Dissecting heterogeneity in malignant pleural mesothelioma through histo-molecular gradients for clinical applications.

Malignant pleural mesothelioma (MPM) is recognized as heterogeneous based both on histology and molecular profiling. Histology addresses inter-tumor and intra-tumor heterogeneity in MPM and describes three major types: epithelioid, sarcomatoid and biphasic, a combination of the former two types. Molecular profiling studies have not addressed intra-tumor heterogeneity in MPM to date. Here, we use a deconvolution approach and show that molecular gradients shed new light on the intra-tumor heterogeneity of MPM, leading to a reconsideration of MPM molecular classifications. We show that each tumor can be decomposed as a combination of epithelioid-like and sarcomatoid-like components whose proportions are highly associated with the prognosis. Moreover, we show that this more subtle way of characterizing MPM heterogeneity provides a better understanding of the underlying oncogenic pathways and the related epigenetic regulation and immune and stromal contexts. We discuss the implications of these findings for guiding therapeutic strategies, particularly immunotherapies and targeted therapies.

Co-occurring Mutations of Tumor Suppressor Genes, LATS2 and NF2, in Malignant Pleural Mesothelioma.

Purpose: To better define malignant pleural mesothelioma (MPM) heterogeneity and identify molecular subtypes of MPM, we focus on the tumor suppressor gene LATS2, a member of the Hippo signaling pathway, which plays a key role in mesothelial carcinogenesis.Experimental Design: Sixty-one MPM primary cultures established in our laboratory were screened for mutations in LATS2 Gene inactivation was modeled using siRNAs. Gene and protein expressions were analyzed by quantitative RT-PCR, Western blot analysis, and reverse phase protein array. Cell proliferation, viability, apoptosis, mobility, and invasion were determined after siRNA knockdown or YAP (verteporfin), mTOR (rapamycin), and mTOR/PI3K/AKT (PF-04691502) inhibitor treatment.Results: The LATS2 gene was altered in 11% of MPM by point mutations and large exon deletions. Genetic data coupled with transcriptomic data allowed the identification of a new MPM molecular subgroup, C2LN, characterized by a co-occurring mutation in the LATS2 and NF2 genes in the same MPM. MPM patients of this subgroup presented a poor prognosis. Coinactivation of LATS2 and NF2 leads to loss of cell contact inhibition between MPM cells. Hippo signaling pathway activity, mTOR expression, and phosphorylation were altered in the C2LN MPM subgroup. MPMs of this new subgroup show higher sensitivity to PF-04691502 inhibitor. The MOK gene was identified as a potential biomarker of the C2LN MPM subgroup and PF-04691502 sensitivity.Conclusions: We identified a new MPM molecular subgroup that shares common genetic and transcriptomic characteristics. Our results made it possible to highlight a greater sensitivity to an anticancer compound for this MPM subgroup and to identify a specific potential biomarker. Clin Cancer Res; 23(12); 3191-202. ©2016 AACR.

Molecular classification of malignant pleural mesothelioma: identification of a poor prognosis subgroup linked to the epithelial-to-mesenchymal transition.

PURPOSE: Despite research efforts to develop more effective diagnostic and therapeutic approaches, malignant pleural mesothelioma (MPM) prognosis remains poor. The assessment of tumor response to therapy can be improved by a deeper phenotypical classification of the tumor, with emphasis on its clinico-biological heterogeneity. The identification of molecular profiles is a powerful approach to better define MPM subclasses and targeted therapies. EXPERIMENTAL DESIGN: Molecular subclasses were defined by transcriptomic microarray on 38 primary MPM cultures. A three-gene predictor, identified by quantitative reverse transcription PCR, was used to classify an independent series of 108 frozen tumor samples. Gene mutations were determined in BAP1, CDKN2A, CDKN2B, NF2, and TP53. Epithelial-to-mesenchymal transition (EMT) markers were studied at the mRNA and protein levels. RESULTS: Unsupervised hierarchical clustering on transcriptomic data defined two robust MPM subgroups (C1 and C2), closely related to prognosis and partly to histologic subtypes. All sarcomatoid/desmoplastic MPM were included in the C2 subgroup. Epithelioid MPM were found in both subgroups, with a worse survival prognosis in the C2 subgroup. This classification and its association with histologic subtypes and survival were validated in our independent series using the three-gene predictor. Similar subgroups were found after classification of other MPM series from transcriptomic public datasets. C1 subgroup exhibited more frequent BAP1 alterations. Pathway analysis revealed that EMT was differentially regulated between MPM subgroups. C2 subgroup is characterized by a mesenchymal phenotype. CONCLUSIONS: A robust classification of MPM that defines two subgroups of epithelioid MPM, characterized by different molecular profiles, gene alterations, and survival outcomes, was established.

Differential mutation profiles and similar intronic TP53 polymorphisms in asbestos-related lung cancer and pleural mesothelioma.

