Distinct Signatures of Tumor-Associated Microbiota and Metabolome in Low-Grade vs. High-Grade Dysplastic Colon Polyps: Inference of Their Role in Tumor Initiation and Progression.

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/ß-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.

Deficiency of ribosomal protein S26, which is mutated in a subset of patients with Diamond Blackfan anemia, impairs erythroid differentiation.

Introduction: Diamond Blackfan anemia (DBA) is a rare congenital disease characterized by defective maturation of the erythroid progenitors in the bone marrow, for which treatment involves steroids, chronic transfusions, or hematopoietic stem cells transplantation. Diamond Blackfan anemia is caused by defective ribosome biogenesis due to heterozygous pathogenic variants in one of 19 ribosomal protein (RP) genes. The decreased number of functional ribosomes leads to the activation of pro-apoptotic pathways and to the reduced translation of key genes for erythropoiesis. Results and discussion: Here we characterized the phenotype of RPS26-deficiency in a cell line derived from human umbilical cord blood erythroid progenitors (HUDEP-1 cells). This model recapitulates cellular hallmarks of Diamond Blackfan anemia including: imbalanced production of ribosomal RNAs, upregulation of pro-apoptotic genes and reduced viability, and shows increased levels of intracellular calcium. Evaluation of the expression of erythroid markers revealed the impairment of erythroid differentiation in RPS26-silenced cells compared to control cells. Conclusions: In conclusion, for the first time we assessed the effect of RPS26 deficiency in a human erythroid progenitor cell line and demonstrated that these cells can be used as a scalable model system to study aspects of DBA pathophysiology that have been refractory to detailed investigation because of the paucity of specific cell types affected in this disorder.

Diagnostics of BAP1-Tumor Predisposition Syndrome by a Multitesting Approach: A Ten-Year-Long Experience.

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: Germline variants in DNA repair genes may steer tailored treatment.

INTRODUCTION: 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. AIM: 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. METHODS: 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. RESULTS: 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). CONCLUSIONS: Overall these data suggest that patients with MPM and DNA repair insufficiency may benefit from this treatment, which induces synthetic lethality.

A new method for investigating microbiota-produced small molecules in adenomatous polyps.

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.

Genetic Predisposition to Myelodysplastic Syndromes: A Challenge for Adult Hematologists.

Myelodysplastic syndromes (MDS) arising in the context of inherited bone marrow failure syndromes (IBMFS) differ in terms of prognosis and treatment strategy compared to MDS occurring in the adult population without an inherited genetic predisposition. The main molecular pathways affected in IBMFS involve telomere maintenance, DNA repair, biogenesis of ribosomes, control of proliferation and others. The increased knowledge on the genes involved in MDS pathogenesis and the wider availability of molecular diagnostic assessment have led to an improvement in the detection of IBMFS genetic predisposition in MDS patients. A punctual recognition of these disorders implies a strict surveillance of the patient in order to detect early signs of progression and promptly offer allogeneic hematopoietic stem cell transplantation, which is the only curative treatment. Moreover, identifying an inherited mutation allows the screening and counseling of family members and directs the choice of donors in case of need for transplantation. Here we provide an overview of the most recent data on MDS with genetic predisposition highlighting the main steps of the diagnostic and therapeutic management. In order to highlight the pitfalls of detecting IBMFS in adults, we report the case of a 27-year-old man affected by MDS with an underlying telomeropathy.

DNA Methylation of FKBP5 as Predictor of Overall Survival in Malignant Pleural Mesothelioma.

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.

Genetic predisposition for malignant mesothelioma: A concise review.

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.

Rare ribosomopathies: insights into mechanisms of cancer.

Long thought to be too big and too ubiquitous to fail, we now know that human cells can fail to make sufficient amounts of ribosomes, causing a number of diseases collectively known as ribosomopathies. The best characterized ribosomopathies, with the exception of Treacher Collins syndrome, are inherited bone marrow failure syndromes, each of which has a marked increase in cancer predisposition relative to the general population. Although rare, emerging data reveal that the inherited bone marrow failure syndromes may be underdiagnosed on the basis of classical symptomology, leaving undiagnosed patients with these syndromes at an elevated risk of cancer without adequate counselling and surveillance. The link between the inherited ribosomopathies and cancer has led to greater awareness that somatic mutations in factors involved in ribosome biogenesis may also be drivers in sporadic cancers. Our goal here is to compare and contrast the pathophysiological mechanisms underpinning ribosomopathies to gain a better understanding of the mechanisms that predispose these disorders to cancer.

