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Vascular endothelial growth factor (VEGF), acidic and basic fibroblast growth factors (FGF-1 and -2), and transforming growth factor beta (TGFbeta) are potent angiogenic cytokines. Malignant mesothelioma of the pleura presents with a high intra-tumoural microvascular density (IMD) which also has prognostic relevance. This study was designed to verify the immunohistochemical expression of the angiogenic cytokines in mesothelioma as well as in non-neoplastic human mesothelial cells and to study the individual as well as the combined expression of these cytokines in mesothelioma in relation to both IMD and prognosis. In addition, four mesothelioma cell lines were studied by ELISA for the secretion of VEGF and FGF-2 in their supernatants and were shown to contain high levels of both of these cytokines. Immunohistochemically, VEGF, FGF-1 and -2, and TGFbeta immunoreactivity was present in 81, 67, 92 and 96 per cent of mesotheliomas, and in 20, 50, 40, and 10 per cent of samples of the non-neoplastic mesothelium, respectively. Co-ordinate expression of the cytokines was observed whereby mesotheliomas expressed more than one cytokine. The combined immunohistochemical expression levels for all four cytokines correlated significantly with both IMD (p=0.01) and prognosis (p=0. 0013). When studied individually, high FGF-2 expression correlated best with more tumour aggressiveness and worse prognosis for mesothelioma (p=0.0011). There was no significant correlation between prognosis and immunoexpression of VEGF (p=0.07), FGF-1 (p=0.3), or TGFbeta (p=0.1), or between IMD and any of the cytokines studied individually. These data support the assertion that selective angiogenic cytokines might contribute to the progressive changes of mesothelioma by tumour angiogenesis., (Copyright 1999 John Wiley & Sons, Ltd.)

Syndecan-1 binds basic fibroblast growth factor (bFGF), modulates neovascularization, plays a role in epithelial differentiation and is up-regulated by WT1. Malignant mesothelioma of the pleura is one of the most aggressive tumours known and expresses high levels of angiogenic growth factors. This study has analysed syndecan-1 expression in mesothelioma tumours and cell lines by immunohistochemistry and immunoblotting, using anti-syndecan-1 antibody directed against the core protein, and has examined its relation to morphology, bFGF, WT1, and intra-tumoural microvascular density (IMD). Shedding of syndecan-1 in the conditioned medium of mesothelioma cell lines was detected in variable amounts. These studies indicate that (1) there is no correlation of syndecan-1 with either bFGF expression or IMD in mesotheliomas in vivo; (2) syndecan-1 is strongly expressed in the epithelial type of mesothelioma and in the epithelial component of biphasic mesotheliomas and the expression is reduced or lost in sarcomatoid differentiation; together with the finding that (3) syndecan-1 correlates with WT1 immuno-expression, this suggests that syndecan-1 might relate to the differentiation state of mesothelial/mesothelioma cells; and (4) syndecan-1-positive tumours are associated with a longer survival (p = 0.02) than mesotheliomas with no or little syndecan-1 expression, on univariate analysis. These findings therefore indicate that syndecan-1 can be an important prognostic indicator in mesotheliomas and its loss may be important in the epithelial-mesenchymal transformation of mesothelioma cells.

Angiogenesis plays an important role in the growth, progression, and metastasis of solid tumours. Malignant mesothelioma (MM) of the pleura is a highly invasive tumour with a poor prognosis. In the present study, microvascular quantification was undertaken on 25 specimens of mesothelioma and 15 specimens of non-neoplastic mesothelium (NNM), by staining for the antigens CD34 and CD31. Areas of highest intratumoural microvascular density (IMD) were identified and counted either manually (mIMD) or on a computerized image analysis system (CIAS; iIMD). The two IMDs were significantly correlated with each other (r = 0.736; P < 0.001). The average IMD for MM was significantly (P < 0.001) higher than in NNM. Moreover, each unit increment in iIMD for MM, when regarded as a continuous variable, was significantly (P = 0.001) associated with an increased hazard of about 4 per cent. When regarded as a categorical variable, the patients in the highest tertile (> 58 vessels/field) had a significantly (P < 0.01; log-rank test) shorter survival than patients in the lowest tertile (< 45 vessels/field). This association was independent of the age of the patient and of the histological type or grade of the MM. No association was noted with p53 immunoexpression. Although the mean vascular area of blood vessels measured on the CIAS did not correlate with survival, assessment of IMDs can be an important independent prognostic indicator in malignant mesothelioma.

The Wilms tumour 1 (WT1) gene is believed to contribute to the growth and differentiation of certain tissues, including mesothelium. This study assessed WT1 gene status by mutational screening in 42 malignant mesotheliomas (MMs) and 3 MM cell lines and detected two tumours with identical heterozygous single nucleotide deletions in intron 7, with no apparent consequence for WT1 function. Furthermore, the expression pattern of the WT1 gene was studied in MMs and related lesions using three anti-WT1 monoclonal antibodies (MAbs). Strong to moderate nuclear immunoreactivity was noted in MM in situ (54/56), cultured mesothelioma cells (4/5), and hyperplastic and normal pleural (non-neoplastic, NNM) specimens. WT1 immunoreactivity was absent in all primary tumours of lung and in pleural metastases from adenocarcinomas of breast and colon; immunoreactivity was present in pleural metastases from renal carcinomas, melanomas, and papillary carcinomas of the ovary. Expression of the WT1 protein in MM was not correlated with survival. Coordinate expression of the WT1 protein and its putative transcriptional target genes was determined by correlating WT1 immunostaining with epidermal growth factor receptor (EGF-R) and insulin-like growth factor 1 receptor (IGF-1R) expression on MM and NNM; no significant correlation was found, irrespective of p53 expression status. Finally, the putative involvement of WT1 in cell-type transition was supported by this study, in that epithelial mesothelioma showed the strongest WT1 immunoreactivity while sarcomatous mesothelioma showed the least.

