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PDF file 97K, List of the 89 Wnt pathway selected genes

Supplementary Figure Legends 1-3 from Microarray-Based Identification of Tenascin C and Tenascin XB, Genes Possibly Involved in Tumorigenesis Associated with Neurofibromatosis Type 1

PDF file 161K, A simplified representation of the three different Wnt signalling pathways: the canonical pathway, the planar cell polarity pathway, and the Wnt/calcium pathway. In the absence of Wnt ligand, the 'destruction complex' composed of the core proteins Axin, adenomatous polyposis coli (APC), and glycogen synthase kinase-3 (GSK3) rapidly phosphorylates cytosolic beta-catenin, targeting it for subsequent proteasome-mediated destruction. Binding of Wnt to Frizzled (FZD) and low-density lipoprotein receptor-related protein 5/6 (LRP5/6) activates the cytosolic protein Dishevelled (DVL), leading to inhibition of the destruction complex. The resulting accumulated beta-catenin can then translocate to the nucleus to activate Wnt-responsive target genes regulated by lymphoid enhancer factor (LEF) and T cell factor (TCF) family transcription factors, leading to various cellular effects. The secreted inhibitor Dickkopf (DKK) can antagonize Wnt signalling by competitively binding to LRP5/6. Secreted FZD-related proteins (SFRPs) and Wnt inhibitory factor (WIF) are thought to antagonize Wnt signalling by sequestering Wnt ligand in the extracellular space. Binding of Wnt isoforms to FZD can trigger beta-catenin-independent downstream signalling events, other so-called non-canonical Wnt pathways that do not require the transcriptional activity of beta-catenin. One branch of non-canonical pathways involves the activation of RHO and RAC small G proteins to regulate the actin cytoskeleton. DVL-associated activator of morphogenesis 1 (DAAM1), when complexed with DVL and RHO, acts through the regulation of RHO-associated protein kinases (ROCK) and the DVL-RAC GTPase complex to affect actin remodelling. Another branch, when activated, is defined by a phospholipase C (PLC)-mediated increase in intracellular Ca2+ levels and Ca2+ fluxes that lead to the activation of Ca2+/calmodulin-dependent protein kinase (CaMK), protein kinase C (PKC), and nuclear factor of activated T cells (NFAT)

Purpose: The hallmark of neurofibromatosis type 1 (NF1) is the onset of dermal or plexiform neurofibromas, mainly composed of Schwann cells. Plexiform neurofibromas can transform into malignant peripheral nerve sheath tumors (MPNST) that are resistant to therapies.Experimental Design: The aim of this study was to identify an additional pathway in the NF1 tumorigenesis. We focused our work on Wnt signaling that is highly implicated in cancer, mainly in regulating the proliferation of cancer stem cells. We quantified mRNAs of 89 Wnt pathway genes in 57 NF1-associated tumors including dermal and plexiform neurofibromas and MPNSTs. Expression of two major stem cell marker genes and five major epithelial–mesenchymal transition marker genes was also assessed. The expression of significantly deregulated Wnt genes was then studied in normal human Schwann cells, fibroblasts, endothelial cells, and mast cells and in seven MPNST cell lines.Results: The expression of nine Wnt genes was significantly deregulated in plexiform neurofibromas in comparison with dermal neurofibromas. Twenty Wnt genes showed altered expression in MPNST biopsies and cell lines. Immunohistochemical studies confirmed the Wnt pathway activation in NF1-associated MPNSTs. We then confirmed that the knockdown of NF1 in Schwann cells but not in epithelial cells provoked the activation of Wnt pathway by functional transfection assays. Furthermore, we showed that the protein expression of active β-catenin was increased in NF1-silenced cell lines. Wnt pathway activation was strongly associated to both cancer stem cell reservoir and Schwann–mesenchymal transition.Conclusion: We highlighted the implication of Wnt pathway in NF1-associated tumorigenesis. Clin Cancer Res; 20(2); 358–71. ©2013 AACR.

Supplementary Tables 1-4 from Microarray-Based Identification of Tenascin C and Tenascin XB, Genes Possibly Involved in Tumorigenesis Associated with Neurofibromatosis Type 1

Purpose: Neurofibromatosis type 1 (NF1) is an autosomal dominant disorder with a complex variety of clinical manifestations. The hallmark of NF1 is the onset of heterogeneous (dermal or plexiform) benign neurofibromas. Plexiform neurofibromas can give rise to malignant peripheral nerve sheath tumors, which are resistant to conventional therapies.Experimental Design: To identify new signaling pathways involved in the malignant transformation of plexiform neurofibromas, we applied a 22,000-oligonucleotide microarray approach to a series of plexiform neurofibromas and malignant peripheral nerve sheath tumors. Changes in the expression of selected genes were then confirmed by real-time quantitative reverse transcription-PCR.Results: We identified two tenascin gene family members that were significantly deregulated in both human NF1-associated tumors and NF1-deficient primary cells: Tenascin C (TNC) was up-regulated whereas tenascin XB (TNXB) was down-regulated during tumor progression. TNC activation is mainly due to the up-regulation of large TNC splice variants. Immunohistochemical studies showed that TNC transcripts are translated into TNC protein in TNC-overexpressing tumors. Aberrant transcriptional activation of TNC seems to be principally mediated by activator protein transcription factor complexes.Conclusion:TNXB and TNC may be involved in the malignant transformation of plexiform neurofibromas. Anti-TNC antibodies, already used successfully in clinical trials to treat malignant human gliomas, may be an appropriate new therapeutic strategy for NF1.

Supplementary Figures 1-3 from Microarray-Based Identification of Tenascin C and Tenascin XB, Genes Possibly Involved in Tumorigenesis Associated with Neurofibromatosis Type 1

Supplementary Figure 1 from Characterization of a Germ-Line Deletion, Including the Entire INK4/ARF Locus, in a Melanoma-Neural System Tumor Family: Identification of ANRIL, an Antisense Noncoding RNA Whose Expression Coclusters with ARF

We have previously detected a large germ-line deletion, which included the entire p15/CDKN2B-p16/CDKN2A-p14/ARF gene cluster, in the largest melanoma-neural system tumor (NST) syndrome family known to date by means of heterozygosity mapping based on microsatellite markers. Here, we used gene dose mapping with sequence-tagged site real-time PCR to locate the deletion end points, which were then precisely characterized by means of long-range PCR and nucleotide sequencing. The deletion was exactly 403,231 bp long and included the entire p15/CDKN2B, p16/CDKN2A, and p14/ARF genes. We then developed a simple and rapid assay to detect the junction fragment and to serve as a direct predictive DNA test for this large French family. We identified a new large antisense noncoding RNA (named ANRIL) within the 403-kb germ-line deletion, with a first exon located in the promoter of the p14/ARF gene and overlapping the two exons of p15/CDKN2B. Expression of ANRIL mainly coclustered with p14/ARF both in physiologic (various normal human tissues) and in pathologic conditions (human breast tumors). This study points to the existence of a new gene within the p15/CDKN2B-p16/CDKN2A-p14/ARF locus putatively involved in melanoma-NST syndrome families and in melanoma-prone families with no identified p16/CDKN2A mutations as well as in somatic tumors. [Cancer Res 2007;67(8):3963–9]

Supplementary Figure 1 Legend from Characterization of a Germ-Line Deletion, Including the Entire INK4/ARF Locus, in a Melanoma-Neural System Tumor Family: Identification of ANRIL, an Antisense Noncoding RNA Whose Expression Coclusters with ARF

