Malignant Serous Effusions Research Articles

Evaluation of a Panel of Molecular Markers for the Diagnosis of Malignant Serous Effusions

Our main goal was to evaluate a panel of molecular markers for the detection of cancer cells in serous effusions and to determine their value as an adjunctive reverse transcription-PCR (RT-PCR) test to cytologic examination. One hundred fourteen serous effusions from 71 patients with tumors and 43 patients with benign diseases were subjected to RT-PCR for expression of carcinoembryonic antigen (CEA), epithelial cell adhesion molecule (Ep-CAM), E-cadherin, mammaglobin, mucin 1 (MUC1) isoforms MUC1/REP, MUC1/Y, and MUC1/Z, calretinin, and Wilms' tumor 1 susceptibility gene. CEA, Ep-CAM, E-cadherin, and mammaglobin were specifically expressed in malignant effusions. The sensitivity of RT-PCR in cytologically negative malignant effusions was 63.1% combining CEA and Ep-CAM (with 100% specificity) and reached 78.9% adding MUC1/Y or MUC1/Z (with 93% specificity). In the whole population of effusions, the combination of cytology with RT-PCR of CEA and Ep-CAM yielded a 90.1% sensitivity, a specificity and a positive predictive value of 100%, and a 86% negative predictive value for malignancy. Adding MUC1/Y or MUC1/Z to the panel, the sensitivity was 94.5% with 93% specificity, 95.7% PPV, and 90.9% negative predictive value. Moreover, CEA and mammaglobin were specifically expressed in epithelial malignancies, and mammaglobin was mainly expressed in effusions from breast carcinoma (97.3% of specificity). A combination of cytology and RT-PCR analysis of CEA and Ep-CAM significantly improved the detection sensitivity of tumor cells in serous effusions. RT-PCR analysis of CEA, Ep-CAM, and mammaglobin in serous effusions could be a beneficial adjunct to cytology for the diagnosis of malignancy.

The value of immunocytochemistry in differentiating benign and malignant serous effusion

一、材料与方法 1.材料:收集我院2003年6月至2004年6月门诊及住院患者96例浆膜腔积液标本,其中胸水72例,腹水18例,心包积液6例.患者男62例,女34例,年龄27～84岁。

Initial diagnosis of breast cancer via cytological examination of a pleural effusion: A rare event facilitated by recognition of an unusual morphological pattern

It is uncommon for breast carcinoma to present as a malignant serous effusion. Here, we describe a case in which the initial diagnosis of an occult invasive ductal carcinoma of the breast was made via cytological examination of a pleural effusion. Recognition of a cribriform architecture with intraluminal necrosis and microcalcifications in a cell block preparation was critical in making that diagnosis. To our knowledge, this specific morphological pattern of breast carcinoma in a cell block preparation from an effusion has not been reported previously.

Indian File Pattern of Adenocarcinoma Cells in Effusions

To determine the prevalence of the Indian file pattern of adenocarcinoma cells in malignant serous effusions. Twenty-five malignant and 10 benign effusion samples were selected. Clinical data, including the sites of the primary neoplasms, were noted. The presence or absence of Indian file arrangement of carcinoma cells and other patterns were recorded. Seven of the 25 positive cases showed Indian file arrangement, whereas none of the negative cases did. Their number did not show any correlation with the type and site of the primary neoplasms. In most of the 7 cases other patterns, such as 3-dimensional balls and papillations, were also present. Cells in Indian file in serous effusions are strongly suggestive of metastatic carcinoma. They are not specific to any particular site of the primary adenocarcinoma.

E-cadherin and calretinin as immunocytochemical markers to differentiate malignant from benign serous effusions.

To investigate the expressions of E-cadherin and calretinin in exfoliated cells of serous effusions and evaluate their values in distinguishing malignant effusions from benign ones. Fresh serous effusion specimens were centrifuged and exfoliated cells were collected. Cells were then processed with a standardized procedure, including paraformaldehyde fixation, BSA-PBS solution washing and smears preparation. E-cadherin and calretinin were detected by immunocytochemistry (ICC). In the exfoliated cells of serous effusions, most of carcinoma cells only expressed E-cadherin, and most of mesothelial cells only expressed calretinin, and benign cells (lymphocytes and granulocytes) did not express either of them. For E-cadherin, 85.7% (30/35) of malignant effusions and 8.1% (3/37) of benign fluids were ICC-positive (P<0.001). The sensitivity of E-cadherin ICC in the diagnosis of malignant effusions was 85.7%, specificity 91.9%, and diagnostic rate 88.9%. For calretinin, 94.6% (35/37) of benign effusions and 11.4% (4/35) of malignant effusions were ICC-positive (P<0.001). The sensitivity of calretinin ICC in the diagnosis of benign effusions was 94.6%, specificity 88.6%, and diagnostic rate 91.7%. For diagnosis of benign and malignant effusions by combining E-cadherin ICC and calretinin ICC, the specificities were up to 100% and 97.1%, respectively. E-cadherin ICC and calretinin ICC are sensitive and specific in differential diagnosis of benign and malignant serous effusion specimens and specificities are evidently improved when both markers are combined.

