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According to the driver–passenger model for colorectal cancer (CRC), the tumor-associated microbiota is a dynamic ecosystem of bacterial species where bacteria with carcinogenic features linked to CRC initiation are defined as “drivers”, while opportunistic bacteria colonizing more advanced tumor stages are known as “passengers”. We reasoned that also gut microbiota-associated metabolites may be differentially enriched according to tumor stage, and be potential determinants of CRC development. Thus, we characterized the mucosa- and lumen-associated microbiota (MAM and LAM, respectively) and mucosa-associated metabolites in low- vs. high-grade dysplastic colon polyps from 78 patients. We show that MAM, obtained with a new biopsy-preserving approach, and LAM differ in composition and α/β-diversity. By stratifying patients for polyp histology, we found that bacteria proposed as passengers by previous studies colonized high-grade dysplastic adenomas, whereas driver taxa were enriched in low-grade polyps. Furthermore, we report altered “mucosa-associated metabolite” levels in low- vs. high-grade groups. Integrated microbiota-metabolome analysis suggests the involvement of the gut microbiota in the production and consumption of these metabolites. Altogether, our findings support the involvement of bacterial species and associated metabolites in CRC mucosal homeostasis in a tumor-stage-specific manner. These distinct signatures may be used to distinguish low-grade from high-grade dysplastic polyps.

Malignant pleural mesothelioma (MPM) is a tumour associated with asbestos exposure. Approximately, 10% of patients with MPM carry a germline pathogenic variant (PV), mostly in DNA repair genes, suggesting the occurrence of inherited predispositions.This article aimed to 1) search for new predisposing genes and assess the prevalence of PVs in DNA repair genes, by next-generation sequencing (NGS) analysis of germline DNA from 113 unselected patients with MPM and 2) evaluate whether these patients could be sensitive to tailored treatments.NGS was performed using a custom panel of 107 cancer-predisposing genes. To investigate the response to selected drugs in conditions of DNA repair insufficiency, we created a three-dimensional-MPM cell model that had a defect in ataxia telangiectasia mutated (ATM), the master regulator of DNA repair.We identified PVs in approximately 7% of patients with MPM (8/113) and a new PV in BAP1 in a further patient with familial MPM. Most of these PVs were in genes involved or supposedly involved in DNA repair (BRCA1, BRIP1, CHEK2, SLX4, FLCN and BAP1). In vitro studies showed apoptosis induction in ATM-silenced/inhibited MPM spheroids treated with an enhancer of zeste homologue 2 inhibitor (tazemetostat).Overall these data suggest that patients with MPM and DNA repair insufficiency may benefit from this treatment, which induces synthetic lethality.

We previously observed increased levels of adenosine-deaminase-acting-on-dsRNA (Adar)-dependent RNA editing during mesothelioma development in mice exposed to asbestos. The aim of this study was to characterize and assess the role of ADAR-dependent RNA editing in mesothelioma. Tumors and mesothelioma primary cultures have higher ADAR-mediated RNA editing compared to mesothelial cells. Unsupervised clustering of editing in different genomic regions revealed heterogeneity between tumor samples as well as mesothelioma primary cultures. ADAR2 expression levels are higher in BRCA1-associated protein 1 wild-type tumors, with corresponding changes in RNA editing in transcripts and 3’UTR. ADAR2 knockdown and rescue models indicated a role in cell proliferation, altered cell cycle, increased sensitivity to antifolate treatment and type-1 interferon signaling upregulation, leading to changes in the microenvironment in vivo. Our data indicate that RNA editing contributes to mesothelioma heterogeneity and highlights an important role of ADAR2 not only in growth regulation in mesothelioma but also chemotherapy response, in addition to regulating inflammatory response downstream of sensing nucleic acid structures.