Peripheral Blood DNA Methylation as Potential Biomarker of Malignant Pleural Mesothelioma in Asbestos-Exposed Subjects.

INTRODUCTION: Malignant pleural mesothelioma (MPM) is an aggressive tumor strongly associated with asbestos exposure. Patients are usually diagnosed when current treatments have limited benefits, highlighting the need for noninvasive early diagnostic tests to monitor asbestos-exposed people. METHODS: We used a genome-wide methylation array to identify, in asbestos-exposed subjects, novel blood DNA methylation markers of MPM in 163 MPM cases and 137 cancer-free controls (82 MPM cases and 68 controls, training set; replication in 81 MPM cases and 69 controls, test set) sampled from the same areas. RESULTS: Evidence of differential methylation between MPM cases and controls was found (more than 800 cytosine-guanine dinucleotide sites, false discovery rate p value (pfdr) < 0.05), mainly in immune system-related genes. Considering the top differentially methylated signals, seven single- cytosine-guanine dinucleotides and five genomic regions of coordinated methylation replicated with similar effect size in the test set (pfdr < 0.05). The top hypomethylated single-CpG (cases versus controls effect size less than -0.15, pfdr < 0.05 in both the training and test sets) was detected in FOXK1 (Forkhead-box K1) gene, an interactor of BAP1 which was found mutated in MPM tissue and as germline mutation in familial MPM. In the test set, comparison of receiver operating characteristic curves and the area under the curve (AUC) of two models, including or excluding methylation, showed a significant increase in case/control discrimination when considering DNA methylation together with asbestos exposure (AUC = 0.81 versus AUC = 0.89, DeLong's test p = 0.0013). CONCLUSIONS: We identified signatures of differential methylation in DNA from whole blood between asbestos exposed MPM cases and controls. Our results provide the rationale to further investigate, in prospective studies, the potential use of blood DNA methylation profiles for the identification of early changes related to the MPM carcinogenic process.

Author Correction: Lymphoblastoid cell lines from Diamond Blackfan anaemia patients exhibit a full ribosomal stress phenotype that is rescued by gene therapy.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has not been fixed in the paper.

Emerging Therapeutic Approaches for Diamond Blackfan Anemia.

Diamond Blackfan Anemia (DBA) is an inherited erythroid aplasia with onset in childhood. Patients carry heterozygous mutations in one of 19 Ribosomal Protein (RP) genes, that lead to defective ribosome biogenesis and function. Standard treatments include steroids or blood transfusions but the only definitive cure is allogeneic Hematopoietic Stem Cell Transplantation (HSCT). Although advances in HSCT have greatly improved the success rate over the last years, the risk of adverse events and mortality is still significant. Clinical trials employing gene therapy are now in progress for a variety of monogenic diseases and the development of innovative stem cell-based strategies may open new alternatives for DBA treatment as well. In this review, we summarize the most recent progress toward the implementation of new therapeutic approaches for this disorder. We present different DNA- and RNA-based technologies as well as new candidate pharmacological treatments and discuss their relevance and potential applicability for the cure of DBA.

Sensitivity to asbestos is increased in patients with mesothelioma and pathogenic germline variants in BAP1 or other DNA repair genes.

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.

Blocking Macrophage Migration Inhibitory Factor Protects Against Cisplatin-Induced Acute Kidney Injury in Mice.

Macrophage migration inhibitory factor (MIF) is elevated in patients with acute kidney injury (AKI) and is suggested as a potential predictor for renal replacement therapy in AKI. In this study, we found that MIF also plays a pathogenic role and is a therapeutic target for AKI. In a cisplatin-induced AKI mouse model, elevated plasma MIF correlated with increased serum creatinine and the severity of renal inflammation and tubular necrosis, whereas deletion of MIF protected the kidney from cisplatin-induced AKI by largely improving renal functional and histological injury, and suppressing renal inflammation including upregulation of cytokines such as interleukin (IL)-1ß, tumor necrosis factor-alpha (TNF-α), IL-6, inducible nitric oxide synthase (iNOS), MCP-1, IL-8, and infiltration of macrophages, neutrophils, and T cells. We next developed a novel therapeutic strategy for AKI by blocking the endogenous MIF with an MIF inhibitor, ribosomal protein S19 (RPS19). Similar to the MIF-knockout mice, treatment with RPS19, but not the mutant RPS19, suppressed cisplatin-induced AKI. Mechanistically, we found that both genetic knockout and pharmacological inhibition of MIF protected against AKI by inactivating the CD74-nuclear factor κB (NF-κB) signaling. In conclusion, MIF is pathogenic in cisplatin-induced AKI. Targeting MIF with an MIF inhibitor RPS19 could be a promising therapeutic potential for AKI.

