Synergy of the microRNA Ratio as a Promising Diagnosis Biomarker for Mucinous Borderline and Malignant Ovarian Tumors.

Epithelial ovarian cancers (EOCs) are a heterogeneous collection of malignancies, each with their own developmental origin, clinical behavior and molecular profile. With less than 5% of EOC cases, mucinous ovarian carcinoma is a rare form with a poor prognosis and a 5-year survival of 11% for advanced stages (III/IV). At the early stages, these malignant forms are clinically difficult to distinguish from borderline (15%) and benign (80%) forms with a better prognosis due to the large size and heterogeneity of mucinous tumors. Improving their diagnosis is therefore a challenge with regard to the risk of under-treating a malignant form or of unnecessarily undertaking radical surgical excision. The involvement of microRNAs (miRNAs) in tumor progression and their potential as biomarkers of diagnosis are becoming increasingly recognized. In this study, the comparison of miRNA microarray expression profiles between malignant and borderline tumor FFPE samples identified 10 down-regulated and 5 up-regulated malignant miRNAs, which were validated by individual RT-qPCR. To overcome normalization issues and to improve the accuracy of the results, a ratio analysis combining paired up-regulated and down-regulated miRNAs was performed. Although 21/50 miRNA expression ratios were significantly different between malignant and borderline tumor samples, any ratio could perfectly discriminate the two groups. However, a combination of 14 pairs of miRNA ratios (double ratio) showed high discriminatory potential, with 100% of accuracy in distinguishing malignant and borderline ovarian tumors, which suggests that miRNAs may hold significant clinical potential as a diagnostic tool. In summary, these ratio miRNA-based signatures may help to improve the precision of histological diagnosis, likely to provide a preoperative diagnosis in order to adapt surgical procedures.

Gene/environment interaction in the susceptibility of Crohn's disease patients to aluminum.

BACKGROUND & AIM: The key role of environmental factors in the pathogenesis of Inflammatory Bowel Diseases (IBD) is recognized. Aluminum is suspected to be a risk factor for IBD. However, mechanisms linking aluminum exposure to disease development are unknown. We examined the role of aluminum transport and subcellular localisation on human colon susceptibility to aluminum-induced inflammation. METHODS: Human colon biopsies isolated from Crohn's disease (CD) or control patients and Caco-2 cells were incubated with aluminum. The effects of aluminum were evaluated on cytokine secretion and transporter expression. The role of aluminum kinetics parameters was studied in Caco-2 using transport inhibitors and in human colon biopsies by assessing genetic polymorphisms of transporters. RESULTS: Aluminum exposure was shown to induce cytokine secretion in colon of CD but not healthy patients. In Caco-2 cells, aluminum internalisation was correlated with inflammatory status. In human colon, analysis of genetic polymorphisms and expression of ABCB1 and SLC26A3 transporters showed that their decreased activity was involved in aluminum-induced inflammation. CONCLUSIONS: We hypothesize that alteration in detoxifying response would lead to a deregulation of intestinal homeostasis and to the expression of IBD. Our study emphasizes the complexity of gene/environment interaction for aluminum adverse health effect, highlighting at risk populations or subtypes of patients. A better understanding of correlations between gene expression or SNP and xenobiotic kinetics parameters would shift the medical paradigm to more personalized disease management and treatment.

Simple gene signature to assess murine fibroblast polarization.

