Single-strand conformational polymorphism (SSCP)-detected p53 gene mutations are a less sensitive marker of malignancy in pleural fluids than p53 immunostaining

p53 immunostaining has been advocated as a marker of malignancy in pleural biopsies and serous fluids. The object of this study was to compare the sensitivity and specificity of p53 immunostaining for the detection of malignant cells in pleural fluids with a technique designed to detect p53 gene mutations in exons 5, 6, 7 and 8 by SSCP and nucleotide sequencing. Five out of eight pleural fluids containing adenocarcinoma showed p53 immunostaining and two of these also showed polymorphisms on SSCP and a mutation on sequencing. None of the 10 benign pleural fluids showed immunostaining for p53 or polymorphisms on SSCP. We believe that the poor sensitivity of p53 gene mutation by SSCP is mainly due to DNA from the background reactive cells 'swamping' the mutant DNA. We do not advocate its use as a diagnostic aid.     

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.     

An immunoperoxidase study of S-100 protein in neoplastic cells in serous effusions. Use as a marker for melanoma

S-100 protein has been demonstrated on histologic sections in a number of neural and nonneural tissues, including a variety of neoplasms. Since pleural or peritoneal effusions are frequently the initial presentation of cancer, a study was undertaken to determine if S-100 protein in exfoliated cancer cells could be used as a marker for melanoma. Cells in 36 serous fluids obtained from 32 patients were retrospectively examined for S-100 protein by the peroxidase-antiperoxidase technique. All samples had been previously studied as Papanicolaou-stained cytology specimens, and 25 samples had been studied by transmission electron microscopy. All benign effusions were negative for S-100 protein. Malignant effusions were negative except for some that contained malignant melanoma cells: two of five pigmented melanomas and both cases of amelanotic melanoma. This study indicates that S-100 protein in malignant cells is a useful marker for malignant melanomas, especially the amelanotic type.     

Significance of immunocytochemical expression of E-cadherin, N-cadherin and CD44 in serous effusions using liquid-based cytology

OBJECTIVE: To assess the diagnostic utility of E-cadherin (E-cad), N-cadherin (N-cad) and CD44 to discriminate adenocarcinoma cells from benign and malignant mesothelial cells in body cavity fluids and to clarify the origin of cancer cells. STUDY DESIGN: A total of 120 ThinPrep (Cytyc Corp., Boxborough, Massachusetts, U.S.A.) cytologic specimens of serous effusions, which included 22 cases of reactive mesothelium, 6 cases of malignant mesothelioma and 92 cases of metastatic adenocarcinoma from various sites, were immunostained for E-cad, N-cad and CD44. RESULTS: Eighty-three of 92 metastatic adenocarcinomas (90.21%) expressed E-cad, while 1 of 6 malignant mesotheliomas and 1 of 22 cases of reactive mesothelium were positive for E-cad. All 6 cases of mesothelioma expressed N-cad, whereas most cases of metastatic adenocarcinomas were negative. CD44 immunoreactivity was seen in 18 of 22 (81.81%) benign effusions and in 21 of 92 (22.82%) metastatic adenocarcinomas. CONCLUSION: The combination of E-cad, N-cad and CD44 appears to be a useful panel for distinguishing metastatic adenocarcinoma, mesothelioma and reactive mesothelium and also for clarifying the exact histogenetic origin of cancer cells. This is of great importance in a few otherwise-insoluble cases because of differences in tumor treatment and prognosis.     

Significance of pericellular lacunae in cell blocks of effusions.

A retrospective study was conducted to assess the usefulness of pericellular lacunae in cell block sections of serous effusions in diagnosis. From January to December 1988, 286 cell blocks were prepared in our laboratory from pleural, pericardial and peritoneal fluids; 62 of them were excluded from this study because of inadequate cellularity, diagnostic uncertainty or lack of a proteinaceous background. The remaining consisted of 148 benign effusions from 128 patients and 76 malignant effusions from 56 patients. A single specimen from each patient was selected and reviewed to assess the presence and number of pericellular lacunae and to determine the relationship of this feature to cell arrangement (single cells versus cell clusters). Pericellular lacunae were found in 42 (75%) of the malignant effusions as compared to 41 (32%) of benign specimens. In the majority of malignant cases with lacunae, this feature was associated with greater than two-thirds of the cells, whereas in benign cases, when present, it was seen in less than one-third of the cells. Neoplasms characterized by large cell clusters more frequently had lacunae than did those with small groups or single cells. Lacunae were not evident in cases of malignant melanoma and lymphoma. We conclude that although pericellular lacunae are more often associated with malignant cells, their presence in itself cannot be used as a reliable indicator of malignancy in body cavity fluids.     