Fifty-seven cases of malignant mesothelioma were analyzed for nuclear diameter, DNA content and ploidy-related parameters, using Feulgen stained paraffin sections with a digital imaging analysis system. Thirty cases had a mean nuclear diameter > 7 microns and 31 cases were classified as near-diploid. A statistically significant correlation between survival and the mean nuclear diameter (P = 0.0006) and between survival and DNA index (P = 0.007) was obtained. For other DNA content parameters (proliferation index, 5c exceeding rate), only one of the two statistical tests showed a significant correlation with survival while the other test was of borderline significance. In this malignant mesothelioma population, the prognosis for patients with the epithelial type was better than for those with sarcomatous tumours (P = 0.01). In this population of patients, about half of the malignant mesotheliomas were aneuploid. The mean nuclear diameter, DNA index analysis and proliferation index analysis of the tumour cells on Feulgen stained paraffin sections can be used as independent prognostic parameters.

The differential diagnosis between malignant mesothelioma and benign pleural hyperplasia constitutes a well-known problem. In the present study we examined unbiased stereological techniques to assess the mean nuclear volume (MNV) using the point-sampled intercepts (PSI) in 37 cases of malignant mesothelioma and in 28 cases of benign pleural hyperplasia. Neither the use of different fixatives nor the histological type of malignant mesothelioma produced any significant difference on the measured nuclear volume. The differences observed between the MNV data obtained from benign pleural hyperplasia and those from any of the three types of malignant mesothelioma were found to be highly significant. All lesions with an MNV larger than 250 microns3 were found in our study to correspond to the malignant mesothelioma type, while an MNV that was smaller than 200 microns3 could only be detected in benign specimens. These observations lead us to propose the MNV measurement using PSI as an additional tool to enhance the differential diagnosis of malignant mesothelioma versus benign pleural hyperplasia.

Expression of the glutathione S-transferase (GST) subclasses alpha, mu and pi was investigated immunohistochemically in 20 normal or hyperplastic mesothelium and in 57 malignant mesothelioma cases. These results were correlated with survival and also with P-170 glycoprotein expression. Nearly all the non-neoplastic mesothelium cases were positive for GST alpha and pi. About half of the non-neoplastic cases were positive for mu. Twenty-nine (51%) malignant mesotheliomas were positive for at least one of the GST species; 21 (37%) showed immunoreactivity for alpha, 18 (31.5%) for mu and 21 (37%) for pi. A total of 54 mesothelioma cases displayed immunoreactivity for the P-170 glycoprotein. For GST pi and GST mu, a statistical significance between expression and increased survival was found (respectively P = 0.012 and 0.024) while for GST alpha no significance was found. The results of this study demonstrate that expression of GST pi correlates positively with increased survival in malignant mesothelioma. It is also concluded that, in mesothelioma, GST and P-170 glycoprotein may contribute to the resistance to cytotoxic drugs frequently observed in these tumours. No correlation between GST and P-170 expression was demonstrated.

An in situ hybridization (ISH) study on paraffin sections of 13 malignant mesotheliomas was performed to detect numerical chromosomal aberrations with biotin-labelled DNA probes specific for the centromeric regions of chromosomes 1, 3, 6, 7, 11, and 17. All chromosomes contributed to numerical changes, which can be summarized as follows: first, a monosomy for chromosome 6 was found in one case; second, in five cases a trisomy for at least one chromosome was detected; and third, in seven cases a pentasomy or a higher polysomy was found for at least one chromosome. Although these data have to be confirmed on a larger group of patients, survival analysis of this group showed no significant difference between the first and second groups taken together and the third group. In this study no specific numerical chromosomal aberrations were identified. Nevertheless, numerical gains appear to be more frequent than has previously been shown by karyotype analysis.

In order to assess its discriminating and prognostic value, we studied immunoreactivity for proliferating nuclear cell antigen (PCNA) in human malignant mesothelioma (31 cases) and in human non-neoplastic mesothelium (33 cases with reactive mesothelium and 20 cases of normal mesothelium) using the murine monoclonal antibody PC10. We also compared it with mitosis counts expressed as the mitotic volume index (MV index). There were differences between malignant mesothelioma, reactive mesothelium, and normal mesothelium for percentage of PCNA immunoreactive cells (mean +/- SD; 27 +/- 9, 9.5 +/- 5.1, and 3.6 +/- 1.6, respectively) and for their MV index (20.3 +/- 4.5, 9.4 +/- 2.1, and 3.6 +/- 0.6, respectively). The median actuarial survival was 10.1 months for patients with less than 25 per cent PCNA immunoreactive cells, 9.4 months for patients with less than 20 mitoses per mm2 of tumoural tissue, 5.9 months for patients with more than 25 per cent PCNA immunoreactive cells, and 5.3 months for patients with more than 20 mitoses per mm2 of tumoural tissue. Our results suggest that PCNA immunoreactivity is useful in discriminating between neoplastic and non-neoplastic mesothelium and that it may have prognostic value in malignant mesothelioma.

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.

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. [ABSTRACT FROM AUTHOR]