[The French mesothelioma network from 1998 to 2013]. / Mésothéliome: les dispositifs en place en France « le réseau mésothéliome ¼ 1998-2013.

Mesothelioma is a rare disease less than 0.3% of cancers in France, very aggressive and resistant to the majority of conventional therapies. Asbestos exposure is nearly the only recognized cause of mesothelioma in men observed in 80% of case. In 1990, the projections based on mortality predicted a raise of incidence in mesothelioma for the next three decades. Nowadays, the diagnosis of this cancer is based on pathology, but the histological presentation frequently heterogeneous, is responsible for numerous pitfalls and major problems of early detection toward effective therapy. Facing such a diagnostic, epidemiological and medico-legal context, a national and international multidisciplinary network has been progressively set up in order to answer to epidemiological survey, translational or academic research questions. Moreover, in response to the action of the French Cancer Program (action 23.1) a network of pathologists was organized for expert pathological second opinion using a standardized procedure of certification for mesothelioma diagnosis. We describe the network organization and show the results during this last 15years period of time from 1998-2013. These results show the major impact on patient's management, and confirm the interest of this second opinion to provide accuracy of epidemiological data, quality of medico-legal acknowledgement and accuracy of clinical diagnostic for the benefit of patients. We also show the impact of these collaborative efforts for creating a high quality clinicobiological, epidemiological and therapeutic data collection for improvement of the knowledge of this dramatic disease.

Overexpression and promoter mutation of the TERT gene in malignant pleural mesothelioma.

Malignant pleural mesothelioma (MPM) is a very aggressive tumor with no known curative treatment. Better knowledge of the molecular mechanisms of mesothelial carcinogenesis is required to develop new therapeutic strategies. MPM, like all cancer cells, needs to maintain telomere length to prevent senescence. Previous studies suggested that the telomere lengthening mechanism in MPM is based mainly on telomerase activity. For this reason, we focused on the key catalytic enzyme, TERT (telomerase reverse transcriptase), by analyzing its gene expression in MPM and by studying the mechanism underlying its upregulation. We used our large collection of MPM composed of 61 MPM in culture and 71 frozen MPM tumor samples. Evaluation of TERT mRNA expression by quantitative RT-PCR showed overexpression in MPM in culture compared with normal mesothelial cells, and in MPM tumor samples compared with normal pleura. We identified a 'hot spot' of mutations in the TERT gene core promoter in both MPM in culture and in MPM tumor samples with an overall frequency of 15%. Furthermore, data clearly identified mutation in the TERT promoter as a mechanism of TERT mRNA upregulation in MPM. In contrast, gene copy number amplification was not associated with TERT overexpression. Then, we analyzed the clinicopathological, etiological and genetic characteristics of MPM with mutations in the TERT promoter. TERT promoter mutations were more frequent in MPM with sarcomatoid histologic subtype (P<0.01), and they were frequently associated with CDKN2A gene inactivation (P=0.03). In conclusion, a subgroup of MPM presents TERT promoter mutations, which lead to TERT mRNA upregulation. This is the first recurrent gain-of-function oncogenic mutations identified in MPM.

[Pleural lymphatics and pleural diseases related to fibres]. / Lymphatiques pleuraux et pathologies liées aux fibres.

It is now well established that some pleural diseases, pleural plaques and malignant mesothelioma are related to asbestos fibre exposure although the mechanism of action of asbestos fibres is not fully understood. The development of artificial mineral fibres and carbon nanotubes, which share some morphological characteristics similar to asbestos fibres, is a present concern in the context of pleural diseases. Pleural plaques develop only in the parietal pleura, and in the 1990s, clinical observations have shown that the early development of mesothelioma also occurred on the parietal pleura. The peculiarity of the parietal pleura in contrast to the visceral pleura is the presence of "stomas" which are communication holes between the pleural cavity and the parietal pleura lymphatics. Morphological observations by thoracoscopy and experimental studies have shown that inhaled fibres translocate to the pleural space and, in human, are present in the parietal pleura at specific anthracotic areas (blackspots). Fibres accumulate on the stomas, up to block and locally induce an inflammatory reaction with cytokines release, that can be the bed of mesothelioma. However, despite the experimental data and observations in human pathology, the mechanisms of fibre translocation into the pleura is not yet clearly established.

Malignant pleural mesothelioma cells resist anoikis as quiescent pluricellular aggregates.