We provide an original multi-stage approach identifying a gene signature to assess murine fibroblast polarization. Prototypic polarizations (inflammatory/fibrotic) were induced by seeded mouse embryonic fibroblasts (MEFs) with TNFα or TGFß1, respectively. The transcriptomic and proteomic profiles were obtained by RNA microarray and LC-MS/MS. Gene Ontology and pathways analysis were performed among the differentially expressed genes (DEGs) and proteins (DEPs). Balb/c mice underwent daily intradermal injections of HOCl (or PBS) as an experimental murine model of inflammation-mediated fibrosis in a time-dependent manner. As results, 1456 and 2215 DEGs, and 289 and 233 DEPs were respectively found in MEFs in response to TNFα or TGFß1, respectively. Among the most significant pathways, we combined 26 representative genes to encompass the proinflammatory and profibrotic polarizations of fibroblasts. Based on principal component analysis, this signature deciphered baseline state, proinflammatory polarization, and profibrotic polarization as accurately as RNA microarray and LC-MS/MS did. Then, we assessed the gene signature on dermal fibroblasts isolated from the experimental murine model. We observed a proinflammatory polarization at day 7, and a mixture of a proinflammatory and profibrotic polarizations at day 42 in line with histological findings. Our approach provides a small-size and convenient gene signature to assess murine fibroblast polarization.

Detection of residual and chemoresistant leukemic cells in an immune-competent mouse model of acute myeloid leukemia: Potential for unravelling their interactions with immunity.

Acute myeloid leukemia (AML) is characterized by blocked differentiation and extensive proliferation of hematopoietic progenitors/precursors. Relapse is often observed after chemotherapy due to the presence of residual leukemic cells, which is also called minimal residual disease (MRD). Subclonal heterogeneity at diagnosis was found to be responsible for MRD after treatment. Patient xenograft mouse models are valuable tools for studying MRD after chemotherapy; however, the contribution of the immune system in these models is usually missing. To evaluate its role in leukemic persistence, we generated an immune-competent AML mouse model of persistence after chemotherapy treatment. We used well-characterized (phenotypically and genetically) subclones of the murine C1498 cell line stably expressing the ZsGreen reporter gene and the WT1 protein, a valuable antigen. Accordingly, these subclones were also selected due to their in vitro aracytidine (Ara-c) sensitivity. A combination of 3 subclones (expressing or not expressing WT1) was found to lead to prolonged mouse survival after Ara-c treatment (as long as 150 days). The presence of residual leukemic cells in the blood and BM of surviving mice indicated their persistence. Thus, a new mouse model that may offer insights into immune contributions to leukemic persistence was developed.

A Spontaneous Model of Experimental Autoimmune Encephalomyelitis Provides Evidence of MOG-Specific B Cell Recruitment and Clonal Expansion.

The key role of B cells in the pathophysiology of multiple sclerosis (MS) is supported by the presence of oligoclonal bands in the cerebrospinal fluid, by the association of meningeal ectopic B cell follicles with demyelination, axonal loss and reduction of astrocytes, as well as by the high efficacy of B lymphocyte depletion in controlling inflammatory parameters of MS. Here, we use a spontaneous model of experimental autoimmune encephalomyelitis (EAE) to study the clonality of the B cell response targeting myelin oligodendrocyte glycoprotein (MOG). In particular, 94% of SJL/j mice expressing an I-As: MOG92-106 specific transgenic T cell receptor (TCR1640) spontaneously develop a chronic paralytic EAE between the age of 60-500 days. The immune response is triggered by the microbiota in the gut-associated lymphoid tissue, while there is evidence that the maturation of the autoimmune demyelinating response might occur in the cervical lymph nodes owing to local brain drainage. Using MOG-protein-tetramers we tracked the autoantigen-specific B cells and localized their enrichment to the cervical lymph nodes and among the brain immune infiltrate. MOG-specific IgG1 antibodies were detected in the serum of diseased TCR1640 mice and proved pathogenic upon adoptive transfer into disease-prone recipients. The ontogeny of the MOG-specific humoral response preceded disease onset coherent with their contribution to EAE initiation. This humoral response was, however, not sufficient for disease induction as MOG-antibodies could be detected at the age of 69 days in a model with an average age of onset of 197 days. To assess the MOG-specific B cell repertoire we FACS-sorted MOG-tetramer binding cells and clonally expand them in vitro to sequence the paratopes of the IgG heavy chain and kappa light chains. Despite the fragility of clonally expanding MOG-tetramer binding effector B cells, our results indicate the selection of a common CDR-3 clonotype among the Igk light chains derived from both disease-free and diseased TCR1640 mice. Our study demonstrates the pre-clinical mobilization of the MOG-specific B cell response within the brain-draining cervical lymph nodes, and reiterates that MOG antibodies are a poor biomarker of disease onset and progression.

p65/RelA NF-κB fragments generated by RIPK3 activity regulate tumorigenicity, cell metabolism, and stemness characteristics.