Germline mutations in the tumor suppressor gene BRCA1-associated protein-1 (

Mesothelioma is an aggressive cancer associated with asbestos exposure. RNA-binding motif protein 8a (RBM8A) mRNA editing increases in mouse tissues upon asbestos exposure. The aim of this study was to further characterize the role of RBM8A in mesothelioma and the consequences of its mRNA editing. RBM8A protein expression was higher in mesothelioma compared to mesothelial cells. Silencing RBM8A changed splicing patterns in mesothelial and mesothelioma cells but drastically reduced viability only in mesothelioma cells. In the tissues of asbestos-exposed mice, editing of Rbm8a mRNA was associated with increased protein immunoreactivity, with no change in mRNA levels. Increased adenosine deaminase acting on dsRNA (ADAR)-dependent editing of Alu elements in the RBM8A 3′UTR was observed in mesothelioma cells compared to mesothelial cells. Editing stabilized protein expression. The unedited RBM8A 3′UTR had a stronger interaction with Musashi (MSI) compared to the edited form. The silencing of MSI2 in mesothelioma or overexpression of Adar2 in mesothelial cells resulted in increased RBM8A protein levels. Therefore, ADAR-dependent editing contributes to maintaining elevated RBM8A protein levels in mesothelioma by counteracting MSI2-driven downregulation. A wider implication of this mechanism for the translational control of protein expression is suggested by the editing of similarly structured Alu elements in several other transcripts., Cells, 10 (12), ISSN:2073-4409

Simple Summary Malignant pleural mesothelioma (MPM) is an aggressive and still incurable cancer. There is an urgent need to identify effective and reliable tools for detecting and diagnosing the early onset of MPM. In our study, we investigated the whole miRNAs expression profile from serum extracellular vesicles to identify early changes related to MPM development. miR-11400, miR-148a-3p, and miR-409-3p levels were increased in pre-clinical MPM patients up to five years before their diagnosis. The three-miRNA pattern showed a good discrimination capacity to distinguish pre-clinical MPM from cancer-free controls. The three miRNAs also displayed high diagnostic capabilities for differentiating between MPM patients and controls. This study identified a potential EV miRNA signature in preclinical MPM up to five years before diagnosis and raises the possibility of early intervention. Malignant pleural mesothelioma (MPM) is an aggressive cancer with a dismal prognosis. Early therapeutic interventions could improve patient outcomes. We aimed to identify a pattern of microRNAs (miRNAs) as potential early non-invasive markers of MPM. In a case-control study nested in the European Prospective Investigation into Cancer and Nutrition cohort, we screened the whole miRNome in serum extracellular vesicles (EVs) of preclinical MPM cases. In a subgroup of 20 preclinical samples collected five years prior MPM diagnosis, we observed an upregulation of miR-11400 (fold change (FC) = 2.6, adjusted p-value = 0.01), miR-148a-3p (FC = 1.5, p-value = 0.001), and miR-409-3p (FC = 1.5, p-value = 0.04) relative to matched controls. The 3-miRNA panel showed a good classification capacity with an area under the receiver operating characteristic curve (AUC) of 0.81 (specificity = 0.75, sensitivity = 0.70). The diagnostic ability of the model was also evaluated in an independent retrospective cohort, yielding a higher predictive power (AUC = 0.86). A signature of EV miRNA can be detected up to five years before MPM; moreover, the identified miRNAs could provide functional insights into the molecular changes related to the late carcinogenic process, preceding MPM development.

Germline mutations in the tumor suppressor gene BRCA1-associated protein-1 (BAP1) lead to BAP1 tumor predisposition syndrome (BAP1-TPDS), characterized by high susceptibility to several tumor types, chiefly melanoma, mesothelioma, renal cell carcinoma, and basal cell carcinoma. Here, we present the results of our ten-year experience in the molecular diagnosis of BAP1-TPDS, along with a clinical update and cascade genetic testing of previously reported BAP1-TPDS patients and their relatives. Specifically, we sequenced germline DNA samples from 101 individuals with suspected BAP1-TPDS and validated pathogenic variants (PVs) by assessing BAP1 somatic loss in matching tumor specimens. Overall, we identified seven patients (7/101, 6.9%) carrying six different germline BAP1 PVs, including one novel variant. Consistently, cascade testing revealed a total of seven BAP1 PV carriers. In addition, we explored the mutational burden of BAP1-TPDS tumors by targeted next-generation sequencing. Lastly, we found that certain tumors present in PV carriers retain a wild-type BAP1 allele, suggesting a sporadic origin of these tumors or a functional role of heterozygous BAP1 in neoplastic development. Altogether, our findings have important clinical implications for therapeutic response of BAP1-TPDS patients.