Pleural fluid accumulation is a frequent clinical observation in diffuse malignant pleural mesothelioma (MPM). The cytological analysis of pleural fluid often reveals the presence of free spheroid aggregates of malignant cells, giving rise to the question of the ability of non-adherent tumor cells to resist the loss of anchorage-induced apoptosis (termed as anoikis), and to develop new tumor foci in the pleural cavity. Here, we show that MPM cells cultured under non-adherent conditions form well-organized aggregates composed of viable cells, which progressively enter in G(0). Although the PI3K/Akt, ERK and SAPK/JNK signaling pathways are activated in adherent MPM cells, loss of anchorage results in the inactivation of these pathways. By comparison, we show that the non-tumoral mesothelial cells MeT-5A enter anoikis in an SAPK/JNK-, Bim- and caspase-9-dependent pathway. The survival of MPM cells can be reversed by activating SAPK/JNK with anisomycin, according to a Bim-dependent mitochondrial pathway. Finally, our findings show that impairment of cell aggregation activates SAPK/JNK and Bim and induces anoikis. Our results underline the importance of intercellular contacts in the anoikis resistance of MPM cells.

Eighth international mesothelioma interest group.

The eighth International Mesothelioma Interest Group (IMIG) meeting was held in Chicago, IL, United States, in 19-22 October 2006 to discuss mesothelioma - the cancer often linked to asbestos exposure. It is a very aggressive malignancy with a median survival of less than 1 year from diagnosis. Millions of people have been exposed worldwide to asbestos, especially during the second half of the twentieth century when asbestos use increased significantly. The tons of asbestos utilized in the past remain a health hazard for current and future generations because asbestos is difficult to be disposed off. This makes asbestos and mesothelioma research a public health issue in addition to a medical problem. Moreover, the very high costs of asbestos litigation have a significant impact on the whole economy. In the United States, up until 2001, defendant companies had paid 54 billion dollars in claims and estimated future liabilities ranged from 145 to 210 billion. Therefore, asbestos research is of great interest to a large audience that includes patients, millions of asbestos-exposed individuals, scientists, physicians, public health officials, politicians, unions of asbestos workers, lawyers and the public at large. During the past few years, there has been significant progress in understanding the process of mineral fiber carcinogenesis and mesothelioma pathogenesis. With improved understanding of the pathogenesis of mesothelioma, new diagnostic, preventive and therapeutic options are being developed. A total of 247 papers were presented at the IMIG: the abstracts of these presentations were published in Lung Cancer, Supplement 1, October 2006. Here, experts in different disciplines critically review some of the most exciting presentations of the IMIG meeting. The result is a comprehensive review of the research field of asbestos carcinogenesis and mesothelioma, and of the progress that has been made in recent years in both basic and clinical sciences.

Transfer into a mesothelioma cell line of tumor suppressor gene p16 by cholesterol-based cationic lipids.

In this work, the tumor suppressor gene p16 was efficiently transferred into FR cells isolated from a patient with malignant mesothelioma using cationic liposomes prepared from trimethyl aminoethane carbamoyl cholesterol (TMAEC-Chol) and triethyl aminopropane carbamoyl cholesterol (TEAPC-Chol). This transfer was performed after preliminary assays were undertaken to find the optimal transfection conditions. Results showed that an efficient transfer of plasmids containing the reporter gene pCMV-beta galactosidase vectorized by TMAEC-Chol/DOPE and TEAPC-Chol/DOPE liposomes into mesothelioma FR cells was obtained as assessed by luminometric measurements of beta-galactosidase activity. Cytotoxicity studied by MTT test showed that at concentrations used for this study, the cationic liposomes have no effect on cell growth. Transfer into mesothelioma FR cells of a plasmid construct containing the tumor suppressor gene p16 was carried out with these liposomes. Western blotting and immunofluorescence showed the presence of p16 in treated cells. An inhibition of cell growth was observed, indicating that efficient tumor suppressor gene transfer can be performed by using cationic liposomes.

Cell cycle checkpoint status in human malignant mesothelioma cell lines: response to gamma radiation.