DNA flow cytometry of pleural effusions. Comparison with pathology for the diagnosis of malignancy

A study was undertaken to determine the potential value of DNA flow cytometry (FCM) for the diagnosis of malignancy in pleural effusions and to compare its results with those of traditional cytomorphology. Forty-one pleural fluids from 37 patients were evaluated by DNA FCM and routine cytologic techniques. Of the 41 pleural fluids, 29 (70.7%) demonstrated an abnormal DNA content by FCM. Two of the 29 pleural fluids had been originally diagnosed as benign by cytology. Cytologic review of these two cases showed no abnormal cells; therefore, there were no false-negative cases based on cytology. In 12 (29.3%) of the 41 fluids, DNA FCM and cytologic evaluation both indicated benign processes. Our preliminary observations indicate that FCM is an accurate and reliable technique that may be of aid in the diagnosis of malignant effusions. The technique may prove to be of special value in the differential diagnosis of reactive mesothelial cells versus malignant mesothelioma as well as for following patients who receive chemotherapy for malignant pleural effusions. DNA FCM may also complement cytomorphologic diagnoses in other serous, exfoliative or aspiration material.     

Diagnostic utility of sialosyl-Tn in discriminating carcinomatous cells from benign mesothelium in body cavity effusions

OBJECTIVE: Detecting malignant cells in the setting of reactive mesothelium can be difficult. Several techniques have been tried but without widespread acceptance. Sialosyl-Tn (STn) is an aberrantly glycosylated precursor of the MN blood group antigen frequently expressed in carcinomas and dysplastic epithelium. We investigated the STn monoclonal antibody for its clinical utility as an isolated stain to discriminate benign mesothelium from malignant cells. STUDY DESIGN: Cell block material from 72 cases of body cavity fluids were immunostained for STn using the avidin-biotin complex method without antigen retrieval. Slides were incubated overnight at 4 degrees C in a humidified chamber. RESULTS: Strong immunoreactivity was noted in 31/40 (77%) carcinomatous cases. Only moderate staining was noted in 1 of 28 (4%) benign effusions and weak staining in 5 (18%) additional benign cases. Specificity was 100%, sensitivity 78%, positive predictive value 100% and negative predictive value 76%. No staining was noted in four noncarcinomatous malignant effusions. CONCLUSION: STn may have diagnostic value in this cytologic setting as part of a diagnostic panel but not as an isolated stain.     

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.     

Characterizing subpopulations of neoplastic cells in serous effusions. The role of immunocytochemistry

OBJECTIVE: To analyze the role of immunochemistry in serous effusions. STUDY DESIGN: We analyzed cell blocks of 18 pleural and 18 peritoneal effusions diagnosed as malignant (18), benign (14) and suspicious (4). They were immunostained by the avidin-biotin complex method with a panel of four monoclonal antibodies--CEA, Ber-EP4, LeuM1 (CD15) and p53--and, for lectins (Ulex europaeus) UEA-l, ConA and ConBr. RESULTS: Seventeen of the 18 cases of adenocarcinoma were positive for CEA (95%), 12 (66.6%) for Ber-EP4, 11 (61%) for CD15 and 11 (61%) for p53. Twelve of the 18 (66.6%) were positive for UEA-1, CEA, Ber-EP4 and CD15. UEA-1 did not react with mesothelial cells. p53 Gave a positive reaction in only one case, reactive mesothelial cells. ConA and ConBr reacted indiscriminately with benign and malignant cells; thus, it was not useful in distinguishing between these cells. CONCLUSION: In this context no antibody used alone is reliable for corroborating a diagnosis, but the selective use of a small panel of three markers (CEA, Ber-EP4 and LeuM1) can be very useful in solving diagnostic difficulties in the cytodiagnosis of serous effusions.     

