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Non-autonomous thyroid nodules are common in the general population with a proportion found to be cancerous. A current challenge in the field is to be able to distinguish benign adenoma (FA) from preoperatively malignant thyroid follicular carcinoma (FTC), which are very similar both histologically and genetically. One controversial issue, which is currently not understood, is whether both tumor types represent different molecular entities or rather a biological continuum. To gain a better insight into FA and FTC tumorigenesis, we defined their molecular profiles by mRNA and miRNA microarray. Expression data were analyzed, validated by qRT-PCR and compared with previously published data sets. The majority of deregulated mRNAs were common between FA and FTC and were downregulated, however FTC showed additional deregulated mRNA. Both types of tumors share deregulated pathways, molecular functions and biological processes. The additional deregulations in FTC include the lipid transport process that may be involved in tumor progression. The strongest candidate genes which may be able to discriminate follicular adenomas and carcinomas, CRABP1, FABP4 and HMGA2, were validated in independent samples by qRT-PCR and immunohistochemistry. However, they were not able to adequately classify FA or FTC, supporting the notion of continuous evolving tumors, whereby FA and FTC appear to show quantitative rather than qualitative changes. Conversely, miRNA expression profiles showed few dysregulations in FTC, and even fewer in FA, suggesting that miRNA play a minor, if any, role in tumor progression.

Non-autonomous thyroid nodules are common in the general population with a proportion found to be cancerous. A current challenge in the field is to be able to distinguish benign adenoma (FA) from preoperatively malignant thyroid follicular carcinoma (FTC), which are very similar both histologically and genetically. One controversial issue, which is currently not understood, is whether both tumor types represent different molecular entities or rather a biological continuum. To gain a better insight into FA and FTC tumorigenesis, we defined their molecular profiles by mRNA and miRNA microarray. Expression data were analyzed, validated by qRT-PCR and compared with previously published data sets. The majority of deregulated mRNAs were common between FA and FTC and were downregulated, however FTC showed additional deregulated mRNA. Both types of tumors share deregulated pathways, molecular functions and biological processes. The additional deregulations in FTC include the lipid transport process that may be involved in tumor progression. The strongest candidate genes which may be able to discriminate follicular adenomas and carcinomas, CRABP1, FABP4 and HMGA2, were validated in independent samples by qRT-PCR and immunohistochemistry. However, they were not able to adequately classify FA or FTC, supporting the notion of continuous evolving tumors, whereby FA and FTC appear to show quantitative rather than qualitative changes. Conversely, miRNA expression profiles showed few dysregulations in FTC, and even fewer in FA, suggesting that miRNA play a minor, if any, role in tumor progression., SCOPUS: ar.j, info:eu-repo/semantics/published

Tryptophan catabolism by indoleamine 2,3-dioxygenase 1 (IDO1) plays a key role in tumoral resistance to immune rejection. In humans, constitutive expression of IDO1 has been observed in several tumor types. However, a comprehensive analysis of its expression in normal and tumor tissues is still required to anticipate the risks and potential benefits of IDO1 inhibitors. Using a newly validated monoclonal antibody to human IDO1, we performed an extensive immunohistochemical analysis of IDO1 expression in normal and tumor tissues. In normal tissues, IDO1 was expressed by endothelial cells in the placenta and lung and by epithelial cells in the female genital tract. In lymphoid tissues, IDO1 was expressed in mature dendritic cells with a phenotype (CD83+, DC-LAMP+, langerin-, CD123-, CD163-) distinct from plasmacytoid dendritic cells. Importantly, IDO1-expressing dendritic cells were not enriched in tumor-draining lymph nodes, in contrast with previously reported findings. IDO1-expressing cells were observed in a large fraction (505/866, 58%) of human tumors. They comprised tumor cells, endothelial cells, and stromal cells in proportions that varied depending on the tumor type. Tumors showing the highest proportions of IDO1-immunolabeled samples were carcinomas of the endometrium and cervix, followed by kidney, lung, and colon. This hierarchy of IDO1 expression was confirmed by gene expression data mined from The Cancer Genome Atlas database. Expression of IDO1 may be used to select tumors likely to benefit from targeted therapy with IDO1 inhibitors.