Neurofibromatosis type 1 (NF1) is a common autosomal dominant disorder. The role of angiogenesis and VEGF pathway in the pathogenesis of neurofibromas and malignant peripheral nerve sheath tumors (MPNSTs) remains poorly understood. We assessed the expression of VEGF and VEGFR family members in cohorts of plexiform neurofibromas (pNF), MPNSTs and MPNST cell lines at transcript [pNF, n = 49; MPNST, n = 34] and protein levels [pNF, n = 21; MPNST, n = 9]. VEGF and VEGFR members were variably expressed in cell lines. VEGFA (p = 3.10

Background Pediatric neurofibromatosis type 1 (NF1)–associated optic pathway gliomas (OPGs) exhibit different clinico-radiological features, treatment, and outcome compared with sporadic OPGs. While NF1-associated OPGs are caused by complete loss-of-function of the NF1 gene, other genetic alterations of the RAS-MAPK pathway are frequently described in the sporadic cases. We identified a group of patients who presented OPGs with typical radiological features of NF1-associated OPGs but without the NF1 diagnostic criteria. We aim to investigate into the possible molecular mechanisms underlying this “NF1-like” pediatric OPGs presentation. Methods We analyzed clinico-radiological features of 16 children with NF1-like OPGs and without NF1 diagnostic criteria. We performed targeted sequencing of the NF1 gene in constitutional samples (n = 16). The RAS-MAPK pathway major genes were sequenced in OPG tumor samples (n = 11); BRAF FISH and IHC analyses were also performed. Results In one patient’s blood and tumor samples, we identified a NF1 nonsense mutation (exon 50: c.7285C>T, p.Arg2429*) with ~8% and ~70% VAFs, respectively, suggesting a mosaic NF1 mutation limited to the brain (segmental NF1). This patient presented signs of neurodevelopmental disorder. We identified a somatic alteration of the RAS-MAPK pathway in eight tumors: four BRAF activating p.Val600Glu mutations, three BRAF:KIAA oncogenic fusions, and one putative gain-of-function complex KRAS indel inframe mutation. Conclusions NF1-like OPGs can rarely be associated with mosaic NF1 that needs specific constitutional DNA analyses for diagnosis. Further studies are warranted to explore unknown predisposition condition leading to the NF1-like OPG presentation, particularly in patients with the association of a neurodevelopmental disorder.

Simple Summary Neurofibromatosis type 1 (NF1) is a genetic disorder caused by pathogenic variants in the NF1 tumor suppressor gene. In 5–10% of NF1 patients, a large heterozygous deletion of the whole NF1 gene is identified, leading to the commonly called “NF1 microdeletion syndrome”. NF1-deleted patients were previously reported to develop a particularly severe form of the disease with frequent cognitive impairment and an increased risk of benign and malignant tumors. Here, we performed a comprehensive clinical assessment of the largest NF1-deleted cohort to date, including 126 NF1 patients with a deletion of the NF1 gene. This work provides new insights into the specific phenotype associated with NF1 deletions and may contribute to improve the follow-up care of NF1 patients. Abstract Complete deletion of the NF1 gene is identified in 5–10% of patients with neurofibromatosis type 1 (NF1). Several studies have previously described particularly severe forms of the disease in NF1 patients with deletion of the NF1 locus, but comprehensive descriptions of large cohorts are still missing to fully characterize this contiguous gene syndrome. NF1-deleted patients were enrolled and phenotypically characterized with a standardized questionnaire between 2005 and 2020 from a large French NF1 cohort. Statistical analyses for main NF1-associated symptoms were performed versus an NF1 reference population. A deletion of the NF1 gene was detected in 4% (139/3479) of molecularly confirmed NF1 index cases. The median age of the group at clinical investigations was 21 years old. A comprehensive clinical assessment showed that 93% (116/126) of NF1-deleted patients fulfilled the NIH criteria for NF1. More than half had café-au-lait spots, skinfold freckling, Lisch nodules, neurofibromas, neurological abnormalities, and cognitive impairment or learning disabilities. Comparison with previously described “classic” NF1 cohorts showed a significantly higher proportion of symptomatic spinal neurofibromas, dysmorphism, learning disabilities, malignancies, and skeletal and cardiovascular abnormalities in the NF1-deleted group. We described the largest NF1-deleted cohort to date and clarified the more severe phenotype observed in these patients.

Introduction Bien que la neurofibromatose de type 1 (NF1) soit une maladie mendelienne a penetrance complete, son expression est extremement variable y compris au sein d’une meme famille. Le role mineur du gene NF1 lui-meme a ete confirme par l’absence de correlation genotype-phenotype, excepte pour quelques rares mutations. La preuve de l’implication de modificateurs genetiques dans la variabilite d’expression de la NF1 a ete apportee par les modeles animaux et les correlations entre apparentes. Nous presentons ici les resultats de la premiere etude d’association genome entier (GWAS) pour la NF1, realisee en collaboration avec le reseau NF-France. Materiel et methodes Une cohorte francaise constituee de 2002 a 2013 a inclus plus de 1500 patients atteints de NF1 et a ete genotypee avec la puce Illumina OmniExpressExome. La caracterisation de la mutation NF1 dans l’ensemble de la cohorte a permis l’exclusion de patients porteurs de mutations associees a une correlation genotype-phenotype connue. Apres controle qualite des donnees genetiques et phenotypiques et imputation des genotypes manquants, ce sont les donnees de plus de 7 millions de variants chez 1333 patients (918 dans l’echantillon de decouverte (ED) et 415 dans l’echantillon de replication (ER)) qui ont ete inclus dans la GWAS. Les tests d’association avec les trois phenotypes cliniques majeurs de la NF1 (neurofibromes cutanes, sous-cutanes et plexiformes) ont ete realises avec un modele a effets mixtes, en considerant l’âge, le sexe, la nature heritee ou de novo de la mutation NF1 et le type de mutation NF1 (tronquante/faux-sens/entrainant une modification de l’epissage) comme effets fixes, et en integrant la matrice genomique d’apparentement pour corriger les biais dus a la presence d’une structure de population. Resultats Seul le trait des neurofibromes plexiformes presente un signal d’association depassant le seuil de signification statistique a l’echelle du genome (p Discussion Les regions genomiques a risque different entre les trois signes cliniques etudies, suggerant que les processus physiopathologiques a l’origine des differents types de neurofibromes sont gouvernes par des mecanismes specifiques a chacun.

Neurofibromatosis type 1 (NF1) is an autosomal dominant disease with complete penetrance but high variable expressivity. NF1 is caused by loss-of-function mutations in the NF1 gene, a negative regulator of the RAS-MAPK pathway. The NF1 gene has one of the highest mutation rates in human disorders, which may explain the outbreak of independent de novo variants in the same family. Here, we report the co-occurrence of pathogenic variants in the NF1 and SPRED1 genes in six families with NF1 and Legius syndrome, using next-generation sequencing. In five of these families, we observed the co-occurrence of two independent NF1 variants. All NF1 variants were classified as pathogenic, according to the American College of Medical Genetics and Genomics and the Association for Molecular Pathology (ACMG-AMP) guidelines. In the sixth family, one sibling inherited a complete deletion of the NF1 gene from her mother and carried a variant of unknown significance in the SPRED1 gene. This variant was also present in her brother, who was diagnosed with Legius syndrome, a differential diagnosis of NF1. This work illustrates the complexity of molecular diagnosis in a not-so-rare genetic disease.

