Asbestos fibres detected by scanning electron microscopy in the gallbladder of patients with malignant pleural mesothelioma (MPM).

Gallbladders from patients affected by both malignant pleural mesothelioma (MPM) and important gallbladder disorders were analyzed to verify the presence of asbestos fibres. Histological thin sections were analyzed by optical microscope and variable pressure scanning electron microscopy coupled with energy dispersive spectroscopy, allowing morphological and chemical characterization of each inorganic phase observed. Fibres of chrysotile and crocidolite, minerals regulated as asbestos, were identified. By immunohistochemical analysis, connective tissue was recognized as the incorporation site. These findings confirm that asbestos fibres can reach the gallbladders of patients with MPM, for whom the development of respiratory diseases confirms asbestos exposure.

CDKN2A and BAP1 germline mutations predispose to melanoma and mesothelioma.

BAP1 germline mutations predispose to a cancer predisposition syndrome that includes mesothelioma, cutaneous melanoma, uveal melanoma and other cancers. This co-occurrence suggests that these tumors share a common carcinogenic pathway. To evaluate this hypothesis, we studied 40 Italian families with mesothelioma and/or melanoma. The probands were sequenced for BAP1 and for the most common melanoma predisposition genes (i.e. CDKN2A, CDK4, TERT, MITF and POT1) to investigate if these genes may also confer susceptibility to mesothelioma. In two out of six families with both mesothelioma and melanoma we identified either a germline nonsense mutation (c.1153C > T, p.Arg385*) in BAP1 or a recurrent pathogenic germline mutation (c.301G > T, p.Gly101Trp) in CDKN2A. Our study suggests that CDKN2A, in addition to BAP1, could be involved in the melanoma and mesothelioma susceptibility, leading to the rare familial cancer syndromes. It also suggests that these tumors share key steps that drive carcinogenesis and that other genes may be involved in inherited predisposition to malignant mesothelioma and melanoma.

XRCC1 and ERCC1 variants modify malignant mesothelioma risk: a case-control study.

Malignant pleural mesothelioma (MPM) is a rare aggressive tumor associated with asbestos exposure. The possible role of genetic factors has also been suggested and MPM has been associated with single nucleotide polymorphisms (SNPs) of xenobiotic and oxidative metabolism enzymes. We have identified an association of the DNA repair gene XRCC1 with MPM in the population of Casale Monferrato, a town exposed to high asbestos pollution. To extend this observation we examined 35 SNPs in 15 genes that could be involved in MPM carcinogenicity in 220 MPM patients and 296 controls from two case-control studies conducted in Casale (151 patients, 252 controls) and Turin (69 patients, 44 controls), respectively. Unconditional multivariate logistic regression was used to estimate odds ratios (ORs) and 95% confidence intervals (95% CIs). Two DNA repair genes were associated with MPM, i.e. XRCC1 and ERCC1. Considering asbestos-exposed only, the risk increased with the increasing number of XRCC1-399Q alleles (Casale: OR=1.44, 95%CI 1.02-2.03; Casale+Turin: OR=1.34, 95%CI 0.98-1.84) or XRCC1 -77T alleles (Casale+Turin: OR=1.33, 95%CI 0.97-1.81). The XRCC1-TGGGGGAACAGA haplotype was significantly associated with MPM (Casale: OR=1.76, 95%CI 1.04-2.96). Patients heterozygotes for ERCC1 N118N showed an increased OR in all subjects (OR=1.66, 95%CI 1.06-2.60) and in asbestos-exposed only (OR=1.59, 95%CI 1.01-2.50). When the dominant model was considered (i.e. ERCC1 heterozygotes CT plus homozygotes CC versus homozygotes TT) the risk was statistically significant both in all subjects (OR=1.61, 95%CI 1.06-2.47) and in asbestos-exposed only (OR=1.56, 95%CI 1.02-2.40). The combination of ERCC1 N118N and XRCC1 R399Q was statistically significant (Casale: OR=2.02, 95%CI 1.01-4.05; Casale+Turin: OR=2.39, 95%CI 1.29-4.43). The association of MPM with DNA repair genes support the hypothesis that an increased susceptibility to DNA damage may favour asbestos carcinogenicity.

Cancer testis antigens expression in mesothelioma: role of DNA methylation and bioimmunotherapeutic implications.

Recent evidences suggest that malignant mesothelioma may be sensitive to immunotherapy; however, little is known about malignant mesothelioma-associated tumour antigens. Focusing on cancer/testis antigens, the expression of well-characterised immunogenic tumour-associated antigens was investigated in malignant mesothelioma cells. At variance with MAGE-4 and NY-ESO-1, malignant mesothelioma cells frequently expressed MAGE-1, -2 and -3, GAGE 1-2, GAGE 1-6, SSX-2 and SSX 1-5, and distinct malignant mesothelioma cells concomitantly expressed at least four cancer/testis antigens. Additionally, the tumour-associated antigens RAGE-1 was expressed at high levels in both benign and malignant mesothelial cells. Lastly, treatment with the DNA hypomethylating agent 5-aza-2'-deoxycytidine induced and up-regulated the expression of the cancer/testis antigen examined in malignant mesothelioma cells. Overall, these findings strongly suggest that cancer/testis antigens-based immunotherapy may represent a suitable therapeutic approach to malignant mesothelioma, and foresee the clinical use of 5-aza-2'-deoxycytidine to design new chemo-immunotherapeutic strategies in malignant mesothelioma patients.