Receptor-interacting protein kinase 3 (RIPK3) can induce necroptosis, apoptosis, or cell proliferation and is silenced in several hematological malignancies. We previously reported that RIPK3 activity independent of its kinase domain induces caspase-mediated p65/RelA cleavage, resulting in N-terminal 1-361 and C-terminal 362-549 fragments. We show here that a noncleavable p65/RelA D361E mutant expressed in DA1-3b leukemia cells decreases mouse survival times and that coexpression of p65/RelA fragments increases the tumorigenicity of B16F1 melanoma cells. This aggressiveness in vivo did not correlate with NF-κB activity measured in vitro. The fragments and p65/RelA D361E mutant induced different expression profiles in DA1-3b and B16F1 cells. Stemness markers were affected: p65/RelA D361E increased ALDH activity in DA1-3b cells, and fragment expression increased melanoma sphere formation in B16/F1 cells. p65/RelA fragments and the D361E noncleavable mutant decreased oxidative or glycolytic cell metabolism, with differences observed between models. Thus, p65/RelA cleavage initiated by kinase-independent RIPK3 activity in cancer cells is not neutral and induces pleiotropic effects in vitro and in vivo that may vary across tumor types.

Bimodal expression of RHOH during myelomonocytic differentiation: Implications for the expansion of AML differentiation therapy.

RhoH is an unusual member of the Rho family of small GTP-binding proteins in that it lacks GTPase activity. Since the RhoH protein is constantly bound by GTP, it is constitutively active and controlled predominantly by changes in quantitative expression. Abnormal levels of RHOH gene transcripts have been linked to a range of malignancies including acute myeloid leukemia (AML). One of the hallmarks of AML is a block in the normal program of myeloid differentiation. Here we investigate how myeloid differentiation is controlled by the quantitative expression of RHOH. Our analysis demonstrates that increasingly mature myeloid cells express progressively lower levels of RHOH. However, as monocytic myeloid cells terminally differentiate into macrophages, RHOH expression is up-regulated. This up-regulation is not apparent in AML where myeloid differentiation is blocked at stages of low RHOH expression. Nevertheless, when the up-regulation of RHOH is forced, then terminal macrophage differentiation is induced and the Cdc42 and Wnt intracellular signalling pathways are repressed. These results indicate that RHOH induction is a driver of terminal differentiation and might represent a means of effecting AML differentiation therapy. The potential of this therapeutic strategy is supported by forced up-regulation of RHOH reducing the ability of AML cells to produce tumours in vivo.

A Novel Missense Single Nucleotide Polymorphism in the GREM1 Gene is Highly Associated with Higher Reproductive Traits in Awassi Sheep.

