Absence of SV40 large T-antigen expression in human mesothelioma cell lines

Simian virus (SV) 40 and SV40-like DNA sequences have recently been detected in several types of human tumors, including malignant mesothelioma. However, the presence of SV40 DNA sequences is not sufficient to account for its possible role in tumor development because the viral proteins must be expressed and ultimately impair the function of relevant cell proteins, such as p53 and pRb. In this study we investigated SV40 large T antigen (SV40 Tag) protein expression in mesothelioma cell lines, established in our laboratory, by Western blotting, immunoprecipitation, and immunocytochemistry using Tag-specific mouse monoclonal antibodies (mAbs) Ab-1 (or Pab 419). By Western blotting of cell extracts, none of the mesothelioma cell lines expressed detectable amounts of SV40 Tag. However, we found that Ab-1 as well as Pab-101, another SV 40 Tag-specific mAb, may generate false-positive signals due to the fact that both antibody preparations are contaminated by a protein of similar size (90 kD) as SV40 Tag and react with the various secondary horseradish peroxidase- conjugated antimouse immunoglobulin Gs tested. The present study suggests that immunodetection of SV40 Tag protein may be puzzling because this contaminating Taglike protein may bind to particular cell structures, thereby generating false-positive signals.     

恶性间皮瘤可能与致瘤性猿猴病毒SV40无关

目的探讨致瘤性猿猴病毒SV40（simian virus 40）是否与中国人恶性间皮瘤的发生相关。方法从蜡块中提取17例恶性间皮瘤组织中的DNA后,用三组引物对SV40大T抗原（TAg）的基因片段分别进行聚合酶链反应（PCR）扩增,另外,用两种SV40相关抗体（Pab101和Ab-2）分别进行免疫组织化学染色,检测肿瘤组织中是否存在SV40 TAg。结果（1）一组引物的PCR反应仅有3例扩增出了SV40TAg的基因片段,其余两组引物的PCR反应均为阴性。（2）两种抗体的免疫组织化学染色均未检测出SV40TAg。结论中国人恶性间皮瘤与SV40感染的关系可能不密切。     

β-catenin expression in benign and malignant pleural disorders

Benign and malignant pleural processes display a large and overlapping spectrum of morphological appearances, and can be difficult to distinguish, histologically, from each other. β-catenin, a participant in the wingless-type (Wnt) transduction pathway, is involved in the pathogenesis of malignant mesothelioma and has received limited evaluation for its ability to serve as a diagnostic aid for distinguishing between individual pleural disorders. We performed immunohistochemistry for β-catenin on 10 pleural malignant mesotheliomas, 10 examples of mesothelial hyperplasia and 18 cases of organizing pleuritis. Although differences were noted in staining intensity between the mesothelioma and mesothelial hyperplasia groups, extensiveness and cellular location were similar. Staining intensity (mean +/- s.d.) in mesotheliomas (2.00 +/- 0.67) was significantly less intense than in mesothelial hyperplasia cases (3.00 +/- 0.00) (p=0.0005). Stromal cell staining was cytoplasmic in all cases, and endothelial cell staining was membranous, submembranous and cytoplasmic. Nuclear expression of β-catenin was not observed in any of the cases studied. This lack of nuclear staining in the stromal cells of organizing pleuritis differs markedly from the previously reported high frequencies of nuclear β-catenin expression in other pleural spindle cell proliferations (desmoid tumors and solitary fibrous tumors). In summary, the current study adds to previous work indicating a role for β-catenin in the genesis of pleural conditions including organizing pleuritis, mesothelial hyperplasia and malignant mesothelioma. Although IHC for β-catenin does not appear to be conclusive for separating benign from malignant mesothelial proliferations, it may be valuable for assisting in the differential diagnosis of mesothelial and spindle cell proliferations in the pleura.     