A functional assay for the clinical annotation of genetic variants of uncertain significance in Diamond-Blackfan anemia.

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.

Recurring mutations in RPL15 are linked to hydrops fetalis and treatment independence in Diamond-Blackfan anemia.

Diamond-Blackfan anemia (DBA) is a rare inherited bone marrow failure disorder linked predominantly to ribosomal protein gene mutations. Here the European DBA consortium reports novel mutations identified in the RPL15 gene in 6 unrelated individuals diagnosed with DBA. Although point mutations have not been previously reported for RPL15, we identified 4 individuals with truncating mutations p.Tyr81* (in 3 of 4) and p.Gln29*, and 2 with missense variants p.Leu10Pro and p.Lys153Thr. Notably, 75% (3 of 4) of truncating mutation carriers manifested with severe hydrops fetalis and required intrauterine transfusions. Even more remarkable is the observation that the 3 carriers of p.Tyr81* mutation became treatment-independent between four and 16 months of life and maintained normal blood counts until their last follow up. Genetic reversion at the DNA level as a potential mechanism of remission was not observed in our patients. In vitro studies revealed that cells carrying RPL15 mutations have pre-rRNA processing defects, reduced 60S ribosomal subunit formation, and severe proliferation defects. Red cell culture assays of RPL15-mutated primary erythroblast cells also showed a severe reduction in cell proliferation, delayed erythroid differentiation, elevated TP53 activity, and increased apoptosis. This study identifies a novel subgroup of DBA with mutations in the RPL15 gene with an unexpected high rate of hydrops fetalis and spontaneous, long-lasting remission.

Lymphoblastoid cell lines from Diamond Blackfan anaemia patients exhibit a full ribosomal stress phenotype that is rescued by gene therapy.

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.

Osteopontin at the Crossroads of Inflammation and Tumor Progression.

Complex interactions between tumor and host cells regulate systemic tumor dissemination, a process that begins early at the primary tumor site and goes on until tumor cells detach themselves from the tumor mass and start migrating into the blood or lymphatic vessels. Metastatic cells colonize the target organs and are capable of surviving and growing at distant sites. In this context, osteopontin (OPN) appears to be a key determinant of the crosstalk between cancer cells and the host microenvironment, which in turn modulates immune evasion. OPN is overexpressed in several human carcinomas and has been implicated in inflammation, tumor progression, and metastasis. Thus, it represents one of the most attracting targets for cancer therapy. Within the tumor mass, OPN is secreted in various forms either by the tumor itself or by stroma cells, and it can exert either pro- or antitumorigenic effects according to the cell type and tumor microenvironment. Thus, targeting OPN for therapeutic purposes needs to take into account the heterogeneous functions of the multiple OPN forms with regard to cancer formation and progression. In this review, we will describe the role of systemic, tumor-derived, and stroma-derived OPN, highlighting its pivotal role at the crossroads of inflammation and tumor progression.

Germline mutations in DNA repair genes predispose asbestos-exposed patients to malignant pleural mesothelioma.

Malignant pleural mesothelioma (MPM) is a rare, aggressive cancer caused by asbestos exposure. An inherited predisposition has been suggested to explain multiple cases in the same family and the observation that not all individuals highly exposed to asbestos develop the tumor. Germline mutations in BAP1 are responsible for a rare cancer predisposition syndrome that includes predisposition to mesothelioma. We hypothesized that other genes involved in hereditary cancer syndromes could be responsible for the inherited mesothelioma predisposition. We investigated the prevalence of germline variants in 94 cancer-predisposing genes in 93 MPM patients with a quantified asbestos exposure. Ten pathogenic truncating variants (PTVs) were identified in PALB2, BRCA1, FANCI, ATM, SLX4, BRCA2, FANCC, FANCF, PMS1 and XPC. All these genes are involved in DNA repair pathways, mostly in homologous recombination repair. Patients carrying PTVs represented 9.7% of the panel and showed lower asbestos exposure than did all the other patients (p = 0.0015). This suggests that they did not efficiently repair the DNA damage induced by asbestos and leading to carcinogenesis. This study shows that germline variants in several genes may increase MPM susceptibility in the presence of asbestos exposure and may be important for specific treatment.