Objective Endometrial carcinoma (EC) is the sixth most common cancer in women and therapies are limited for advanced and recurrent disease. Patient-derived tumor xenograft (PDTX) models are becoming popular tools in translational research because of their histological and genetic similarity to the original tumors and the ability to predict therapeutic response to treatments. Here, we established and characterized a panel of 24 EC PDTX models which includes the major histological and genetic subtypes observed in patients. Methods Fresh tumor tissues collected from primary, metastatic and recurrent type I and type II EC patients were engrafted in immunocompromised mice. Histology, vimentin, and cytokeratin expression were evaluated, together with Microsatellite instability (MSI), mutation profiling by Whole Exome Sequencing and copy number profiling by Whole Genome Low Coverage Sequencing. The efficacy of both PI3K and MEK inhibitors was evaluated in a model of endometrioid carcinoma harboring PTEN, PIK3CA and KRAS mutations. Results We observed good similarity between primary tumors and the corresponding xenografts, at histological and genetic level. Among the engrafted endometrioid models, we found a significant enrichment of MSI and POLE mutated tumors, compared to non-engrafted samples. Combination treatment with NVP-BEZ235 and AZD6244 showed the possibility to stabilize the tumor growth in one model originated from a patient who already received several lines of chemotherapy. Conclusion The established EC PDTX models, resembling the original human tumors, promise to be useful for preclinical evaluation of novel combination and targeted therapies in specific EC subgroups

Background/Purpose: Toll-like Receptor (TLR)3 is an endosomal TLR that binds double-stranded viral RNA and endogenous double-stranded RNA-like structures. Recently, it has been reported that UV irradiation causes changes in self noncoding RNAs, which induce TLR3-dependent inflammatory responses resulting in solar injury. Since the TLR3 gene is located in 4q35, a chromosomic region associated with susceptibility to systemic lupus erythematosus (SLE) in patients with anti-Ro/SSA antibodies (Ab), and since photosensitivity is a major clinical symptom in patients carrying these autoantibodies, we investigated the role of TLR3 in the pathogenesis of SLE. Methods: The variations in the genomic sequence of TLR3 were first explored in a population of Western-European SLE patients versus age- and gender-matched healthy controls, using 8 Tag SNP from the HapMap database. In addition, the distribution of TLR3 rs3775291 alleles and genotypes were assessed in two independent populations of Western- and Southern-European SLE patients (n = 197 and 282, respectively) and controls (n = 262 and 291, respectively). Functional experiments were performed on peripheral blood mononuclear cells (PBMC) and monocyte-derived dendritic cells (moDC) generated from PBMC of healthy individuals categorized according to their TLR3 rs3775291 genotype. Anti-Ro/SSA-specific T cells were derived from PBMC of healthy individuals by repeated stimulations with autologous Ro/SSA-pulsed moDC. Results: Out of the 8 TLR3 Tag SNP, 2 are significantly associated with SLE in Western-European anti-Ro/SSA Ab-positive patients. None of these intronic SNP have a functional impact. However, one of them is in linkage disequilibrium with rs3775291, a SNP that encodes an amino-acid substitution in the ligand-binding pocket of TLR3. We found a positive association between rs3775291 and susceptibility to SLE in patients with anti-Ro/SSA Ab, in two independent populations of SLE patients and controls. We confirmed that the

Rituximab has been shown to be successful in the treatment of rheumatoid arthritis (RA), and this unexpected finding indicates that B cells have an important role in this disease. The present study was undertaken to investigate the mechanism of action of rituximab in RA.

OBJECTIVE: Mutations in genes encoding succinate dehydrogenase and its anchoring subunits (SDH genes) are at the origin of hereditary head and neck paraganglioma (PGL) and a subset of apparently sporadic pheochromocytoma. METHODS: We describe a family including three patients harbouring bilateral head and neck PGL diagnosed before 25 years of age. Multiple hypervascular hepatic lesions were subsequently discovered in two of them. In both, liver biopsy confirmed the diagnosis of PGL. In addition, in one patient, MRI disclosed multiple target-like lesions of the spine, highly suggestive of metastatic PGL. Family history was compatible with autosomal dominant inheritance with possible maternal imprinting. RESULTS: Combined single-strand conformation polymorphism and heteroduplex analysis followed by sequencing did not show any mutation of the coding parts of SDHB, SDHC, SDHD, RET or VHL genes. Screening of copy number alterations and loss of heterozygosity in the three affected family members showed no deletion or amplification of the SDH, RET and VHL genes. Furthermore, succinate dehydrogenase activity measured in a liver PGL sample was not significantly decreased in the affected patient as compared with controls, underscoring the exclusion of the SDH genes. CONCLUSIONS: To our knowledge, this is the first reported family of hereditary head and neck PGL with metastatic dissemination in the liver and the spine. A large body of evidence supports the absence of mutations in SDH, RET and VHL genes, which suggests the existence of a yet unknown gene at the origin of this particular form of familial PGL.