Diagnostic utility of BCA-225 in detecting adenocarcinoma in serous effusions

OBJECTIVE: To determine the diagnostic value of the BCA-225 antibody in discriminating adenocarcinoma from benign mesothelium in body cavity effusions. STUDY DESIGN: One hundred four cases of unequivocally benign (34 cases) and malignant (70 cases) serous effusions with cell block material were immunostained for BCA-225 using the ABC method without antigen retrieval. The percentage of positively staining cells in each case was estimated in a blind fashion. RESULTS: BCA-225 stained at least 10% of morphologically malignant cells in 28 of 32 (88%) breast carcinomas and 58 of 67 (87%) adenocarcinomas overall. Neuroendocrine carcinomas (two cases) and one mesothelioma were positive in < or = 5% of their respective tumor cells. Of 34 benign cases, 6 (18%) exhibited positive staining, albeit in rare, morphologically benign cells. CONCLUSION: BCA-225 is able to discriminate adenocarcinoma from reactive mesothelium in cell block preparations and may prove useful as part of an antibody panel.     

Diagnostic value of CA 15-3 antibody in detecting metastatic adenocarcinoma

OBJECTIVE: To determine the diagnostic value of CA 15-3 in detecting metastatic adenocarcinoma in body fluids using PreservCyt solution (Cytyc Corp., Boxborough, Massachusetts, U.S.A.) as collection fluid. STUDY DESIGN: Cytospin slides prepared from 72 cases with unequivocally benign or malignant diagnosis were studied. Of the cases studied, 34 were breast carcinomas, and 17 were benign pleural effusions. Slides were stained for CA 15-3 by using the avidin-biotin complex method. Cases were evaluated for the presence of membranous or cytoplasmic staining. The percentage of cells exhibiting strong staining was estimated for both breast carcinoma and all adenocarcinomas as a group. These results were compared with CA 15-3 staining exhibited by benign mesothelium. RESULTS: Ninety-one percent of the breast cancer cases studied showed a positive reaction with CA 15-3, while 6% of the benign mesothelium cases were positive (p < 0.01). The sensitivity of CA 15-3 was 91 % for breast carcinoma and 80% for all adenocarcinomas. Specificity was 94% for breast carcinoma and for all adenocarcinoma. CONCLUSION: CA 15-3 is a sensitive and specific marker for diagnosing adenocarcinoma in cytologic specimens using PreservCyt solution as collection fluid.     

Clinicopathological study of metallothionein immunohistochemical expression, in benign, borderline and malignant ovarian epithelial tumors

Metallothioneins (MTs) are a family of cystein-rich metal-binding proteins, which are expressed in normal cells during fetal and postnatal life but also in a variety of human neoplasms. MT expression in human tumors has been linked to resistance to anticancer drugs and differentiation and progression in some types of tumors. This study examined the immunohistochemical expression of MTs in benign, borderline and malignant tumors of ovarian surface epithelium and the possible correlations with clinicopathological parameters and survival. A total of 87 cases with diagnosis of ovarian surface epithelial tumors were included. Specifically, 21 cases of benign cystadenomas (11 serous and 10 mucinous), 14 borderline (low malignant potential tumors, 8 mucinous and 6 serous) and 52 cases of ovarian cancer were analysed. Immunohistochemical expression of MT (cut-off level > 10% of tumor cells) was clearly associated with malignancy. A statistically significant correlation was found between the expression of MT in cancer cases and benign tumors (p < 0.0001) and cancer cases and borderline tumors p = 0.003. In cancer cases a difference was observed between grade I and III (p = 0.002). There was no correlation of MT overexpression with survival in the small number of ovarian carcinoma patients where it was analysed. MT constitutes a marker that characterizes aggressiveness and a high malignant potential in ovarian epithelial tumors. In diagnostic problems MT may help distinguish between benign, borderline and malignant tumors.     