Knowledge of the function of the cell cycle checkpoints in tumour cells may be important to develop treatment strategies for human cancers. The protein p53 is an important factor that regulates cell cycle progression and apoptosis in response to drugs. In human malignant mesothelioma, p53 is generally not mutated, but may be inactivated by SV40 early region T antigen (SV40 Tag). However, the function of p53 has not been investigated in mesothelioma cells. Here, we investigated the function of the cell cycle checkpoints in six human mesothelioma cell lines (HMCLs) by studying the cell distribution in the different phases of the cell cycle by flow cytometry, and expression of cell cycle proteins, p53, p21(WAF1/CIP1) and p27(KIP1). In addition, we studied p53 gene mutations and expression of SV40 Tag. After exposure to gamma-radiation, HMCLs were arrested either in one or both phases of the cell cycle, demonstrating a heterogeneity in cell cycle control. G1 arrest was p21(WAF1/CIP1)- and p53-dependent. Lack of arrest in G1 was not related to p53 mutation or binding to SV40 Tag, except in one HMCL presenting a missense mutation at codon 248. These results may help us to understand mesothelioma and develop new treatments.

Control of cell cycle progression in human mesothelioma cells treated with gamma interferon.

Recombinant human interferon gamma (r-hu-IFNgamma) exerts both antitumoral activity in the early stages of human malignant mesothelioma and a cytostatic effect in human mesothelioma (HM) cell lines in vitro. The antiproliferative effect of interferons (IFNs) reported in a variety of cells has been attributed to several mechanisms. In order to progress in the understanding of HM cell growth modulation by r-hu-IFNgamma, modifications of cell cycle progression and expression of key cell cycle regulator proteins in response to r-hu-IFNgamma were examined. Nine HM cell lines were studied, including one resistant to the antiproliferative effect of r-hu-IFNgamma. Except in the resistant cell line r-hu-IFNgamma produced an arrest in the G1 and G2-M phases of the cell cycle, associated with a reduction in both cyclin A and cyclin dependent kinase inhibitors (CDKIs) expression. Moreover cyclin B1/cdc2 activity was decreased. The present study provides the first evidence of a G2-arrest in r-hu-IFNgamma-treated HM cell lines and indicates that HM cell lines, despite their tumorigenic origin still support cell cycle control. The cell cycle arrest induced by r-hu-IFNgamma seems to depend on cyclin regulation through p21(WAF1/CIP1)- and p27(Kip1)-independent mechanisms and is not directly related to the induced DNA damage.

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.

Hepatocyte growth factor/scatter factor enhances the invasion of mesothelioma cell lines and the expression of matrix metalloproteinases.

Hepatocyte growth factor/scatter factor (HGF/SF) is a multifunctional factor involved both in development and tissue repair, as well as pathological processes such as cancer and metastasis. It has been identified in vivo in many types of tumours together with its tyrosine kinase receptor, Met. We show here that exogenous HGF/SF acts as a strong chemoattractant for human mesothelioma cell lines. The factor also enhanced cell adhesion to and invasion into Matrigel. The mesothelioma cell lines synthesized a panel of matrix metalloproteinases critical for tumour progression such as MMP-1, 2, 3, 9 and membrane-bound MT1-MMP. HGF/SF stimulated the expression of MMP-1, 9 and MT1-MMP and had a slight effect on expression of the MMP inhibitor TIMP-1 but not TIMP-2. However, there was no simple correlation between the levels of MMPs and TIMPs of the cell lines and their different invasion properties or between HGF/SF stimulatory effects on MMP expression and invasion. In addition, effects of protease inhibitors on invasion suggested that serine proteases were also expressed in human mesothelioma cell lines and were involved in HGF/SF-induced invasion. The results show a predominant role for HGF/SF in mesothelioma cell invasion, stimulating simultaneously adhesion, motility, invasion and regulation of MMP and TIMP levels.

Investigations of DNA Damage Produced by Asbestos Fibers in Rat Pleural Mesothelial Cells.

Asbestos fibers have long been considered nongenotoxic because of the absence of point asbestos-treated cells mutations in several assays. More recently, some studies have demonstrated that various kinds of DNA damage were observed in asbestos-treated cells, namely, base hydroxylation, DNA adducts, and DNA breaks. The occurrence of DNA breakage has been mostly suggested by indirect methods. We investigated DNA damage produced by Rhodesian chrysotile fibers in a model of normal rat pleural mesothelial cells (RPMC) developed in our laboratory, by different approaches. DNA breakage and repair of DNA damage were evidenced by the enhancement of the activity of poly(ADP)ribose polymerase (PARP) and of unscheduled DNA synthesis, and were suggested by cell cycle arrest in asbestos-exposed RPMC. In growing cells, a cell cycle arrest was observed at the G1-S transition. DNA breakage was also demonstrated by the formation of comets in single-cell gel electrophoresis (SCGE) assay. The results showed that comets were generated by asbestos, as assessed by the significant increase in mean tail length and mean tail moment after exposure to asbestos. In RPMC-TSV40 (i.e., RPMC in which the protein was p53 inactivated by infection with a retroviral recombinant encoding the large T antigen of the SV40 virus), the comet parameters were moderately enhanced. Regarding previous results of an impairment of DNA repair in RPMC-TSV40, the SCGE results suggest that chrysotile caused DNA breakage in both RPMC and RPMC-TSV40. Moreover, these findings lead to the hypothesis that DNA repair was impaired in RPMC-TSV40.