Neurofibromatosis type 1 (NF1) is an autosomal dominant disease with complete penetrance but high variable expressivity. NF1 is caused by loss-of-function mutations in the NF1 gene, a negative regulator of the RAS-MAPK pathway. The NF1 gene has one of the highest mutation rates in human disorders, which may explain the outbreak of independent de novo variants in the same family. Here, we report the co-occurrence of pathogenic variants in the NF1 and SPRED1 genes in six families with NF1 and Legius syndrome, using next-generation sequencing. In five of these families, we observed the co-occurrence of two independent NF1 variants. All NF1 variants were classified as pathogenic, according to the American College of Medical Genetics and Genomics and the Association for Molecular Pathology (ACMG-AMP) guidelines. In the sixth family, one sibling inherited a complete deletion of the NF1 gene from her mother and carried a variant of unknown significance in the SPRED1 gene. This variant was also present in her brother, who was diagnosed with Legius syndrome, a differential diagnosis of NF1. This work illustrates the complexity of molecular diagnosis in a not-so-rare genetic disease.

Background Heterozygous NSD1 mutations were identified in 60%–90% of patients with Sotos syndrome. Recently, mutations of the SETD2 and DNMT3A genes were identified in patients exhibiting only some Sotos syndrome features. Both NSD1 and SETD2 genes encode epigenetic ‘writer’ proteins that catalyse methylation of histone 3 lysine 36 (H3K36me). The DNMT3A gene encodes an epigenetic ‘reader’ protein of the H3K36me chromatin mark. Methods We aimed at confirming the implication of DNMT3A and SETD2 mutations in an overgrowth phenotype, through a comprehensive targeted-next generation sequencing (NGS) screening in 210 well-phenotyped index cases with a Sotos-like phenotype and no NSD1 mutation, from a French cohort. Results Six unreported heterozygous likely pathogenic variants in DNMT3A were identified in seven patients: two nonsense variants and four de novo missense variants. One de novo unreported heterozygous frameshift variant was identified in SETD2 in one patient. All the four DNMT3A missense variants affected DNMT3A functional domains, suggesting a potential deleterious impact. DNMT3A -mutated index cases shared similar clinical features including overgrowth phenotype characterised by postnatal tall stature (≥+2SD), macrocephaly (≥+2SD), overweight or obesity at older age, intellectual deficiency and minor facial features. The phenotype associated with SETD2 mutations remains to be described more precisely. The p.Arg882Cys missense de novo constitutional DNMT3A variant found in two patients is the most frequent DNMT3A somatic mutation in acute leukaemia. Conclusions Our results illustrate the power of targeted NGS to identify rare disease-causing variants. These observations provided evidence for a unifying mechanism (disruption of apposition and reading of the epigenetic chromatin mark H3K36me) that causes an overgrowth syndrome phenotype. Further studies are needed in order to assess the role of SETD2 and DNMT3A in intellectual deficiency without overgrowth.

The tumor suppressor gene neurofibromin 1 (NF1) is a major regulator of the RAS‐MAPK pathway. NF1 mutations occur in lung cancer but were not extensively explored. We hypothesized that NF1‐mutated tumors could define a specific population with a distinct clinical and molecular profile. We performed NF1 sequencing using next generation sequencing (NGS) in 154 lung adenocarcinoma surgical specimens with known KRAS, EGFR, TP53, BRAF, HER2, and PIK3CA status, to evaluate the molecular and clinical specificities of NF1‐mutated lung cancers. Clinical data were retrospectively collected, and their associations with molecular profiles assessed. In this series, 24 tumors were NF1 mutated (17.5%) and 11 were NF1 deleted (8%). There was no mutation hotspot. NF1 mutations were rarely associated with other RAS‐MAPK pathway mutations. Most of patients with NF1 alterations were males (74.3%) and smokers (74.3%). Overall survival and disease‐free survival were statistically better in patients with NF1 alterations (N = 34) than in patients with KRAS mutations (N = 30) in univariate analysis. Our results confirm that NF1 is frequently mutated and represents a distinct molecular and clinical subtype of lung adenocarcinoma.

International audience; Aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor involved in xenobiotic metabolism. Plexiform neurofibromas (PNFs) can transform into malignant peripheral nerve sheath tumors (MPNSTs) that are resistant to existing therapies. These tumors are primarily composed of Schwann cells. In addition to neurofibroma-tosis type 1 (NF1) gene inactivation, further genetic lesions are required for malignant transformation. We have quantified the mRNA expression levels of AHR and its associated genes in 38 human samples. We report that AHR and the biosynthetic enzymes of its en-dogenous ligand are overexpressed in human biopsies of PNFs and MPNSTs. We also detect a strong nuclear AHR staining in MPNSTs. The inhibition of AHR by siRNA or antagonists, CH-223191 and tri-methoxyflavone, induces apoptosis in human MPNST cells. Since AHR dysregulation is observed in these tumors, we investigate AHR involvement in Schwann cell physiology. Hence, we studied the role of AHR in myelin structure and myelin gene regulation in Ahr −/− mice during myelin development. AHR ablation leads to lo-comotion defects and provokes thinner myelin sheaths around the axons. We observe a dysregulation of myelin gene expression and myelin developmental markers in Ahr −/− mice. Interestingly, AHR does not directly bind to myelin gene promoters. The inhibition of AHR in vitro and in vivo increased β-catenin levels and stimulated the binding of β-catenin on myelin gene promoters. Taken together, our findings reveal an endogenous role of AHR in peripheral myeli-nation and in peripheral nerve sheath tumors. Finally, we suggest a potential therapeutic approach by targeting AHR in nerve tumors. AHR | myelin | nerve | MPNST | neurofibroma

Introduction Les tumeurs malignes des gaines nerveuses peripheriques (TMGN) sont des sarcomes agressifs developpes a partir des neurofibromes (NF) profonds chez environ 10 % des patients presentant une neurofibromatose 1 (NF1). Il a ete suggere que l’angiogenese induite par le vascular endothelial growth factor (VEGF) via son recepteur VEGFR pourrait jouer un role cle dans le developpement de ces tumeurs. Materiel et methodes Nous avons etudie l’expression du VEGF et de son recepteur par immunohistochimie (VEGFA et VEGFR1) et RT-PCR quantitative (VEGF A/B/C/D et VEGFR1/2/3) dans les cellules tumorales (cellules de Schwann ou fibroblastiques) d’une serie de 13 TMGN en comparaison a 26 NF profonds (17 avec architecture plexiforme [NFP] et 19 avec architecture purement diffuse [NFD]) de patients NF1. La densite microvasculaire (nombre de capillaires sur 20 champs a ×400) et l’activation des cellules endotheliales (pourcentage de cellules endotheliales pAKT+ et pERK+ sur 10 capillaires) ont ete quantifiees sur coupes de tissus. Resultats L’etude immunohistochimique a montre une plus forte expression de VEGFR1 dans les TMGN et les NFP comparativement aux NFD (p = 0,05) et de VEGFA uniquement dans les TMGN (p = 0,01). L’etude en qRT-PCR a montre une surexpression des transcrits des isoformes du VEGF et du VEGFR dans les TMGN, en particulier de VEGFC (p = 0,03) et VEGFR2 (p Fig. 1 ). Discussion Les cellules tumorales des TMGN co-expriment des isoformes du VEGF et de leur recepteur VEGFR, ce qui avait deja ete rapporte dans l’etude de lignees cellulaires. Cette expression apparait plus importante que dans les NF mais s’accompagne paradoxalement d’une reduction de la densite microvasculaire. Cela suggere que l’axe VEGF-VEGFR pourrait jouer un role oncogene plus par activation autocrine ou paracrine des cellules tumorales, que par la stimulation de l’angiogenese dans l’environnement tumoral. Conclusion Ces donnees semblent confirmer l’hypothese d’une boucle autocrine impliquant les proteines de l’angiogenese dans le developpement des tumeurs et en particulier les TMGN chez les patients NF1, lesquelles pourraient etre de nouvelles cibles therapeutiques.

