免疫细胞化学方法对胸腔积液中恶性肿瘤细胞的分类与诊断

目的应用免疫细胞化学对胸腔积液中的肺非小细胞癌分类与恶性间皮瘤的鉴别诊断。方法利用液基薄层细胞学自动涂片技术方法对筛查出的胸腔积液可疑瘤细胞及瘤细胞标本1158例进行细胞包埋连续切片,分别作肺非小细胞癌（NSCLC）肿瘤细胞标记物CK7、CK5＆6、TTF-1、E—Ca及恶性间皮瘤标记物MC（MesothelialCell,MC）、CR（Calfetinin,CR）、P53、Vimentin免疫细胞化学染色。结果1158例胸腔积液患者确诊为肺腺癌581例,鳞癌509例,腺鳞癌48例,恶性间皮瘤20例。TTF-1在腺癌中有明显高表达,阳性表达率为92．43％;CK58L6在鳞癌中有明显高表达,阳性表达率为97．45％;MC、CR在恶性间皮瘤中有明显高表达,阳性表达率为100．00％和95．00％。结论液基细胞学与免疫细胞化学技术相结合在胸腔积液鉴别诊断中有很重要的临床意义,CK7、CK58L6、TTF-1、E—ca联合应用可用于胸腔积液中NSCLC之间的分类与诊断,CK58L6、MC、CR、P53、Vimentin联合应用可用于胸腔积液中间皮瘤的定性诊断,值得在临床细胞病理学诊断中推广应用。     

CK5/6 in effusions: no difference between mesothelioma and pulmonary and nonpulmonary adenocarcinoma

OBJECTIVE: To test the performance of CK5/6 for the differentiation between mesothelioma, adenocarcinoma and benign mesothelia/proliferations in effusion cytology. STUDY DESIGN: CKS/6 immunocytochemistry was applied to ethanol-fixed cytospin preparations from 74 benign and malignant effusions. RESULTS: Reactivity was seen in 7 of 8 mesotheliomas and in 9 of 11 benign mesothelial proliferations but also in 11 of l7 pulmonary adenocarcinomas and in 12 of 31 adenocarcinomas of nonpulmonary origin. Reactivity was also found in 3 of 5 non-small cell lung carcinomas and 1 of 1 squamous carcinoma. CONCLUSION: CK5/6 reactivity was found in a considerable proportion of metastatic adenocarcinomas of pulmonary and nonpulmonary origin. The high reactivity rate in pulmonary adenocarcinomas disagrees with the results obtained with histologic sections from solid tumor tissue, and CK5/6 seems to be of very limited value as an additional marker in effusion cytology.     

SM047 immunoreactivity in peritoneal fluids

SM047 is a recently developed monoclonal antibody generated against an ovarian adenocarcinoma cell line. A recent immunohistochemical study has shown that SM047 is strongly expressed in tissue sections of most ovarian serous adenocarcinomas. This study aimed to ascertain whether SM047 staining is of value in cytological preparations of peritoneal fluid. A total of 206 consecutive peritoneal fluids were stained immunocytochemically with SM047, CA125, monoclonal carcinoembryonic antigen (mCEA), Ber-EP4 and cytokeratins (CK7 and 20). SM047 positivity was present in reactive mesothelial cells in 117 of 141 (83%) benign cases in which these were present. SM047 positive tumour cells were present in 22 of 23 (96%) ovarian serous adenocarcinomas and in small numbers of gastric adenocarcinomas (two of three), mesotheliomas (one of two) and pancreatic adenocarcinomas (one of one). All six colorectal and two breast adenocarcinomas were negative with SM047. Reactive mesothelial cells in all cases were positive with CK7 and in most cases with CA125. They were negative with CEA, Ber-EP4 and CK20. All adenocarcinomas were positive with Ber-EP4 and mesothelial cells were always negative. All colorectal adenocarcinomas were positive with CK20. This study shows that SM047 staining may be of value in the diagnosis of an ovarian serous adenocarcinoma in peritoneal fluids. Negative staining helps to exclude a primary ovarian serous adenocarcinoma and is characteristic of colorectal adenocarcinoma. The small numbers of other malignancies in the study precludes a judgement of the value of SM047 staining in these neoplasms. SM047 staining may be useful, as part of a larger panel, in the work up of patients with peritoneal effusions.     