SV40 replication in human mesothelial cells induces HGF/Met receptor activation: a model for viral-related carcinogenesis of human malignant mesothelioma.

Recent studies suggested that simian virus 40 (SV40) may cause malignant mesothelioma, although the pathogenic mechanism is unclear. We found that in SV40-positive malignant mesothelioma cells, the hepatocyte growth factor (HGF) receptor (Met) was activated. In human mesothelial cells (HMC) transfected with full-length SV40 DNA (SV40-HMC), Met receptor activation was associated with S-phase entry, acquisition of a fibroblastoid morphology, and the assembly of viral particles. Coculture experiments revealed the ability of SV40-HMC to infect permissive monkey cells (CV-1), HMC, and murine BNL CL cells. Cocultured human and murine SV40-positive cells expressed HGF, showed Met tyrosine phosphorylation and S-phase entry, and acquired a spindle-shaped morphology (spBNL), whereas CV-1 cells were lysed. Cocultured HMC inherited from SV40-HMC the infectivity, as they induced lysis in cocultured CV-1 cells. Treatment with suramin or HGF-blocking antibodies inhibited Met tyrosine phosphorylation in all large T antigen (Tag)-positive cells and reverted the spindle-shaped morphology of spBNL. This finding indicated that Met activation and subsequent biological effects were mediated by an autocrine HGF circuit. This, in turn, was causally related to Tag expression, being induced by transfection with the SV40 early region alone. Our findings suggest that when SV40 infects HMC it causes Met activation via an autocrine loop. Furthermore, SV40 replicates in HMC and infects the adjacent HMC, inducing an HGF-dependent Met activation and cell-cycle progression into S phase. This may explain how a limited number of SV40-positive cells may be sufficient to direct noninfected HMC toward malignant transformation.

Interleukin-2 induces cell cycle perturbations leading to cell growth inhibition and death in malignant mesothelioma cells in vitro.

Previous report indicated that Interleukin-2 (IL-2) is able to inhibit the growth of IL-2-receptor-positive cancer cell lines without any involvement of the immune system, through IL-2-induced alterations of the cell cycle kinetics. In this study we provide evidence that IL-2 exerts anti-proliferative effect on three human malignant mesothelioma (MMe) cells in vitro, while no effects were observed on normal human mesothelial cell (HMC) primary cultures. The growth inhibitory effect of IL-2 on neoplastic cells appeared to depend on the baseline proliferative status of these cells. Indeed, in highly proliferating MMe cells, we observed a reduction of malignant cells in the S-phase of the cell cycle, with an accumulation in G0/G1, followed by apotosis for longer incubations or exposure to higher doses. On the contrary, in MMe cells proliferating at lower rate, IL-2 induces only a late cytotoxic effect, leading to apoptosis, without significantly affecting the cell cycle. IL-2Rbeta mRNA was detectable by RT-PCR in all MMe cells, IL-2Ralpha mRNA in one only out the three assayed and IL-2Rgamma mRNA in none. In addition, mRNA specific for the IL-2Rbeta-associated Jak-1 tyrosine kinase was expressed in all MMe cell lines, further suggesting that IL-2Rbeta may play a role in the observed effects. Very low, albeit detectable, levels of IL-2Rbeta chain appeared to be expressed at the cell surface of MMe cells by indirect immunofluorescence and FACS analyses. Finally, Ca(++) fluxes were rapidly induced when MMe cells were exposed to exogenous IL-2.

Newly marketed tissue markers for malignant mesothelioma: immunoreactivity of rabbit AMAD-2 antiserum compared with monoclonal antibody HBME-1 and a review of the literature on so-called antimesothelioma antibodies.