Bull’s Eye (Target) Inclusions in Neoplastic Cells in Malignant Serous Effusions

To study the prevalence and significance of bull's eye (target) inclusions in neoplastic cells in malignant serous effusions. We reviewed malignant pleural, peritoneal and pericardial effusions from 289 patients who had proven cancer at known primary sites. The ages of the patients ranged from 5 to 72 years; 166 were male and 123 female. Bull's eye inclusions are an uncommon finding and appeared in only 13 cases of metastatic adenocarcinoma of the breast, stomach, colon, lung, ovary, pancreas and urinary bladder. They were positively stained with periodic acid-Schiff stain with diastase. The inclusions were not seen in cells of nonadenocarcinomatous neoplasms, such as squamous cell carcinoma, oat cell (small cell) carcinoma, neuroblastoma, lymphoma and germ cell tumors. Bull's eye inclusions are found in about 5% of malignant serous effusions containing cells of metastatic adenocarcinoma. The primary site of an adenocarcinoma cannot be deduced on the basis of the presence of inclusions.

Immunohistochemical detection of cytotoxic lymphocytes in malignant serous effusions.

The use of ancillary techniques to aid in the diagnosis of metastatic carcinoma in serous effusions has been the subject of numerous studies. In this article, we study 35 cases of malignant effusions (metastatic adenocarcinoma) and 20 benign effusions using a panel of immunohistochemical markers to determine whether changes in the subpopulations of accompanying lymphoid cells can be detected with this technique and whether such changes are associated with the presence of malignancy. We noted a significant increase in cytotoxic lymphocytes, defined as the percentage of all lymphoid cells staining with an antibody to TIA-1 (an antigen localized to the cytotoxic granule membranes of cytotoxic T cells and natural killer cells) in malignant compared with benign effusions (23% vs. 12%; P < 0.05). In addition, nearly all cases in which cytotoxic lymphocytes composed > 20% of the lymphoid cell population contained metastatic tumor. Thus, immunohistochemical staining for TIA-1 can reliably detect cytotoxic lymphocytes in cell blocks of serous effusions; in addition, a relative increase in their number is associated with the presence of malignancy.

Occurrence of intercellular spaces (windows) in metastatic adenocarcinoma in serous fluids: a cytomorphologic, histochemical, and ultrastructural study.

The application of cytomorphologic criteria to the examination of serous effusions allows the reliable diagnosis of malignancy in the majority of cases. One feature observed in tissue fragments previously thought to be indicative of mesothelial origin is the presence of intercellular windows, presumably due to long surface microvilli. In this study, however, we examined cytologic preparations of 143 effusion and body-cavity washing specimens and noted distinct intercellular window formation within tissue fragments of adenocarcinoma in 13% of the cases studied. Stains for mucicarmine, Alcian blue with hyaluronidase pretreatment, and periodic acid-Schiff following diastase digestion on corresponding cell block material demonstrated that intercellular mucin contributes to such window formation in greater than half of these cases. Thus the presence of intercellular windows within tissue fragments does not, in isolation, preclude the diagnosis of malignancy in serous effusions.

Tumor markers in the diagnosis of malignant serous effusions.

To assess the possibility of increasing the detection rates of cytological examination in malignant effusions by the selection of specific tumor markers for a given type of tumor, we measured CEA, CA 19.9, CA 15.3, MCA, PSA, and AFP in malignant effusions from 89 patients with the following primary malignancies: colon, stomach, breast, liver, prostate, lung, and kidney. Cytological examination was positive in only 35 of 89 patients (40%), while the tumor markers were positive in 72 of 89 cases (80%). However, apart from small cell lung and kidney cancers, where the lack of a specific tumor marker resulted in no advantage, in the other types of tumors, the specific marker for each tumor identified correctly malignant effusions in 72 of 74 cases (97%). In fact, CEA was positive in 11 of 11 effusions induced by colorectal cancer; CA 19.9 in 28 of 30 gastric cancer effusions, while MCA and CA 15.3 were positive in breast cancer effusions (16/22 and 20/22). Finally, elevated AFP and PSA indicated hepatocellular and prostate cancer, respectively. In conclusion, in cancer patients with elevated effusion levels of specific tumor markers, the effusions could be considered of a malignant nature even without cytologically demonstrable tumor cells.