Identification of Malignant Cells In Serous Effusions Using A Panel of Monoclonal Antibodies Ber-Ep4, Mca-B-12 and Ema

A panel of three monoclonal antibodies (MoAbs) was tested on 29 benign and 53 malignant effusions with the aim of investigating its usefulness for the discrimination between benign and malignant lesions. The panel consisted of MoAbs directed against epithelial membrane antigen (EMA); MCA-b-12, reacting with a 350 kD glycoprotein with mucin-like characteristics present on human breast cancer cells and various other normal and neoplastic tissues, and Ber-EP4, directed against a 34 and 39 kD glycopeptide on human epithelial cells but not on mesothelium. Fifty-two (98%) of the malignant effusions reacted with EMA, 49 (92%) with MCA-b-12 and 44 (83%) with Ber-EP4. Fourteen per cent of benign effusions reacted with EMA, 17% with MCA-b-12 and 7% with Ber-EP4. All seven effusions obtained from patients with a malignant mesothelioma reacted with EMA, six of the seven cases staining intensively. None of the seven stained with Ber-EP4. MCA-b-12 did not react with the cells in one case of malignant mesothelioma. The results suggest that the combination of EMA and Ber-EP4 may be used to discriminate between benign and malignant cells and possibly also between adenocarcinoma and malignant mesothelioma. MCA-b-12 followed in general the reaction pattern of EMA, although often with a less intense staining reaction, making this antibody unsuitable for inclusion in the panel.     

Diagnostic utility of GLUT-1 expression in the cytologic evaluation of serous fluids

OBJECTIVE: To evaluate the extent to which adenocarcinomas in body cavity fluids express GLUT-1 in comparison to currently available markers for adenocarcinomas. STUDY DESIGN: Archival paraffin-embedded cell blocks of serous fluids from 25 cases of benign effusions containing reactive mesothelial cells and 39 cases of malignant effusions with metastatic adenocarcinoma (11 ovarian, 11 pulmonary, 9 gastrointestinal and 8 breast) were retrieved from the surgical pathology files. All cases were stained with antibodies for GLUT-1, Ber-Ep4, B72.3 and CEA. Positive staining was defined as distinct linear membrane staining for GLUT-1 and Ber-EP4, cytoplasmic staining for CEA, and cytoplasmic or membrane staining for B72.3. Strong staining in at least 10% of the tumor cells was required in order to consider the case positive for the particular marker. RESULTS: GLUT-1 was expressed in 72% (28 of 39) of cases of malignant effusions: 100% (11 of 11) from the ovary, 91% (10 of 11) from the lung, 67% (6 of 9) from the gastrointestinal tract and 12% (1 of 8) from the breast. None (0 of 25) of the benign effusions expressed GLUT-1. Malignant effusions expressed CEA in 74% (29 of 39), Ber-Ep4 in 85% (33 of 39), and B72.3 in 62% (24 of 39). Benign effusions expressed CEA in 3 cases and B72.3 in 2 cases. CONCLUSION: GLUT-1 is a useful marker that can be applied to cytologic specimens. It can be used as a reliable component of an antibody panel to distinguish reactive mesothelial cells from metastatic adenocarcinoma in particular adenocarcinomas of body cavity effusions, in particular adenocarcinomas of ovarian and pulmonary origin.     

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     

MN/CA9: a potential gene marker for detection of malignant cells in effusions.

Many cancers cause malignant effusions. The presence of malignant cells in effusions has implications in diagnosis, tumour staging and prognosis. The detection of malignant cells currently presents a challenge for cytopathologists. New adjunctive methods are needed. Although the effusions provide excellent materials for molecular assay, the available molecular markers are extremely limited, which hinders its clinical application. MN/CA9 has proved to be a valuable marker in many cancers such as lung, breast, colon, kidney, etc. The present study was to evaluate MN/CA9 as a new molecular marker for the detection of cancer cells in pleural effusions. Seventy-one pleural effusions including 59 malignant effusions from patients with cancer, and 12 patients with benign diseases as a control, were subjected to RT-PCR for detection of MN/CA9 gene expression. MN/CA9 gene expression was detected in 53/59 (89.8%) pleural effusions from cancer patients (15/16 for breast cancers, 10/11 for lung cancers, 4/4 for ovary cancers, 2/3 for colon-rectal cancers, 5/6 for cancers of unknown site, 7/8 for mesothelioma and 10/11 for other cancers). Furthermore, MN/CA9 was positive in 13/18 (72.2%) of cytologically negative effusions of cancer patients. MN/CA9 was detected in only 1/12 (8.3%) effusions from the control patients (p < 0.01). The sensitivity and specificity of MN/CA9 gene expression were, respectively, 89.8% and 91.7%. Our preliminary results suggest that MN/CA9 could be a potential marker for the detection of malignant cells in effusions. A large-scale study is needed to confirm these results.     