No treatment is available for early-onset forms of metachromatic leukodystrophy (MLD), a lysosomal storage disease caused by autosomal recessive defect in arylsulfatase A (ARSA) gene causing severe demyelination in central and peripheral nervous systems. We have developed a gene therapy approach, based on intracerebral administration of AAVrh.10-hARSA vector, coding for human ARSA enzyme. We have previously demonstrated potency of this approach in MLD mice lacking ARSA expression. We describe herein the preclinical efficacy, safety, and biodistribution profile of intracerebral administration of AAVrh.10-hARSA to nonhuman primates (NHPs). NHPs received either the dose planned for patients adjusted to the brain volume ratio between child and NHP (1×dose, 1.1×10(11) vg/hemisphere, unilateral or bilateral injection) or 5-fold this dose (5×dose, 5.5×10(11) vg/hemisphere, bilateral injection). NHPs were subjected to clinical, biological, and brain imaging observations and were euthanized 7 or 90 days after injection. There was no toxicity based on clinical and biological parameters, nor treatment-related histological findings in peripheral organs. A neuroinflammatory process correlating with brain MRI T2 hypersignals was observed in the brain 90 days after administration of the 5×dose, but was absent or minimal after administration of the 1×dose. Antibody response to AAVrh.10 and hARSA was detected, without correlation with brain lesions. After injection of the 1×dose, AAVrh.10-hARSA vector was detected in a large part of the injected hemisphere, while ARSA activity exceeded the normal endogenous activity level by 14-31%. Consistently with other reports, vector genome was detected in off-target organs such as liver, spleen, lymph nodes, or blood, but not in gonads. Importantly, AAVrh.10-hARSA vector was no longer detectable in urine at day 7. Our data demonstrate requisite safe and effective profile for intracerebral AAVrh.10-hARSA delivery in NHPs, supporting its clinical use in children affected with MLD.

Constitutional dominant loss-of-function mutations in the SPRED1 gene cause a rare phenotype referred as neurofibromatosis type 1 (NF1)-like syndrome or Legius syndrome, consisted of multiple café-au-lait macules, axillary freckling, learning disabilities and macrocephaly. SPRED1 is a negative regulator of the RAS MAPK pathway and can interact with neurofibromin, the NF1 gene product. Individuals with NF1 have a higher risk of haematological malignancies. SPRED1 is highly expressed in haematopoietic cells and negatively regulates haematopoiesis. SPRED1 seemed to be a good candidate for leukaemia predisposition or transformation. We performed SPRED1 mutation screening and expression status in 230 paediatric lymphoblastic and acute myeloblastic leukaemias (AMLs). We found a loss-of-function frameshift SPRED1 mutation in a patient with Legius syndrome. In this patient, the leukaemia blasts karyotype showed a SPRED1 loss of heterozygosity, confirming SPRED1 as a tumour suppressor. Our observation confirmed that acute leukaemias are rare complications of the Legius syndrome. Moreover, SPRED1 was significantly decreased at RNA and protein levels in the majority of AMLs at diagnosis compared with normal or paired complete remission bone marrows. SPRED1 decreased expression correlated with genetic features of AML. Our study reveals a new mechanism which contributes to deregulate RAS MAPK pathway in the vast majority of paediatric AMLs.

Background: Ex vivo colospheres have been previously characterised as a colorectal cancer (CRC) well-rounded multicellular model, exclusively formed by carcinoma cells, and derived from fresh CRC tissue after mechanical dissociation. The ability to form colospheres was correlated with tumour aggressiveness. Their three-dimensional conformation prompted us to further investigate their potential interest as a preclinical cancer tool. Methods: Patient-derived CRC xenografts were used to produce numerous colospheres. Mechanism of formation was elucidated by confocal microscopy. Expression analysis of a panel of 64 selected cancer-related genes by real-time qRT–PCR and hierarchical clustering allowed comparison of colospheres with parent xenografts. In vitro and in vivo assays were performed for migration and chemosensitivity studies. Results: Colospheres, formed by tissue remodelling and compaction, remained viable several weeks in floating conditions, escaping anoikis through their strong cell–cell interactions. Colospheres matched the gene expression profile of the parent xenograft tissue. Colosphere-forming cells migrated in collagen I matrix and metastasised when subrenally implanted in nude mice. Besides, the colosphere responses to 5-fluorouracil and irinotecan, two standard drugs in CRC, reproduced those of the in vivo original xenografts. Conclusion: Colospheres closely mimic biological characteristics of in vivo CRC tumours. Consequently, they would be relevant ex vivo CRC models.

The commonest tumors associated with neurofibromatosis type 1 (NF1) are benign peripheral nerve sheath tumors, called neurofibromas. Malignant transformation of neurofibromas into aggressive MPNSTs may occur with a poor patient prognosis. A cooperative role of SUZ12 or EED inactivation, along with NF1, TP53, and CDKN2A loss-of-function, has been proposed to drive progression to MPNSTs. An exome sequencing analysis of eight MPNSTs, one plexiform neurofibroma, and seven cutaneous neurofibromas was undertaken. Biallelic inactivation of the NF1 gene was observed in the plexiform neurofibroma and the MPNSTs, underlining that somatic biallelic NF1 inactivation is likely to be the initiating event for plexiform neurofibroma genesis, although it is unlikely to be sufficient for the subsequent MPNST development. The majority (5/8) of MPNSTs in our analyses demonstrated homozygous or heterozygous deletions of CDKN2A, which may represent an early event following NF1 LOH in the malignant transformation of Schwann cells from plexiform neurofibroma to MPNST. Biallelic somatic alterations of SUZ12 was also found in 4/8 MPNSTs. EED biallelic alterations were detected in 2 of the other four MPNSTs, with one tumor having a homozygous EED deletion. A missense mutation in the chromatin regulator KDM2B was also identified in one MPNST. No TP53 point mutations were found in this study, confirming previous data that TP53 mutations may be relatively rare in NF1-associated MPNSTs. Our study confirms the frequent biallelic inactivation of PRC2 subunits SUZ12 and EED in MPNSTs, and suggests the implication of KDM2B.

International audience; Approximately 30-50% of individuals with Neurofibromatosis type 1 develop benign peripheral nerve sheath tumors, called plexiform neurofibromas (PNFs). PNFs can undergo malignant transformation to highly metastatic malignant peripheral nerve sheath tumors (MPNSTs) in 5-10% of NF1 patients, with poor prognosis. No effective systemic therapy is currently available for unresectable tumors. In tumors, the NF1 gene deficiency leads to Ras hyperactivation causing the subsequent activation of the AKT/mTOR and Raf/MEK/ERK pathways and inducing multiple cellular responses including cell proliferation. In this study, three NF1-null MPNST-derived cell lines (90-8, 88-14 and 96-2), STS26T sporadic MPNST cell line and PNF-derived primary Schwann cells were used to test responses to AZD8055, an ATP-competitive "active-site" mTOR inhibitor. In contrast to rapamycin treatment which only partially affected mTORC1 signaling, AZD8055 induced a strong inhibition of mTORC1 and mTORC2 signaling in MPNST-derived cell lines and PNF-derived Schwann cells. AZD8055 induced full blockade of mTORC1 leading to an efficient decrease of global protein synthesis. A higher cytotoxic effect was observed with AZD8055 compared to rapamycin in the NF1-null MPNST-derived cell lines with IC50 ranging from 70 to 140 nM and antiproliferative effect was confirmed in PNF-derived Schwann cells. Cell migration was impaired by AZD8055 treatment and cell cycle analysis showed a G0/G1 arrest. Combined effects of AZD8055 and PD0325901 MEK inhibitor as well as BRD4 (BromoDomain-containing protein 4) inhibitors showed a synergistic antiproliferative effect. These data suggest that NF1-associated peripheral nerve sheath tumors are an ideal target for AZD8055 as a single molecule or in combined therapies.