Malignant pleural mesothelioma (MPM) is a rare and aggressive neoplasm. Patients are usually diagnosed when current treatments have limited benefits, highlighting the need for noninvasive tests aimed at an MPM risk assessment tool that might improve life expectancy. Three hundred asbestos-exposed subjects (163 MPM cases and 137 cancer-free controls), from the same geographical region in Italy, were recruited. The evaluation of asbestos exposure was conducted considering the frequency, the duration and the intensity of occupational, environmental and domestic exposure. A genome-wide methylation array was performed to identify novel blood DNA methylation (DNAm) markers of MPM. Multiple regression analyses adjusting for potential confounding factors and interaction between asbestos exposure and DNAm on the MPM odds ratio were applied. Epigenome-wide analysis (EWAS) revealed 12 single-CpGs associated with the disease. Two of these showed high statistical power (99%) and effect size (&gt, 0.05) after false discovery rate (FDR) multiple comparison corrections: (i) cg03546163 in FKBP5, significantly hypomethylated in cases (Mean Difference in beta values (MD) = −0.09, 95% CI = −0.12|−0.06, p = 1.2 × 10−7), and (ii) cg06633438 in MLLT1, statistically hypermethylated in cases (MD = 0.07, 95% CI = 0.04|0.10, p = 1.0 × 10−6). Based on the interaction analysis, asbestos exposure and epigenetic profile together may improve MPM risk assessment. Above-median asbestos exposure and hypomethylation of cg03546163 in FKBP5 (OR = 20.84, 95% CI = 8.71|53.96, p = 5.5 × 10−11) and hypermethylation of cg06633438 in MLLT1 (OR = 11.71, 95% CI = 4.97|29.64, p = 5.9 × 10−8) genes compared to below-median asbestos exposure and hyper/hypomethylation of single-CpG DNAm, respectively. Receiver Operation Characteristics (ROC) for Case-Control Discrimination showed a significant increase in MPM discrimination when DNAm information was added in the model (baseline model, BM: asbestos exposure, age, gender and white blood cells), area under the curve, AUC = 0.75, BM + cg03546163 at FKBP5. AUC = 0.89, 2.1 × 10−7, BM + cg06633438 at MLLT1. AUC = 0.89, 6.3 × 10−8. Validation and replication procedures, considering independent sample size and a different DNAm analysis technique, confirmed the observed associations. Our results suggest the potential application of DNAm profiles in blood to develop noninvasive tests for MPM risk assessment in asbestos-exposed subjects.

Simple Summary Our study is the first one to investigate DNA methylation changes in white blood cells (WBCs) from easily accessible peripheral blood as malignant pleural mesothelioma (MPM) survival biomarker. The Cox proportional hazards regression model highlighted that the methylation status of the CpG dinucleotide cg03546163 is an independent marker of prognosis in MPM patients with a better performance than traditional inflammation-based scores such as lymphocyte-to-monocyte ratio (LMR). Biological validation and replication showed that epigenetic changes at the FKBP5 gene were robustly associated with overall survival (OS) in MPM cases. The identification of simple and valuable prognostic markers for MPM will enable clinicians to select patients who are most likely to benefit from aggressive therapies and avoid subjecting non-responder patients to ineffective treatment. Abstract Malignant pleural mesothelioma (MPM) is an aggressive tumor with median survival of 12 months and limited effective treatments. The scope of this study was to study the relationship between blood DNA methylation (DNAm) and overall survival (OS) aiming at a noninvasive prognostic test. We investigated a cohort of 159 incident asbestos exposed MPM cases enrolled in an Italian area with high incidence of mesothelioma. Considering 12 months as a cut-off for OS, epigenome-wide association study (EWAS) revealed statistically significant (p value = 7.7 × 10−9) OS-related differential methylation of a single-CpG (cg03546163), located in the 5′UTR region of the FKBP5 gene. This is an independent marker of prognosis in MPM patients with a better performance than traditional inflammation-based scores such as lymphocyte-to-monocyte ratio (LMR). Cases with DNAm < 0.45 at the cg03546163 had significantly poor survival compared with those showing DNAm ≥ 0.45 (mean: 243 versus 534 days; p value< 0.001). Epigenetic changes at the FKBP5 gene were robustly associated with OS in MPM cases. Our results showed that blood DNA methylation levels could be promising and dynamic prognostic biomarkers in MPM.