Immunohistochemical distribution patterns of epidermal growth factor receptor in malignant mesothelioma and non-neoplastic mesothelium

Summary An immunohistochemical study of the epidermal growth factor (EGF) receptor in non-neoplastic pleural mesothelium (35 cases) and in human malignant mesothelioma (36 cases) was made, using a murine monoclonal antibody OM-11-951. All malignant mesotheliomas and non-neoplastic pleural biopsies exhibited a strong cytoplasmic immunoreactivity in mesothelial cells. Nuclear immunoreactivity was detected in mesothelial cells of all specimens of both malignant and non-neoplastic pleura. No statistically significant differences were found between malignant mesothelioma and non-neoplastic pleural mesothelium. There were differences, between the three subtypes of mesothelioma, in the number of cells that exhibited nuclear staining. Statistically significant differences were noted between the epithelial subtype and the mesenchymal subtype (P< 0.005), epithelial subtype versus the mixed cell type (P< 0.005) and between the mesenchymal component of the mixed cell type and the mesenchymal type (P<0.0005). We conclude that there is strong expression of EGF receptor in both malignant mesothelioma and in non-neoplastic pleural mesothelium. Different staining patterns are seen when comparing the different subtypes of mesotheliomas with each other. EGF receptor expression cannot be used to distinguish between malignant and benign mesothelium.     

Immunoreactivity for p53 protein in malignant mesothelioma and non-neoplastic mesothelium.

The presence of p53 protein in non-neoplastic pleural mesothelium (40 cases) and in human malignant mesothelioma (36 cases) was assessed immunohistochemically using the antibodies DO7, CM-1, and PAb240. In a quarter of the malignant mesotheliomas, there was nuclear immunoreactivity for p53 protein with both the DO7 and CM-1 antibodies. There were no statistically significant differences between the various mesothelioma subtypes (P > 0.05). No immunoreactivity was found with the PAb240 antibody, suggesting absence of mutant-type p53 protein. Nonneoplastic mesothelium was not immunoreactive with any of the antibodies. We conclude that there is immunoreactivity for p53 protein in some mesotheliomas. p53 protein immunoreactivity could be used to discriminate between neoplastic and reactive mesothelium.     

Expression of cytokeratin 5/6 in epithelial neoplasms: an immunohistochemical study of 509 cases.

Cytokeratin 5/6 (CK 5/6) immunoreactivity has been observed in the vast majority of cases of malignant mesothelioma but only rarely in pulmonary adenocarcinomas. Thus, CK 5/6 has been used to distinguish malignant mesothelioma from pulmonary adenocarcinoma. However, the utility of CK 5/6 in distinguishing pleural malignant mesothelioma from pleural metastases from nonpulmonary adenocarcinoma, as well as peritoneal malignant mesothelioma from peritoneal metastatic adenocarcinoma, has not yet been adequately addressed because the tissue expression of CK 5/6 in nonpulmonary neoplasms has not been well defined. We have studied the CK 5/6 expression in 509 cases of various epithelial tumors by immunohistochemistry. We found that the vast majority of cases of squamous cell carcinoma, basal cell carcinoma, thymoma, salivary gland tumor, and biphasic malignant mesothelioma were positive for CK 5/6. In addition, CK 5/6 immunoreactivity was detected in 15 of 24 cases (62%) of transitional cell carcinoma, in 5 of 10 cases (50%) of endometrial adenocarcinoma, in about one third of cases of pancreatic adenocarcinoma (38%) and breast adenocarcinoma (31%), and in one quarter of cases of ovarian adenocarcinomas (25%). Our study confirms the diagnostic utility of CK 5/6 immunohistochemistry in distinguishing biphasic mesothelioma from pulmonary adenocarcinoma but raises caution about its use for the differential diagnosis of pleural or peritoneal malignant mesothelioma versus pleural or peritoneal metastatic nonpulmonary adenocarcinoma, because many types of nonpulmonary adenocarcinomas may be positive for CK 5/6.     