Driver molecular alterations are found in >20% of non-small cell lung cancers (NSCLCs). They specifically target the RAS-MAPK pathway, including the EGFR, KRAS, and BRAF oncogenes. NF1 is a tumor suppressor gene that encodes neurofibromin, an inhibitor of the RAS-MAPK pathway. NF1 mutation detection is challenging owing to its large size, the presence of numerous pseudogenes, and the absence of mutation hotspot. According to The Cancer Genome Atlas data (TCGA), NF1 somatic mutations are found in ~15% of lung cancer. However, NF1 mutations in NSCLCs are not extensively explored in NSCLC to date. We hypothesized that NF1 alterations could define a specific NSCLC population with distinct clinical and molecular profiles. We performed NF1 analysis using next-generation sequencing in NSCLC surgical specimens with known KRAS, EGFR, TP53, BRAF, HER2, and PIK3CA status. We evaluated the specificities of NF1-mutated NSCLCs. Then, we established of NF1-mutated cellular models with different NF1 wild-type (WT) cell lines. We chose two NSCLC cell lines (A549 and NCI H-1703, ATCC), and one nontumorigenic human bronchial epithelial cell line (HBE4-E6/E7-C1, ATCC). Mono- and biallelic NF1 mutations were established using CRISPR-Cas9 and nickase CRISPR-Cas9 technologies. In vitro functional tests and drug screening were performed using these isogenic cell models. In our series of 138 lung adenocarcinoma specimens, 25 tumors showed NF1 mutations (18%) and 11 showed NF1 deletions (8%). NF1 mutations were rarely associated with other mutations. Most of patients with NF1 alterations were males (72%) and smokers (75%). Overall survival and disease-free survival were statistically better in patients with NF1 alteration patients (N=35) than in KRAS mutated patients (N=30) in univariate analysis. There were more NF1 mutations in patients treated by neoadjuvant chemotherapy (p = 0.01). Then, we established cellular models of NF1-mutated NSCLC, using nickase and CRISPR-Cas9 technology. In HBE4-E6/E7-C1 cells, mono- and biallelic NF1 mutations were generated. Loss of NF1 expression was confirmed by Western blot: partial and total loss-of-expression of neurofibromin was found in monoallelic and biallelic NF1 mutated cell lines, respectively. Using Western blot, we showed that pERK/ERK ratio was higher in NF1-mutated cell lines versus WT cell lines, confirming that NF1 loss-of-function triggered RAS-MAPK pathway activation. Transcriptome analysis confirmed this activation. Pharmacologic screen (including MEK inhibitors) in this isogenic NSCLC model will enable us to assess specific vulnerabilities due to NF1 mono- or biallelic mutations. Our results confirm that NF1 is frequently mutated and represents a distinct subtype of NSCLCs. A better comprehension of functional consequences of NF1 mutations, including mono- and biallelic alterations, may open new avenues for NSCLC therapy. Citation Format: Camille Tlemsani, Nicolas Pecuchet, Aurélia Gruber, Audrey Mansuet-Lupo, Diane Damotte, Marco Alifano, Jennifer Varin, Ingrid Laurendeau, Armelle Luscan, Benoit Rousseau, Beatrice Parfait, Ivan Bieche, Audrey Briand, Benoit Terris, Helene Blons, Karen Leroy, Dominique Vidaud, Michel Vidaud, Pierre Laurent-Puig, Eric Pasmant. Characterization of molecular and functional consequences of somatic NF1 mutations in non-small cell lung cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5511.

The hallmark of neurofibromatosis type 1 is the development of dermal and plexiform neurofibromas. Neurofibromatosis type 1 patients with plexiform neurofibromas are at risk of developing malignant peripheral nerve sheath tumors. We applied a 22,000-oligonucleotide microarray transcriptomic approach to a series of plexiform neurofibromas in comparison with dermal neurofibromas, and results were confirmed with real-time quantitative reverse transcription-polymerase chain reaction. Thirteen genes were upregulated and 10 were downregulated in plexiform neurofibromas. The upregulated genes mainly encode molecules involved in cell adhesion, extracellular matrix, fibrogenesis, and angiogenesis. Several CCN gene family members were dysregulated in neurofibromatosis type 1 tumorigenesis; the angiogenic gene CCN1/CYR61 was specifically upregulated in the plexiform neurofibromas; CCN4/WISP1 was upregulated, and CCN3/NOV and CCN5/WISP2 were downregulated in paired comparisons of plexiform neurofibroma and malignant peripheral nerve sheath tumor from the same patients. CCN1 and CCN3 proteins were detected by immunohistochemistry in neurofibromatosis type 1-associated tumors. Upregulation of S100A8, S100A9, and CD36 was also observed and suggests a role of this pathway in inflammation-associated genesis of plexiform neurofibromas. In summary, a limited number of pathways are potentially involved in plexiform neurofibroma development. Some of the genes identified, particularly CCN1, might be useful diagnostic or prognostic markers or form the basis for novel therapeutic strategies.

Gene expression technologies allow the analysis of gene networks whose expression is associated with specific pathological conditions compared with normal tissue. We hypothesized that histologically normal tissue obtained in different ways (percutaneous or surgical liver biopsies), usually used as normal controls in gene expression studies, could have different gene expression patterns. Group A comprised percutaneous liver biopsies in 14 patients with mildly elevated alanine aminotransferase in whom all causes of liver disease had been ruled out. Group B comprised 14 surgical liver biopsies of nontumoral livers. All 28 specimens were histologically normal. Real-time quantitative reverse-transcription polymerase chain reaction were used to compare the messenger RNA expression of 240 selected genes in these two groups. Expression of 26 of the 240 genes was significantly different between groups A and B; 23 genes were up-regulated in group A, while three were down-regulated in group B. The most notable changes occurred in the inflammatory response family genes. Eight genes discriminated perfectly between groups A and B: seven up-regulated genes (PAI1, THBS1, IL8, PTGS2, CXCR4, JUN, and FOS), and one down-regulated gene (IHH). In chronic hepatitis C liver samples, a lower or higher expression of a IL8 was found depending on whether the controls were obtained percutaneously or surgically. Conclusion: Our study demonstrates that histologically normal liver tissue obtained in two different ways (percutaneous or surgical) has different gene expression patterns emphasizing the importance of an adequate selection of histologically normal controls to prevent discordant results in gene expression studies. (Hepatology 2008.)