Susceptibility to autoimmune disorders results from the interaction of multiple genetic factors that regulate the threshold of autoreactivity. Genome-wide microsatellite screens and large-scale single nucleotide polymorphism (SNP) association studies have identified chromosomal loci that are associated with specific disorders including systemic lupus erythematosus, rheumatoid arthritis, juvenile arthritis, multiple sclerosis, and diabetes. Numerous candidate gene association studies have in turn investigated the association of specific genes within these chromosomal regions, with susceptibility to autoimmune diseases (e.g. FcgammaReceptors, TYK2 and systemic lupus). More recently, large-scale differential gene expression studies performed on selected tissues from patients with autoimmune disorders, have led to the identification of gene signatures associated with the activation of specific pathways in these diseases (e.g. interferon signature in lupus). In the future, integrated analyses of gene (and protein) expression together with SNP data will allow us to sketch an intelligible picture of the genesis of autoimmunity in humans. This review sets out to illustrate how the most recent advances in the field of systemic lupus erythematosus, rheumatoid arthritis and juvenile arthritis have led to a better understanding of these disorders.

Atrial septal defect (ASD) is one of the most frequent congenital heart defects (CHDs) with a variable phenotypic effect depending on the size of the septal shunt. We identified two pedigrees comprising 20 members segregating isolated autosomal dominant secundum ASD. By genetic mapping, we identified the gene-encoding alpha-cardiac actin (ACTC1), which is essential for cardiac contraction, as the likely candidate. A mutation screen of the coding regions of ACTC1 revealed a founder mutation predicting an M123V substitution in affected individuals of both pedigrees. Functional analysis of ACTC1 with an M123V substitution shows a reduced affinity for myosin, but with retained actomyosin motor properties. We also screened 408 sporadic patients with CHDs and identified a case with ASD and a 17-bp deletion in ACTC1 predicting a non-functional protein. Morpholino (MO) knockdown of ACTC1 in chick embryos produces delayed looping and reduced atrial septa, supporting a developmental role for this protein. The combined results indicate, for the first time, that ACTC1 mutations or reduced ACTC1 levels may lead to ASD without signs of cardiomyopathy.