SV40-like DNA sequences in pleural mesothelioma,bronchopulmonary carcinoma,and non-malignant pulmonary diseases

Pleural and pulmonary malignancies are usually associated with well-known carcinogen exposure. Recently, the presence of simian virus 40 (SV40)-like DNA sequences has been detected in brain and bone-related human cancers and in pleural mesothelioma. In order to determine whether SV40-like DNA sequences are also present in bronchopulmonary carcinoma and non-malignant lung samples, 125 frozen pleural and pulmonary samples (including 21 mesotheliomas, 63 bronchopulmonary carcinomas, 8 other tumours, and 33 non-malignant samples) and 38 additional samples distant from tumours were studied for the occurrence of SV40-like DNA sequences by polymerase chain reaction (PCR) amplification followed by hybridization with specific probes. Sequences related to SV40 large T antigen (Tag) were present in 28·6 per cent of bronchopulmonary carcinomas, 47·6 per cent of mesotheliomas, and 16·0 per cent of cases with non-neoplastic pleural and pulmonary disease. No statistically significant difference in the occurrence of these DNA sequences was found between malignant mesothelioma and bronchopulmonary carcinoma, but a significantly higher number of mesothelioma cases exhibited SV40-like DNA sequences in comparison with cases of non-malignant pleural or pulmonary disease (P<0·04). Among cases positive for SV40-like DNA sequences, a history of asbestos exposure was found in 3 out of 12 bronchopulmonary carcinomas and 8 out of 10 mesotheliomas. Immunohistochemistry using monoclonal antibodies directed against Tag did not demonstrate nuclear staining. The DNA sequences were not related to BK virus sequences, but three samples were positive with probes hybridizing with JC virus DNA sequences. In conclusion, this study demonstrates the presence of SV40-like DNA sequences in pulmonary neoplasms and in non-malignant lung tissues. It appears that the presence of SV40-like DNA is not unique to cancer. © 1998 John Wiley & Sons, Ltd.     

SV40 large T antigen-specific human T cell memory responses

The continued presence of simian virus 40 (SV40), a monkey polyomavirus, in man is confirmed by the regular detection of SV40-specific antibodies in 5-10% of children who are unlikely to have received contaminated polio-vaccines. The aim of our experiments was to find cellular immunological evidence of SV40 infection in humans by testing memory T cell responses to SV40 large T antigen (Tag). As there is some indication that the virus may be present in malignant pleural mesothelioma (MPM) cells, we analyzed T cell responses in MPM patients and in healthy donors. The frequencies of responding T cells to overlapping Tag peptides were tested by cytokine flow cytometry. CD8+ T cells from 4 of 32 MPM patients responded (above twofold of control) to SV40 Tag peptides, while no positive responses were detected in 12 healthy donors. Within SV40 Tag we identified three 15 amino acid-long immunogenic sequences and one 9 amino acid-long T cell epitope (p138) (138FPSELLSFL146), the latter including a HLA-B7-restriction motif. T cell responses to p138 were SV40-specific as T cells stimulated with p138 did not cross-react with the corresponding sequences of Tag of human polyomaviruses BKV and JCV. Similarly, the relevant BKV and JCV Tag peptides did not generate T cell responses against SV40 TAg p138. Peptide-stimulated T cells also killed SV40 Tag-transfected target cells. This article demonstrates the presence, and provides a detailed analysis, of SV40-specific T cell memory in man.     

Assessment of cell cycle state may facilitate the histopathological diagnosis of malignant mesothelioma

AIMS: The differentiation of benign pleural conditions from malignant mesothelioma may be difficult, especially with a small biopsy. We have tested the hypothesis that assessment of the cell cycle status is of value in the histopathological diagnosis of such biopsies, by comparing 33 malignant mesotheliomas with 36 cases of reactive mesothelial hyperplasia and reactive pleural fibrosis. METHODS AND RESULTS: Biopsies were investigated for proliferative status by immunostaining for a novel antibody, MCM2, all of which showed nuclear expression of MCM2 at higher frequency than Ki67 (P < 0.0001). Counts in areas of maximum tumour staining showed significantly higher labelling indices (LIMax) in epithelioid and sarcomatoid mesotheliomas compared with reactive mesothelial hyperplasia and reactive pleural fibrosis (P < 0.0001 for both). Average counts (LIAve) revealed a significant increase in epithelioid mesothelioma compared with reactive mesothelial hyperplasia (P < 0.0001). CONCLUSION: We consider MCM2 to be a useful adjunct in the differential diagnosis of malignant mesothelioma.     