We describe a large germline deletion removing the NF1 locus, identified by heterozygosity mapping based on microsatellite markers, in an 8-year-old French girl with a particularly severe NF1 contiguous gene syndrome. We used gene-dose mapping with sequence-tagged site real-time PCR to locate the deletion end points, which were precisely characterized by means of long-range PCR and nucleotide sequencing. The deletion is located on chromosome arm 17q and is exactly 7 586 986 bp long. It encompasses the entire NF1 locus and about 100 other genes, including numerous chemokine genes, an attractive in silico-selected cerebrally expressed candidate gene (designated NUFIP2, for nuclear fragile X mental retardation protein interacting protein 2; NM_020772) and four microRNA genes. Interestingly, the centromeric breakpoint is located in intron 4 of the PIPOX gene (pipecolic acid oxidase; NM_016518) and the telomeric breakpoint in intron 5 of the GGNBP2 gene (gametogenetin binding protein 2; NM_024835) coding a transcription factor. As PIPOX and GGNBP2 have the same transcriptional orientation, we postulated, and then confirmed, the existence of a chimeric transcript. This transcript, and/or haploinsufficiency of one or several deleted genes, could explain the clinical severity of the syndrome in this patient.

Objectives— As SDF-1 and its cognate receptor CXCR4 play a key role in the survival and mobilization of immature cells, we examined whether preconditioning of endothelial progenitor cells (EPCs) with SDF-1 could further promote their capacity to enhance angiogenesis. Methods and Results— EPC exposure to 100 ng/mL SDF-1 for 30 min induced a proangiogenic phenotype, with cell migration and differentiation into vascular cords in Matrigel and increased their therapeutic potential in a nude mouse model of hindlimb ischemia. This pretreatment enhanced EPC adhesion to activated endothelium in physiological conditions of blood flow by stimulating integrin-mediated EPCs binding to endothelial cells. Pretreated EPCs showed significantly upregulated surface α4 and αM integrin subunit expression involved in the homing of immature cells to a neovasculature and enhanced FGF-2 and promatrix metalloproteinase (MMP)-2 secretion. All these effects were significantly attenuated by EPC incubation with AMD-3100, a CXCR4 antagonist, by prior HSPGs disruption and by HUVEC incubation with anti–intercellular adhesion molecule1 (ICAM-1) and anti–vascular cell adhesion molecule (VCAM) blocking antibodies. Pretreated EPCs adhered very rapidly (within minutes) and were resistant to shear stresses of up to 2500s −1 . Conclusions— SDF-1 pretreatment during EPC expansion stimulates EPC adhesion to endothelial cells and thus augments the efficiency of cell therapy for ischemic vascular diseases.

OBJECTIVE—In search of functional polymorphisms associated with the genetics of insulin resistance, we studied a variant in the promoter of PIK3CB, the gene coding for the catalytic p110β subunit of phosphatidylinositol (PI) 3-kinase, a major effector of insulin action. RESEARCH DESIGN AND METHODS—The rs361072 C/T variant was selected among single nucleotide polymorphisms of the PIK3CB region because we suspected that its common C allele (allelic frequency ∼50% in Europeans) could create a GATA-binding motif and was genotyped in five cohorts of obese (n = 1,876) and two cohorts of nonobese (n = 1,490) European children. To estimate insulin resistance in these children, the homeostasis model assessment for insulin resistance (HOMA-IR) index was measured in strict nutritional conditions. GATA-binding and functional effects of rs361072 were explored in transfected cell lines and in lymphocytes from obese children. RESULTS—The rs361072 C/T variant was associated with HOMA-IR in the obese children cohorts (1.7 × 10−12 < P < 2 × 10−4 for C/C vs. T/T using regression analysis). HOMA-IR averaged 3.3 ± 0.1 in C/C and 4.5 ± 0.2 in T/T obese children (P = 4.5 × 10−6 by ANOVA). C/T patients had intermediate values. As shown by the interaction between BMI and genotype (P = 2.1 × 10−9), the association of rs361072 with HOMA-IR depended on BMI and was only marginal in nonobese children (P = 0.04). At the molecular level, the C allele of rs361072 was found to create a GATA-binding site able to increase transcription of PIK3CB. CONCLUSIONS—We postulate that the C allele of rs361072 is a causal variant capable of attenuating insulin resistance in obese children through increased expression of p110β.

Background and Aims The gold standard treatment of chronic hepatitis C is the combination of pegylated interferon plus ribavirin. Considering side effects and treatment cost, prediction of treatment response before therapy is important. The aim of our study was to identify a liver gene signature to predict sustained virological response in patients with chronic hepatitis C. Patients and Methods Group A (training set): 40 patients with chronic hepatitis C including 14 non responders and 26 sustained responders. Group B (validation set): 29 patients including 9 non responders and 20 sustained responders. Eleven responder-relapsers were also included. We selected 58 genes from the literature, associated with liver gene expression dysregulation during chronic hepatitis C. Real-time quantitative RT-PCR assays was used to analyse the mRNA expression of these 58 selected genes in liver biopsy specimens taken from the patients before treatment. Results From the Group A data, we identified three genes whose expression was significantly increased in non responders compared to sustained responders: IFI-6-16/G1P3, IFI27 and ISG15/G1P2. These three genes also showed significant differences in their expression profiles between non responders and responders in the independent sample (Group B). Supervised class prediction analysis identified a two-gene (IFI27 and CXCL9) signature, which accurately predicted treatment response in 79.3% (23/29) of patients from the validation set (Group B), with a predictive accuracy of 100% (9/9) and of 70% (14/20) in non responders and sustained responders, respectively. Expression profiles of responder-relapsers did not differ significantly from those of non responders and responders and 73% (8/11) of them were predicted as responders with the two-gene classifier. Conclusion Non responders and responders have different liver gene expression profiles before treatment. The most notable changes mainly occurred in the interferon stimulated genes. Treatment response could be predicted with a two-gene signature (IFI27 and CXCL9).

Background/Aims Endoplasmic reticulum (ER)-related unfolded protein response (UPR) is mediated by PKR-like ER kinase (PERK), ATF6 and IRE1. PERK phosphorylates eukaryotic translation initiation factor-2α (eIF2α) to attenuate protein synthesis, including in NF-κB-dependent antiapoptotic proteins. We hypothesized that an altered UPR in the liver may sensitize cirrhotic livers to LPS-induced, TNFα-mediated apoptosis. Thus, we examined in vivo UPR and NF-κB activity in livers from cirrhotic and normal LPS-challenged rats. Methods Livers were harvested in rats that did or did not receive LPS. Results Under baseline conditions, no UPR was found in normal livers while PERK/eIF2α and ATF6 pathways were activated in cirrhotic livers. After LPS, in normal livers, the PERK/eIF2α pathway was transiently activated. ATF6 and IRE1 were activated. In cirrhotic livers, the PERK/eIF2α pathway remained elevated. ATF6 and IRE1 pathways were altered. LPS-induced, NF-κB-dependent antiapoptotic proteins increased in normal livers whereas their expression was blunted at the posttranscriptional level in cirrhotic livers. Conclusions Cirrhotic livers exhibit partial UPR activation in the basal state and full UPR, although altered, after LPS challenge. Sustained eIF2α phosphorylation, a hallmark of cirrhotic liver UPR, is associated with a lack of LPS-induced accumulation of NF-κB-dependent antiapoptotic proteins which may sensitize cirrhotic livers to LPS/TNFα-mediated apoptosis.