Monoclonal antibodies in ovarian cancer

Monoclonal antibodies recognising a number of antigens are used in the diagnosis and treatment of ovarian carcinoma. Conventional histology and cytology can be supplemented by immunohistology. Antigens shed from tumours are routinely measured in patients sera by monoclonal based assays to monitor disease progression. Radiolabelled antibodies can be used to localise tumour in vivo by gamma camera imaging or intraoperatively using a hand held probe. Antibodies conjugated to radionuclides have been successfully used in the treatment of patients with small volume residual disease and malignant serous effusions.

Cytologic diagnosis of malignant mesothelioma in serous effusions using an antimesothelial-cell antibody.

Differentiating mesothelioma, reactive mesothelium, and adenocarcinoma in serous effusions is often difficult, despite the application of ancillary techniques in support of the traditional cytomorphologic criteria. A polyclonal antimesothelial-cell antibody recently developed by our group was evaluated as a histogenetic marker on a series of primary (n = 12) and metastatic (n = 12) malignant effusions. Immunostaining was performed on paraffin sections from cell blocks. All mesothelioma effusions stained positive for the antibody, whereas, in contrast, all metastatic carcinoma specimens failed to react. These results (100 percent specificity and 100% sensitivity for mesothelioma) provide a basis for a reliable use of the antibody in the cytologic examination of suspicious or malignant serous effusions.

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.

In vitro and in vivo regulation of tumor antigen expression by human recombinant interferons

In vitro treatment with either type I or type II Interferon (IFN) can selectively enhance the expession of several tumor antigens, such as the carcinoembryonic antigen (CEA) and the tumor-associated glycoprotein-72 (TAG-72) in different human carcinoma cell lines and result in enhanced level of monoclonal antibody (MAb) binding to the cell surface. In vivo animal studies demonstrated that treatment of athymic mice with a type I interferon [i.e. interferon-α(A)]significantly increased the expression of a 90 kDa tumor antigen which improved the targeting of a MAb to the carcinoma xenograft. More recent studies reported that in vitro IFN treatment of human adenocarcinoma cells isolated from human malignant serous effusions selectively increased the expression of TAG-72 and CEA. One can envision that the ability of these cytokines to upregulate the level of expression of human tumor antigens presents an important experimental model in which to study the regulation of markers often correlated with epithelial cell differentiation. In addition, the increase of selective MAb-defined antigens may also be exploited in an adjuvant setting to localize higher amounts of MAbs to the tumor cell surface and, thereby, improve the effectiveness of a MAb for tumor diagnosis and, possibly, therapy.

Detection of three sialylated tumor-associated antigens in malignant serous effusions.

One hundred thirty-seven fluid samples from patients with malignant diseases, including 58 with lung cancer and 36 with gastric carcinoma, were screened for three tumor-associated carbohydrate antigens. The antigens tested were sialylated Lewis (SLEX) antigen, sialylated SSEA-1 (SLX) antigen, and sialylated Lewis (CA19-9) antigen. Thirty-four fluid samples from patients with benign diseases including 20 with tuberculous pleurisy were also tested for these three antigens as a control. SLEX antigen had the highest positivity rate among the three antigens both in the pleural effusions of patients with pulmonary adenocarcinoma and for all types of lung cancer (64.1% and 46.6%, respectively). In cancers of digestive system, the percentage of SLEX positivity was almost same as that for CA19-9, and higher than that for SLX. A combination assay using the three antigens resulted in an increased rate of detection of tumor-associated antigens (68% of 58 lung cancers including 83% of 39 adenocarcinomas, as well as 77% of 63 digestive system cancers including 84% of 36 gastric carcinomas). Gel filtration of pleural effusions or bile using sephacryl S-1000 showed that these three antigens were eluted in the void volume, indicating a molecular weight of greater than 2 X 10(6). Thus, all 3 antigens possibly exist on carrier protein with a high molecular weight. These observations suggest the potential application of these antigens to distinguish between benign and malignant fluid, and to monitor the effects of the treatment of various types of cancer.

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.

Tumour associated antigens in diagnosis of serous effusions.