Cytopathologic differential diagnosis of malignant mesothelioma, adenocarcinoma and reactive mesothelial cells: A logistic regression analysis

Distinguishing malignant mesothelioma, adenocarcinoma and reactive mesothelial proliferation in both cytologic and surgical pathologic specimens is often a diagnostic challenge. Conventional cytomorphologic assessment is an important step in the differential diagnosis of these entities.The pleural effusion cytologies from 40 cases of malignant mesothelioma, 40 cases of adenocarcinoma and 30 cases of reactive mesothelial proliferation diagnosed between 1997 and 2007 were reviewed. Twenty-seven cytologic features which are regarded as useful in the differential diagnosis of mesothelioma, adenocarcinoma and benign mesothelial proliferation were assessed. These cytologic features were subjected to a stepwise logistic regression analysis. Three features were selected to distinguish malignant mesothelioma from adenocarcinoma: giant atypical mesothelial cell (P = 0.0001), nuclear pleomorphism (P = 0.0001) and acinar structures (P = 0.0001), the latter two being characteristics of adenocarcinoma. The variables selected to differentiate malignant mesothelioma from reactive mesothelial cells were: cell ball formation (P = 0.0001), cell in cell engulfment (P = 0.0001) and monolayer cell groups (P = 0.0001), the latter being a feature of benign mesothelial proliferation. When these selected variables were subjected to a stepwise logistic regression analysis, the logistic model correctly predicted 90% of cases of benign mesothelial proliferation versus 97.5% of malignant mesothelioma and 92.5% of malignant mesothelioma versus 92.5% of adenocarcinoma.Conventional cytomorphologic assessment is the first step to establish an accurate diagnosis in pleural effusions. Several cytologic features have predictive value to separate malignant mesothelioma from adenocarcinoma and reactive mesothelial proliferation.     

Cellient™ automated cell block versus traditional cell block preparation: a comparison of morphologic features and immunohistochemical staining

Traditional cell block (TCB) sections serve as an important diagnostic adjunct to cytologic smears but are also used today as a reliable preparation for immunohistochemical (IHC) studies. There are many ways to prepare a cell block and the methods continue to be revised. In this study, we compare the TCB with the Cellient? automated cell block system. Thirty‐five cell blocks were obtained from 16 benign and 19 malignant nongynecologic cytology specimens at a large university teaching hospital and prepared according to TCB and Cellient protocols. Cell block sections from both methods were compared for possible differences in various morphologic features and immunohistochemical staining patterns. In the 16 benign cases, no significant morphologic differences were found between the TCB and Cellient cell block sections. For the 19 malignant cases, some noticeable differences in the nuclear chromatin and cellularity were identified, although statistical significance was not attained. Immunohistochemical or special stains were performed on 89% of the malignant cases (17/19). Inadequate cellularity precluded full evaluation in 23% of Cellient cell block IHC preparations (4/17). Of the malignant cases with adequate cellularity (13/17), the immunohistochemical staining patterns from the different methods were identical in 53% of cases. The traditional and Cellient cell block sections showed similar morphologic and immunohistochemical staining patterns. The only significant difference between the two methods concerned the lower overall cell block cellularity identified during immunohistochemical staining in the Cellient cell block sections. Diagn. Cytopathol. 2010. © 2010 Wiley‐Liss, Inc.     

Simian virus 40 sequences and expression of the viral large T antigen oncoprotein in human pleomorphic adenomas of parotid glands

Simian virus 40 (SV40) sequences of the early region coding for the large T antigen (Tag) oncoprotein were investigated in DNA samples from human pleomorphic adenoma (PA) of parotid glands. Specific SV40 sequences were detected, by PCR and filter hybridization with an internal oligoprobe, in 28 of 45 (62%) human PA specimens. None of the DNA samples from 11 normal salivary gland tissues was SV40-positive. DNA sequence analysis, carried out in all PCR amplified products from SV40-positive PA specimens, confirmed the SV40 specificity and indicated that PCR products had a sequence not distinguishable from SV40 DNA wild-type strain 776. SV40 Tag expression was revealed by immunohistochemistry with the specific monoclonal antibody Pab 101 in PA thin sections with a highly sensitive technical approach which retrieved the nuclear viral oncoprotein in 26 out of 28 (93%) samples previously found SV40-positive by PCR. Detection of SV40 sequences and Tag expression in human PA suggests that this oncogenic virus may play a role as a cofactor in the onset and/or progression of this benign neoplasm, or that SV40 DNA could replicate and express the Tag in PA cells.     