Given the interest in defining biomarkers of asbestos exposure and to provide insights into asbestos-related and cell-specific mechanisms of neoplasia, the identification of gene alterations in asbestos-related cancers can help to a better understanding of exposure risk. To understand the aetiology of asbestos-induced malignancies and to increase our knowledge of mesothelial carcinogenesis, we compared genetic alterations in relevant cancer genes between lung cancer, induced by asbestos and tobacco smoke, and malignant pleural mesothelioma (MPM), a cancer related to asbestos, but not to tobacco smoke. TP53, KRAS, EGFR and NF2 gene alteration analyses were performed in 100 non-small cell lung cancer (NSCLC) patients, 50 asbestos-exposed and 50 unexposed patients, matched for age, gender, histology and smoking habits. Detailed assessment of asbestos exposure was based on both specific questionnaires and asbestos body quantification in lung tissue. Genetic analyses were also performed in 34 MPM patients. TP53, EGFR and KRAS mutations were found in NSCLC with no link with asbestos exposure. NF2 was only altered in MPM. Significant enhancement of TP53 G:C to T:A transversions was found in NSCLC from asbestos-exposed patients when compared with unexposed patients (P = 0.037). Interestingly, TP53 polymorphisms in intron 7 (rs12947788 and rs12951053) were more frequently identified in asbestos-exposed NSCLC (P = 0.046) and MPM patients than in unexposed patients (P < 0.001 and P = 0.012, respectively). These results emphasise distinct genetic alterations between asbestos-related thoracic tumours, but identify common potential susceptibility factors, i.e. single nucleotide polymorphisms in intron 7 of TP53. While genetic changes in NSCLC are dominated by the effects of tobacco smoke, the increase of transversions in TP53 gene is consistent with a synergistic effect of asbestos. These results may help to define cell-dependent mechanisms of action of asbestos and identify susceptibility factors to asbestos.

Syntenic relationships between genomic profiles of fiber-induced murine and human malignant mesothelioma.

Malignant mesothelioma (MM) is an aggressive tumor with a poor prognosis mainly linked to past asbestos exposure. Murine models of MM based on fiber exposure have been developed to elucidate the mechanism of mesothelioma formation. Genomic alterations in murine MM have now been partially characterized. To gain insight into the pathophysiology of mesothelioma, 16 murine and 35 human mesotheliomas were characterized by array-comparative genomic hybridization and were screened for common genomic alterations. Alteration of the 9p21 human region, often by biallelic deletion, was the most frequent alteration in both species, in agreement with the CDKN2A/CDKN2B locus deletion in human disease and murine models. Other shared aberrations were losses of 1p36.3-p35 and 13q14-q33 and gains of 5p15.3-p13 regions. However, some differences were noted, such as absence of recurrent alterations in mouse regions corresponding to human chromosome 22. Comparison between altered recurrent regions in asbestos-exposed and non-asbestos-exposed patients showed a significant difference in the 14q11.2-q21 region, which was also lost in fiber-induced murine mesothelioma. A correlation was also demonstrated between genomic instability and tumorigenicity of human mesothelioma xenografts in nude mice. Overall, these data show similarities between murine and human disease, and contribute to the understanding of the influence of fibers in the pathogenesis of mesothelioma and validation of the murine model for preclinical testing.

p16INK4A inactivation mechanisms in non-small-cell lung cancer patients occupationally exposed to asbestos.

Epidemiological studies have shown that asbestos fibers constitute the major occupational risk factor and that asbestos acts synergistically with tobacco smoking to induce lung cancer. Although some somatic gene alterations in lung cancer have been linked to tobacco smoke, few data are available on the role of asbestos fibers. P16/CDKN2A is an important tumor suppressor gene that is frequently altered in lung cancer via promoter 5'-CpG island hypermethylation and homozygous deletion, and rarely via point mutation. Many studies suggest that tobacco smoking produces P16/CDKN2A promoter hypermethylation in lung cancer, but the status of this gene in relation to asbestos exposure has yet to be determined. The purpose of this study was to investigate the mechanism of P16/CDKN2A alterations in lung cancer in asbestos-exposed patients. P16/CDKN2A gene status was studied in 75 human non-small-cell lung cancer (NSCLC) cases with well-defined smoking habits, and detailed assessment of asbestos exposure, based on occupational questionnaire and determination of asbestos bodies in lung tissue. The results of this study confirm published data on the effect of tobacco smoke on P16/CDKN2A gene alterations, characterized by significantly higher P16/CDKN2A promoter hypermethylation in heavy smokers (more than 40 pack-years (P-Y)) than in smokers of less than 40 P-Y. These results also demonstrate a higher incidence of loss of heterozygosity and homozygous deletion in asbestos-exposed cases, after adjustment for age and cumulative tobacco consumption, than in unexposed cases (P=0.0062). This study suggests that P16/CDKN2A gene inactivation in asbestos-exposed NSCLC cases mainly occurs via deletion, a feature also found in malignant mesothelioma, a tumor independent of tobacco smoking but associated with asbestos exposure, suggesting a possible relationship with an effect of asbestos fibers.

Mesothelioma: Do asbestos and carbon nanotubes pose the same health risk?