Diamond–Blackfan anemia (DBA) is a rare genetic hypoplasia of erythroid progenitors characterized by mild to severe anemia and associated with congenital malformations. Clinical manifestations in DBA patients are quite variable and genetic testing has become a critical factor in establishing a diagnosis of DBA. The majority of DBA cases are due to heterozygous loss‐of‐function mutations in ribosomal protein (RP) genes. Causative mutations are fairly straightforward to identify in the case of large deletions and frameshift and nonsense mutations found early in a protein coding sequence, but diagnosis becomes more challenging in the case of missense mutations and small in‐frame indels. Our group recently characterized the phenotype of lymphoblastoid cell lines established from DBA patients with pathogenic lesions in RPS19 and observed that defective pre‐rRNA processing, a hallmark of the disease, was rescued by lentiviral vectors expressing wild‐type RPS19. Here, we use this complementation assay to determine whether RPS19 variants of unknown significance are capable of rescuing pre‐rRNA processing defects in these lymphoblastoid cells as a means of assessing the effects of these sequence changes on the function of the RPS19 protein. This approach will be useful in differentiating pathogenic mutations from benign polymorphisms in identifying causative genes in DBA patients.

The intestinal microbiota is composed of a large number of different bacteria that produce a variety of metabolites. Colorectal cancer, which typically develops from adenomatous polyps, is highly influenced by microbiota. Since a variety of molecular changes may occur as these polyps transform from benign tumor to malignant carcinoma, the ability to study the microbiota-produced metabolites can lead to new discoveries about the development and progression of this cancer. However, to address the complexity of the microbiota-produced molecules, novel methods are needed. To this aim, in the present work, we developed a high-throughput metabolomics method to capture the metabolic complexity of the microbiota metabolome adherent to adenomatous polyps and adenocarcinoma. For the first time, the method enables the simultaneous quantification of almost 300 metabolites, while preserving the integrity of the original sample. The metabolomics approach was analytically validated and had excellent performances in terms of recovery, linearity, specificity, intra- and inter-day precision, limits of detection, and quantification. Furthermore, the clinical potential of the method was demonstrated in adenoma collected for a colorectal adenoma study.

Malignant mesothelioma (MM) is an aggressive cancer associated with asbestos exposure . Studies of familial malignant pleural mesothelioma (MPM) have suggested the existence of a genetic predisposition . Information on the role of genetic risk factors in the development of MM has been growing in the last years, and both low- and high-risk genetic factors have been identified, but genetic factors alone (without any exposure to asbestos or other mineral fibers) have never been shown to induce MM. Low-risk genetic factors have been identified in studies that systematically analyzed the whole genome. When considered alone these low-risk genetic factors carry a relative risk of MPM that is 10- to 15-fold lower than that carried by asbestos exposure; however, a large number of these factors in combination may increase the impact of asbestos exposure. High-risk genetic factors include truncating variants in the tumor suppressor BAP1 and in other tumor suppressor genes belonging to DNA repair pathways. Heterozygous germline variants in these genes may favor carcinogenesis if a second somatic variant occurs that impairs the wild-type allele. This impairment can cause genetic instability due to the suppression of a specific DNA repair pathway, and transformation. This genetic predisposition may have translational consequences, as it may predict patient response to drugs that induce tumor-specific synthetic lethality .

The definition of the mesothelioma genome is expected to have a great impact toward the development of new drugs and therapeutic approaches with a view to precision medicine. A few studies reported that the malignant pleural mesothelioma (MPM) genomic landscape is characterized by a much greater number of genomic losses than point mutations. Inactivating gene fusions, copy losses, and protein-truncating variants (PTVs) mostly affect tumor suppressor genes, above all BAP1, NF2, CDKN2A, and SETD2. Some of them may be exploited to design novel therapeutic strategies. Germline mutations in some of these genes represent MPM-predisposing risk factors. These mutations require a second hit, i.e., asbestos exposure, to induce carcinogenesis. The most studied of these genes is BAP1. Germline mutations in other tumor suppressor genes, mostly involved in DNA repair, have also been identified in about 10% of MPM patients. These patients are more sensitive to asbestos exposure than those who do not carry such mutations and may benefit of specific treatments. Additionally, epigenetic mechanisms, such as methylation or miRNA alterations, may modify gene expression and drive carcinogenesis. The same abnormalities may be used as disease biomarkers or therapeutic targets.