GREM1 (gremlin1) is a known inhibitor for BMP15 (bone morphogenetic protein 15) family, but its genetic diversity in sheep is unknown. The present study was conducted to analyze the polymorphism of GREM1 gene using PCR- single-strand conformation polymorphism (SSCP) and DNA sequencing methods and to assess the possible association of GREM1 gene polymorphism with reproductive traits in Awassi ewes. A total of 224 ewes, 124 producing singles and 100 producing twins, were included in the study. Two SSCP patterns were detected in two amplified loci within the exon 2. Two exonic novel single nucleotide polymorphism (SNP)s were identified, c.74 T > G (the silent SNP p.Met123 =) and c.30 T > A with (the missense SNP p.Ile237Phe). Statistical analyses indicated a non-significant (P > 0.05) association of p.Met123 = with the analyzed reproductive traits of fecundity, prolificacy, litter size, and twinning rate. Meanwhile, p.Ile237Phe SNP exhibited a highly significant (P < 0.01) association with the measured reproductive traits, in which ewes with TA genotype (with p.Ile237Phe SNP) exhibited higher litter size, twinning ratio, fecundity, and prolificacy than those with TT genotype (without p.Ile237Phe SNP). The deleterious impact of p.Ile237Phe SNP was observed by the means of ten different state-of-the-art in silico tools that predicted a highly damaging effect of p.Ile237Phe SNP on the structure, function, and stability of gremlin1. In conclusion, the results of our study suggest that p.Ile237Phe SNP has a remarkable negative impact on the gremlin1 structure, function, and stability. Since gremlin1 is a known inhibitor of reproductive performance, a consequent higher reproductive performance was observed in ewes with damaged gremlin1 (with p.Ile237Phe SNP) than those with non-damaged gremlin1 (without p.Ile237Phe SNP). Therefore, it can be stated that the implementation of the novel p.Ile237Phe SNP in the GREM1 gene could be a useful marker in marker-assisted selection. This manuscript is the first one to describe GREM1 gene variations in sheep.

Two co-inherited novel SNPs in the MC4R gene related to live body weight and hormonal assays in Awassi and Arabi sheep breeds of Iraq.

Melanocortin-4 receptor (MC4R) gene plays a key role in the regulation of body weight and energy homeostasis. This study aims to evaluate the association of single nucleotide polymorphisms (SNPs) of the MC4R gene with live body weight and hormonal assays in two breeds of sheep that differ in productive performance, Awassi and Arabi. All known coding sequences of the MC4R gene were covered in this study. DNA samples from 150 animals (Awassi and Arabi breed) were genotyped by PCR-single-strand conformation polymorphism (PCR-SSCP) to assess their pattern of genetic variation. Concerning exon 1, clear heterogeneity was detected with three different SSCP-banding patterns. The sequencing reactions confirmed these variations by detecting the presence of the two novel SNPs, 107G/C and 138A/C, and three genotypes, GC, AC and AA. The 107G/C SNP was detected in GC genotype, while the 138A/C was detected on both GC and AC genotypes. The other SSCP-banding pattern (AA genotype) did not show any detectable unique variation. Both SNPs were closely and strongly linked in both breeds (D' and r2 values were 1.00), which signifies that both loci were co-inherited as one unit. Association analysis indicated that both breeds with GC/AC haplotype showed higher live body weight (37.250 ± 0.790) relative to the GG/AA (30.244 ± 0.968) and CC/CC (47.231 ± 1.230) haplotypes (p < .05). Concerning the genotyping of exon 2, only 362 bp showed heterogeneity with a missense mutation, with no significant association (p > .05) with the measured traits. In conclusion, the two novel SNPs (107G/C and 138 A/C) were highly associated with live body weight in both breeds. Haplotype analysis confirmed that these two novel SNPs were in strong linkage disequilibrium (LD) and could be used as genetic markers for sheep phenotypic trait improvement.

Co-inherited novel SNPs of the LIPE gene associated with increased carcass dressing and decreased fat-tail weight in Awassi breed.

The lipase E hormone-sensitive (LIPE) enzyme is one of the lipolytic enzymes, and it plays a key role in the regulation of adipose tissue deposition. This study was conducted to investigate the possible association between the LIPE gene variations and the main body weight measurements in Awassi sheep. A total of 160 of sexually mature Awassi rams (Ovis aries) that aged between 2 and 3 years were included in the present study. Genomic DNA was extracted and two specific PCR amplicons were designed to amplify two coding regions within the LIPE gene. Genotyping experiments were performed using polymerase chain reaction-single-strand conformational polymorphism (PCR-SSCP). Two different SSCP banding patterns were identified, CC and CD in exon 2, and AA and AT in exon 9. Five novel single-nucleotide polymorphisms (SNPs) were detected by sequencing, namely g.151C > A and g.198C > T in exon 2, and g.213G > C, g.226G > T, and g.232A > C in exon 9. Haplotype block analysis showed strong linkage disequilibrium values between the two SNPs in exon 2 and the three SNPs in exon 9. Association analysis of haplotypes with carcass traits demonstrated a significantly higher dressing percentage (P < 0.05) and lower fat tail weight (FTW) in CACT and GCGTAC haplotypes made these haplotypes more favorable for human consumption. The current research is the first one to report a tight association between the LIPE genetic polymorphism and the dressing percentage and FTW traits, suggesting a pivotal role played by these co-inherited SNPs in the metabolism of carcass traits in sheep.

