesothelioma cells in male patients.     

Carbohydrate antigen expression in primary tumors, metastatic lesions, and serous effusions from patients diagnosed with epithelial ovarian carcinoma: evidence of up-regulated Tn and Sialyl Tn antigen expression in effusions

The object of this study was the investigation of carbohydrate antigen expression in malignant epithelial cells and benign mesothelial cells in serous effusions from patients diagnosed with epithelial ovarian carcinomas. In addition, to compare antigen expression in carcinoma cells in effusions with those of corresponding primary tumors and metastatic lesions. Sections from 63 malignant effusions from ovarian carcinoma patients and 15 reactive effusions were immunohistochemically stained, using 5 monoclonal antibodies for Lewis(y), Sialyl Lewis(x), Tn, and Sialyl Tn antigens. Tissue sections (n = 97) from corresponding primary ovarian carcinomas and metastatic lesions, as well as from 12 malignant mesotheliomas, were additionally stained using the above panel. Staining for the 4 antigens was seen in carcinoma cells in serous effusions in the majority of cases (range = 71% to 85%). In contrast, immunoreactivity was detected in mesothelial cells in only 6% to 23% of the specimens studied (P < .001 for all 5 markers). With the exception of B3 antibody against Lewis(y) antigen, malignant mesotheliomas stained negative, infrequently showing focal immunoreactivity. An up-regulation of Tn and Sialyl Tn expression was detected in carcinoma cells in effusions when compared with both primary tumors (P < .003 and P < .007, respectively) and metastatic lesions (P < .034 and .041, respectively). Cancer-associated carbohydrate antigens can thus be used as an adjunct in the differentiation between malignant epithelial and reactive mesothelial cells. Ovarian carcinoma cells in effusions show up-regulation of Tn and Sialyl Tn, possibly representing a transient phenotypic alteration facilitating metastasis.     

Immunocytochemical reaction of Ca1 and HMFG2 monoclonal antibodies with cells from serous effusions.

The Ca1 antibody was used in an alkaline phosphatase immunocytochemical method on cells obtained from 150 specimens of pleural and ascitic fluids. The results were compared with the routine cytology report based on the light microscopical appearances. The Ca1 antibody identified tumour cells in 51 of 57 specimens with malignant cells. The exceptions were four small cell carcinomas, one malignant lymphoma, and one adenocarcinoma. A further seven specimens reported as containing atypical cells but without conclusive evidence of malignancy were Ca1 positive. The Ca1 antibody did not give a positive reaction with benign mesothelial cells. Similar results were obtained with the HMFG2 antibody and malignant cells, but in eight of 18 benign effusions it reacted with mesothelial cells.     

Malignant lymphoma in pleural effusions: an immunocytochemical cell surface analysis

Nine malignant pleural effusions due to lymphoma were immunocytochemically analyzed with the peroxidase-antiperoxidase adhesive slide assay for detection of cell surface antigens using a broad panel of monoclonal antibodies. The study population included one case of hairy-cell leukemia; four cases of B cell non-Hodgkin's lymphoma (B-NHL), low malignant grade; two cases of B-NHL, high malignant grade; one case of Hodgkin's disease; and one case of plasmacytoma. In the cases of B cell lymphoma, high percentages of B cells with monoclonal staining for kappa were found. In hairy-cell leukemia, the hairy cells reacted with the monoclonal antibodies CD20, CD25, HLA-DR, CD45, and HLA-1. In Hodgkin's disease, the Hodgkin cells reacted with CD15, CD20, CD25, CD30, Tü9, and OKT9. The plasmacytoma case showed tumor cells negative for CD20, HLA-DR, and CD45; partially positive for CD38; positive for HLA-1; monoclonally positive for lambda; and negative for heavy-chain immunoglobulins. The analysis of nonmalignant lymphocyte subpopulations revealed CD4/CD8 ratios similar to those in effusions of other etiologies. The percentages of natural killer cells (Leu-7-positive and CD16-positive) were small and also similar to percentages in effusions of other etiologies. We conclude that immunocytochemical analysis of pleural effusions allows a clear recognition of B lymphoma cells and also of Hodgkin and hairy cells and that the distribution of nonmalignant lymphocyte subsets is indistinguishable from those found in other malignant and nonmalignant effusions.     

