Determination of the Potential Tumor-Suppressive Effects of Gsdme in a Chemically Induced and in a Genetically Modified Intestinal Cancer Mouse Model.

Gasdermin E (GSDME), also known as deafness autosomal dominant 5 (DFNA5) and previously identified to be an inducer of regulated cell death, is frequently epigenetically inactivated in different cancer types, suggesting that GSDME is a tumor suppressor gene. In this study, we aimed to evaluate the tumor-suppressive effects of GSDME in two intestinal cancer mouse models. To mimic the silencing of GSDME by methylation as observed in human cancers, a Gsdme knockout (KO) mouse was developed. The effect of GSDME on tumorigenesis was studied both in a chemically induced and in a genetic intestinal cancer mouse model, as strong evidence shows that GSDME plays a role in human colorectal cancer and representative mouse models for intestinal cancer are available. Azoxymethane (AOM) was used to induce colorectal tumors in the chemically induced intestinal cancer model (n = 100). For the genetic intestinal cancer model, Apc1638N/+ mice were used (n = 37). In both experiments, the number of mice bearing microscopic proliferative lesions, the number and type of lesions per mouse and the histopathological features of the adenocarcinomas were compared between Gsdme KO and wild type (WT) mice. Unfortunately, we found no major differences between Gsdme KO and WT mice, neither for the number of affected mice nor for the multiplicity of proliferative lesions in the mice. However, recent breakthroughs on gasdermin function indicate that GSDME is an executioner of necrotic cell death. Therefore, it is possible that GSDME may be important for creating an inflammatory microenvironment around the tumor. This is in line with the trend towards more severe inflammation in WT compared to Gsdme KO mice, that we observed in our study. We conclude that the effect of GSDME in tumor biology is probably more subtle than previously thought.

Tumor-specific genetic variants can be detected in circulating cell-free DNA of malignant pleural mesothelioma patients.

OBJECTIVES: Patients diagnosed with malignant pleural mesothelioma (MPM) face a poor prognosis, with an overall survival plateauing at a median of one year. This can be explained by difficulties in early diagnosis, effective treatment and treatment monitoring. Circulating cell-free tumor DNA (ctDNA) is emerging as an interesting biomarker addressing some of these issues. So far, the development of ctDNA in MPM lags behind that in other tumors. In this study, the possibility of tracing tumor-specific genetic variants, identified in MPM tissue, in circulating DNA of the corresponding patients is investigated. MATERIALS AND METHODS: Whole exome sequencing was performed on paired tumor and germline DNA of ten MPM patients, of which five were treatment naïve. For each patient, a tumor-specific variant was selected and traced in tumor, germline and circulating DNA using droplet digital PCR in two independent runs. RESULTS: All but one tumor-specific variants, selected after whole exome sequencing, were validated on primary tumor tissue using droplet digital PCR analysis. Patient-specific, selected variants could be detected in circulating DNA of three MPM patients, either in one or both independent droplet digital PCR runs. Mutated fractions in circulating DNA ranged from 0.28 to 0.9%. Interestingly, all patients whose circulating DNA samples contained tumor-specific variants, were treatment naïve. CONCLUSION: We demonstrated for the first time the presence of ctDNA within circulating DNA of treatment naïve MPM patients. This finding opens perspectives towards the use of ctDNA as a biomarker for (early and differential) diagnosis, treatment and treatment monitoring of MPM, which all remain challenging.

Molecular analysis of an asbestos-exposed Belgian family with a high prevalence of mesothelioma.

Familial clustering of malignant mesothelioma (MM) has been linked to the presence of germline mutations in BAP1. However, families with multiple MM patients, without segregating BAP1 mutation were described, suggesting the existence of other predisposing genetic factors. In this study, we report a previously undescribed Belgian family, in which BAP1 was found to be absent in the epithelial malignant mesothelial cells of the index patient. Whole exome analysis did not reveal a germline or somatic BAP1 variant. Also, no germline or somatic copy number changes in the BAP1 region could be identified. However, germline variants, predicted to be damaging, were detected in 11 other 'Cancer census genes' (i.e. MPL, RBM15, TET2, FAT1, HLA-A, EGFR, KMT2C, BRD3, NOTCH1, RB1 and MYO5A). Of these, the one in RBM15 seems to be the most interesting given its low minor allele frequency and absence in the germline DNA of the index patient's mother. The importance of this 'Cancer census gene' in familial MM clustering needs to be evaluated further. Nevertheless, this study strengthens the suspicion that, next to germline BAP1 alterations, other genetic factors might predispose families to the development of MM.

Large-scale copy number analysis reveals variations in genes not previously associated with malignant pleural mesothelioma.

Malignant pleural mesothelioma (MPM) is an aggressive tumor that is often causally associated with asbestos exposure. Comparative genomic hybridization techniques and arrays demonstrated a complex set of copy number variations (CNVs) in the MPM-genome. These techniques however have a limited resolution, throughput and flexibility compared to next-generation sequencing platforms. In this study, the presence of CNVs in the MPM-genome was investigated using an MPM-cohort (N = 85) for which genomic microarray data are available through 'The Cancer Genome Atlas' (TCGA). To validate these results, the genomes of MPMs and matched normal samples (N = 21) were analyzed using low-pass whole genome sequencing on an 'Illumina HiSeq' platform. CNVs were detected using in-house developed analysis pipelines and frequencies of copy number loss and gain were calculated. In both datasets, losses on chromosomes 1, 3, 4, 6, 9, 13 and 22 and gains on chromosomes 1, 5, 7 and 17 were found in at least 25% and 15% of MPMs, respectively. Besides the well-known MPM-associated genes, CDKN2A, NF2 and BAP1, other interesting cancer-associated genes were listed as frequently involved in a copy number loss (e.g. EP300, SETD2 and PBRM1). Moreover, four cancer-associated genes showed a high frequency of copy number gain in both datasets (i.e. TERT, FCGR2B, CD79B and PRKAR1A). A statistically significant association between overall survival and the presence of copy number loss in the CDKN2A-containing region was observed in the TCGA-set. In conclusion, recurrent CNVs were detected in both datasets, occurring in regions harboring known MPM-associated genes and genes not previously linked to MPM.

The Genetic Landscape of Malignant Pleural Mesothelioma: Results from Massively Parallel Sequencing.

Malignant pleural mesothelioma (MPM) is a rare yet aggressive tumor that is causally associated with-mostly professional-asbestos exposure. Given the long latency between exposure and disease, and because asbestos is still being used, MPM will remain a global health issue for decades to come. Notwithstanding the increasing incidence of MPM and the fact that patients with MPM face a poor prognosis, currently available treatment options are limited. To enable the development of novel targeted therapies, identification of the genetic alterations underlying MPM will be crucial. The first studies reporting on the genomic background of MPM identified recurrent somatic mutations in a number of tumor suppressor genes (i.e., cyclin-dependent kinase inhibitor 2A gene [CDKN2A], neurofibromin 2 (merlin) gene [NF2], and BRCA1 associated protein 1 gene [BAP1]). More recently, massively parallel sequencing strategies have been used and have provided a more genome-wide view on the genetic landscape of MPM. This review summarizes their results, describing alterations that cluster mainly in four pathways: the tumor protein p53/DNA repair, cell cycle, mitogen-activated protein kinase, and phosphoinisitide 3-kinase (PI3K)/AKT pathways. As these pathways are important during tumor development, they provide interesting candidates for targeting with novel drugs.