The value of ThinPrep and cytospin preparation in pleural effusion cytological diagnosis of mesothelioma and adenocarcinoma

The diagnosis of malignant mesothelioma requires an integration of the clinical presentation, radiological studies, and immunohistochemical stain of histological sections. Cytological diagnosis on pleural effusions of mesothelioma and pulmonary adenocarcinoma is highly desirable but debatable. A spectrum of cytological features has been found to be associated more commonly with malignant mesothelioma (e.g., peripheral cytoplasmic skirt, bubbly cytoplasm, cyanophilic cytoplasm, and scalloped border of cell balls) vs. adenocarcinoma (e.g., two-cell population, inspissated cytoplasmic material, cytoplasmic vacuole, angulated and indented nuclei, and smooth border of cell balls) to only name a few. The current study is designed to assess whether the introduction of a liquid-based technology such as ThinPrep (TP) can provide additional diagnostic value in addition to the conventional cytospin Diff-Quik (DQ) preparations. Pleural effusion specimens were prepared with split samples for DQ-stained cytospin and Papanicolaou-stained liquid-based TP. Fifteen pleural effusion samples with immunohistologically confirmed malignant mesothelioma and 13 pleural effusion samples of immunohistologically confirmed pulmonary adenocarcinomas were retrieved from our files. Both DQ cytospin- and Papanicolaou-stained TP slides were evaluated for the known cytological features associated with malignant mesothelioma (25 cytological features) and adenocarcinoma (22 cytological features) without knowledge of the original cytological and histological diagnoses. The McNemar test was used to compare these two cytological preparations for both malignant mesothelioma and pulmonary adenocarcinoma. In the malignant mesothelioma group, 4 of 25 cytological features evaluated, bubbly cytoplasm (P = 0.002), vacuolated cytoplasm (P = 0.005), cell-in-cell arrangement (P = 0.007) and irregular nuclear contour (P = 0.083), were seen more frequently in the DQ cytospin preparation, as opposed to only one feature, nuclear size enlargement (P = 0.008), more readily seen using TP. In the pulmonary adenocarcinoma group, only 1 of 22 cytological features evaluated, presence of angulated or indented nuclei (P = 0.025), was seen more frequently in DQ as opposed to two features, presence of two- cell population (P = 0.04) and presence of micropapillary structures (P = 0.1), were seen more readily in TP. All other cytological features evaluated distinguishing mesotheliomas (20 features) and pleural adenocarcinomas (19 features) were seen equally readily in both types of specimen preparation techniques. This study suggests that the liquid-based TP preparation of pleural effusions does not appear to provide additional diagnostic value when compared with the DQ cytospin preparation in the cytological distinction between mesothelioma and adenocarcinoma in pleural effusions. Most cytological features evaluated, 20 of 25 (mesothelioma) and 19 of 22 (adenocarcinoma), can be seen in both preparation techniques.     

A comparison of epidermal growth factor receptor expression in malignant peritoneal and pleural mesothelioma

An evaluation of epidermal growth factor receptor (EGFR) phenotypic expression in malignant pleural and peritoneal mesothelioma was undertaken, using immunohistochemical (IHC) and fluorescence in situ hybridization (FISH) analysis. Thirty-eight malignant mesothelioma (MM) specimens were subjected to IHC staining and FISH to evaluate the expression of EGFR protein and gene status. Overall positive IHC reaction was detected in 20/38 (53%) cases, in 11/22 (50%) pleural MM, and in 9/16 (56%) peritoneal MM. Our study confirmed that EGFR membranous expression is a common feature in MM, but not in benign mesothelial lesion. Thirty-seven cases did not show a gene copy number gain. Only one case showed a copy number gain. The protein overexpression of EGFR was not related to a gene copy number gain.     