Marker profile of different phases in the transition of normal human ovarian epithelium to ovarian carcinomas.

To investigate whether early changes in the transformation of normal ovarian epithelial cells into tumor cells can be detected with monoclonal antibodies, a comparative immunohistochemical study was performed on normal human ovarian mesothelial cells, cystomas, cystadenomas, ovarian carcinomas, as well as granulosa cell tumor. Using monoclonal antibodies against different keratin subtypes, it was shown that mesothelial cells, ovarian cysts, cystadenomas, and carcinomas all reacted positively with broad-spectrum anti-keratin monoclonal antibodies (MAbs), as well as with MAbs to keratins 7, 8, 18, and 19. Keratins 4 and 13 were not found in mesothelial cells, but positive groups of cells were identified in several cystomas, adenomas, and carcinomas. While mesothelial cells did not react with the pan-epithelial marker BW495/36, invaginating metaplastic mesothelial cells, inclusion cysts, cystomas, adenomas, and carcinomas showed an increasing reactivity with BW495/36, with an increasing degree of malignancy. The reactivity of MAbs against ovarian carcinoma-associated antigens (OV-TL 3, OC 125, MOv 18, and OV-TL 10) was limited to weak staining reaction in some mesothelial cells but were found to be positive on more than 50% of the ovarian cystadenomas and more than 90% of the ovarian carcinomas. Thecal and granulosa cells of primordial, primary, and secondary follicles all reacted positively with antibodies to the broad-spectrum keratins OV-TL 12/5 and RCK 102, and to keratins 8 and 18, but not with keratins 4, 7, 13, and 19. These keratins decreased or disappeared in granulosa cells of mature follicles (Graafian follicles), whereas granulosa cell tumors did not react with anti-keratin antibodies. The reactivity of BW 495/36 was negative or limited to traces in some granulosa cells. Ovarian carcinoma-associated antigens were not expressed in granulosa cells or granulosa cell tumors. The data indicate that mesothelial cells undergoing metaplastic changes finally resulting in ovarian cystadenomas (and carcinomas) initiate the synthesis of a 200-kd glycoprotein recognized by MAb (BW 495/36), the production of ovarian carcinoma associated antigens, in addition to focal production of keratin 4 and/or 13, as seen in several samples. The granulosa cell tumors decrease or switch off their keratin production and remain negative for the 200-kd glycoprotein and the ovarian carcinoma-associated antigens.     

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.     

Monoclonal antibody Ber-EP4: Its use in the differential diagnosis of malignant mesothelioma and carcinoma in cell blocks of malignant effusions and FNA specimens

Formal sublimate-fixed cell blocks derived from 129 malignant pleural (and some peritoneal) effusions, 8 benign effusions with reactive mesothelial cells, and 23 FNA specimens, were immunostained with monoclonal antibody Ber-EP4 to assess its ability to distinguish malignant mesothelioma (MM) from carcinoma. Only 2 of 44 (4%) well-characterized MM were Ber-EP4⁺, while none of 8 benign mesothelial proliferations reacted with the antibody. Fifty-seven percent of 23 pulmonary adenocarcinomas (AC) and 60% of 43 pulmonary carcinomas of all other histological types were Ber-EP4⁺. Of 40 metastatic AC originating from breast, gastrointestinal tract, ovary, endometrium, and kidney, 80% were Ber-EP4⁺. The predictive value of positive Ber-EP4 staining in distinguishing AC from MM was 96%. The predictive value of a negative Ber-EP4 in excluding MM was 70%, when the differential diagnosis was adenocarcinoma. These results suggest that Ber-EP4 is helpful in differentiating MM and AC if used together with other discriminating antibodies. © 1994 Wiley-Liss, Inc.     