Mesothelioma Symposium 11.30–12.30 Tuesday 16 September 2003. Cytopathology of malignant mesothelioma

The diagnosis of malignant mesothelioma on the cytology of serous effusions is a two‐phase process. First is to determine that the effusion is malignant based on morphological features such as a highly cellular fluid with many large three dimensional cell aggregates, and/or the recognition of minor malignant criteria including prominent cell engulfment, uniformly present very prominent nucleoli, or the finding of very large (giant) cells. In cell block sections, strong positive staining with EMA often with cell membrane accentuation provides compelling support for a cytological diagnosis of malignancy. Second is to recognize that the malignant cells have a mesothelial phenotype and do not represent metastatic malignancy (usually adenocarcinoma). Criteria in support of mesothelioma include the lack of a ‘two cell’ population, that is one native (mesothelial) and one foreign (metastatic), cells with abundant dense staining cytoplasm, the presence of ‘windows’ where mesothelioma cells lie in close apposition and intracytoplasmic glycogen presenting either as small peripheral vacuoles on MGG stained smears or large yellow refractile crescents on Papanicolaou stained smears. In addition, mesothliomas often possess connective tissue stromal cores occurring as either well‐formed collagen within papillary aggregates or lying free as pink (MGG) or light green (Pap) amorphous material in the background of the smear or in loose association with mesothelioma cells. Finally small orange staining squamous‐like cells can occasionally be identified and sometimes this may be a very prominent finding and has resulted in the false impression of a squamous cell carcinoma. Almost certainly these cells represent apoptotic tumour cells. The connective tissue mucin hyaluronic acid may be found as a net‐like pattern in the smear background or as large hard‐edged magenta‐stained vacuoles on MGG‐stained smears. Cell block sections provide architectural information and it is usually possible to separate mesothelioma aggregates with their cuboidal cells, central nuclei and abundant dense cytoplasm arranged in solid, papillary or hollow clusters from those of adenocarcinoma with less dense, often foamy cytoplasm, often composed of columnar cells with elongated nuclei. Aggregate form in adenocarcinoma can be variable but true acini are a rare finding. These cell block sections provide an ideal medium for histochemistry (PAS with and without diastase digestion) and immunocytochemistry. By using a panel of antibodies (Calretinin and CK 5/6, BerEp4, CEA, B72.3) it is almost always possible to distinguish mesothelioma from metastatic adenocarcinoma. Calretinin and CK 5/6 positive staining and absent staining with BerEp4, CEA and B72.3 is considered diagnostic of mesothelioma.     

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.     

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.     

Carcinoembryonic antigen in effusions. A diagnostic adjunct to cytology

A total of 189 effusion specimens (100 benign and 89 malignant) submitted for cytologic examination were assayed for carcinoembryonic antigen (CEA) by an enzyme immunoassay to determine whether the addition of CEA evaluation to cytologic study would improve the diagnostic accuracy for the detection of malignancy. The sensitivity and specificity were 78% and 90%, respectively, for a cytologic diagnosis of malignancy and 68% and 99%, respectively, for a positive CEA (greater than 5 ng/mL). CEA assay was negative in the most common epithelial malignancies of the female genital tract (15 of 17 cases), mesotheliomas (5), lymphomas (7) and alveolar-cell carcinoma of lung (1). CEA assay was positive in 55 of 89 cases of malignancy, including 14 cases with cytologically negative malignant effusions. The CEA assay sensitivity for lung carcinoma (95% for adenocarcinoma, 100% for oat-cell carcinoma and 100% for carcinosarcoma), breast carcinoma (95%), and gastrointestinal carcinoma (100%) were all over 90%. No significant difference in the levels of CEA was noted between gastrointestinal and lung adenocarcinomas. Oat-cell carcinomas and squamous-cell carcinomas had lower values. In cases of an effusion with an unknown primary, an elevated CEA in the fluid is diagnostic of metastatic carcinoma arising from the breast, lung or gastrointestinal tract.     