A complementary DNA (cDNA) library was constructed from a human malignant mesothelioma (MM) cell line and a cDNA fragment encoding for a cytoplasmic mesothelial protein recognized by the polyclonal antibody AMAD-1 was then cloned and expressed in Escherichia coli. The purified recombinant protein was used to raise a novel antibody, named AMAD-2, in rabbits. This antibody reacted with normal mesothelium and most MM (15 of 17) on paraffin sections and featured a cytoplasmic labeling. Conversely, AMAD-2 immunostaining of normal and tumor tissues from body sites other than serosal membranes was limited with respect to the proportion of positive specimens and usually less conspicuous than in MM. AMAD-2 immunoreactivity was subsequently compared with staining for HBME-1, another newly marketed antimesothelial monoclonal antibody, concerning the ability to distinguish pleural MM from metastatic pleural tumors of epithelial type. A granular cytoplasmic immunoreactivity for AMAD-2 was present in 50% or more of tumor cells in all 84 MM, regardless of histological type, but also in 3 (7%) of 42 pleural metastases, albeit only focally. HBME-1 was shown in 63 of 66 epithelial MM and in the epithelial component of all 8 mixed MM, with a prevailingly membranous pattern, usually homogeneous and strong, whereas none of the 10 sarcomatous MM was positive. HBME-1 was also expressed in 6 (14%) of 42 pleural metastases in a cytoplasmic or membranous pattern. Compared with HBME-1, AMAD-2 showed a higher degree of specificity and sensitivity for MM. AMAD-2 still proved to be superior to HBME-1, also when sarcomatoid MM were excluded from the assessment. This finding supports the view that AMAD-2 is an antibody highly, although not entirely, specific for the mesothelial lineage, whereas HBME-1 is probably a cell marker more closely related to the epithelial differentiation of MM. Therefore, AMAD-2 is preferable as a positive tissue marker to be incorporated in the optimal immunohistochemical panel for the diagnosis of MM.

The in-vitro hematopoietic capacity of the adult human mesothelial cell: a model of cell differentiation induced by the structure of the microenvironment.

Upon exposure to collagen sponges, cultured adult human mesothelial cells were shown to differentiate into hematopoietic cells similar to those of the red bone marrow. This transformation was confirmed by morphological analysis and by cell immunoreactivity toward specific antibodies directed to antigens of the hematopoietic cell lines at various stages of differentiation. Besides demonstrating that the pluri-potentiality of the mesothelium persist into adulthood, this observation suggests that the process of differentiation may also be influenced by the structural organization of the microenvironment hosting the mesothelial cells.

Quantitative analysis of nucleoli and nucleolar organizer regions in cultured primary human normal, reactive and malignant mesothelial cells.

The number and the size of silver-stained intranuclear granules, which correspond to the nucleolus and nucleolar organizer regions, have been determined by means of quantitative methods in cultured primary human mesothelial cells obtained from normal, reactive and malignant mesothelium. The mean values per nucleus of the number, the total area, the average area, and the relative area of the silver-stained granules and the mean nuclear area were determined for each of the three conditions. Normal, reactive and malignant mesothelial cells differed significantly in all the features. These findings at the optical level reflect the differing rate of the nucleolar biosynthetic activity related to the different biological properties of the three cell types, and the features can be useful morphometric descriptors in the diagnostic pathology of the mesothelium.

Mitogenic effects of a mesothelial cell growth factor: evidence for a potential autocrine regulation of normal and malignant mesothelial cell proliferation.

We have investigated the growth-factor-like activity of a approximately 200-kDa, IP 8.3, cytoplasmic glycoprotein, the expression of which appears to be restricted to normal and malignant human mesothelium. This substance stimulated the growth of human mesothelioma cell cultures at greater rates than did foetal calf serum, but it failed to induce proliferation of lung carcinoma cell cultures. In addition, we have tried to trace the biosynthetic pathway of this mitogenic factor in normal human mesothelial cells by means of immuno-electron microscopy with a polyclonal antibody directed against this molecule. Positive immunogold labelling was found in the lumina of the cisternae of the endoplasmic reticulum, to a lesser extent on the outer surface of the plasma membrane, and also in structures corresponding to the coated pits. These ultrastructural findings are consistent with the hypothesis of the glycosylation of the newly synthesized protein in the endoplasmic reticulum and the subsequent uptake of the secreted molecule, which accumulates in the coated pits before internalization. The results suggest that this mitogenic glycoprotein could play a role in an autocrine growth control mechanism influencing mesothelial cell proliferation.

Changes in nucleolar transcriptional activity in hepatitis B virus-associated chronic liver diseases. Preliminary results from a quantitative study of silver-stained nucleoli.

Modifications of gene expression may occur in hepatitis B virus (HBV)-related chronic liver diseases, possibly also involving ribosomal RNA (rRNA) genes contained in the nucleolus. Changes in the level of transcriptional activity of rRNA genes are reflected by variations in the number and/or size of the nucleoli. Therefore a quantitative analysis of the silver-stained nucleoli (AgNus) was performed in a small series of liver needle biopsies from patients with HBV+ chronic persistent hepatitis (CPH) (n = 3), HBV+ chronic active hepatitis (CAH) (n = 3) and HBV+ cirrhosis (CIR) (n = 3). In each case, 100 hepatocytes were selected. The number of the nucleoli (AgNuN), their total area (tAgNuA), the average area of each nucleolus (xAgNuA), the nuclear area (NA) and the percentage ratio of tAgNuA related to NA (rAgNuA) were determined for each hepatocyte nucleus. The pooled mean values of all the features were significantly different (p less than 0.001) among the case groups. The results point towards a remarkable increase of nucleolar activity in CAH in comparison with CPH, whereas an additional increment of this activity is associated with the progress from CAH to CIR.