Histological examination of clots from cytological specimens: is this a worthwhile procedure?

A comparison of conventional cytology and clot histology was made on 174 serous fluids, fine needle aspirates (FNA) and other non‐gynaecological specimens. In eight cases (4.5%) this clot material contained malignant cells or cells suspicious of malignancy despite the absence of suspicious or malignant cells in conventionally prepared smears from the same specimen. In 11 cases (6.3%) the clot was negative, although conventional smears contained malignant or suspicious cells. A χ² test showed that there was no statistically significant difference between the number of positive diagnostic scores in each group with χ² of 0.223 (degrees of freedom=1), P=0.637. Examination of clot material from serous fluids and FNA aspirates is as effective as examination of conventional cytological preparations. Processing of clots from cytological aspirates for histological examination should be more widely adopted, and is applicable in all cytopathology laboratories.     

p53 immunohistochemistry for distinguishing reactive mesothelium from low grade ovarian carcinoma

OBJECTIVE: To determine the utility of immunohistochemical staining for p53 in cell block material for distinguishing reactive mesothelium from borderline or low grade ovarian carcinoma. STUDY DESIGN: Paraffin-embedded cell blocks from paracentesis and pelvic wash fluid of 44 cases of ovarian carcinoma and 20 cases containing only reactive mesothelium were immunostained for p53 using monoclonal antibody DO-7. Tumor grades ranged from borderline to high grade and were serous papillary (33), clear cell (3), mucinous (2), endometrioid (2), mixed serous papillary/clear cell (3) and undifferentiated (1). The three authors independently evaluated the staining, including estimation of the percentage and intensity of positive nuclear staining. RESULTS: A separation of positive from negative cases was seen when staining intensity was considered the critical parameter; moderate to strong staining was considered truly positive. Seventy-three percent (8/11) of borderline tumors, 80% (8/10) of low grade tumors and 65% (15/23) of intermediate to high grade tumors showed moderate to strong positivity. Percentage of staining was a less-reliable parameter as 25% of negative cases were positive by this assessment. CONCLUSION: p53 Immunohistochemistry, using monoclonal antibody DO-7 combined with standard morphologic evaluation, may be useful in distinguishing benign reactive mesothelium from borderline or low grade ovarian carcinoma.     

Diagnostic DNA-flow- vs. -image-cytometry in effusion cytology.

AIMS: To determine the sensitivity and specificity of flow- and image-cytometry for the detection of DNA-aneuploidy as a marker for malignant cells in effusions. METHODS: 200 effusions (80 tumor cell-positive, 74 negative and 46 cytologically equivocal) were stained with DAPI-SR for DNA-flow- and with Feulgen-Pararosaniline for -image-cytometry. They were measured using a PAS-flow-cytometer and an AutoCyte-QUIC-DNA-workstation according to the ESACP consensus reports for DNA-flow- and -image-cytometry, respectively [7,23,29,49]. RESULTS: Sensitivity of DNA-aneuploidy for the identification of malignant cells was 32.1% for DNA-flow- and 75.0% for -image-cytometry, specificity of -euploidy in benign cells was 100.0% for both methods. Positive predictive value of DNA-aneuploidy for the identification of malignant cells was 100.0% for both techniques, negative predictive value of DNA-euploidy was 48.6% for DNA-flow- and 72.0% for -image-cytometry. CONCLUSIONS: Searching for DNA-aneuploidy as a diagnostic marker for neoplastic cells in serous effusions image-cytometry revealed superior sensitivity as compared with monoparametric flow cytometry.