The use of tumour associated antigens in the diagnosis of serous effusions was studied in 76 patients with benign and 200 patients with malignant disease. Tissue polypeptide antigen (TPA), alpha fetoprotein, and CA 125 were found to be of little value. At cut off points of 3 ng/ml, 10 U/ml, and 30 U/ml, respectively, carcinoembryonic antigen (CEA), biliary glycoprotein I (BGP I), and CA 19-9 discriminated between benign and malignant serous effusions with a sensitivity of between 24% and 67%. The immunocytochemical staining for these markers resulted in malignant cells being detected in 18% to 33% of cases. Various combinations of conventional cytological examination, effusion fluid tumour marker determination, and immunocytochemical analysis identified malignant cells in serous effusions in up to 72% of cases; conventional cytology alone detected tumour cells in only 30%.

The use of immunoperoxidase panels for the cytologic diagnosis of malignancy in serous effusions

The diagnostic value of an immunoperoxidase panel composed of antisera to carcinoembryonic antigen (CEA), epithelial membrane antigens (EMA), and high- and low-molecular-weight cytokeratins was evaluated on 39 consecutive pleural and peritoneal fluid specimens and correlated with routine cytologic and histochemical studies. The cases were classified into two groups: malignant (epithelial and small-cell undifferentiated carcinomas) and benign effusions. We found that the CEA and EMA antisera identified 91% and 100% of the epithelial malignancies, respectively. Mucin stains were positive in only 41% (mucicarmine) and 59% (Alcian blue) of these cases. The battery of cytokeratin markers identified malignant cells from 45%-100% of the cases but showed considerable positive staining of mesothelial cells. A selective review of the literature is presented along with recommendations for technical improvement in immunoperoxidase studies. We believe that an immunoperoxidase panel can be of considerable value in the cytologic diagnosis of epithelial malignancies in effusions. The panel composed of CEA and EMA can prove helpful in a routine cytology practice. Although the cytokeratin marker identified malignant cells, the concomitant immunostaining of mesothelial cells limits its utility. The commercially available panel can be a potential aid in improving the accuracy of serous fluid cytologic examination by decreasing both false-positive and false-negative diagnoses and thereby helping to prevent delays in diagnosis and treatment.

USE OF TWO EPITHELIUM-SPECIFIC MONOCLONAL ANTIBODIES FOR DIAGNOSIS OF MALIGNANCY IN SEROUS EFFUSIONS

Two monoclonal antibodies, HMFG2 and AUA1, raised against epithelial-cell determinants were used to detect carcinoma cells in smears of serous effusions. Both antibodies react strongly with malignant epithelial cells but not with normal mesothelial or endothelial cells. A third antibody, 11-4.1, which does not react with any human tissue, was used as a negative control. An immunoperoxidase-staining technique was applied to smears of cells from serous effusions. The immunohistochemical diagnosis was in agreement with the cytological diagnosis. In cases with equivocal cytology in which the diagnosis became clear in later samples, the immunohistochemical diagnosis gave the correct result. With this combination of tumour-associated monoclonal antibodies a confident diagnosis of malignancy in serous effusions can be made.

Use of antisera to epithelial membrane antigen for the cytodiagnosis of malignancy in serous effusions.

A new human antigen, designated epithelial membrane antigen (EMA), has recently been described on surface membranes of a wide variety of normal epithelium but not on connective tissue cells. The antigen is only weakly expressed on normal or reactive mesothelium. Increased expression of the antigen has been observed in most neoplasms of epithelial origin and in malignant mesothelioma. We have investigated the possibility of using this difference in the expression of the antigen to distinguish between mesothelial cells and malignant cells in cytological smears of serous effusions. This distinction cannot always be made on morphological grounds alone and problems of differential diagnosis are encountered in about 15% of all specimens of serous effusions sent for cytological examination. Using antisera to EMA we have applied an indirect immunoalkaline phosphatase technique to alcohol-fixed smears prepared from serous effusions and have found that intense staining of the antigen is confined to effusions from patients in whom there is either clinical or cytological evidence of malignancy. The technique proved to be especially useful in cytologically equivocal cases, where there were problems of differential diagnosis.

Cellular contamination during automatic and manual staining of cytological smears.

Three routine cervical smears were contaminated with adenocarcinoma cells from a malignant serous effusion which was being processed simultaneously in an automatic staining machine. Further investigation showed that cells were transferred in up to 17% of cases. However, with manual staining, the figure could be as high as 30%. The possibility of misleading cellular transfer should therefore always be considered. Serious fluids should not be processed with routine cervical smears.