Functional Analysis of Somatic Mutations Affecting Receptor Tyrosine Kinase Family in Metastatic Colorectal Cancer.

Besides the detection of somatic receptor tyrosine kinases (RTK) mutations in tumor samples, the current challenge is to interpret their biological relevance to give patients effective targeted treatment. By high-throughput sequencing of the 58 RTK exons of healthy tissues, colorectal tumors, and hepatic metastases from 30 patients, 38 different somatic mutations in RTKs were identified. The mutations in the kinase domains and present in both tumors and metastases were reconstituted to perform an unbiased functional study. Among eight variants found in seven RTKs (EPHA4-Met726Ile, EPHB2-Val621Ile, ERBB4-Thr731Met, FGFR4-Ala585Thr, VEGFR3-Leu1014Phe, KIT-Pro875Leu, TRKB-Leu584Val, and NTRK2-Lys618Thr), none displayed significantly increased tyrosine kinase activity. Consistently, none of them induced transformation of NIH3T3 fibroblasts. On the contrary, two RTK variants (FGFR4-Ala585Thr and FLT4-Leu1014Phe) caused drastic inhibition of their kinase activity. These findings indicate that these RTK variants are not suitable targets and highlight the importance of functional studies to validate RTK mutations as potential therapeutic targets.

The stem cell-associated gene expression signature allows risk stratification in pediatric acute myeloid leukemia.

Despite constant progress in prognostic risk stratification, children with acute myeloid leukemia (AML) still relapse. Treatment failure and subsequent relapse have been attributed to acute myeloid leukemia-initiating cells (LSC), which harbor stem cell properties and are inherently chemoresistant. Although pediatric and adult AML represent two genetically very distinct diseases, we reasoned that common LSC gene expression programs are shared and consequently, the highly prognostic LSC17 signature score recently developed in adults may also be of clinical interest in childhood AML. Here, we demonstrated prognostic relevance of the LSC17 score in pediatric non-core-binding factor AML using Nanostring technology (ELAM02) and RNA-seq data from the NCI (TARGET-AML). AML were stratified by LSC17 quartile groups (lowest 25%, intermediate 50% and highest 25%) and children with low LSC17 score had significantly better event-free survival (EFS: HR = 3.35 (95%CI = 1.64-6.82), P < 0.001) and overall survival (OS: HR = 3.51 (95%CI = 1.38-8.92), P = 0.008) compared with patients with high LSC17 scores. More importantly, the high LSC17 score was an independent negative EFS and OS prognosticator determined by multivariate Cox model analysis (EFS: HR = 3.42 (95% CI = 1.63-7.16), P = 0.001; OS HR = 3.02 (95%CI = 1.16-7.85), P = 0.026). In conclusion, we have demonstrated the broad applicability of the LSC17 score in the clinical management of AML by extending its prognostic relevance to pediatric AML.

Isolation and characterization of two canine melanoma cell lines: new models for comparative oncology.