Carbon nanotubes (CNTs), the product of new technology, may be used in a wide range of applications. Because they present similarities to asbestos fibres in terms of their shape and size, it is legitimate to raise the question of their safety for human health. Recent animal and cellular studies suggest that CNTs elicit tissue and cell responses similar to those observed with asbestos fibres, which increases concern about the adverse biological effects of CNTs. While asbestos fibres' mechanisms of action are not fully understood, sufficient results are available to develop hypotheses about the significant factors underlying their damaging effects. This review will summarize the current state of knowledge about the biological effects of CNTs and will discuss to what extent they present similarities to those of asbestos fibres. Finally, the characteristics of asbestos known to be associated with toxicity will be analyzed to address the possible impact of CNTs.

Clinico-pathological features and somatic gene alterations in refractory ceramic fibre-induced murine mesothelioma reveal mineral fibre-induced mesothelioma identities.

Although human malignant mesothelioma (HMM) is mainly caused by asbestos exposure, refractory ceramic fibres (RCFs) have been classified as possibly carcinogenic to humans on the basis of their biological effects in rodents' lung and pleura and in cultured cells. Hence, further investigations are needed to clarify the mechanism of fibre-induced carcinogenicity and to prevent use of harmful particles. In a previous study, mesotheliomas were found in hemizygous Nf2 (Nf2(+/-)) mice exposed to asbestos fibres, and showed similar alterations in genes at the Ink4 locus and in Trp53 as described in HMM. Here we found that Nf2(+/-) mice developed mesotheliomas after intra-peritoneal inoculation of a RCF sample (RCF1). Clinical features in exposed mice were similar to those observed in HMM, showing association between ascite and mesothelioma. Early passages of 12 mesothelioma cell cultures from ascites developed in RCF1-exposed Nf2(+/-) mice demonstrated frequent inactivation by deletion of genes at the Ink4 locus, and low rate of Trp53 point and insertion mutations. Nf2 gene was inactivated in all cultures. In most cases, co-inactivation of genes at the Ink4 locus and Nf2 was found and, at a lower rate, of Trp53 and Nf2. These results are the first to identify mutations in RCF-induced mesothelioma. They suggest that nf2 mutation is complementary of p15(Ink4b), p16(Ink4a) and p19(Arf) or p53 mutations and show similar profile of gene alterations resulting from exposure to ceramic or asbestos fibres in Nf2(+/-) mice, also consistent with the one found in HMM. These somatic genetic changes define different pathways of mesothelial cell transformation.

Similar tumor suppressor gene alteration profiles in asbestos-induced murine and human mesothelioma.

Human malignant mesothelioma (HMM) is an aggressive malignancy mainly caused by exposure to asbestos fibers. Here we investigated tumor suppressor genes in mesothelioma cells from tumoral ascites developed in mice exposed to asbestos (asb) fibers and in 12 HMM cell cultures. Mutations in Nf2, p16/Cdkn2a, p19/Arf and Trp53 genes and protein expression of p15/Cdkn2b and Cdk4 were analyzed in 12 cultures from mice hemizygous for Nf2 (asb-Nf2(KO3/+)) and 4 wild type counterparts (asb-Nf2(+/+)). We have found frequent inactivations of p16/Cdkn2a, p19/Arf (or P14/ARF) and p15/Cdkn2b, coinactivation of p16/Cdkn2a and p15/Cdkn2b and low rate of Trp53 mutations in both asb-Nf2(KO3/+) and asb-Nf2(+/+) mesothelioma cells. In both mouse and human mesothelioma cells, inactivation of the hortologous genes p16/Cdkn2a or P16/CDKN2A was due to deletions at the Ink4/Arf locus encompassing p19/Arf or P14/ARF, respectively. Loss of heterozygosity at the Nf2 locus was detected in 10 of 11 asb-Nf2(KO3/+) cultures and Nf2 gene rearrangement in one asb-Nf2(+/+) culture. These data show that the profile of TSG alterations in asbestos-induced mesothelioma is similar in mice and humans. Thus, the mouse mesothelioma model could be useful for human risk assessment, taking into account interindividual variations in genetic sensitivity to carcinogens.

Hemizygosity of Nf2 is associated with increased susceptibility to asbestos-induced peritoneal tumours.

Biallelic NF2 gene inactivation is frequently found in human malignant mesothelioma. In order to assess whether NF2 hemizygosity may enhance susceptibility to asbestos fibres, we investigated the Nf2 status in mesothelioma developed in mice presenting a heterozygous mutation of the Nf2 gene (Nf2(KO3/+)), after intraperitoneal inoculation of crocidolite fibres. Asbestos-exposed Nf2(KO3/+) mice developed tumoural ascites and mesothelioma at a higher frequency than their wild-type (WT) counterparts (P&<0.05). Six out of seven mesothelioma cell lines established from neoplastic ascitic fluids of Nf2(KO3/+) mice exhibited loss of the WT Nf2 allele and no neurofibromatosis type 2 protein expression was found in these cells. The results show the importance of the NF2 gene in mesothelial oncogenesis, the potential association of asbestos exposure and tumour suppressor gene inactivation, and suggest that NF2 gene mutation may be a susceptibility factor to asbestos.