The prevalence of congenital heart defects is approximately 1% of all births, yet the causative factors remain largely uncharacterized. For the majority, the physiopathogenesis is believed to be multifactorial, hindering the identification of causative factors. However, several genes have been identified for septation defects that are part of a syndrome. Yet, in non-syndromic septal defects it has been difficult to identify predisposing genetic factors. When I started this thesis project, only one gene had been identified to be responsible for non-syndromic septal defects. We collected families in which two or more individuals were affected with non-syndromic cardiac septal defects. In five families, arrhythmia was associated with ASD/VSD. We screened the CSX/NKX2-5 gene, previously identified to be responsible for ASD and PR prolongation, and identified 3 novel missense mutations. In parallel, we screened the CSX/NKX2-5 gene in sporadic and familial cases of other cardiopathies, but additional mutations were not found. Substitutions in this gene seem to be a rare cause of cardiopathies without conduction defect. The absence of CSX/NKX2-5 mutations in two families suggest locus heterogeneity. We also examined whether the VEGF gene, which modifies the cardiac phenotype in del22q11 patients, could be responsible for the phenotypic variability observed in the three CSX/NKX2-5 mutated families. No statistically significant association was observed. We also evaluated the role of five candidate genes (GATA4, FOG2, CRELD1, HEY2 and BMP4) in a series of 66 patients affected with structural cardiac malformations, especially septal defects. Twenty nine nucleotide changes were identified. Based on their presence on dbSNP, dbEST and their position in multiple alignements, they were not considered to be mutations. However, we cannot exclude that some of them have an effect on RNA stability or abnormal splicing. We conclude that none of these five genes is a major cause of struct, Les anomalies cardiaques ont une fréquence de 1% chez les nouveaux-nés. Leurs causes peuvent être multiples, mais pour la plupart l'étiologie n'a pas encore été élucidée. Cependant plusieurs gènes ont déjà été identifiés dans les défauts de septation non-syndromiques. En ce qui concerne les anomalies touchant la septation et ne faisant pas partie d'un syndrome, très peu est encore connu. Lorsque j'ai commencé mon travail de thèse, un seul gène était impliqué dans les défauts de septation non-syndromiques. Parmi les familles collectées, cinq familles présentant des défauts de septation associés à des troubles de conduction nous ont particulièrement intéressés. Nous avons criblé le gène CSX/NKX2-5, dont des mutations avaient été décrites auparavant comme étant responsables de CIA associées à des prolongements de l'intervalle PR. Ceci nous a permis d'identifier trois nouvelles mutations dans le gène CSX/NKX2-5. En parallèle, nous avons étudié la fréquence des mutations dans ce gène parmi des patients sporadiques et des cas familiaux atteints de diverses cardiopathies. Comme aucune nouvelle mutation n'a été identifiée, ce gène ne semble donc pas être fréquemment impliqué dans les cardiopathies non associées à des troubles de conduction. L'absence de mutations dans les deux autres familles suggère une hétérogénéité de locus. Nous avons de plus étudié si le gène VEGF, qui modifie le phénotype cardiaque chez les patients atteints du syndrome de DiGeorge, pouvait être responsable de la variabilité phénotypique observée dans les trois familles ayant des mutations dans le gène CSX/NKX2-5. Aucune association significative n'a été observée. Nous avons aussi évalué l'implication de cinq gènes candidats (GATA4, FOG2, CRELD1, HEY2 et BMP4) dans une série de 66 patients atteints de malformations cardiaques structurelles, constituées en grande partie par des défauts de septation. Vingt neuf changements nucléotidiques ont été identifiés. Aucun de ces changements nucléotidiques n'a ét, (SBIM 3)--UCL, 2005

Mutations in the glomulin gene result in dominantly inherited vascular lesions of the skin known as glomuvenous malformations (GVMs). These lesions are histologically distinguished by their distended vein-like channels containing characteristic 'glomus cells', which appear to be incompletely or improperly differentiated vascular smooth muscle cells (VSMCs). The function of glomulin is currently unknown. We studied glomulin expression during murine development (E9.5 days post-coitum until adulthood) by non-radioactive in situ hybridization. Glomulin was first detected at E10.5 dpc in cardiac outflow tracts. Later, it showed strong expression in VSMCs as well as a limited expression in the perichondrium. At E11.5-14.5 dpc glomulin RNA was most abundant in the walls of the large vessels. At E16.5 dpc expression was also detectable in smaller arteries and veins. The high expression of glomulin in murine vasculature suggests an important role for glomulin in blood vessel development and/or maintenance, which is supported by the vascular phenotype seen in GVM patients with mutations in this gene.

We have identified an antigen recognized by autologous CTL on the lung carcinoma cells of a patient who enjoyed a favorable clinical evolution, being alive 10 years after partial resection of the primary tumor. The antigenic peptide is presented by HLA-A2 molecules and encoded by a mutated sequence in the gene coding for malic enzyme, an essential enzyme that converts malate to pyruvate, In the tumor cell lint derived from the patient, only the mutated malic enzyme allele is expressed, because of a loss of heterozygosity in the region of chromosome 6 that contains this locus, Tetramers of soluble HLA-A2 molecules loaded with the antigenic peptide stained similar to0.4% of the patient's blood CD8 T cells. When these cells were stimulated in clonal conditions, 25% of them proliferated, and the resulting clones were lytic and specific for the mutated malic enzyme peptide, T-cell receptor analysis indicated that almost all of these antimalic CTLs shared the same receptor. Antimalic T cells Here consistently found in blood samples collected from the patient between 1990 and 1999, at frequencies ranging from 0.1 to 0.4% of the CD8 cells. Their frequency appeared to double within 2 weeks after intradermal inoculation of lethally irradiated autologous tumor cells. These results indicate that nonmelanoma cancer patients may also have a high frequency of blood CTLs directed against a tumor-specific antigen.