BACKGROUND: Metastatic melanoma is one of the most aggressive forms of cancer in humans. Among its types, mucosal melanomas represent one of the most highly metastatic and aggressive forms, with a very poor prognosis. Because they are rare in Caucasian individuals, unlike cutaneous melanomas, there has been fewer epidemiological, clinical and genetic evaluation of mucosal melanomas. Moreover, the lack of predictive models fully reproducing the pathogenesis and molecular alterations of mucosal melanoma makes its treatment challenging. Interestingly, dogs are frequently affected by melanomas of the oral cavity that are characterized, as their human counterparts, by focal infiltration, recurrence, and metastasis to regional lymph nodes, lungs and other organs. In dogs, some particular breeds are at high risk, suggesting a specific genetic background and strong genetic drivers. Altogether, the striking homologies in clinical presentation, histopathological features, and overall biology between human and canine mucosal melanomas make dogs invaluable natural models with which to investigate tumor development, including tumor ætiology, and develop tailored treatments. METHODS: We developed and characterized two canine oral melanoma cell lines from tumors isolated from dog patients with distinct clinical profiles; with and without lung metastases. The cells were characterized using immunohistochemistry, pharmacology and genetic studies. RESULTS: We have developed and immunohistochemically, genetically, and pharmacologically characterized. Two cell lines (Ocr_OCMM1X & Ocr_OCMM2X) were produced through mouse xenografts originating from two clinically contrasting melanomas of the oral cavity. Their exhaustive characterization showed two distinct biological and genetic profiles that are potentially linked to the stage of malignancy at the time of diagnosis and sample collection of each melanoma case. These cell lines thus constitute relevant tools with which to perform genetic and drug screening analyses for a better understanding of mucosal melanomas in dogs and humans. CONCLUSIONS: The aim of this study was to establish and characterize xenograft-derived canine melanoma cell lines with different morphologies, genetic features and pharmacological sensitivities that constitute good predictive models for comparative oncology. These cell lines are relevant tools to advance the use of canine mucosal melanomas as natural models for the benefit of both veterinary and human medicine.

Optimization of Routine Testing for MET Exon 14 Splice Site Mutations in NSCLC Patients.

INTRODUCTION: Genomic alterations affecting splice sites of MNNG HOS transforming gene (MET) exon 14 were recently identified in NSCLC patients. Objective responses to MET tyrosine kinase inhibitors have been reported in these patients. Thus, detection of MET exon 14 splice site mutations represents a major challenge. So far, most of these alterations were found by full-exome sequencing or large capture-based next-generation sequencing (NGS) panels, which are not suitable for routine diagnosis. METHODS: Aiming to provide a molecular testing method applicable in routine practice, we first developed a fragment-length analysis for detecting deletions in introns flanking MET exon 14. Second, we designed an optimized targeted NGS panel called CLAPv1, covering the MET exon 14 and flanking regions in addition to the main molecular targets usually covered in genomic testing. In patients with MET exon 14 mutations, MET gene amplification, gene copy number and MET receptor expression were also determined. RESULTS: Among 1514 formalin-fixed paraffin-embedded NSCLC samples, nonoptimized NGS allowed detection of MET exon 14 mutations in only 0.3% of the patients, and fragment length analysis detected deletions in 1.1% of the patients. Combined, the optimized CLAPv1 panel and fragment-length analysis implemented for routine molecular testing revealed MET exon 14 alterations in 2.2% of 365 additional NSCLC patients. MET gene amplification or high gene copy number was observed in 6 of 30 patients (20%) harboring MET exon 14 mutations. CONCLUSIONS: These results show that optimized targeted NGS and fragment-length analysis improve detection of MET alterations in routine practice.

Sub-clonal analysis of the murine C1498 acute myeloid leukaemia cell line reveals genomic and immunogenic diversity.