Cytology, immunopathology and flow cytometry in the diagnosis of pleural and peritoneal effusions

There were 106 pleural and peritoneal effusions studied in order to investigate the contribution of immunocytochemistry and flow cytometry to routine cytologic diagnosis. A panel of antibodies (to cytokeratin, vimentin, human milk fat globule, epithelial membrane antigen and carcinoembryonic antigen) was applied to aceton-fixed slides, using immunoperoxydase and immunofluorescence methods. Flow cytometry was performed using a double labeling method, i.e., propidium iodide for DNA staining and keratin for labeling of epithelial cells. In this way DNA aneuploidy was more evident in the histograms when the fluid contained many reactive nonepithelial cells (lymphocytes). A designation of marker profiles was made for the three most frequently occurring diagnoses, i.e., reactive mesothelial proliferation (I), adenocarcinoma (II), and malignant mesothelioma (III). For the differentiation between adenocarcinoma and malignant mesothelioma, carcinoembryonic antigen was the most useful marker as 75% of the adenocarcinomas was carcinoembryonic antigen-positive and the malignant mesotheliomas were consistently negative. Furthermore, evident DNA-aneuploidy strongly supported the diagnosis of adenocarcinoma, as most malignant mesotheliomas were DNA-euploid, even though occasional DNA-aneuploidy was found in malignant mesotheliomas when different effusions from the same patient were examined. For the differentiation between reactive mesothelial cells and malignant mesothelioma human milk fat globule and/or epithelial membrane antigen, in this study proved to be most reliable, their presence strongly indicating malignancy. It is stressed that the method used (fixation, antibodies, washing procedures) can influence these findings. In 16 patients (17%) performing immunopathology and/or flow cytometry meant an important contribution to diagnosis.     

Immunocytochemical panel for the identification of malignant cells in serous effusions.

The cytologic diagnosis of malignancy in serous effusions can be challenging. An immunocytochemical (ICC) panel using commercially available antibodies (to carcinoembryonic antigen [CEA], epithelial membrane antigen [EMA], B72.3, Leu-M1, cytokeratin [CK], leukocyte common antigen [LCA], S-100 protein, and vimentin) was applied to cell blocks fixed in methyl Carnoy's solution that were from 55 consecutive pleural, peritoneal, and pericardial fluid specimens. The results were correlated with data from clinical records and routine cytologic studies. Final cytologic diagnoses included 26 of adenocarcinoma and 1 of mesothelioma. The remaining 28 cases were considered to be benign (reactive) proliferations. EMA, CEA, B72.3, and Leu-M1 were present in 96%, 77%, 58%, and 42% of adenocarcinomas, respectively. These determinants were absent in the mesothelioma and the reactive effusions, although anti-CEA yielded strong background staining of inflammatory cells. The CK markers identified malignant cells in 93% of cases, but consistently stained mesothelial cells as well. Antivimentin strongly labeled mesothelial cells in all cases, with weak to absent staining of malignant cells. In 3 of 26 carcinoma cases (12%), the ICC panel identified malignant cells that were not recognized initially on routine cytologic examination. In 1 of 26 cases (4%), the panel was falsely negative. Use of this approach can improve the diagnostic accuracy of cytologic examination of serous fluids. The ICC panel is especially helpful when atypical mesothelial proliferation is present, or in cases that are clinically suspect for malignancy, but cytologically negative because there are only a few malignant cells, or those that are cytologically bland.     

Localization of a membrane glycoprotein in benign fibrocystic disease and infiltrating duct carcinomas of the human breast with the use of a monoclonal antibody to guinea pig milk fat globule membrane.

With monoclonal antibody D-274, raised against guinea pig milk fat globule membrane, the distribution of mucinlike glycoproteins of Mrs greater than or equal to 400,000 was determined in benign fibrocystic disease and infiltrating duct carcinoma of the human breast. These glycoproteins, called collectively PAS-I, were detected in 19 out of 20 cases of benign fibrocystic disease and in at least 26 out of 47 cases of infiltrating duct carcinoma. PAS-I was concentrated on luminal surfaces of ducts and alveoli in morphologically differentiated regions of the tumors. In areas where the glandular nature of the tissue was less evident in infiltrating duct carcinoma, the PAS-I determinant recognized by antibody D-274 was present on irregular luminal surfaces and in the cytoplasm. There was a negative correlation between the short-term recurrence (less than 2 years) of infiltrating duct carcinoma and the detection of strong positive staining with antibody D-274. The results are discussed with reference to recent studies on PAS-I in human breast tissue using monoclonal antibodies raised against human milk fat globule membrane.     