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.     

Positive effusion cytology as the initial presentation of malignancy

During a period of four years (1981 to 1984), 641 ascitic, 860 pleural and 47 pericardial fluid specimens were examined cytologically. Of these, 154 ascitic samples, 174 pleural specimens and 10 pericardial effusions, obtained, respectively, from 108, 133 and 7 patients, were found to contain malignant cells. In 7 patients, ascites, and in 18 cases, pleural effusions were the first indication of cancer. None of the positive pericardial fluids was the initial presentation of malignancy. The cytologic findings and follow-up data on these 25 patients are the subject of this study. The most common type of neoplasm in these effusions was adenocarcinoma (86% of the ascitic and 78% of the pleural fluids). Most of the malignant neoplasms in ascitic fluids were derived from ovarian tumors (5 of 7) while those in pleural effusions came mainly from lung tumors (12 of 18). Mammary carcinoma, which was the most common malignant tumor found in cases of pleural effusions, did not present initially with an effusion in any of our cases. The cytologic diagnosis was confirmed in all cases by either biopsy or strong clinical evidence. The prognosis in patients who initially presented with an effusion was poor. All of the patients with an adequate follow-up died within 29 months in cases of ascites and within 19 months in cases of pleural effusions.     

Cytomorphological and immunocytochemical study of non-Hodgkin's lymphoma in pleural effusion and ascitic fluid

INTRODUCTION: Non-Hodgkin's lymphoma (NHL) is often complicated by pleural effusion and ascites. The present study is an attempt to categorize the lymphomatous effusions according to the WHO classification, using archival material. METHODS: May-Grünwald-Giemsa and Papanicolaou-stained smears of 31 lymphomatous effusion specimens were reviewed. Of these, detailed cytological assessment was done on 12 pleural effusions and ten ascitic fluid specimens from 22 patients using the WHO lymphoma classification system. Immunocytochemical studies were performed in 21 specimens. RESULTS: Based on cytomorphological features, the 22 lymphomatous effusion specimens were categorized into lymphoplasmacytoid lymphoma (1), follicle centre cell (FCC) grade-1 (centrocytic) lymphoma (3), FCC grade-2 (centrocytic-centroblastic) lymphoma (3), FCC grade-3 (centroblastic) lymphoma (4), large cell immunoblastic lymphoma (4), lymphoblastic lymphoma (2), anaplastic large cell lymphoma (3) and miscellaneous types (2). Immunocytochemically, the lymphoma cells were T-cell (positive for CD3) and B-cell type (CD20 positive) in five and six cases respectively. CONCLUSION: Cytological examination of pleural effusion and ascitic fluid samples, supported by immunocytochemical studies, may be useful for the classification of lymphomas under the WHO system.     

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.     

Utility of HBME-1 immunostaining in serous effusions

Utility of HBME-1 immunostaining in serous effusions
HBME-1 is an anti-mesothelial cell monoclonal antibody derived from human mesothelioma cells. We investigated 227 body cavity effusions to test its utility in differentiating mesothelioma from adenocarcinoma. HBME-1 outlined cell membranes in non-neoplastic mesothelial cells. Thick surface staining was observed on all mesotheliomas. HBME-1 reactivity was also detected in 24% of metastatic carcinomatous effusions. Most ovarian carcinomas (83%) reacted with this antibody, showing surface staining. Cytoplasmic HBME-1 immunoreactivity was observed in a small proportion of non-ovarian adenocarcinomas (14%). Despite its limited specificity, HBME-1 might be added to the battery of other markers of epithelial and/or mesothelial differentiation to be used in cases of suspected mesothelioma. Evaluation of suspicious cells should include careful study of the pattern of immunostaining.     

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.     