BACKGROUND: In acute myeloid leukaemia (AML)-affected patients, the presence of heterogeneous sub-clones at diagnosis has been shown to be responsible for minimal residual disease and relapses. The role played by the immune system in this leukaemic sub-clonal hierarchy and maintenance remains unknown. As leukaemic sub-clone immunogenicity could not be evaluated in human AML xenograft models, we assessed the sub-clonal diversity of the murine C1498 AML cell line and the immunogenicity of its sub-clones in immune-competent syngeneic mice. METHODOLOGY: The murine C1498 cell line was cultured in vitro and sub-clonal cells were generated after limiting dilution. The genomic profiles of 6 different sub-clones were analysed by comparative genomic hybridization arrays (CGH). The sub-clones were then injected into immune-deficient and - competent syngeneic mice. The immunogenicities of the sub-clones was evaluated through 1) assessment of mouse survival, 2) determination of leukaemic cell infiltration into organs by flow cytometry and the expression of a fluorescent reporter gene, 3) assessment of the CTL response ex vivo and 4) detection of residual leukaemic cells in the organs via amplification of the genomic reporter gene by real-time PCR (qPCR). RESULTS: Genomic analyses revealed heterogeneity among the parental cell line and its derived sub-clones. When injected individually into immune-deficient mice, all sub-clones induced cases of AML with different kinetics. However, when administered into immune-competent animals, some sub-clones triggered AML in which no mice survived, whereas others elicited reduced lethality rates. The AML-surviving mice presented efficient anti-leukaemia CTL activity ex vivo and eliminated the leukaemic cells in vivo. CONCLUSION: We showed that C1498 cell sub-clones presented genomic heterogeneity and differential immunogenicity resulting either in immune escape or elimination. Such findings could have potent implications for new immunotherapeutic strategies in patients with AML.

BAP1 Is Altered by Copy Number Loss, Mutation, and/or Loss of Protein Expression in More Than 70% of Malignant Peritoneal Mesotheliomas.

INTRODUCTION: Malignant mesothelioma is a deadly disease that is strongly associated with asbestos exposure. Peritoneal mesotheliomas account for 10% of all the cases. BRCA1 associated protein 1 (BAP1) is a deubiquitinating hydrolase that plays a key role in various cellular processes. Germline and somatic inactivation of BRCA1 associated protein 1 gene (BAP1) is frequent in pleural mesothelioma; however, little is known about its status in peritoneal mesothelioma. METHODS: Taking advantage of the extensive French National Network for the Diagnosis of Malignant Pleural Mesothelioma and Rare Peritoneal Tumors and the French National Network for the Treatment of Rare Peritoneal Surface Malignancies, we collected biological material and clinical and epidemiological data for 46 patients with peritoneal mesothelioma. The status of BAP1 was evaluated at the mutational and protein expression levels and combined with our previous data on copy number alterations assessed in the same samples. RESULTS: We detected mutations in 32% of the malignant peritoneal mesotheliomas analyzed. In addition, we have previously reported that copy number losses occurred in 42% of the samples included in this series. Overall, 73% of the malignant peritoneal mesotheliomas analyzed carried at least one inactivated BAP1 allele, but only 57% had a complete loss of its protein nuclear expression. Better overall survival was observed for patients with BAP1 mutations (p = 0.04), protein expression loss (p = 0.016), or at least one of these alterations (p = 0.007) independently of tumor histological subtype, age, and sex. CONCLUSIONS: As in pleural mesothelioma, inactivation of BAP1 is frequent in peritoneal mesotheliomas. We found that BAP1 protein nuclear expression is a good prognostic factor and a more reliable marker for the complete loss of BAP1 activity than mutation or copy number loss.

RNA-binding disturbances as a continuum from spinocerebellar ataxia type 2 to Parkinson disease.