Immunocytochemical panel for distinguishing between adenocarcinomas and reactive mesothelial cells in effusion cell blocks

The aim of our study was to determine the value of a panel that consisted of one epithelial marker (MOC‐31) and two mesothelial markers (D2‐40 and calretinin) for distinguishing between reactive mesothelial cells (RMCs) and adenocarcinomas (ACs) in effusion fluids. A total of 118 cell block specimens from pleural and peritoneal effusions, including 88 ACs and 30 benign effusions with RMCs were stained with antibodies against MOC‐31, D2‐40, and calretinin. MOC‐31 membranous activity was observed in all samples from ACs, regardless of the primary tumor site. All benign effusion samples with RMCs were negative for MOC‐31. All benign effusion samples with RMCs exhibited membranous staining for D2‐40, and one AC case had focal reactivity for D2‐40. Almost all benign effusions reacted positively with calretinin. Staining was noted in both the cytoplasm and the nucleus in the majority of cases. Scattered tumor cells had weak calretinin positivity in two AC cases. Background RMCs in AC effusions were consistently positive for D2‐40 and calretinin. In general, D2‐40 identified more RMCs than calretinin. The staining combination of positive for MOC‐31 and negative for D2‐40 or calretinin were 100% specific and 99% sensitive for ACs. Our data suggest that immunohistochemical studies performed on cell blocks with MOC‐31, D2‐40, and calretinin were useful in the differentiation between ACs and RMCs. D2‐40 was a more sensitive marker for RMCs than calretinin. Diagn. Cytopathol. 2009. © 2009 Wiley‐Liss, Inc.     

Immunocytochemical profile of benign and carcinomatous effusions. A practical approach to difficult diagnosis.

One of the great challenges in the cytodiagnosis of effusions is the distinction between reactive mesothelium/histiocytes and cancer cells. This is notably true in patients having undergone radiation and/or chemotherapy. To establish whether monoclonal antibodies (MoAbs) could be used as reliable diagnostic adjuvants, the authors retrospectively and blindly studied 60 cases diagnosed by standard cytologic criteria (malignant, benign, and equivocal), with a panel of seven readily available MoAbs (cytokeratins, vimentin, EMA, B72.3, alpha-CEA, HMFG-2, and Leu-M1) and the lectin Ulex europaeus I. All 18 (100%) malignant cases showed reactivity with EMA and HMFG, whereas 17 (95%) and 11 (61%) reacted with B72.3 and alpha-CEA, respectively. Combinations of (1) EMA + B72.3, (2) EMA + alpha-CEA, and (3) EMA + alpha-CEA + B72.3 displayed positivity in 17 (95%), 11 (61%), and 10 (56%) malignant cases, respectively. Of the 18 benign cases, 7 reacted with HMFG and 2 each with EMA and B72.3. Only one case (5.5%) reacted with both EMA and B72.3. Based on these results, the 24 equivocal cases were regrouped into 14 malignant and 10 benign cases. Follow-up effusions obtained within the ensuing three months in all these patients allowed the authors to unequivocally confirm the diagnosis in all but five. The combination of EMA and B72.3 MoAbs detected malignant cells in 95% of the cases, with a 3.5% incidence of false positive cases in this study. A panel of EMA, B72.3, and alpha-CEA MoAbs should prove the most useful and simple approach to the correct diagnosis in most questionable effusions. Some of the potential pitfalls are discussed.     

Distinction between cells in serous effusions using a panel of antibodies

Summary In serous effusions the distinction between reactive mesothelial cells and malignant cells (especially adenocarcinoma cells and malignant mesothelial cells) is frequently a cause of diagnostic difficulty. The present paper describes the immunocytochemical staining of cells in 76 effusions from 71 patients with different malignancies. In 91% of the effusions obtained from patients with adenocarcinomas, the cells stained positive for anti-EMA, 94% for anti-CEA, 64% for anti-LeuM1-antigen and 75% for anti-keratins. In more than 90% of the cases the reactive mesothelial cells stained positive for anti-keratins, but not for anti-EMA, anti-CEA or anti-LeuM 1-antigen. It is concluded that a panel of the antibodies against EMA, CEA, LeuM 1-antigen and keratins is valuable in the distinction between adenocarcinoma cells, malignant mesothelial cells and reactive mesothelial cells in serous effusions.     