Pleuro-pulmonary tumours detected by clinical and chest X-ray analyses in rats transplanted with mesothelioma cells.

New strategies for cancer therapy must be developed, especially in severe neoplasms such as malignant pleural mesothelioma. Animal models of cancer, as close as possible to the human situation, are needed to investigate novel therapeutical approaches. Orthotopic transplantation of cancer cells is then relevant and efforts should be made to follow up tumour evolution in animals. In the present study, we developed a method for the orthotopic growth of mesothelioma cells in the pleural cavity of Fischer 344 and nude rats, along with a procedure for clinical survey. Two mesothelioma cell lines, of rat and human origin, were inoculated by transthoracic puncture. Body weight determination and chest X-ray analyses permitted the follow-up of tumour evolution by identifying different stages. Autopsies showed that tumours localized on the whole pleural cavity (diaphragm, parietal pleura), mediastinum and pericardium. Tumour morphology and antigenic characteristics were consistent with those of the inoculated cells and were similar in both types of rats inoculated with the same cell type. These results demonstrate that mesothelioma formation in rats can be followed up by clinical and radiographic survey after gentle intrathoracic inoculation of mesothelioma cells, thus allowing the definition of stages of interest for further experimental trials.

Changes in expression and microheterogeneity of the genetic variants of human alpha1-acid glycoprotein in malignant mesothelioma.

Human alpha1-acid glycoprotein (AAG), an acute-phase plasma protein, is heterogeneous in the native state and polymorphic in the desialylated state. The AAG heterogeneity is mainly explained by a variable glycan chain composition in its five glycosylation sites. The AAG polymorphism is due to the presence of genetic variants. Three main variants are observed for AAG, ORM1 F1, ORM1 S and ORM2 A, which have a separate genetic origin. In this paper, we have used different isoelectric focusing (IEF) methods and chromatography on immobilized metal affinity adsorbent to study the relative occurrence of the genetic variants of AAG in relation to changes in microheterogeneity, in plasma and pleural effusions of patients with malignant mesothelioma (MM). The results were compared to those obtained with the variants in plasma of healthy individuals. Significant changes in variant distribution were observed in the MM samples, that corresponded to a rise in the proportion of the ORM1 variants and a fall in that of the ORM2 variant. However, the concentration in MM plasma increased for both variants. The AAG in MM plasma and effusion fluids was found to be more heterogeneous on IEF than AAG of healthy plasma. The evidence of stronger concentrations of both the high and low pI forms of AAG in the MM samples suggested two kinds of changes in charge heterogeneity. These two changes were shown to be attributed to different variants--i.e. the high pI forms to ORM1 F1 and S and the low pI forms to ORM2 A, after fractionation of AAG by chromatography on immobilized copper(II) ions. These results indicate specific changes in both the expression and glycosylation for each AAG variant, according to its separate genetic origin, in MM.

Detection of SV40-like DNA sequences in pleural mesothelioma, bronchopulmonary carcinoma and other pulmonary diseases.

Mesotheliomas are pleural-based tumours mainly associated with asbestos exposure (70% of cases) and the incidence is still raising. Recently, a possible viral connection was reported and 60% of mesotheliomas were demonstrated to contain and express SV40-like DNA sequences. In this study, the presence of SV40-like DNA sequences were investigated in mesotheliomas (15 tissue samples and six cell lines) and in 63 additional bronchopulmonary carcinomas, one parietal osteosarcoma and non-malignant lung samples as well as in organizing pleuritis (8). Finally, 163 samples were analysed by the polymerase chain reaction (PCR) with a set of primers PYV.for and PYV.rev to amplify a 173 bp region of the SV40 Tag. and a 179 bp region JC virus (JCV) as well as a 182 bp region BK virus (BKV). PCR amplification and hybridization with a probe specific for SV40 Tag revealed that 47.6% of mesotheliomas, 28.6% of primary bronchopulmonary carcinomas and 16% of non-neoplasic lung diseases contained SV40-like DNA sequences. No statistically significant difference in the occurrence of these DNA sequences was found between malignant mesothelioma and bronchopulmonary carcinoma. However, a significantly higher number of mesothelioma cases exhibited SV40- like DNA sequences in comparison with non-malignant pleural and pulmonary tissues. The DNA sequences were not related to BK and JC virus sequences. These results indicate that SV40-like DNA sequences are present in mesotheliomas as well as in bronchopulmonary carcinomas and non-malignant pleuropulmonary diseases.