Identification of the primary site of metastatic adenocarcinoma in serous effusions. Value of an immunocytochemical panel added to the clinical arsenal

OBJECTIVE: To use an immunocytochemical panel as ancillary method to identify the origin of adenocarcinomas in serous effusions. STUDY DESIGN: Serous effusion samples examined cytologically in the Department of Surgical Pathology, A. C. Camargo Hospital, between 1966 and 1990, were investigated. Of 4,297 cases, 2,317 were associated with adenocarcinoma, and 1,099 were positive for adenocarcinoma by cytologic examination. We selected a total of 248 cases of different origins to subject to immunoreactions. A panel composed of CA-125, CA-19.9, HBME-1, lactoferrin and BRST 2 was tested for the efficiency of these antibodies under two conditions: the panel alone and associated with clinical data, such as anatomic localization of the effusion (pleural or ascitic) and patient sex and age. RESULTS: BRST 2 and lactoferrin were both positive in 29.9% of cases of adenocarcinoma of breast origin; CA-125 and HBME-1 were 28.6% and 25.0% positive in cases of adenocarcinoma of the ovaries, respectively. These immunoreactivities were highly specific when compared to the others. The statistical significance of the results was improved by information on the anatomic location of the effusions and patient sex. CONCLUSION: Our data strongly indicate that BRST 2 and lactoferrin are important components of an immunocytochemical panel used to identify carcinomas of breast origin. Similarly, CA-125 and HBME-1 may be useful in suggesting the ovaries as possible primary sites.     

Ultrastructure of pleural mesothelioma and pulmonary adenocarcinoma in malignant effusions as compared with reactive mesothelial cells.

OBJECTIVE: To determine the ultrastructural features of diffuse malignant pleural mesothelioma cells in cytologic specimens from pleural effusions. STUDY DESIGN: We retrospectively studied 35 pleural effusions: 12 diffuse malignant pleural mesotheliomas (8 epithelial type, 4 biphasic type), 12 pulmonary adenocarcinomas and 11 cases of reactive mesothelial cells. RESULTS: In the cytoplasm, reactive and malignant mesothelial cells had more-abundant intermediate filaments (P < .05, P < .01) and fewer free ribosomes (P < .001, P < .001) than adenocarcinoma cells. Reactive mesothelial cells had fewer mitochondria than mesothelioma cells (P < .05). Mesothelioma cells had longer, thinner microvilli on the cell surfaces (P < .001); length/diameter ratios of microvilli were 19.1 +/- 7.0 (mesothelioma) vs. 9.1 +/- 2.2 (adenocarcinoma) and 9.2 +/- 2.4 (mesothelial cells). Giant intercellular junctions (desmosomes or desmosomelike structures > 1 micron in length) were found in eight cases of mesothelioma. Core filaments or rootlets in microvilli were present in two cases of adenocarcinoma. CONCLUSION: Because cytologic specimens from pleural effusions were easy to obtain, we think ultrastructural cytology is useful in distinguishing mesothelioma from adenocarcinoma and benign effusions.     

Overexpression of HER2/neu oncoprotein in cytologic specimens

OBJECTIVE: To assess the rate of HER2/neu overexpression in cytologic specimens by immunocytochemistry (ICC) and compare these results in matched surgical specimens by immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH), when available. STUDY DESIGN: All cytologic specimens processed for HER2/neu evaluation by ICC (72 cases) and available corresponding histologic specimens (16 cases) were retrieved from our files. ICC was applied to previously Papanicolaou stained, routine fine needle aspirations specimens (64 cases) and cytocentrifuged, alcohol-fixed, fluid specimens (8 cases). FISH was performed on 6 histologic specimens. RESULTS: Overexpression of HER2/neu was seen in 7/22 breast cancers (31.8%), 3/18 pulmonary adenocarcinomas (16.6%), 2/5 colorectal adenocarcinomas (40%), 1/2 adenocarcinomas of the biliary system (50%), 1/3 thyroid papillary carcinomas (33.3%) and 1/3 prostate adenocarcinomas (33.3%). Sixteen cases had IHC in matched histologic specimens: 14 (87.5%) cases were concordant (11 negative and 3 positive in both specimens), 1 case was negative in the cytologic specimen and positive in the histologic specimen (with no amplification by FISH), and 1 case was positive in the cytologic specimen and negative in the histologic specimen (not informative by FISH). CONCLUSION: Our data suggest that overexpression of HER2/neu oncoprotein can be successfully detected in routine cytologic specimens, providing a simple, fast and cost-effective method of selecting patients for specific treatment.     

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.