Halofuginone, an inhibitor of collagen synthesis, appears to be a promising antitumoral drug in preclinical studies. We used a relevant rat model of autochthonous, chemically induced, spontaneously metastasizing hepatocellular carcinoma (HCC) to test the efficacy of halofuginone on tumor progression and matrix metalloproteinase (MMP) expression. Following sequential administration of diethylnitrosamine and M nitrosomorpholine for 14 weeks, all animals developed HCC and then received halofuginone or its solvent for 10 weeks. The final number of liver tumors was lower in the halofuginone group than in the solvent group (57.2 ± 4.6 vs 68 ± 5.0; P < .01). The percentage of the lung surface infiltrated by metastasis was much smaller in the halofuginone group (0.3 ± 0.2%) than in the solvent group (13.5 ± 10.1%a; P < .02). MM P-9 activity was decreased in the halofuginone group by 89% and 63% in non-neoplastic parts of the liver and tumor, respectively. The percentage of active MMP-2 was reduced by 90% in non-neoplastic parts of the liver and by 61% in tumors. This was likely subsequent to a decreased expression of both MMP-14 and tissue inhibitor of matrix metal loproteinase-2, which are required for proMMP-2 activation. These results, obtained from a clinically relevant model, further suggest the potential benefit of halofuginone in HCC.

Background & Aims: The molecular mechanisms of hepatocellular carcinoma have been studied, but little is known of the changes in liver gene expression during the different stages of chronic hepatitis C virus (HCV) infection, in particular the transition from mild to moderate fibrosis. Methods: We used real-time quantitative RT PCR to study the messenger RNA expression of 240 selected genes in 2 pools of liver specimens according to the stages of fibrosis (Metavir score; mild fibrosis=F1 and septal fibrosis=F2). Genes whose expression differed between pools (F2 vs F1) by at least 2-fold were selected. In addition, the expression level of these selected genes then was assessed in each of the 62 individual samples (F4, n=6; F3, n=17; F2, n=21; vs F1, n=18). Results: The 22 genes that were up-regulated in the 21 F2 samples relative to the 18 F1 samples mainly encoded genes involved in cytoskeleton ( KRT 19 and SCG 10 ), growth factors/cytokines ( CXCL6 , interleukin 8 [ IL8 ], IL1A , IL2 , and CXCL10 ), or growth factor receptors ( CCR2 , CXCR3 , and CXCR4 ), or were involved in extracellular matrix production ( COL1A1 , CHI3L , and SPP1 ), in extracellular matrix remodeling ( TIMP1 , MMP7 , and MMP9 ), and in cell junction ( ITGA2 and CLDN 4 ). When hierarchically clustering the F2 and F1 samples according to the expression of the 11 most discriminatory genes ( KRT 19 , COL1A1 , STMN2 , CXCL6 , CCR2 , TIMP1 , IL8 , IL1A , ITGA2 , CLDN 4 , and IL2 ), the patient population was categorized into 2 subgroups: F1 and F2. Specifically, 15 of 18 F1 (83%) and 19 of 21 F2 (90%) were classified correctly ( P −5 ). We also studied the messenger RNA expression of these 240 selected genes in normal liver in comparison with F1. Genes dysregulated in the transition from normal liver to F1 mainly were interferon-inducible genes, and therefore were very different from those dysregulated in the transition from F1 to F2. Conclusions: Genes involved in extracellular matrix turnover and immune response are implicated in the transition from mild to moderate fibrosis. Eleven of the genes could form the basis for the gene expression signature of mild versus moderate fibrosis in patients with chronic hepatitis C.

The molecular mechanisms of acute hepatitis C virus (HCV) infection, end-stage hepatitis (cirrhosis), and hepatocellular carcinoma have been extensively studied, but little is known of the changes in liver gene expression during the early stages of liver fibrosis associated with chronic HCV infection, that is, the transition from normal liver (NL) of uninfected patients to the first stage of liver fibrosis (F1-CH-C). To obtain insight into the molecular pathogenesis of F1-CH-C, we used real-time quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) to study the mRNA expression of 240 selected genes in liver tissue with F1-CH-C, in comparison with NL. The expression of 54 (22.5%) of the 240 genes was significantly different between F1-CH-C and NL; 46 genes were upregulated and 8 were downregulated in F1-CH-C. The most noteworthy changes in gene expression mainly affected the transcriptional network regulated by interferons (IFNs), including both IFN-alpha/beta-inducible genes (STAT1, STAT2, ISGF3G/IRF9, IFI27, G1P3, G1P2, OAS2, MX1) and IFN-gamma-inducible genes (CXCL9, CXCL10, CXCL11). Interesting, upregulation of IFN-alpha/beta-inducible genes (but not IFN-gamma-inducible genes) was independent of histological scores (grade and stage of fibrosis) and HCV characteristics (hepatic HCV mRNA levels and the HCV genotype), and was specific to HCV (as compared to hepatitis B virus (HBV)). Other genes dysregulated in F1-CH-C, albeit less markedly than IFN-alpha/beta- and IFN-gamma-inducible genes, were mainly involved in the activation of lymphocytes infiltrating the liver (IFNG, TNF, CXCL6, IL6, CCL8, CXCR3, CXCR4, CCR2), cell proliferation (p16/CDKN2A, MKI67, p14/ARF), extracellular matrix remodeling (MMP9, ITGA2), lymphangiogenesis (XLKD1/LYVE), oxidative stress (CYP2E1), and cytoskeleton microtubule organization (STMN2/SCG10). Thus, a limited number of signaling pathways, and particularly the transcriptional network regulated by interferons, are dysregulated in the first stage of HCV-induced liver fibrosis. Some of the genes identified here could form the basis for new approaches aimed at refining IFN-based therapies for chronic HCV infection.

Purpose: Neurofibromatosis type 1 (NF1) is an autosomal dominant disorder with a complex variety of clinical symptoms. The hallmark of NF1 is the development of heterogeneous benign neurofibromas, which may appear as dermal neurofibromas or plexiform neurofibromas. NF1 patients with plexiform neurofibromas are at risk of developing malignant peripheral nerve sheath tumors. Experimental Design: To obtain additional insight into the molecular pathogenesis of plexiform neurofibromas, we used real-time quantitative reverse transcription-PCR assays to quantify the mRNA expression of 349 selected genes in plexiform neurofibromas in comparison with dermal neurofibromas and patient-matched malignant peripheral nerve sheath tumors. Results: Thirty genes were significantly up-regulated in plexiform neurofibromas compared with dermal neurofibromas. None were down-regulated. The up-regulated genes mainly encoded transcription factors and growth factors and secreted proteins, cytokines, and their receptors, pointing to a role of paracrine and autocrine signaling defects in the genesis of plexiform neurofibromas. We also identified a gene expression profile, based on MMP9, FLT4/VEGFR3, TNFRSF10B/TRAILR2, SHH, and GLI1, which discriminated those plexiform neurofibromas most likely to undergo malignant transformation. Conclusion: Our study has identified a limited number of signaling pathways that could be involved, when altered, in plexiform neurofibroma development. Some of the up-regulated genes could be useful diagnostic or prognostic markers or form the basis of novel therapeutic strategies.

International audience; Human placenta extracts are widely used in clinical and fundamental research, particularly to study the hormonal and exchange functions of the placenta. However, very little is known about the distribution of the main hormone mRNAs in the placenta as a whole. Total placenta extracts are heterogeneous in their cellular components, as they contain material of both fetal and maternal origin, and in their structure. Results vary greatly depending upon the location of the biopsy and the number of biopsies performed. We used real-time quantitative RT-PCR to determine whether transcripts corresponding to the main hormones secreted by the human placenta (e.g. hCG, HPL and PGH) are equally distributed within and between term placentae. We also measured cytokeratin 7 transcripts, which are specifically expressed in the trophoblast, and transcripts corresponding to nuclear receptors PPAR and RXR. A comparison of the results obtained with 12 different samples from each of four normal term placentae revealed that the amounts of transcripts differ considerably within and between each placenta. This emphasizes the need to study large numbers of samples when looking for significant differences in gene expression.