Heterogeneity of mesothelioma cell lines as defined by altered genomic structure and expression of the NF2 gene.

Germ-line mutations in the neurofibromatosis 2 (NF2) gene cause a susceptibility to the development of schwannoma and meningioma, 2 mostly benign tumors of neural crest origin. Bi-allelic inactivation of this gene has been observed in sporadic schwannomas and meningiomas. The NF2 gene may also be somatically inactivated in human malignant mesotheliomas (HMMs). Surprisingly, patients with an NF2 germ-line mutation have not been reported to be at an increased risk for this highly invasive tumor of mesodermal origin. To investigate in HMMs the silencing mechanism of the NF2 gene, we have analyzed its structure and expression in a series of 18 cell lines derived from HMMs. NF2 gene alterations were identified at a genomic level in 7 cell lines and were associated with a marked decrease in the concentration of the NF2 transcript. This decrease was also observed in 4 additional cell lines with no identified NF2 mutation. The 11 cell lines presented evidence suggesting deletion of one NF2 allele. None of these enabled the detection of normal or truncated forms of the NF2 protein by immunoprecipitational immunoblot analyses. In the 7 remaining cell lines, NF2 mRNA and NF2 protein were easily detectable. Among the latter, 4 lines were heterozygous for several chromosome 22 microsatellite loci, suggesting the presence of 2 NF2 alleles. Taken together, our data indicate that silencing of the NF2 gene is restricted to a subset of mesothelioma cell lines. The availability of established cell lines with different characterized NF2 status provides a powerful tool to explore the mechanism by which the NF2 protein exerts its tumor suppressive activity.

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.

Expression of HGF/SF in mesothelioma cell lines and its effects on cell motility, proliferation and morphology.

The expression of hepatocyte growth factor/scatter factor (HGF/SF) was studied in 12 mesothelioma cell lines characterized by either an epithelioid or a fibroblast-like phenotype. Conditioned media from these lines were analysed by bioassay and ELISA, and HGF/SF was detected in three cell lines, all with a fibroblast-like or mixed morphology. None of eight epithelioid cell lines expressed the factor. Thus, for these cell lines, the ability to secrete HGF/SF correlated with the cell phenotype. Following on from these observations, two cell lines, BR and BT, with a fibroblast-like and an epithelioid phenotype, respectively, were further investigated. Both cell lines expressed the Met receptor but only BR secreted HGF/SF. Both cell lines responded to exogenous HGF/SF treatment by a change of morphology but in different ways: BR became more elongated and bipolar, while BT formed more spread-out cell colonies. HGF/SF acted as a paracrine effector on the epithelioid BT cells and stimulated both cell-spreading and proliferation. Interestingly, BT cells spread but did not scatter in response to exogenous HGF/SF. In contrast BR cells showed only some stimulation of cell motility with HGF/SF and no increase in cell proliferation was observed. Because HGF/SF was previously found in the pleural effusion fluids of patients with malignant mesothelioma and in paraffin-embedded tumour tissues, it is concluded that HGF/SF may well stimulate the growth and spread of malignant mesothelioma in vivo by paracrine and/or autocrine mechanisms.

Shedding of CD44 from PMA-differentiated U-937 cells is enhanced by treatment with mineral particles.

In this report, we show that enhanced shedding of CD44 might contribute to the down-regulation of this receptor observed after phagocytosis of MnO2 particles by PMA-differentiated U-937. The apparent Mr of the soluble CD44 detected in culture supernatants was slightly lower than that of the membrane form suggesting that shedding resulted from proteolytic cleavage. Increased shedding of CD44 was also noted with other mineral particles (chrysotile and DQ12) but to a lower extent whereas some (TiO2 and amosite) had no effect on this process. These results indicate that shedding enhancement was particle-specific rather than a general consequence of phagocytosis. The ability of the particles to enhance CD44 shedding was not directly dependent on their cytotoxic potency. Different patterns of reactivity were noted with CD11b, suggesting that the underlying mechanisms are specific.