恶性胸膜间皮瘤13例病理学报道及分析

目的：探讨恶性胸膜间皮瘤的临床及病理学特点。方法：对天津市海河医院13例恶性胸膜间皮瘤患者的临床资料和病理学特征进行回顾性分析。结果：13例患者男性为主(69.2%),中位年龄55岁,主要症状为胸闷(76.7%),放射学主要表现为胸腔积液(69.2%),7例(53.8%)确诊通过胸腔镜获得标本。病理表现上皮样型12例,肉瘤样型1例;免疫组织化学染色calretinin及D2-40阳性率较高(100%、76.7%),CEA及TTF-1阳性率均为0。结论：恶性胸膜间皮瘤恶性程度高,应尽早行胸腔镜检查;除常规染色外,免疫组化染色可帮助鉴别诊断。     

Immunoreactivity for bcl-2 protein in malignant mesothelioma and non-neoplastic mesothelium

Immunohistochemical study of bcl-2 protein immunoreactivity in human non-neoplastic mesothelium (44 cases) and in malignant mesothelioma (62 cases) using a murine monoclonal antibody (clone 124) showed cytoplasmic immunoreactivity for bcl-2 protein in five cases of malignant mesothelioma. Non-neoplastic mesothelium was not immunoreactive. Immunoreactivity for bcl-2 protein does not add useful prognostic information in malignant mesothelioma since survival times of bcl-2 positive and bcl-2 negative cases did not differ. Nevertheless, the detection of bcl-2 protein in malignant mesothelioma might be useful for the differentiation from reactive mesothelium.     

Wilms' Tumor 1 Susceptibility (WT1) Gene Products are Selectively Expressed in Malignant Mesothelioma

The distinction between malignant mesothelioma and other neoplastic processes involving the pleura is difficult, partly due to the lack of specific markers expressed on mesothelioma. Because of evidence suggesting that the Wilms' tumor susceptibility gene (WT1), unlike ot her tumor suppressor genes, is restricted mostly to mesenchymaly derived tissues, we hypothesized that the WTI gene products could serve as a potential marker for mesothelioma. The expression of WTI mRNA was analyzed in 19 malignant mesothelioma cell lines and 9 tumors and compared with the expression of WT1 in 10 non-small cell lung cancer lines and 9 lung cancer specimens. WTI mRNA was detectable by Northern analysis in 16 of 19 mesothelioma cell lines and in 5 of 8 malignant mesothelioma tumors. In contrast, WTI mRNA was not detected by Northern analysis in non-small cell lung cancer lines or carcinomas. Immunoprecipitation with an anti-WT1 monoclonal antibody showed that a52-to 54-kdprotein was present in 4 mesothelioma cell lines. Immunostanig with this antibody localizd the WT1 protein to the nucleus in two mesothelioma lines and in 20 of 21 mesothelioma tumors examined. This distinctive pattern of nuclear immunoreactivity was absent in 26 non-mesothelioma tumors involving the lung, including 20 non-small cell lung carcinomas. The detection of WTI mRNA or protein may thus provide a specific molecular or immunohistochemical marker for differentiation of mesothelioma from other pleural tumors, inparticular, adenocarcinoma.     

Role of fragile histidine triad protein expression in pathogenesis of malignant pleural mesothelioma

AIM:To investigate the relationship of fragile histidine triad (FHIT) and Ki-67 expression with clinicopathological variables of patients with malignant pleural mesothelioma (MPM). METHODS: Formalin-fixed, paraffin-embedded tissue sections of 30 asbestos induced MPM (epithelial and biphasic) patients were examined for FHIT and Ki-67 expression using immunohistochemical techniques and results were compared with clinicopathological variables. RESULTS: Immunohistochemical study results were as follows: 12 (40%) cases showed low FHIT expression and 18 (60%) showed high expression. There was no significant relationship between FHIT and age, gender or histological subtypes (p > 0.05). Ki-67 expression was 'low' in 13 (43.3%) cases and 'high' in 17 (56.7%) cases. No correlation could be demonstrated between Ki-67 expression and age, gender or histological subtypes (p > 0.05). No significant association was observed between FHIT and Ki-67 expression in MPM. CONCLUSION: The results support the role of FHIT as a tumour suppressor gene in asbestos induced MPM. There is no significant correlation between FHIT and cell proliferation marker expressions in malignant pleural mesothelioma. Therefore, it can be concluded that loss of FHIT does not interfere with tumour proliferation. This can be accepted as evidence for an early role of FHIT loss in carcinogenesis; however, it needs to be strengthened by further studies.