免疫组织化学法诊断恶性淋巴瘤的研究——(Ⅱ,石蜡切片)

应用ABC免疫组化方法标记10多种单克隆抗体,用常规石蜡切片对100例临床诊断为恶性淋巴瘤的病例进行了研究。结果证明,在石蜡切片中能对恶性淋巴瘤发生阳性反应。否认了以往认为抗淋巴细胞各种单克隆抗体仅能用于冰冻切片新鲜淋巴组织的论点,检出淋巴瘤81例(B细胞性71例、T细胞性10例),何杰金氏病9例。单克隆抗体MT-1、CUHL-1、对T细胞性淋巴瘤呈阳性反应,MB-1、MB-2、LN-2、LN-3、L-26对B细胞性淋巴瘤呈阳性反应。Leu-M_1对何杰金氏病的诊断可起相当大的作用。     

A panel approach to the evaluation of the sensitivity and specificity of antibodies for the diagnosis of routinely processed histologically undifferentiated human neoplasms

The evaluation of antibodies for diagnostic purposes on routinely processed sections of histologically undiagnosable neoplasms presents special problems. The authors have addressed this problem by constructing a panel of putatively mutually exclusive antibodies and testing it on sections of anaplastic neoplasms. Tissue sections of 120 routinely processed histologically undifferentiated large cell human neoplasms with a histologic differential diagnosis of carcinoma versus lymphoma and/or melanoma were stained with a panel of antibodies composed of monoclonal antikeratin AE1, monoclonal antileukocyte common antigens PD7/26 and 2B11, and rabbit anti-S-100 protein. Only cases not diagnosable by routine morphologic examination were included. PD7/26 and/or 2B11 were positive in 61 cases (supporting lymphoma), AE1 was positive in 17 cases (supporting carcinoma), and anti-S-100 was positive in 25 cases (supporting melanoma). Seventeen neoplasms failed to react with any of the panel antibodies. None of the neoplasms reacted with antibodies directed against two or more different antigens. These results indicate excellent specificity (100%) and good sensitivity (86%) of the panel antibodies on histologically undifferentiated neoplasms. These results are significant on two levels: first, as a test of the panel approach to evaluate antibodies on anaplastic neoplasms, and, second, as a demonstration of the diagnostic utility of the specific panel the authors have employed in such cases.     

Adenomatoid tumours: a mucin histochemical and immunohistochemical study

We have examined 16 cases of adenomatoid tumour using a panel of monoclonal and polyclonal antibodies and also stained them for the presence of hyaluronidase-sensitive alcianophilic material. Fourteen cases expressed cytokeratins and a proportion of these also expressed S-100 protein, neuron-specific enolase, vimentin, and human milk fat globule protein 2. The same 14 cases also showed hyaluronidase-sensitive staining with alcian blue. No expression of factor VIII-related antigen was seen in these cases. We conclude that this provides further evidence of a mesothelial origin of these tumours. The remaining two cases did not express cytokeratins and no hyaluronidase-sensitive staining with alcian blue was seen. They did however express factor VIII-related antigen. Although they were morphologically indistinguishable from the other 14 cases, we suggest that they should be more properly regarded as angiomas.     

Large cell lymphoma of bone. A report of three cases of B-cell origin

Clinicopathological and immunohistological features of three cases of large cell lymphoma of bone are reported. On histological grounds, all the cases were diagnosed as histiocytic lymphomas (Rappaport) or primary centroblastic lymphomas, polymorphic subtype (Kiel). On immunophenotyping, malignant cells strongly reacted with the anti-leucocyte antibodies PD7/26 and ROS-220C, thereby indicating their lymphomatous nature, and expressed the B-cell antigens CD19 and CD22. Further studies are warranted to determine whether the B-cell phenotype observed in our cases is typical of the majority of primary large cell lymphomas of bone. Immunohistological analysis with monoclonal antibodies is expected to be of great value not only in defining the immunological phenotype of this rare pathological entity, but also in differentiating it from other neoplasms that involve the skeleton, either primarily or secondarily.