Stem cell therapy with human mesenchymal stem cells (hMSCs) represents a promising strategy in spinal cord injury (SCI). However, both systemic and parenchymal hMSCs administrations show significant drawbacks as a limited number and viability of stem cells in situ. Biomaterials able to encapsulate and sustain hMSCs represent a viable approach to overcome these limitations potentially improving the stem cell therapy. In this study, we evaluate a new agarose/carbomer based hydrogel which combines different strategies to optimize hMSCs viability, density and delivery of paracrine factors. Specifically, we evaluate a new loading procedure on a lyophilized scaffold (soaked up effect) that reduces mechanical stress in encapsulating hMSCs into the hydrogel. In addition, we combine arginine-glycine-aspartic acid (RGD) tripeptide and 3D extracellular matrix deposition to increase the capacity to attach and maintain healthy hMSCs within the hydrogel over time. Furthermore, the fluidic diffusion from the hydrogel toward the injury site is improved by using a cling film that oriented efficaciously the delivery of paracrine factors in vivo. Finally, we demonstrate that an improved combination as here proposed of hMSCs and biomimetic hydrogel is able to immunomodulate significantly the pro-inflammatory environment in a SCI mouse model, increasing M2 macrophagic population and promoting a pro-regenerative environment in situ.

Malignant pleural mesothelioma (MPM) is an aggressive tumor with median survival of 12 months and limited effective treatments. The scope of this study was to study the relationship between blood DNA methylation (DNAm) and overall survival (OS) aiming at a noninvasive prognostic test. We investigated a cohort of 159 incident asbestos exposed MPM cases enrolled in an Italian area with high incidence of mesothelioma. Considering 12 months as a cut-off for OS, epigenome-wide association study (EWAS) revealed statistically significant (p value = 7.7 &times, 10&minus, 9) OS-related differential methylation of a single-CpG (cg03546163), located in the 5&prime, UTR region of the FKBP5 gene. This is an independent marker of prognosis in MPM patients with a better performance than traditional inflammation-based scores such as lymphocyte-to-monocyte ratio (LMR). Cases with DNAm &lt, 0.45 at the cg03546163 had significantly poor survival compared with those showing DNAm &ge, 0.45 (mean: 243 versus 534 days, p value&lt, 0.001). Epigenetic changes at the FKBP5 gene were robustly associated with OS in MPM cases. Our results showed that blood DNA methylation levels could be promising and dynamic prognostic biomarkers in MPM.

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Pathogenic germline variants in the BAP1 tumor suppressor gene can cause a cancer syndrome called BAP1 tumor predisposition syndrome (BAP1-TPDS), which is characterized by predisposition to mesothelioma, melanoma, renal cell carcinoma, basal cell carcinoma, and other tumors. Other genes that may predispose to mesothelioma are CDKN2A and DNA repair genes. Asbestos exposure has often been reported in patients with malignant pleural mesothelioma (MPM) and germline variants in BAP1, but this exposure has never been quantified. We aimed to search for germline variants in BAP1 among 25 new Italian probands with suspected BAP1-TPDS, summarize the prevalence of these variants in 39 Italian patients with familial MPM and other tumors recruited over a 5-year period, and compare cumulative asbestos exposure in 14 patients with MPM and pathogenic germline variants in BAP1, CDKN2A, or DNA repair genes with that of 67 patients without germline variants in 94 cancer-predisposing genes. We report here a new pathogenic germline variant in BAP1: c.783 + 2 T > C. The prevalence of pathogenic germline variants in BAP1 was 7.7% among patients with familial MPM (3/39). Patients with pathogenic germline variants in BAP1, CDKN2A, or DNA repair genes showed lower cumulative asbestos exposure than patients without germline variants in 94 cancer-predisposing genes (P = .00002). This suggests an interaction between genetic risk factors and asbestos in the development of mesothelioma.

Diamond Blackfan anaemia (DBA) is a congenital bone marrow failure syndrome characterised by selective red cell hypoplasia. DBA is most often due to heterozygous mutations in ribosomal protein (RP) genes that lead to defects in ribosome biogenesis and function and result in ribosomal stress and p53 activation. The molecular mechanisms underlying this pathology are still poorly understood and studies on patient erythroid cells are hampered by their paucity. Here we report that RP-mutated lymphoblastoid cell lines (LCLs) established from DBA patients show defective rRNA processing and ribosomal stress features such as reduced proliferation, decreased protein synthesis, and activation of p53 and its target p21. These phenotypic alterations were corrected by gene complementation. Our data indicate that DBA LCLs could be a useful model for molecular and pharmacological investigations.