Background & Aims: "Telangiectatic focal nodular hyperplasia" designate atypical lesions considered as variants of focal nodular hyperplasia (FNH). However, because "telangiectatic FNH" share several morphologic patterns with hepatocellular adenomas, classification of such lesions deserve further clarification. Therefore, the aim of the present study was to reconsider the classification of telangiectatic FNH with the help of a molecular approach. Methods: Ten telangiectatic FNH, 6 typical FNH, and 6 hepatocellular adenomas were studied. DNA, RNA, and protein from each lesion were extracted. Clonality was assessed by the study of the X chromosome inactivation pattern (HUMARA assay). Angiopoietin ( ANGPT-1 and ANGPT-2 ) mRNA, genes the expression of which is typically modified in FNH, were quantified by a real-time RT-PCR procedure. Protein profiles were analyzed by SELDI-TOF PROTEINCHIP (Cyphergen Biosystem, Inc., Fremont, CA) technology. Results: Although all informative cases of FNH (5 of 6) and hepatocellular adenomas (6 of 6) were polyclonal and monoclonal, respectively, clonal analysis showed a nonrandom pattern of X chromosome inactivation consistent with a monoclonal lesion in 6 of 8 cases of telangiectatic FNH. The mean value of the ANGPT-1/ANGPT-2 mRNA ratio was 21.4 in FNH, 2.6 in adenomas, and 2.1 in telangiectatic FNH ( P ≤ 0.001 in telangiectatic FNH vs. FNH). SELDI-TOF PROTEINCHIP profiling and hierarchical clustering analysis showed that all except 1 telangiectatic FNH clustered within the group of hepatocellular adenomas. Conclusions: These results show that telangiectatic FNH display a molecular pattern closer to that of hepatocellular adenomas than to FNH and suggest that these lesions should instead be referred to as "telangiectatic hepatocellular adenomas."

Basic fibroblast growth factor (FGF-2) activates its high-affinity receptors (FGFRs) but also acts through interaction with heparan sulfate proteoglycans (HSPG). Exogenous polysaccharides also modulate the angiogenic activity of FGF-2. We investigated the effect and mechanism of action of a low molecular weight fucoidan derivative (LMWF) on tube formation by human endothelial cells. LMWF has a better arterial antithrombotic potential in animals than low molecular weight heparin (LMWH). After stimulation of human umbilical vein endothelial cells (HUVEC) by FGF-2 and LMWF (or LMWH), we observed 1) using flow cytometry, an increase in the amount of the alpha6 integrin subunit; 2) using quantitative reverse transcription-polymerase chain reaction, an increase in alpha6 mRNA (higher with LMWF than with LMWH); and 3) using a Matrigel model, an increase in vascular tube formation (also higher with LMWF than with LMWH). A direct link between alpha6 overexpression and vascular tube formation was confirmed by use of an anti-alpha6 antibody: in its presence, there was no capillary network formation on Matrigel. Unexpectedly, an anti-FGFR blocking antibody had no effect on alpha6 over-expression, whereas stripping off the heparan sulfate with heparitinases abolished overexpression. Overall, our data suggest that FGF-2 stimulates alpha6 over-expression in HUVEC, through HSPG but independently from FGFR, and that LMWF (or LMWH) modulates this interaction. Expression of heparan sulfate proteoglycan increases after ischemic injury. Given its antithrombotic properties and its ability to potentiate tube formation of endothelial cells, LMWF may have to be considered for revascularization of ischemic areas.

The aim of this study was to develop and validate a molecular index for the diagnosis of hepatocellular carcinoma (HCC) based on genes whose specificity and level of expression are the most discriminating for the diagnosis of HCC. The level of expression of 219 genes was assessed with a real-time reverse transcription-polymerase chain reaction approach in a training set of samples including normal livers (15), cirrhosis (12), and HCC (16). The most informative genes were selected for the molecular index. This index was prospectively validated in a new set of 40 samples (testing set) and in a set of 45 cirrhotic macronodules. 44 out of the 219 genes were differentially expressed in HCC. 13 out of these 44 genes were finally selected for the molecular index according to their diagnostic performance and after exclusion of most redundant genes. Using this index, 42 out of 43 samples of the training set and 39 out of the 40 samples of the testing set were correctly ranked as HCC or not HCC (normal liver or cirrhosis). The index also enabled correct ranking of 44 out of 45 cirrhotic macronodules into 2 groups: benign (including macroregenerative and dysplastic macronodules) and malignant macronodules. This molecular diagnostic index is an efficient tool both for identification of overt HCC as well as minute lesions (cirrhotic macronodules). It might be useful to correctly diagnose borderline lesion or small well-differentiated hepatocellular carcinomas whose diagnosis is often difficult on a histopathological basis.

Cirrhosis is considered to be the precursor of most hepatocellular carcinomas. To gain insight into the early molecular mechanisms of liver carcinogenesis, this study compared, using real-time quantitative reverse transcriptase-polymerase chain reaction (RT-PCR), the expression levels of 31 selected genes in normal livers, cirrhotic nodules, and hepatocellular carcinomas. Since cirrhosis is composed of a mixture of polyclonal and monoclonal nodules, gene expression levels were also compared according to the clonal status of the cirrhotic nodules. The expression of eight of the 31 genes studied was significantly increased (NEGF2, ANGPT1, ARF, KRT19, SFN, CLDN4, MMP7, and ETV4) in cirrhotic nodules compared with normal liver, while only one was decreased (LYVE1). The same trend of variation was observed in cirrhosis and hepatocellular carcinomas for all of these genes except KRT19. When gene expression variation was compared according to the clonal status of cirrhotic nodules, only the LYVE1 expression level was significantly different. The LYVE1 gene expression level decreased progressively from polyclonal cirrhotic nodules to monoclonal cirrhotic nodules (polyclonal nodules 0.39 +/- 0.25; monoclonal nodules 0.20 +/- 0.14; p < 0.05) and to hepatocellular carcinoma (0.07 +/- 0.1). In conclusion, this study highlights the fact that among genes strongly dysregulated in hepatocellular carcinoma, some are already abnormally expressed in cirrhosis. The decrease in the expression level of one of these genes, LYVE1, was associated with monoclonality in cirrhotic nodules.

KIS is the only known protein kinase that possesses an RNA recognition motif. This original structure indicates a role for KIS in the maturation of RNAs possibly by phosphorylating and regulating the activities of RNA associated factors. Another function of KIS has recently been unravelled--it negatively regulates the cdk inhibitor p27Kip1 and thus promotes cell cycle progression through G1. In order to explore the functional expression of this kinase, we quantified its mRNA in a wide range of rat and human tissues, during development and in tumors. In both species, the highest level of KIS gene expression was in adult neural tissues. Interestingly, within the adult rat brain, KIS mRNA is enriched in several areas including the substantia nigra compacta and nuclei of the brain stem. Furthermore, KIS gene expression increases dramatically during brain development. Altogether our results point to a ubiquitous function for KIS together with a particular implication during neural differentiation or in the function of mature neural cells. No dysregulation of KIS gene expression was detected in human tumors from breast, bladder, prostate, liver and kidney origins. On the other hand, the KIS gene was overexpressed in NF1-associated plexiform neurofibromas and malignant peripheral nerve sheath tumors (MPNSTs) as compared to dermal neurofibroma which suggests a possible implication of KIS in the genesis of NF1-associated tumors.