CAG triplet expansions in Ataxin-2 gene (ATXN2) cause spinocerebellar ataxia type 2 and have a role that remains to be clarified in Parkinson's disease (PD). To study the molecular events associated with these expansions, we sequenced them and analyzed the transcriptome from blood cells of controls and three patient groups diagnosed with spinocerebellar ataxia type 2 (herein referred to as SCA2c) or PD with or without ATXN2 triplet expansions (named SCA2p). The transcriptome profiles of these 40 patients revealed three main observations: i) a specific pattern of pathways related to cellular contacts, proliferation and differentiation associated with SCA2p group, ii) similarities between the SCA2p and sporadic PD groups in genes and pathways known to be altered in PD such as Wnt, Ephrin and Leukocyte extravasation signaling iii) RNA metabolism disturbances with "RNA-binding" and "poly(A) RNA-binding" as a common feature in all groups. Remarkably, disturbances of ALS signaling were shared between SCA2p and sporadic PD suggesting common molecular dysfunctions in PD and ALS including CACNA1, hnRNP, DDX and PABPC gene family perturbations. Interestingly, the transcriptome profiles of patients with parkinsonian phenotypes were prevalently associated with alterations of translation while SCA2c and PD patients presented perturbations of splicing. While ATXN2 RNA expression was not perturbed, its protein expression in immortalized lymphoblastoid cells was significantly decreased in SCA2c and SCA2p versus control groups assuming post-transcriptional biological perturbations. In conclusion, the transcriptome data do not exclude the role of ATXN2 mutated alleles in PD but its decrease protein expression in both SCA2c and SCA2p patients suggest a potential involvement of this gene in PD. The perturbations of "RNA-binding" and "poly(A) RNA-binding" molecular functions in the three patient groups as well as gene deregulations of factors not yet described in PD but known to be deleterious in other neurological conditions, suggest the existence of RNA-binding disturbances as a continuum between spinocerebellar ataxia type 2 and Parkinson's disease.

Polymorphisms in the Mannose-Binding Lectin Gene are Associated with Defective Mannose-Binding Lectin Functional Activity in Crohn's Disease Patients.

Mannose-binding lectin, together with mannose-associated serine proteases, activates the lectin pathway of the complement system and subsequent inflammatory mechanisms. An association between mannose-binding lectin deficiency and anti-Saccharomyces cerevisiae antibody levels is observed in Crohn's disease and this deficiency is frequently associated with a severe Crohn's disease phenotype. In the present study, we assessed the relationship between serum concentrations of mannose-binding lectin, mannose-binding lectin functional activity, MBL2 and NOD2 polymorphisms, anti-S. cerevisiae antibody levels and clinical Crohn's disease phenotype in 69 Crohn's disease patients and 30 age- and sex-matched healthy controls. The results show that the MBL2 variant rs5030737 at codon 52 was associated with a low level of mannose-binding lectin and impaired mannose-binding lectin-mannose-associated serine protease (MBL-MASP) functional activity in Crohn's disease patients. This MBL2 variant was also associated with a higher level of anti-S. cerevisiae antibodies. In addition, the NOD2 variant rs2066844, which is associated with susceptibility to Crohn's disease, was significantly correlated with an impairment in MBL-MASP functional activity. These results provide evidence that Crohn's disease patients have an impairment in MBL-MASP functional activity and that this defect is associated with MBL2 and NOD2 variants.

Genomic copy number alterations in 33 malignant peritoneal mesothelioma analyzed by comparative genomic hybridization array.

Malignant peritoneal mesotheliomas (MPM) are rare, accounting for approximately 8% of cases of mesothelioma in France. We performed comparative genomic hybridization (CGH) on frozen MPM samples using the Agilent Human Genome CGH 180 K array. Samples were taken from a total of 33 French patients, comprising 20 men and 13 women with a mean (range) age of 58.4 (17-76) years. Asbestos exposure was reported in 8 patients (24.2%). Median (range) overall survival (OS) was 39 (0-119) months. CGH analysis demonstrated the presence of chromosomal instability in patients with MPM, with a genomic pattern that was similar to that described for pleural mesothelioma, including the loss of chromosomal regions 3p21, 9p21, and 22q12. In addition, novel genomic copy number alterations were identified, including the 15q26.2 region and the 8p11.22 region. Median OS was associated with a low peritoneal cancer index (P=.011), epithelioid subtype (P=.038), and a low number of genomic aberrations (P=.015), all of which constitute good prognostic factors for MPM. Our results provide new insights into the genetic and genomic background of MPM. Although pleural and peritoneal mesotheliomas have different risk factors, different therapeutics, and different prognosis; these data provide support to combine pleural and peritoneal mesothelioma in same clinical assays.